Gut microbiota and metabolic function in leeches with distinct feeding niches

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Understanding the composition of microbial communities in leech guts may reveal important insights into their ecological adaptations and feeding strategies. Objective This study aimed to compare the gut microbiota of hematophagous (blood-feeding) and non-hematophagous leeches, to identify microbial signatures associated with dietary divergence and niche specialization. Methods Gut contents were collected from representative species of hematophagous and non-hematophagous leeches. Microbial community composition was analyzed via 16S rRNA gene sequencing. Results A total of 751 microbial species were identified, encompassing 535 genera, 332 families, 203 orders, 86 classes, and 39 phyla. At the phylum level, Proteobacteria and Firmicutes were significantly higher relative more abundant in hematophagous leeches, whereas Bacteroidetes predominated in non-hematophagous counterparts. At the genus level, hematophagous leeches exhibited higher abundances of Elstera , norank_f__Rhodospirillaceae, Aeromonas , f__Rhodospirillales, o__Peptostreptococcales-Tissierellales, and unclassified_o__Oscillospirales. In contrast, unclassified_f__Comamonadaceae, Nubsella , Cetobacterium , Mucispirillum , norank_f__Peptostreptococcaceae, and Bacteroides were enriched in non-hematophagous leeches. Functional prediction analysis revealed significant differences in five key metabolic pathways between the two groups: lipid transport and metabolism, amino acid transport and metabolism, nucleotide transport and metabolism, translation and ribosomal structure biogenesis, and coenzyme transport and metabolism. Conclusion Distinct taxonomic and functional profiles characterize the gut microbiota of hematophagous and non-hematophagous leeches. These results provide microbiological evidence for dietary specialization in leeches and offer a scientific basis for guiding domestication and artificial breeding strategies. Gut microbiota Leeches Feeding strategies 16S rRNA sequencing Microbial metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Microorganisms are ubiquitous, inhabiting diverse environments and thriving within complex ecosystems. Among these the gut harbors, the highest density of microbial cells, forming intricate and dynamic communities collectively termed the gut microbiota [ 1 ] . Extensive research has demonstrated that the gut microbiota plays a crucial role in synthesizing essential nutrients, such as B vitamins and amino acids ,which the host cannot produce endogenously [ 2 ] . As a vital physiological component across species, the gut microbiota influences host metabolism, immune function, and homeostasis. Its composition is shaped by a multitude of environmental factors, including pH, oxygen availability, nutrient supply, water activity, and temperature, all of which influence microbial survival, populations dynamics, functionality, and ecological interactions [ 3 ] . Leeches (class Hirudinea, phylum Annelida) are predominantly freshwater invertebrates, exhibiting a broad range of feeding strategies, from hematophagous parasitism to predation on small aquatic invertebrates [ 4 ] . Hematophagous leeches possess specialized anterior suckers equipped with three chitinous jaws, which allow them to create a triradiate incision in their host’s skin. During feeding, they secrete potent anticoagulants, such as hirudin, decorsin, and destabilase, into the wound, preventing blood coagulation within their digestive tract [ 5 , 6 ] . Notably, symbiotic gut bacteria play an essential role in facilitating the long-term storage and digestion of ingested blood by producing antimicrobial compounds that inhibit spoilage and enzymes (e.g., hemolysins, collagenases, and proteases), that aid in nutrient breakdown and absorption [ 7 – 9 ] . Non-hematophagous leeches typically prey on small invertebrates such as, snails and earthworms. Interestingly, previous studies have shown that the heavy metal content (e.g., Cr, Pb, Zn, Cu) in non-hematophagous leeches is negatively correlated with sediment pH, total nitrogen, and organic matter, but positively correlated with the water pH. This suggests that their gut microbiota may contribute to heavy metal tolerance and potentially promote ecosystem stability [ 10 ] . Analogous studies in earthworms has revealed that amendments such as corn cob charcoal can significantly increase the abundance of nitrogen-cycling bacteria in the gut, thereby enhancing organic matter degradation and supporting environmental resilience [ 11 ] . Investigating the gut microbiota of non-hematophagous leeches may thus offer new insights into microbial mechanisms of heavy metal tolerance and environmental adaptation. Leeches have evolved highly specialized feeding habits that differ significantly from other annelids, with most species exhibiting narrow dietary preferences limited to either hematophagy, bodily fluid consumption, or predation, each accompanied by distinct morphological adaptations [ 12 ] . Hirudo nipponia possesses a relatively large oral opening, equipped with three crescent-shaped jaws [ 13 ] . Each jaw bears a row of fine denticles along the chitinous ridge, and further identified conical papillae at the jaw tips, which function in secretion, facilitating skin penetration and anticoagulant release during blood-feeding [ 14 ] . In contrast to hematophagous species, members of the genus Whitmania (e.g., Whitmania pigra ) specialize in feeding on molluscan body fluids, exhibiting modified jaw morphology where the three jaws bear only two rows of undifferentiated tooth plates adapted for tissue maceration and fluid extraction from prey like snails [ 13 ] . This divergence in jaw morphology reflects the distinct feeding mechanisms between hematophagous and fluid-feeding leeches. In a taxonomic study of two leech groups, Whitmania pigra and Hirudo nipponia were found to share a common ancestor (~ 50 MYA), while Hirudo manillensis diverged earlier. This suggests that blood-feeding behavior was likely present in the ancestral leech but was lost in the Whitmania pigra lineage [ 15 ] . This suggests that during long-term evolution, environmental and genetic factors drove significant divergence in feeding strategies between these two leech groups, while certain behavioral traits remained conserved. Genomic studies of Zeylanicobdella arugamensis reveal a closer phylogenetic relationship with Hirudo nipponia , Whitmania acranulata , Whitmania pigra , and Whitmania laevis , forming a sister clade [ 16 ] . Ancestral Whitmania species may have retained parasitic behavior, with its loss in some lineages likely linked to shifts in food availability, further highlighting the genetic similarities underlying their divergent ecological adaptations. Despite these emerging findings, comparative studies examining the gut microbiota of hematophagous and non-hematophagous leeches remain scarce. This study employs16S rRNA gene sequencing to analyze and compare the gut microbial community of wild hematophagous and non-hematophagous leeches. By exploring the relationships between feeding niches and gut microbiota composition, we aim to elucidate the microbial basis of dietary specialization in leeches and provide a scientific foundation for conservation, domestication, and sustainable aquaculture practices. 2. Materials and methods 2.1 Sample collection Wild leech specimens were collected from shallow waters of the Chaobai River (Baodi District, Tianjin, China; coordinates: 117˚16'31"E, 39˚41'43"N). Three healthy adult individuals were selected from each group: hematophagous leeches ( Hirudo nipponia and Hirudo tianjinensis ) and non-hematophagous leeches ( Whitmania pigra , Whitmania laevis , and Whitmania acranulata ). Prior to processing, each leech was surface-sterilized by immersion in 75% ethanol for 1 minute, followed by three sequential rinses in sterile water, each lasting 1 minute. After sterilization, specimens were transferred individually into 1.5 mL centrifuge tubes, flash-frozen in liquid nitrogen, and stored at − 80 ℃ until further analysis. 2.2 DNA extraction amplification and sequencing Total microbial DNA was extracted from frozen leech samples using the E.Z.N.A.® Soil DNA Kit (OMEGA, USA) following the manufacturer’s protocol. DNA integrity was verified via 1% agarose gel electrophoresis, and DNA concentration and purity (A260/A280 ratio) were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). 2.3 16S rRNA gene The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [ 17 ] . PCR was performed under the following conditions: initial denaturation at 95 ℃ for 3 minutes, followed by 30 cycles of 95 ℃ for 30 seconds, 45 ℃ for 30 seconds, 72 ℃ for 45 seconds, with a final extension at 72 ℃ for 10 minutes. Triplicate PCR reactions were pooled for each sample, and amplicons were purified using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). The purified PCR products were quantified using the QuantiFluor™-ST system (Promega, USA), normalized, and sequenced on the Illumina MiSeq PE300 platform by Shanghai Meiji Biological Pharmaceutical Technology Co., Ltd. 2.4 Bioinformatics and statistical analysis Paired-end reads were merged using FLASH ( https://ccb.jhu.edu/software/FLASH/index.shtml , v1.2.11) and quality-filtered using Fastp ( https://github.com/OpenGene/fastp , v0.19.6) Operational taxonomic units (OTUs) were clustered at a 97% similarity using UPARSE. Diversity indices and sequencing depth were assessed based on OTU clustering results. Taxonomic classification sequences was performed using the Bayesian algorithm of the RDP Classifier ( https://sourceforge.net/projects/rdp-classifier , v2.13) with reference to the Silva 16S rRNA database ( https://www.arb-silva.de , v138). Bacterial community composition at various taxonomic levels was analyzed using Qiime ( http://qiime.org/install/index.html , v1.9.1). Statistical analyses were conducted using the Kruskal-Wallis and Wilcoxon rank-sum test, threshold of p < 0.05. Principal Component Analysis (PCA) was performed via the Majorbio Cloud Platform ( https://cloud.majorbio.com ), and functional prediction of microbial communities was conducted using PICRUSt ( http://picrust.github.io/picrust , v 1.1.0). 3. Results 3.1 Sequencing data and species statistics Illumina MiSeq sequencing of the gut microbiota from hematophagous and non-hematophagous leeches generated a total of 1,130,336 raw reads. After quality control and, sequence, 1,107,010 high-quality reads (average length: 225 bp) were retained for further analysis. Rarefaction curves based on the Sob index plateaued with increasing sequencing depth (Fig. 1 A), indicating sufficient coverage and comprehensive representation of microbial communities. Comparative analysis revealed, that hematophagous leeches harbored significantly lower gut microbiota diversity (as measured by ACE index) compared to non-hematophagous leeches, likely reflecting dietary niches specialization. Clustering at 97% sequence similarity identified 751 microbial species, encompassing 535 genera, 332 families, 203 orders, 86 classes, and 39 phyla. Venn analysis revealed 142 OTUs between the two leech types (Fig. 1 B), highlighting both common and distinct divergent microbial features. Ten bacterial phyla were found in both leech groups, including Proteobacteria, Bacteroidota, Firmicutes, Myxococcota, Fusobacteriota, Patescibacteria, Verrucomicrobiota, Desulfobacterota, Synergistota, and Spirochaetota. At the phylum level, hematophagous leeches were dominated by Proteobacteria (63%), Firmicutes (13%), Bacteroidota (10%), and Myxococcota (10%), whereas non-hematophagous leeches showed enrichment in Proteobacteria (39%), Bacteroidota (33%), and Myxococcota (10%) (Figs. 1 C and 1 D). At the genus level, hematophagous leeches were characterized by norank_f__Rhodospirillaceae (17%), Elstera (12%), Aeromonas (11%), unclassified_o__Oscillospirales (7%), unclassified_f__Rhizobiaceae (6.5%), and unclassified_f__Rhodospirillaceae (6%). In contrast, non-hematophagous leeches were dominated by unclassified_f__Comamonadaceae (13%), P3OB-42 (10%), Bacteroides (8%), Nubsella (6%), unclassified_c__Alphaproteobacteria (5%), unclassified_o__Bacteroidales (5%), Phreatobacter (4%), and Parabacteroides (3%). (Figs. 1 E and 1 F). Shared genera such as P3OB-42 and Nubsella suggest the existence of a core microbiota that support fundamental gut functions across different feeding strategies. 3.2 Alpha and beta diversity analysis of gut microbiota Alpha diversity metrics revealed that non-hematophagous leeches had significantly higher values across all indices: Chao1 (2479.65) ACE (2530.25), Sobs (2220), and Shannon (25.3), compared to hematophagous leeches Chao1 (395.6), ACE (408.92), Sobs (362), (408.92), and Shannon (11.96), and indices (Figs. 2 A– 2 D). This suggests a more diverse and evenly distributed gut microbial community in non-hematophagous leeches. Beta diversity analysis revealed clear distinctions between the gut microbiota of the two leech types. Principal Coordinates Analysis (PCoA) showed that PCO1 and PCO2 together explained 45.14% of the observed variation. While non-hematophagous leeches exhibited tight clustering, indicating greater intra-group similarity, hematophagous leeches displayed more dispersed patterns, suggesting higher inter-individual variation (Fig. 2 E). Community dispersion analysis revealed 33.21% of variation, indicating that dietary habit plays a significant role in shaping the gut microbial communities. Non- hematophagous leeches exhibited higher microbial abundance than hematophagous leeches (Fig. 2 F). 3.3 LEfSe and LDA analysis LEfSe analysis identified significant differences in gut microbial composition between the two leech types. The phylogenetic tree illustrated the species divergence across different taxonomic levels, highlighting distinctions in the gut microbiota between two types of leeches. A total of 63 microbial taxa at varying taxonomic levels were identified to be significantly different in non-hematophagous leeches compared to hematophagous ones (Figure. 3A). Using LEfSe analysis (LDA score threshold = 2), significantly divergent microorganisms between the two dietary groups were identified. Among these, the most prominently enriched taxa included o__Bacteroidales, o__Burkholderiales, f__Comamonadaceae, f__Rhodospirillaceae, and g__norank_f__Rhodospirillaceae (Figure. 3B). These results underscore the influence of long-term dietary and ecological divergence on gut microbiota composition, with non-hematophagous leeches exhibiting significantly higher overall gut microbial diversity. 3.4 KEGG functional prediction PICRUSt2-based KEGG pathway analysis indicated that both types shared expression of nine core metabolic pathways, including general metabolism, secondary metabolite biosynthesis, microbial metabolism, amino acid biosynthesis, carbon metabolism, ABC transporters, two-component systems, quorum sensing, and ribosome (Fig. 4 A). Despite their different diets, these conserved pathways suggest that both leech types maintain similar basic metabolic regulatory mechanisms. However, notably differences were observed in pathways related to lipid, amino acid, nucleotide, and coenzyme transport and metabolism, as well as translation and ribosomal structure biogenesis (Fig. 4 B). Analysis of metabolic pathway-related enzymes revealed higher abundances of DNA-directed DNA polymerase, NADH: ubiquinone oxidoreductase, and DNA-directed RNA polymerase in blood-feeding leeches compared to non-blood-feeding species. Conversely, non-blood-feeding leeches exhibited elevated levels of chaperonin ATPase, histidine kinase, and peptidylprolyl isomerase. These enzyme-specific expression patterns are not solely attributed to differences in gut microbiota but may also be linked to leech feeding habits (Fig. 4 C). These differences likely reflect adaptations to distinct nutritional strategies: hematophagous leeches rely on microbiota capable of degrading blood proteins, whereas non-hematophagous leeches are adapted to environments where they prey on various small invertebrates, with metabolic capacities favoring energy storage and assimilation of diverse animal-derived nutrients. Thus, gut microbiota plays a pivotal role in shaping feeding strategies and, ecological adaptability in leeches. 3.5 Genus-level microbial differences and co-occurrence patterns Evolutionary analysis of microbial genera revealed distinct associations in the two leech types hematophagous leeches, Treponema was closely related to Akkermansia and Peptoniphilus , with Peptoniphilus also closely associated with related to Fretibacterium . In non-hematophagous leeches, notable relationships included Denitratisoma - Aquabacterium , Wolinella-Desulfovibrio , Nubsella - Acetobacteroides , and Rikenella - Mucinivorans (Fig. 5 A). The observed evolutionary proximity among these microbial genera may be attributed not only to genetic relatedness, but also appears inextricably linked to their host’s feeding ecology. Co-occurrence network analysis showed that approximately 22% of microbiota were shared between the two leech types, 17% were unique to hematophagous leeches, and 61% were enriched in non-hematophagous leeches (Fig. 5 B). These findings further support the idea that environmental and dietary factors shape distinct but overlapping microbial ecosystems. 4. Discussion In this study, we observed distinct gut microbiota compositions between hematophagous and non-hematophagous leeches. At the phylum level, the gut microbiota of hematophagous leeches was dominated by Proteobacteria (63%), Firmicutes (13%), Bacteroidota (10%), and Myxococcota (10%). In contrast, non-hematophagous leeches harbored Proteobacteria (39%), Bacteroidota (33%), and Myxococcota (10%), with a notably lower abundance of Firmicutes. Firmicutes are essential for digestion complex organic matter and enhanced energy utilization, prior studies have shown that the Firmicutes/Bacteroidota (F/B) ratio is closely associated with the host’s metabolic state, influencing factors such as lipid deposition, nutrient absorption, and overall physiological condition [ 18 ] . Typically, a higher F/B ratio correlates with increased energy extraction from diet, while a lower ratio supports microbial balance and metabolic efficiency, and is also linked to the host growth rate, fat deposition, and susceptibility to diseases [ 19 ] . Previous studies suggest that changes in specific Firmicutes, Bacteroidetes, and Actinobacteria OTUs may elevate swine niacin levels, subsequently modulating key digestive enzyme (amylase, trypsin, and lipase) activities [ 20 ] . Lactobacillus reuteri ATCC PTA 4659 promotes intestinal barrier function through upregulation of epithelial heat shock proteins (HSP25 and HSP70) [ 21 ] . This probiotic strain enhances mucosal integrity by reinforcing tight junction proteins, thereby optimizing nutrient absorption efficiency in leech hosts. In this study, hematophagous leeches exhibited a significantly higher abundance of Firmicutes compared to non-hematophagous species, potentially reflecting their adaptation to a protein- and hemoglobin-rich blood diet. The higher Firmicutes content may facilitate efficient digestion and nutrient uptake. In contract, non-hematophagous leeches, which feed on small invertebrates and microbial matter, showed a higher abundance of Bacteroidota, which are effective in degrading complex carbohydrates and polysaccharides, while maintaining gut microbial homeostasis. Among Proteobacteria, genera such as Bifidobacterium contribute to physiological balance and immune modulation. However, other members, including Escherichia coli and Salmonella , are pathogenic, while some, like Rhodospirillum , possess nitrogen fixation capabilities [ 22 ] . Several genera within Proteobacteria, such as Bacteroides , Rhodospirillum , Bifidobacterium , Faecalibacterium , and Enterobacter , ferment indigestible oligosaccharide carbohydrates, leading to the production of short-chain fatty acids (SCFAs) such as butyrate, propionate, and acetate. These SCFAs serve as a rich energy source for the host [ 23 , 24 ] . The higher Proteobacteria abundance in hematophagous leeches may enhance their ability to digest and absorb lipids derived from blood. Bacteroidota play a key role in maintaining microbial balance and supporting metabolic processes. For instance, Bacteroides thetaiotaomicron can stimulate the expression of small proline-rich protein 2A (sprr2A), which is essential for maintaining epithelial villus tight junctions [ 25 ] . In this study, Bacteroidota were significantly more abundant in the guts of non-hematophagous leeches, suggesting their involvement in carbohydrate and cellulose digestion. A study found that Bacteroidota is associated with metabolic dysfunction-associated steatohepatitis (MASH). Specifically, an enzyme annotated as a β-lactamase in Bacteroidota uniformis was identified to catalyze the biosynthesis of 3-succinylated bile acids [ 26 ] . Similar patterns have been observed in earthworms, where Bacteroidota, Firmicutes, Actinobacteria, and Proteobacteria dominate before sludge feeding, while after feeding, the composition shifts, with increases in Firmicutes and Proteobacteria and reductions in Bacteroidota and Actinobacteria [ 27 ] aligning with our findings. At the genus level, specific microbial groups appeared to play functional roles. In hematophagous leeches, g__norank_f__Rhodospirillaceae was the most abundant. These photosynthetic bacteria produce, B vitamins, pantothenic acid, coenzyme Q, and pigments such as bacteriochlorophyll and carotenoids, which possess bioactive and recyclable properties [ 28 ] . Such metabolites may support host gut functions. Elstera , also enriched in hematophagous leeches, contributes to energy production via both aerobic and fermentative pathways and supports maintenance of the gut’s anaerobic environment [ 29 ] . Its abundance has been shown to increase under glucose exposure, from 0% to 12.7%, suggesting its role in carbon metabolism. Aeromonas another genus found in hematophagous leeches, produces a wide variety of heterogeneous virulence factors, such as membrane components, toxins, and enzymes, which can cause intestinal infections and, if uncontrolled, may result in systemic effects [ 30 ] . Notably, Aeromonas species exhibit anticancer properties. In particular, Aeromonas veronii has been identified as a promising L-glutaminase producer, with this enzyme showing therapeutic efficacy against acute lymphoblastic leukemia (ALL) [ 31 ] . However these microorganisms also have the potential to cause intestinal infections, which, if not effectively controlled, may progress and disseminate systemically, resulting in associated clinical symptoms [ 32 ] . Additionally, the high abundance of unclassified_o__Oscillospirales, commonly found in herbivores and omnivores, has been associated with intestinal inflammation. Environmental factors such as light exposure can significantly alter gut microbiota composition, reducing Lactobacillus , Butyricicoccus , and Selenomonas , while increasing Bifidobacterium , Oscillospirales , and others [ 33 ] . In non-hematophagous leeches, g__unclassified_f__Comamonadaceae was the most abundant group. This family is involved in glucose and lipid metabolism. It was found that the lack of the PPARδ gene exacerbated the progression of alcoholic fatty liver disease induced by a high-fat diet, leading to a decrease in the abundance of norank_f__Eubacterium_coprostanoligenes_group and Alloprevotella , while the abundance of Acidobacteria , unclassified_f__Comamonadaceae, unclassified_c__Alphaproteobacteria, unclassified_f__Beijerinckiaceae, unclassified_f__Caulobacteraceae, unclassified_c__Bacteroidia, and Bosea increased [ 34 ] . The presence of Nubsella in hematophagous leeches may enhance microbial diversity and environmental adaptability, as shown in studies where its absence reduced host fitness in Phasmotaenia lanyuhensis [ 35 ] . The presence of Nubsella enhances microbial diversity and improves the host’s environmental adaptability, while Bacteroides shows significantly higher abundance in the gut microbiota of hematophagous leeches. Members of this family are potential colonizers of the gut and constitute a major portion of the gut microbiota [ 36 ] . These Gram-negative obligate anaerobes play various roles in the human gut microbiota and are key participants in maintaining the intestinal microbial food web [ 37 ] . Gut microbial differences are often reflected in host metabolic pathways [ 38 ] . Glucose and lipid metabolites are transported intra- and intercellularly and transformed into functional molecules in specific organelles [ 39 ] . Studies have shown that intestinal lipoprotein cholesterol regulates hepatic cholesterol biosynthesis and serves as the sole route for eliminating serum cholesterol via bile acid conversion, highlighting the intestine’s central role in cholesterol metabolism [ 40 ] . In this study, pathways related to lipid transport and metabolism were more prominently expressed in hematophagous leeches, likely due to the higher abundance of lipid-assimilating Proteobacteria. Amino acid transport and metabolism pathways were more active in non-hematophagous leeches, consistent with their elevated Bacteroidota levels. Amino acids not only serve as protein building blocks and energy sources but also act as precursors for bioactive metabolites and regulators of key cellular signaling pathways [ 41 ] . In intestinal microbiota studies of Rhizomys spp , Bacteroidota exhibited notably high abundance. KEGG analysis revealed carbohydrate and amino acid metabolism as the most enriched pathways [ 42 ] , likely mediated by Bacteroidota metabolic functions. Nucleotides are crucial for the transfer and transformation of chemical energy within cells, most notably in the form of the energy currency ATP [ 43 ] . In addition, nucleotides serve as precursors for certain hormones, act as signaling molecules, and are essential components of DNA and RNA [ 44 ] . Comparative analysis revealed significantly elevated expression of nucleotide transport and metabolism pathways in the intestinal tissues of non-hematophagous leeches relative to their hematophagous counterparts, potentially associated with increased Firmicutes abundance. For example, Clostridium thermocellum is capable of cellulose degradation, and this bacterium can modulate glucose transporter A or glucose transporter B, thereby providing energy support for nucleic acid transport and metabolic pathways in the organism [ 45 ] . The study reveals that DNA-directed DNA polymerase exhibits higher expression in blood-feeding leeches, likely due to the dominance of Proteobacteria (e.g., Aeromonas hydrophila , a major cause of bacterial septicemia) in their gut microbiota [ 46 ] . This enzyme may slow replication fork progression during DNA damage response, ensuring stable general metabolism and maintaining a healthy gut microenvironment [ 47 ] . In non-blood-feeding leeches, higher Bacteroidota abundance is observed, potentially linked to their diet (e.g., snails, earthworms), which elevates intestinal lipid levels and increases the risk of inflammatory bowel disease (IBD). Concurrently, elevated Chaperonin ATPase expression in these leeches correlates with mitochondrial dysfunction and IBD [ 48 ] . The interplay between these factors helps balance the gut microbiota and microenvironment. The functional capacity of the gut microbiota is primarily determined by its composition, which is influenced by multiple factors including host genetic background, health status, social behavior, and environmental habitat conditions [ 49 ] . In conclusion, differences in gut microbiota composition between hematophagous and non-hematophagous leeches influence metabolic profiles, energy utilization strategies, and environmental adaptability. These microbial communities play essential roles in shaping the host’s physiological and metabolic functions. 5. Conclusions The composition of the intestinal microbiota plays a pivotal role in shaping host physiological activities. In this study, Proteobacteria were the dominant phylum in hematophagous leeches, whereas Bacteroidota were more prevalent in non-hematophagous leeches. Notably, hematophagous leeches exhibited the highest relative abundance of Proteobacteria, while non-hematophagous leeches harbored a greater proportion of unclassified bacterial taxa. Alpha diversity metrics, including the Chao1 and Shannon indices, revealed significantly higher microbial diversity in non-hematophagous leeches compared to their hematophagous counterparts. From a functional perspective, lipid transport and metabolism pathways were more prominently expressed in hematophagous leeches, aligning with their protein- and lipid-rich blood-based diet. In contrast, pathways related to amino acid transport and metabolism were more active in non-hematophagous leeches, reflecting their adaptation to diverse organic matter diets. These findings suggest that non-hematophagous leeches possess a broader metabolic flexibility and stronger environmental adaptability. In summary, this study provides novel insights into the gut microbial communities of leeches with different feeding strategies and highlights their potential roles in host metabolism and ecological adaptation. These results offer a theoretical foundation for future research on leech domestication, conservation, and sustainable utilization. Declarations Funding This research was funded by the National Natural Science Foundation of China (32260132), the Yunnan Provincial University Serving Key Industry Science and Technology Special Project (FWCY-ZD2024009), the Joint Special Project for Basic Research of Local Universities in Yunnan Province (202301BA070001-105 and 202101BA070001-164) , the Frontier Research Team of Kunming University 2023, and the Yunnan International Joint Laboratory with South and Southeast Asia for the Integrated Development of Animal-Derived Anti-Thrombosis Chinese Medicine (202503AP140025). Data availability statement The raw sequencing data generated in this study have been deposited in the China National Center for Bioinformation (CNCB, https://ngdc.cncb.ac.cn) under the accession number CRA029752. Author contributions Y.C., J.L., H.C., and Z.L.: Conceptualization, Supervision, Writing - original draft, Writing - review & editing. Z.L., and X.T.: Funding acquisition, Project administration. 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Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 16 Jan, 2026 Reviews received at journal 05 Jan, 2026 Reviews received at journal 05 Jan, 2026 Reviewers agreed at journal 16 Dec, 2025 Reviewers agreed at journal 16 Dec, 2025 Reviewers invited by journal 16 Dec, 2025 Editor assigned by journal 29 Nov, 2025 Submission checks completed at journal 29 Nov, 2025 First submitted to journal 17 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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14:02:41","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116246,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8136685/v1/e6a8002b3ced11e2be5962a3.html"},{"id":98625149,"identity":"ad0aac26-ef51-45de-8773-74117568e417","added_by":"auto","created_at":"2025-12-19 17:08:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2871580,"visible":true,"origin":"","legend":"\u003cp\u003eComparative gut microbiota analysis between hematophagous and non-hematophagous leeches. (A) Rarefaction curves showing sufficient sequencing depth. (B) Venn diagram showing 142shared OTUs. (C, D) Phylum-level taxonomic composition. (E, F) Genus-level community structures.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8136685/v1/82433eb42c7bce1f8ad63944.png"},{"id":98522841,"identity":"782abd8b-44a3-4751-83bf-7ea6e2fad4ce","added_by":"auto","created_at":"2025-12-18 14:02:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1523462,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha and beta diversity of gut microbiota. (A-D) Alpha diversity indices: Chao1, Ace, Sobs, and Shannon. (E) Community dispersion analysis. (F) Principal Coordinates Analysis (PCoA).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8136685/v1/3ee1d300a843864357984165.png"},{"id":98522842,"identity":"b87e4c81-77d3-41fa-8375-3982a93d42c6","added_by":"auto","created_at":"2025-12-18 14:02:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6454008,"visible":true,"origin":"","legend":"\u003cp\u003eLEfSe and LDA analysis of microbial taxa. (A) LEfSe plot identifying significantly different taxa. (B) LDA histogram illustrating group-specific biomarkers.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8136685/v1/ca67c0d67a54af829adfb9fd.png"},{"id":98522845,"identity":"3c5276b4-05bd-4865-b093-553cd78651a9","added_by":"auto","created_at":"2025-12-18 14:02:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":847344,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional prediction of gut microbiota. (A) Heatmap of core metabolic pathway. (B) COG classification showing functional differences. (C) Heatmap of core\u003cstrong\u003e \u003c/strong\u003eenzyme.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8136685/v1/d37fffa3b63841049c0361e2.png"},{"id":98522849,"identity":"e9dde6fc-7a80-49b8-8b0e-da8dcb04aec4","added_by":"auto","created_at":"2025-12-18 14:02:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1680164,"visible":true,"origin":"","legend":"\u003cp\u003eGenus-level differences and microbial co-occurrence. (A) Circular phylogenetic tree of gut microbiota. (B) Co-occurrence network showing shared and unique taxa.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8136685/v1/e766ecffe910fe1507295a03.png"},{"id":107351049,"identity":"c0331dea-6339-41b9-b401-74ce74fe546b","added_by":"auto","created_at":"2026-04-20 16:08:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13357082,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8136685/v1/c1dd7280-ddad-477c-8ae4-c59b350087c5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gut microbiota and metabolic function in leeches with distinct feeding niches","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicroorganisms are ubiquitous, inhabiting diverse environments and thriving within complex ecosystems. Among these the gut harbors, the highest density of microbial cells, forming intricate and dynamic communities collectively termed the gut microbiota\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Extensive research has demonstrated that the gut microbiota plays a crucial role in synthesizing essential nutrients, such as B vitamins and amino acids ,which the host cannot produce endogenously\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. As a vital physiological component across species, the gut microbiota influences host metabolism, immune function, and homeostasis. Its composition is shaped by a multitude of environmental factors, including pH, oxygen availability, nutrient supply, water activity, and temperature, all of which influence microbial survival, populations dynamics, functionality, and ecological interactions\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLeeches (class Hirudinea, phylum Annelida) are predominantly freshwater invertebrates, exhibiting a broad range of feeding strategies, from hematophagous parasitism to predation on small aquatic invertebrates\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Hematophagous leeches possess specialized anterior suckers equipped with three chitinous jaws, which allow them to create a triradiate incision in their host\u0026rsquo;s skin. During feeding, they secrete potent anticoagulants, such as hirudin, decorsin, and destabilase, into the wound, preventing blood coagulation within their digestive tract\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Notably, symbiotic gut bacteria play an essential role in facilitating the long-term storage and digestion of ingested blood by producing antimicrobial compounds that inhibit spoilage and enzymes (e.g., hemolysins, collagenases, and proteases), that aid in nutrient breakdown and absorption\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNon-hematophagous leeches typically prey on small invertebrates such as, snails and earthworms. Interestingly, previous studies have shown that the heavy metal content (e.g., Cr, Pb, Zn, Cu) in non-hematophagous leeches is negatively correlated with sediment pH, total nitrogen, and organic matter, but positively correlated with the water pH. This suggests that their gut microbiota may contribute to heavy metal tolerance and potentially promote ecosystem stability\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Analogous studies in earthworms has revealed that amendments such as corn cob charcoal can significantly increase the abundance of nitrogen-cycling bacteria in the gut, thereby enhancing organic matter degradation and supporting environmental resilience\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Investigating the gut microbiota of non-hematophagous leeches may thus offer new insights into microbial mechanisms of heavy metal tolerance and environmental adaptation.\u003c/p\u003e \u003cp\u003eLeeches have evolved highly specialized feeding habits that differ significantly from other annelids, with most species exhibiting narrow dietary preferences limited to either hematophagy, bodily fluid consumption, or predation, each accompanied by distinct morphological adaptations\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eHirudo nipponia\u003c/em\u003e possesses a relatively large oral opening, equipped with three crescent-shaped jaws\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Each jaw bears a row of fine denticles along the chitinous ridge, and further identified conical papillae at the jaw tips, which function in secretion, facilitating skin penetration and anticoagulant release during blood-feeding\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn contrast to hematophagous species, members of the genus \u003cem\u003eWhitmania\u003c/em\u003e (e.g., \u003cem\u003eWhitmania pigra\u003c/em\u003e) specialize in feeding on molluscan body fluids, exhibiting modified jaw morphology where the three jaws bear only two rows of undifferentiated tooth plates adapted for tissue maceration and fluid extraction from prey like snails\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. This divergence in jaw morphology reflects the distinct feeding mechanisms between hematophagous and fluid-feeding leeches.\u003c/p\u003e \u003cp\u003eIn a taxonomic study of two leech groups, \u003cem\u003eWhitmania pigra\u003c/em\u003e and \u003cem\u003eHirudo nipponia\u003c/em\u003e were found to share a common ancestor (~\u0026thinsp;50 MYA), while \u003cem\u003eHirudo manillensis\u003c/em\u003e diverged earlier. This suggests that blood-feeding behavior was likely present in the ancestral leech but was lost in the \u003cem\u003eWhitmania pigra\u003c/em\u003e lineage\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. This suggests that during long-term evolution, environmental and genetic factors drove significant divergence in feeding strategies between these two leech groups, while certain behavioral traits remained conserved. Genomic studies of \u003cem\u003eZeylanicobdella arugamensis\u003c/em\u003e reveal a closer phylogenetic relationship with \u003cem\u003eHirudo nipponia\u003c/em\u003e, \u003cem\u003eWhitmania acranulata\u003c/em\u003e, \u003cem\u003eWhitmania pigra\u003c/em\u003e, and \u003cem\u003eWhitmania laevis\u003c/em\u003e, forming a sister clade\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Ancestral \u003cem\u003eWhitmania\u003c/em\u003e species may have retained parasitic behavior, with its loss in some lineages likely linked to shifts in food availability, further highlighting the genetic similarities underlying their divergent ecological adaptations.\u003c/p\u003e \u003cp\u003eDespite these emerging findings, comparative studies examining the gut microbiota of hematophagous and non-hematophagous leeches remain scarce.\u003c/p\u003e \u003cp\u003eThis study employs16S rRNA gene sequencing to analyze and compare the gut microbial community of wild hematophagous and non-hematophagous leeches. By exploring the relationships between feeding niches and gut microbiota composition, we aim to elucidate the microbial basis of dietary specialization in leeches and provide a scientific foundation for conservation, domestication, and sustainable aquaculture practices.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection\u003c/h2\u003e \u003cp\u003eWild leech specimens were collected from shallow waters of the Chaobai River (Baodi District, Tianjin, China; coordinates: 117˚16'31\"E, 39˚41'43\"N). Three healthy adult individuals were selected from each group: hematophagous leeches (\u003cem\u003eHirudo nipponia\u003c/em\u003e and \u003cem\u003eHirudo tianjinensis\u003c/em\u003e) and non-hematophagous leeches (\u003cem\u003eWhitmania pigra\u003c/em\u003e, \u003cem\u003eWhitmania laevis\u003c/em\u003e, and \u003cem\u003eWhitmania acranulata\u003c/em\u003e). Prior to processing, each leech was surface-sterilized by immersion in 75% ethanol for 1 minute, followed by three sequential rinses in sterile water, each lasting 1 minute. After sterilization, specimens were transferred individually into 1.5 mL centrifuge tubes, flash-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80 ℃ until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 DNA extraction amplification and sequencing\u003c/h2\u003e \u003cp\u003eTotal microbial DNA was extracted from frozen leech samples using the E.Z.N.A.\u0026reg; Soil DNA Kit (OMEGA, USA) following the manufacturer\u0026rsquo;s protocol. DNA integrity was verified via 1% agarose gel electrophoresis, and DNA concentration and purity (A260/A280 ratio) were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 16S rRNA gene\u003c/h2\u003e \u003cp\u003eThe V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 515F (5\u0026prime;-GTGCCAGCMGCCGCGGTAA-3\u0026prime;) and 806R (5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;)\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. PCR was performed under the following conditions: initial denaturation at 95 ℃ for 3 minutes, followed by 30 cycles of 95 ℃ for 30 seconds, 45 ℃ for 30 seconds, 72 ℃ for 45 seconds, with a final extension at 72 ℃ for 10 minutes.\u003c/p\u003e \u003cp\u003eTriplicate PCR reactions were pooled for each sample, and amplicons were purified using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). The purified PCR products were quantified using the QuantiFluor\u0026trade;-ST system (Promega, USA), normalized, and sequenced on the Illumina MiSeq PE300 platform by Shanghai Meiji Biological Pharmaceutical Technology Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Bioinformatics and statistical analysis\u003c/h2\u003e \u003cp\u003ePaired-end reads were merged using FLASH (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccb.jhu.edu/software/FLASH/index.shtml\u003c/span\u003e\u003cspan address=\"https://ccb.jhu.edu/software/FLASH/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, v1.2.11) and quality-filtered using Fastp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OpenGene/fastp\u003c/span\u003e\u003cspan address=\"https://github.com/OpenGene/fastp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, v0.19.6) Operational taxonomic units (OTUs) were clustered at a 97% similarity using UPARSE. Diversity indices and sequencing depth were assessed based on OTU clustering results.\u003c/p\u003e \u003cp\u003eTaxonomic classification sequences was performed using the Bayesian algorithm of the RDP Classifier (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sourceforge.net/projects/rdp-classifier\u003c/span\u003e\u003cspan address=\"https://sourceforge.net/projects/rdp-classifier\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, v2.13) with reference to the Silva 16S rRNA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arb-silva.de\u003c/span\u003e\u003cspan address=\"https://www.arb-silva.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, v138). Bacterial community composition at various taxonomic levels was analyzed using Qiime (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://qiime.org/install/index.html\u003c/span\u003e\u003cspan address=\"http://qiime.org/install/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, v1.9.1).\u003c/p\u003e \u003cp\u003eStatistical analyses were conducted using the Kruskal-Wallis and Wilcoxon rank-sum test, threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Principal Component Analysis (PCA) was performed via the Majorbio Cloud Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and functional prediction of microbial communities was conducted using PICRUSt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://picrust.github.io/picrust\u003c/span\u003e\u003cspan address=\"http://picrust.github.io/picrust\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, v 1.1.0).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sequencing data and species statistics\u003c/h2\u003e \u003cp\u003eIllumina MiSeq sequencing of the gut microbiota from hematophagous and non-hematophagous leeches generated a total of 1,130,336 raw reads. After quality control and, sequence, 1,107,010 high-quality reads (average length: 225 bp) were retained for further analysis. Rarefaction curves based on the Sob index plateaued with increasing sequencing depth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating sufficient coverage and comprehensive representation of microbial communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eComparative analysis revealed, that hematophagous leeches harbored significantly lower gut microbiota diversity (as measured by ACE index) compared to non-hematophagous leeches, likely reflecting dietary niches specialization. Clustering at 97% sequence similarity identified 751 microbial species, encompassing 535 genera, 332 families, 203 orders, 86 classes, and 39 phyla. Venn analysis revealed 142 OTUs between the two leech types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), highlighting both common and distinct divergent microbial features.\u003c/p\u003e \u003cp\u003eTen bacterial phyla were found in both leech groups, including Proteobacteria, Bacteroidota, Firmicutes, Myxococcota, Fusobacteriota, Patescibacteria, Verrucomicrobiota, Desulfobacterota, Synergistota, and Spirochaetota. At the phylum level, hematophagous leeches were dominated by Proteobacteria (63%), Firmicutes (13%), Bacteroidota (10%), and Myxococcota (10%), whereas non-hematophagous leeches showed enrichment in Proteobacteria (39%), Bacteroidota (33%), and Myxococcota (10%) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eAt the genus level, hematophagous leeches were characterized by norank_f__Rhodospirillaceae (17%), \u003cem\u003eElstera\u003c/em\u003e (12%), \u003cem\u003eAeromonas\u003c/em\u003e (11%), unclassified_o__Oscillospirales (7%), unclassified_f__Rhizobiaceae (6.5%), and unclassified_f__Rhodospirillaceae (6%). In contrast, non-hematophagous leeches were dominated by unclassified_f__Comamonadaceae (13%), \u003cem\u003eP3OB-42\u003c/em\u003e (10%), \u003cem\u003eBacteroides\u003c/em\u003e (8%), \u003cem\u003eNubsella\u003c/em\u003e (6%), unclassified_c__Alphaproteobacteria (5%), unclassified_o__Bacteroidales (5%), \u003cem\u003ePhreatobacter\u003c/em\u003e (4%), and \u003cem\u003eParabacteroides\u003c/em\u003e (3%). (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Shared genera such as \u003cem\u003eP3OB-42\u003c/em\u003e and \u003cem\u003eNubsella\u003c/em\u003e suggest the existence of a core microbiota that support fundamental gut functions across different feeding strategies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Alpha and beta diversity analysis of gut microbiota\u003c/h2\u003e \u003cp\u003eAlpha diversity metrics revealed that non-hematophagous leeches had significantly higher values across all indices: Chao1 (2479.65) ACE (2530.25), Sobs (2220), and Shannon (25.3), compared to hematophagous leeches Chao1 (395.6), ACE (408.92), Sobs (362), (408.92), and Shannon (11.96), and indices (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This suggests a more diverse and evenly distributed gut microbial community in non-hematophagous leeches.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBeta diversity analysis revealed clear distinctions between the gut microbiota of the two leech types. Principal Coordinates Analysis (PCoA) showed that PCO1 and PCO2 together explained 45.14% of the observed variation. While non-hematophagous leeches exhibited tight clustering, indicating greater intra-group similarity, hematophagous leeches displayed more dispersed patterns, suggesting higher inter-individual variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eCommunity dispersion analysis revealed 33.21% of variation, indicating that dietary habit plays a significant role in shaping the gut microbial communities. Non- hematophagous leeches exhibited higher microbial abundance than hematophagous leeches (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3 LEfSe and LDA analysis\u003c/h2\u003e \u003cp\u003eLEfSe analysis identified significant differences in gut microbial composition between the two leech types. The phylogenetic tree illustrated the species divergence across different taxonomic levels, highlighting distinctions in the gut microbiota between two types of leeches. A total of 63 microbial taxa at varying taxonomic levels were identified to be significantly different in non-hematophagous leeches compared to hematophagous ones (Figure. 3A). Using LEfSe analysis (LDA score threshold\u0026thinsp;=\u0026thinsp;2), significantly divergent microorganisms between the two dietary groups were identified. Among these, the most prominently enriched taxa included o__Bacteroidales, o__Burkholderiales, f__Comamonadaceae, f__Rhodospirillaceae, and \u003cem\u003eg__norank_f__Rhodospirillaceae\u003c/em\u003e (Figure. 3B). These results underscore the influence of long-term dietary and ecological divergence on gut microbiota composition, with non-hematophagous leeches exhibiting significantly higher overall gut microbial diversity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4 KEGG functional prediction\u003c/h2\u003e \u003cp\u003ePICRUSt2-based KEGG pathway analysis indicated that both types shared expression of nine core metabolic pathways, including general metabolism, secondary metabolite biosynthesis, microbial metabolism, amino acid biosynthesis, carbon metabolism, ABC transporters, two-component systems, quorum sensing, and ribosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite their different diets, these conserved pathways suggest that both leech types maintain similar basic metabolic regulatory mechanisms. However, notably differences were observed in pathways related to lipid, amino acid, nucleotide, and coenzyme transport and metabolism, as well as translation and ribosomal structure biogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAnalysis of metabolic pathway-related enzymes revealed higher abundances of DNA-directed DNA polymerase, NADH: ubiquinone oxidoreductase, and DNA-directed RNA polymerase in blood-feeding leeches compared to non-blood-feeding species. Conversely, non-blood-feeding leeches exhibited elevated levels of chaperonin ATPase, histidine kinase, and peptidylprolyl isomerase. These enzyme-specific expression patterns are not solely attributed to differences in gut microbiota but may also be linked to leech feeding habits (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThese differences likely reflect adaptations to distinct nutritional strategies: hematophagous leeches rely on microbiota capable of degrading blood proteins, whereas non-hematophagous leeches are adapted to environments where they prey on various small invertebrates, with metabolic capacities favoring energy storage and assimilation of diverse animal-derived nutrients. Thus, gut microbiota plays a pivotal role in shaping feeding strategies and, ecological adaptability in leeches.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Genus-level microbial differences and co-occurrence patterns\u003c/h2\u003e \u003cp\u003eEvolutionary analysis of microbial genera revealed distinct associations in the two leech types hematophagous leeches, \u003cem\u003eTreponema\u003c/em\u003e was closely related to \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003ePeptoniphilus\u003c/em\u003e, with \u003cem\u003ePeptoniphilus\u003c/em\u003e also closely associated with related to \u003cem\u003eFretibacterium\u003c/em\u003e. In non-hematophagous leeches, notable relationships included \u003cem\u003eDenitratisoma\u003c/em\u003e-\u003cem\u003eAquabacterium\u003c/em\u003e, \u003cem\u003eWolinella-Desulfovibrio\u003c/em\u003e, \u003cem\u003eNubsella\u003c/em\u003e-\u003cem\u003eAcetobacteroides\u003c/em\u003e, and \u003cem\u003eRikenella\u003c/em\u003e-\u003cem\u003eMucinivorans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The observed evolutionary proximity among these microbial genera may be attributed not only to genetic relatedness, but also appears inextricably linked to their host\u0026rsquo;s feeding ecology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCo-occurrence network analysis showed that approximately 22% of microbiota were shared between the two leech types, 17% were unique to hematophagous leeches, and 61% were enriched in non-hematophagous leeches (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These findings further support the idea that environmental and dietary factors shape distinct but overlapping microbial ecosystems.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we observed distinct gut microbiota compositions between hematophagous and non-hematophagous leeches. At the phylum level, the gut microbiota of hematophagous leeches was dominated by Proteobacteria (63%), Firmicutes (13%), Bacteroidota (10%), and Myxococcota (10%). In contrast, non-hematophagous leeches harbored Proteobacteria (39%), Bacteroidota (33%), and Myxococcota (10%), with a notably lower abundance of Firmicutes.\u003c/p\u003e \u003cp\u003eFirmicutes are essential for digestion complex organic matter and enhanced energy utilization, prior studies have shown that the Firmicutes/Bacteroidota (F/B) ratio is closely associated with the host\u0026rsquo;s metabolic state, influencing factors such as lipid deposition, nutrient absorption, and overall physiological condition\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Typically, a higher F/B ratio correlates with increased energy extraction from diet, while a lower ratio supports microbial balance and metabolic efficiency, and is also linked to the host growth rate, fat deposition, and susceptibility to diseases\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Previous studies suggest that changes in specific Firmicutes, Bacteroidetes, and Actinobacteria OTUs may elevate swine niacin levels, subsequently modulating key digestive enzyme (amylase, trypsin, and lipase) activities\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eLactobacillus reuteri\u003c/em\u003e ATCC PTA 4659 promotes intestinal barrier function through upregulation of epithelial heat shock proteins (HSP25 and HSP70)\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. This probiotic strain enhances mucosal integrity by reinforcing tight junction proteins, thereby optimizing nutrient absorption efficiency in leech hosts. In this study, hematophagous leeches exhibited a significantly higher abundance of Firmicutes compared to non-hematophagous species, potentially reflecting their adaptation to a protein- and hemoglobin-rich blood diet. The higher Firmicutes content may facilitate efficient digestion and nutrient uptake. In contract, non-hematophagous leeches, which feed on small invertebrates and microbial matter, showed a higher abundance of Bacteroidota, which are effective in degrading complex carbohydrates and polysaccharides, while maintaining gut microbial homeostasis.\u003c/p\u003e \u003cp\u003eAmong Proteobacteria, genera such as \u003cem\u003eBifidobacterium\u003c/em\u003e contribute to physiological balance and immune modulation. However, other members, including \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e, are pathogenic, while some, like \u003cem\u003eRhodospirillum\u003c/em\u003e, possess nitrogen fixation capabilities\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Several genera within Proteobacteria, such as \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eRhodospirillum\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eFaecalibacterium\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e, ferment indigestible oligosaccharide carbohydrates, leading to the production of short-chain fatty acids (SCFAs) such as butyrate, propionate, and acetate. These SCFAs serve as a rich energy source for the host\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. The higher Proteobacteria abundance in hematophagous leeches may enhance their ability to digest and absorb lipids derived from blood.\u003c/p\u003e \u003cp\u003eBacteroidota play a key role in maintaining microbial balance and supporting metabolic processes. For instance, \u003cem\u003eBacteroides thetaiotaomicron\u003c/em\u003e can stimulate the expression of small proline-rich protein 2A (sprr2A), which is essential for maintaining epithelial villus tight junctions\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In this study, Bacteroidota were significantly more abundant in the guts of non-hematophagous leeches, suggesting their involvement in carbohydrate and cellulose digestion. A study found that \u003cem\u003eBacteroidota\u003c/em\u003e is associated with metabolic dysfunction-associated steatohepatitis (MASH). Specifically, an enzyme annotated as a β-lactamase in \u003cem\u003eBacteroidota uniformis\u003c/em\u003e was identified to catalyze the biosynthesis of 3-succinylated bile acids\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Similar patterns have been observed in earthworms, where Bacteroidota, Firmicutes, Actinobacteria, and Proteobacteria dominate before sludge feeding, while after feeding, the composition shifts, with increases in Firmicutes and Proteobacteria and reductions in Bacteroidota and Actinobacteria\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e aligning with our findings.\u003c/p\u003e \u003cp\u003eAt the genus level, specific microbial groups appeared to play functional roles. In hematophagous leeches, \u003cem\u003eg__norank_f__Rhodospirillaceae\u003c/em\u003e was the most abundant. These photosynthetic bacteria produce, B vitamins, pantothenic acid, coenzyme Q, and pigments such as bacteriochlorophyll and carotenoids, which possess bioactive and recyclable properties\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Such metabolites may support host gut functions. \u003cem\u003eElstera\u003c/em\u003e, also enriched in hematophagous leeches, contributes to energy production via both aerobic and fermentative pathways and supports maintenance of the gut\u0026rsquo;s anaerobic environment\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Its abundance has been shown to increase under glucose exposure, from 0% to 12.7%, suggesting its role in carbon metabolism.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAeromonas\u003c/em\u003e another genus found in hematophagous leeches, produces a wide variety of heterogeneous virulence factors, such as membrane components, toxins, and enzymes, which can cause intestinal infections and, if uncontrolled, may result in systemic effects\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Notably, Aeromonas species exhibit anticancer properties. In particular, \u003cem\u003eAeromonas veronii\u003c/em\u003e has been identified as a promising L-glutaminase producer, with this enzyme showing therapeutic efficacy against acute lymphoblastic leukemia (ALL)\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. However these microorganisms also have the potential to cause intestinal infections, which, if not effectively controlled, may progress and disseminate systemically, resulting in associated clinical symptoms\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Additionally, the high abundance of unclassified_o__Oscillospirales, commonly found in herbivores and omnivores, has been associated with intestinal inflammation. Environmental factors such as light exposure can significantly alter gut microbiota composition, reducing \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eButyricicoccus\u003c/em\u003e, and \u003cem\u003eSelenomonas\u003c/em\u003e, while increasing \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eOscillospirales\u003c/em\u003e, and others\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn non-hematophagous leeches, \u003cem\u003eg__unclassified_f__Comamonadaceae\u003c/em\u003e was the most abundant group. This family is involved in glucose and lipid metabolism. It was found that the lack of the PPARδ gene exacerbated the progression of alcoholic fatty liver disease induced by a high-fat diet, leading to a decrease in the abundance of norank_f__Eubacterium_coprostanoligenes_group and \u003cem\u003eAlloprevotella\u003c/em\u003e, while the abundance of \u003cem\u003eAcidobacteria\u003c/em\u003e, unclassified_f__Comamonadaceae, unclassified_c__Alphaproteobacteria, unclassified_f__Beijerinckiaceae, unclassified_f__Caulobacteraceae, unclassified_c__Bacteroidia, and \u003cem\u003eBosea\u003c/em\u003e increased \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The presence of \u003cem\u003eNubsella\u003c/em\u003e in hematophagous leeches may enhance microbial diversity and environmental adaptability, as shown in studies where its absence reduced host fitness in \u003cem\u003ePhasmotaenia lanyuhensis\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe presence of \u003cem\u003eNubsella\u003c/em\u003e enhances microbial diversity and improves the host\u0026rsquo;s environmental adaptability, while Bacteroides shows significantly higher abundance in the gut microbiota of hematophagous leeches. Members of this family are potential colonizers of the gut and constitute a major portion of the gut microbiota\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. These Gram-negative obligate anaerobes play various roles in the human gut microbiota and are key participants in maintaining the intestinal microbial food web\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGut microbial differences are often reflected in host metabolic pathways\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Glucose and lipid metabolites are transported intra- and intercellularly and transformed into functional molecules in specific organelles\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that intestinal lipoprotein cholesterol regulates hepatic cholesterol biosynthesis and serves as the sole route for eliminating serum cholesterol via bile acid conversion, highlighting the intestine\u0026rsquo;s central role in cholesterol metabolism\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. In this study, pathways related to lipid transport and metabolism were more prominently expressed in hematophagous leeches, likely due to the higher abundance of lipid-assimilating Proteobacteria.\u003c/p\u003e \u003cp\u003eAmino acid transport and metabolism pathways were more active in non-hematophagous leeches, consistent with their elevated Bacteroidota levels. Amino acids not only serve as protein building blocks and energy sources but also act as precursors for bioactive metabolites and regulators of key cellular signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. In intestinal microbiota studies of \u003cem\u003eRhizomys spp\u003c/em\u003e, Bacteroidota exhibited notably high abundance. KEGG analysis revealed carbohydrate and amino acid metabolism as the most enriched pathways\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, likely mediated by Bacteroidota metabolic functions.\u003c/p\u003e \u003cp\u003eNucleotides are crucial for the transfer and transformation of chemical energy within cells, most notably in the form of the energy currency ATP\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. In addition, nucleotides serve as precursors for certain hormones, act as signaling molecules, and are essential components of DNA and RNA\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Comparative analysis revealed significantly elevated expression of nucleotide transport and metabolism pathways in the intestinal tissues of non-hematophagous leeches relative to their hematophagous counterparts, potentially associated with increased Firmicutes abundance. For example, \u003cem\u003eClostridium thermocellum\u003c/em\u003e is capable of cellulose degradation, and this bacterium can modulate glucose transporter A or glucose transporter B, thereby providing energy support for nucleic acid transport and metabolic pathways in the organism\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe study reveals that DNA-directed DNA polymerase exhibits higher expression in blood-feeding leeches, likely due to the dominance of Proteobacteria (e.g., \u003cem\u003eAeromonas hydrophila\u003c/em\u003e, a major cause of bacterial septicemia) in their gut microbiota\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. This enzyme may slow replication fork progression during DNA damage response, ensuring stable general metabolism and maintaining a healthy gut microenvironment\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. In non-blood-feeding leeches, higher Bacteroidota abundance is observed, potentially linked to their diet (e.g., snails, earthworms), which elevates intestinal lipid levels and increases the risk of inflammatory bowel disease (IBD). Concurrently, elevated Chaperonin ATPase expression in these leeches correlates with mitochondrial dysfunction and IBD\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. The interplay between these factors helps balance the gut microbiota and microenvironment.\u003c/p\u003e \u003cp\u003eThe functional capacity of the gut microbiota is primarily determined by its composition, which is influenced by multiple factors including host genetic background, health status, social behavior, and environmental habitat conditions\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. In conclusion, differences in gut microbiota composition between hematophagous and non-hematophagous leeches influence metabolic profiles, energy utilization strategies, and environmental adaptability. These microbial communities play essential roles in shaping the host\u0026rsquo;s physiological and metabolic functions.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe composition of the intestinal microbiota plays a pivotal role in shaping host physiological activities. In this study, Proteobacteria were the dominant phylum in hematophagous leeches, whereas Bacteroidota were more prevalent in non-hematophagous leeches. Notably, hematophagous leeches exhibited the highest relative abundance of Proteobacteria, while non-hematophagous leeches harbored a greater proportion of unclassified bacterial taxa. Alpha diversity metrics, including the Chao1 and Shannon indices, revealed significantly higher microbial diversity in non-hematophagous leeches compared to their hematophagous counterparts.\u003c/p\u003e \u003cp\u003eFrom a functional perspective, lipid transport and metabolism pathways were more prominently expressed in hematophagous leeches, aligning with their protein- and lipid-rich blood-based diet. In contrast, pathways related to amino acid transport and metabolism were more active in non-hematophagous leeches, reflecting their adaptation to diverse organic matter diets. These findings suggest that non-hematophagous leeches possess a broader metabolic flexibility and stronger environmental adaptability.\u003c/p\u003e \u003cp\u003eIn summary, this study provides novel insights into the gut microbial communities of leeches with different feeding strategies and highlights their potential roles in host metabolism and ecological adaptation. These results offer a theoretical foundation for future research on leech domestication, conservation, and sustainable utilization.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (32260132), the Yunnan Provincial University Serving Key Industry Science and Technology Special Project (FWCY-ZD2024009), the Joint Special Project for Basic Research of Local Universities in Yunnan Province (202301BA070001-105 and 202101BA070001-164) , the Frontier Research Team of Kunming University 2023, and the Yunnan International Joint Laboratory with South and Southeast Asia for the Integrated Development of Animal-Derived Anti-Thrombosis Chinese Medicine (202503AP140025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data generated in this study have been deposited in the China National Center for Bioinformation (CNCB, https://ngdc.cncb.ac.cn) under the accession number CRA029752.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C., J.L., H.C., and Z.L.: Conceptualization, Supervision, Writing - original draft, Writing - review \u0026amp; editing. Z.L., and X.T.: Funding acquisition, Project administration. X.T., D.Y., and X.C.: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. X.Y., and Q.H.: Data curation, Visualization. D.Y., and S.F.: Formal analysis, Visualization, Validation. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLozupone CA, Stombaugh JI, Gordon JI, et al. 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BMC Microbiol. 2020;20(1):68.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gut microbiota, Leeches, Feeding strategies, 16S rRNA sequencing, Microbial metabolism","lastPublishedDoi":"10.21203/rs.3.rs-8136685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8136685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe gut microbiota has co-evolved with its host and plays a vital role in maintaining physiological homeostasis and health. Understanding the composition of microbial communities in leech guts may reveal important insights into their ecological adaptations and feeding strategies.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aimed to compare the gut microbiota of hematophagous (blood-feeding) and non-hematophagous leeches, to identify microbial signatures associated with dietary divergence and niche specialization.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eGut contents were collected from representative species of hematophagous and non-hematophagous leeches. Microbial community composition was analyzed via 16S rRNA gene sequencing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 751 microbial species were identified, encompassing 535 genera, 332 families, 203 orders, 86 classes, and 39 phyla. At the phylum level, Proteobacteria and Firmicutes were significantly higher relative more abundant in hematophagous leeches, whereas Bacteroidetes predominated in non-hematophagous counterparts. At the genus level, hematophagous leeches exhibited higher abundances of \u003cem\u003eElstera\u003c/em\u003e, norank_f__Rhodospirillaceae, \u003cem\u003eAeromonas\u003c/em\u003e, f__Rhodospirillales, o__Peptostreptococcales-Tissierellales, and unclassified_o__Oscillospirales. In contrast, unclassified_f__Comamonadaceae, \u003cem\u003eNubsella\u003c/em\u003e, \u003cem\u003eCetobacterium\u003c/em\u003e, \u003cem\u003eMucispirillum\u003c/em\u003e, norank_f__Peptostreptococcaceae, and \u003cem\u003eBacteroides\u003c/em\u003e were enriched in non-hematophagous leeches. Functional prediction analysis revealed significant differences in five key metabolic pathways between the two groups: lipid transport and metabolism, amino acid transport and metabolism, nucleotide transport and metabolism, translation and ribosomal structure biogenesis, and coenzyme transport and metabolism.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eDistinct taxonomic and functional profiles characterize the gut microbiota of hematophagous and non-hematophagous leeches. These results provide microbiological evidence for dietary specialization in leeches and offer a scientific basis for guiding domestication and artificial breeding strategies.\u003c/p\u003e","manuscriptTitle":"Gut microbiota and metabolic function in leeches with distinct feeding niches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 14:02:36","doi":"10.21203/rs.3.rs-8136685/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-16T05:40:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T23:19:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T17:40:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202320365701268532202024003479126934653","date":"2025-12-16T14:01:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261111711329760030072451745869256625065","date":"2025-12-16T13:03:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-16T10:57:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-29T14:31:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-29T14:30:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-11-17T14:31:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d4c257d-96c5-4071-9183-e85383e0e669","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:05:57+00:00","versionOfRecord":{"articleIdentity":"rs-8136685","link":"https://doi.org/10.1186/s12866-026-05062-z","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-04-15 15:57:37","publishedOnDateReadable":"April 15th, 2026"},"versionCreatedAt":"2025-12-18 14:02:36","video":"","vorDoi":"10.1186/s12866-026-05062-z","vorDoiUrl":"https://doi.org/10.1186/s12866-026-05062-z","workflowStages":[]},"version":"v1","identity":"rs-8136685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8136685","identity":"rs-8136685","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}