SEARCHCROSS-SECTIONALveterinary medicine, animal science

cross-sectional·veterinary medicine, animal science, microbiology, agriculture·PMC10804495

Sex, health status and habitat alter the community composition and assembly processes of symbiotic bacteria in captive frogs

BMC Microbiology · 6 authors, 3 centres

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POPULATIONCaptive black-spotted frogs (Pelophylax nigromaculata) from a farm in Anhui Province, China, including 8 females and 8 males (7 months old), and soil samples from three habitat types (frog-aggregation soil, native soil, soybean soil).

INTERVENTIONComparison of gut microbiota based on sex (female vs. male) and health status (healthy vs. infected with meningitis-like infectious disease, MID).

COMPARISONGut microbiota of female vs. male frogs; healthy vs. MID-infected frogs; soil microbiota among frog-aggregation soil (AS), native soil (NS), and soybean soil (SS).

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TL;DR

This study found that sex, health status, and habitat significantly shape the gut and soil bacterial communities in captive black-spotted frogs. Healthy frogs had more robust gut bacterial metabolism than those infected with meningitis-like infectious disease (MID), and sex influenced microbial network complexity and community assembly processes. These findings provide insights for sustainable frog farming and conservation by highlighting the importance of managing host-microbiota interactions.

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**Background:** Frogs are economically important for agricultural ecosystems, but meningitis-like infectious disease (MID) poses a threat. The gut microbiota of frogs plays roles in nutrient cycling and immunity, yet the effects of sex and health on these communities are poorly understood, and the relationship between frog habitat and soil microbes remains unclear. This study aimed to determine how frog sex, health status, and habitat influence symbiotic bacteria and community assembly mechanisms to guide sustainable frog farming and conservation.

**Methods:** Sixteen adult black-spotted frogs (Pelophylax nigromaculata) (8 females, 8 males, 7 months old) were collected from a farm in Xuanzhou District, Anhui Province, China in February 2020. Based on sex and health status (MID infection), they were divided into four groups: male healthy (MH, n=4), female healthy (FH, n=4), male unhealthy (MNH, n=4), and female unhealthy (FNH, n=4). Additionally, soil samples were collected from three habitat types: frog-aggregation soil (AS), native soil (NS), and soybean soil (SS), each with three replicates. Microbial DNA was extracted from gut contents and soil, and the V3-V4 regions of the 16S rRNA gene were sequenced using Illumina MiSeq. Bioinformatic analysis included alpha diversity (Chao1, Shannon, Simpson, Phylogenetic Diversity), beta diversity (NMDS, PERMANOVA, ANOSIM), taxonomic composition, co-occurrence network analysis (Spearman correlations, r>|0.9|, p<0.01), functional prediction (PICRUSt for gut, FAPROTAX for soil), and community assembly analysis (βNTI and RCbray).

**Key Results:** A total of 1,018,720 high-quality sequences were obtained from frog gut samples, yielding 1,214 ASVs. Soil samples contained 4,461 ASVs. Alpha diversity showed no significant differences in gut bacterial richness or Shannon index between sexes or health statuses (t-test, p>0.05). However, beta diversity revealed significant separation: between female and male groups (ANOSIM: Bray-Curtis, r=0.650, p=0.031) and between healthy and unhealthy groups (ANOSIM: Bray-Curtis, r=0.740, p=0.044). Soil bacterial communities also differed significantly among habitats (r=0.942, p=0.005). At the genus level, Ruminococcaceae was significantly enriched in females, while Laribacter was enriched in males (Kruskal-Wallis, p<0.05). Parabacteroides was significantly higher in unhealthy frogs, whereas Odoribacter and Akkermansia were higher in healthy frogs. Co-occurrence network analysis showed that healthy females had more complex networks than healthy males, but diseased males exhibited the greatest network complexity (MNH: 1251 links, 12 modules, modularity 0.911; MH: 440 links, 4 modules, modularity 0.817). In soil, AS had the most complex network (50 nodes, 472 connections, average degree 19.265). Functional prediction (PICRUSt) indicated that healthy frogs had significantly higher relative abundances of Carbohydrate Biosynthesis, Secondary Metabolite Biosynthesis, C1 Compound Utilization and Assimilation, and Nucleic Acid Processing compared to unhealthy frogs. In soil, FAPROTAX showed that SS was enriched in human_pathogens_all, nitrogen_respiration, nitrate_respiration, nitrogen_fixation, and iron_respiration, while AS was enriched in sulfur_respiration, hydrocarbon_degradation, methylotrophy, and dark_sulfur oxidation. Community assembly analysis revealed that deterministic processes dominated in male frogs (average βNTI=2.17) and in MNH (average βNTI=3.15), while stochastic processes dominated in females (average βNTI=0.21) and in both health groups (H and NH). In soil, deterministic processes were more influential in SS.

**Clinical Implications:** This study demonstrates that sex and health status significantly alter the gut microbiota composition, network complexity, and community assembly in captive frogs, with MID infection suppressing metabolic functions. The findings suggest that managing frog sex ratios and health could improve gut microbial stability and resistance to disease. Additionally, different habitat soils (especially soybean soil) shape distinct bacterial communities with enhanced nitrogen cycling functions, which could be leveraged to improve soil fertility and frog farming sustainability. These insights support the development of targeted probiotic or environmental interventions to enhance frog health and agricultural productivity, contributing to sustainable amphibian conservation and farming practices.

PICO

PPOPULATION

Captive black-spotted frogs (Pelophylax nigromaculata) from a farm in Anhui Province, China, including 8 females and 8 males (7 months old), and soil samples from three habitat types (frog-aggregation soil, native soil, soybean soil).

IINTERVENTION

Comparison of gut microbiota based on sex (female vs. male) and health status (healthy vs. infected with meningitis-like infectious disease, MID).

OOUTCOME

Gut bacterial β-diversity, taxonomy, co-occurrence network complexity, predicted metabolic functions, and community assembly processes; soil bacterial diversity, composition, network structure, and functional predictions.

STUDY TYPE

cross-sectional

SPECIALTY

veterinary medicine

SUMMARISED BY

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100% · A

ORIGINAL PAPER

PMC10804495

Sex, health status and habitat alter the community composition and assembly processes of symbiotic bacteria in captive frogs

BMC Microbiology

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ABSTRACT

Background Frogs are critical economic animals essential to agricultural ecosystem equilibrium. However, Meningitis-like Infectious Disease (MID) often affects them in agricultural settings. While frog-associated microbiota contribute to elemental cycling and immunity, the effects of frog sex and health on gut bacteria remain understudied, and the relationship between frog habitat and soil microbes is unclear. We aimed to determine how frog sex, health status and habitat influence symbiotic bacteria and community assembly mechanism to provide guidance for sustainable frog farming and conservation. Results We employed 16S rRNA sequencing to investigate gut microbiota differences in relation to frog sex and health status. We also compared symbiotic communities in frog-aggregation, native and soybean soil on the farm. Results showed that gut bacterial β-diversity and taxonomy were markedly influenced by frog sex and health. Healthy frogs had more robust gut bacterial metabolism than frogs infected with MID. Cooccurrence network analysis revealed that healthy female frogs had more complex microbial network structure than males; however, diseased males showed the greatest network complexity. The assembly mechanism of gut bacteria in male frogs was dominated by deterministic processes, whereas in female frogs it was dominated by stochastic processes. Among symbiotic bacteria in frog habitat soils, deterministic processes predominantly shaped the community assembly of soybean soil. In particular, soybean soil was enriched in pathogens and nitrogen functions, whereas frog-aggregation soil was markedly increased in sulphur respiration and hydrocarbon degradation. Conclusion Our study reveals that sex mainly alters the interaction network and assembly mechanism of frog intestinal bacteria; MID infection significantly inhibits the metabolic functions of intestinal bacteria. Furthermore, diverse frog habitat soils could shape more symbiotic bacteria to benefit frog farming. Our findings provide new horizons for symbiotic bacteria among frogs, which could contribute to sustainable agriculture and ecological balance. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-023-03150-y.

CITATION EXPORT

Senlin Liu, Sewar Imad, Sarfraz Hussain et al.. Sex, health status and habitat alter the community composition and assembly processes of symbiotic bacteria in captive frogs. BMC Microbiology. (2024). https://doi.org/10.1186/s12866-023-03150-y

AUTHORS

6 · 3 CENTRES

Senlin Liu, Sewar Imad, Sarfraz Hussain, Shuiqing Xiao, Xiaowei Yu, Hui Cao

1grid.27871.3b0000 0000 9750 7019College of Life Sciences/Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture and Rural Affair, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu, 210095 People’s Republic of China
2https://ror.org/01a77tt86grid.7372.10000 0000 8809 1613School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, UK
3https://ror.org/05nkgk822grid.411862.80000 0000 8732 9757Jiangxi Normal University, Nanchang, JX China