Utilizing HCR-FISH to investigate the status of anaerobic methanotrophic archaea in cold seep sediments

doi:10.3969/j.issn.1001-909X.2025.01.003

Abstract

Abstract:

The anaerobic oxidation of methane (AOM) is a pivotal component of elemental cycling within cold seep sediments. This process is usually performed by anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), which usually exist as symbionts. However, pure cultures of ANME have not yet been obtained, and their slow metabolism hinders further exploration and research into their metabolic characteristics and collaborative mechanisms. In this study, we utilized hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) technology and high-throughput 16S rRNA gene sequencing to investigate the composition and state of ANME communities at different depths of the sediments in the black microbial mat area of the South China Sea Formosa cold seep. The results showed that ANME-1 and ANME-2 were the dominant groups in the sampled Formosa cold seep sediments. Specifically, ANME-2 was found to form consortia with SRB, while no such associations were detected for ANME-1. This observation suggested that ANME-2 and SRB primarily engage in symbiotic AOM processes, highlighting potential differences in physiological roles and methane metabolism pathways between ANME-1 and ANME-2. Furthermore, in sediment samples of all layers, the diameters of ANME-2/SRB consortia were predominantly concentrated between 3-10 μm. Correlation analysis indicated a significant link between the distribution of consortium diameters and environmental factors such as sulfate concentration in the sediment, underscoring the impact of environmental factors on the growth of ANME/SRB consortia. Additionally, using HCR-FISH, we further discovered the presence of multiple consortium clusters in the Formosa cold seep sediment, characterized by orderly connected and uniform-sized consortium, implying possible connections or cooperative relationships among consortia. This study revealed the presence and distribution patterns of ANME groups and sizes of symbiotic microbial consortia in sediment samples from different depths of the Formosa cold seep, laying the foundation for further understanding methane metabolism mechanisms and ecological functions of different ANME groups in situ cold seep sediments.

Key words: cold seep, sediment, ANME, SRB, AOM, microbial consortia, HCR-FISH

CLC Number:

X172
P744.4

HE Maoyu, WANG Jing, LI Sihan, LIANG Lewen. Utilizing HCR-FISH to investigate the status of anaerobic methanotrophic archaea in cold seep sediments[J]. Journal of Marine Sciences, 2025, 43(1): 22-33.

Figures/Tables 8

Tab.1 Information of sediment samples in Formosa cold seep used in this study

样品编号	沉积物描述	沉积物柱状样深度/cm	样品数量/个
D231-1	还原性沉积物外部对照	14	7
D231-2	还原性沉积物上边缘交界	30	15
D231-3	还原性沉积物中心1	20	10
D231-4	还原性沉积物中心2	18	9
D231-5	还原性沉积物中心下边缘交界	22	11
D231-6	还原性沉积物白色菌席	18	9
合计			61

Tab.2 HCR-FISH probes used in this study

探针名称		靶向类群	探针序列(5’-3’)	来源
起始探针	ARCH915-H	Archaea	CCGAATACAAAGCATCAACGACTAGAAAAAAGTGCTCCCCCGCCAATTCCT	文献[8]
	EUB338-R	Bacteria	TACGCCCTAAGAATCCGAACCCTATGAAATAGCTGCCTCCCGTAGGAGT	文献[16]
	ANME-1-350-H	ANME-1	CCGAATACAAAGCATCAACGACTAGAAAAAAAGTTTTCGCGCCTGATGC	文献[16]
	ANME-2-538-H	ANME-2	CCGAATACAAAGCATCAACGACTAGAAAAAAGGCTACCACTCGGGCCGC	本研究
	ANME-3-1249-H	ANME-3	CCGAATACAAAGCATCAACGACTAGAAAAAATCGGAGTAGGGACCCATT	本研究
	DSS658-R	Desulfosarcina-Desulfococcus	TACGCCCTAAGAATCCGAACCCTATGAAATATCCACTTCCCTCTCCCAT	本研究
扩增探针	H1		CATAGGGTTCGGATTCTTAGGGCGTAGCAGCATCAATACGCCCTAAGAATCC	文献[21]
	H2		TACGCCCTAAGAATCCGAACCCTATGGGATTCTTAGGGCGTATTGATGCTGC	文献[21]
	R1		TCTAGTCGTTGATGCTTTGTATTCGGCGACAGATAACCGAATACAAAGCATC	文献[21]
	R2		CCGAATACAAAGCATCAACGACTAGAGATGCTTTGTATTCGGTTATCTGTCG	文献[21]

Fig.1 Composition and correlation of ANME and SRB groups in the sediment samples of Formosa cold seep (Figure a illustrates the variation of ANME groups with depth in site D231-4, where “Others” in the figure represents archaea other than ANME and ANME with ASV abundances less than 1%. Figure b depicts the variation of SRB groups with depth in site D231-4, where “Others” denotes bacteria other than SRB and SRB with ASV abundances less than 1%. Figure c presents a Pearson correlation heatmap of ANME and SRB groups across all depth intervals from station D231, based on 61 sediment samples. In the heatmap, * indicates p<0.05, and ** indicates p<0.01.)

Fig.2 HCR-FISH images of ANME-SRB consortia in the sediment samples of Formosa cold seep (Figure a illustrates microbial consortia composed of archaea and bacteria. Figure b displays ANME-SRB consortia consisting of ANME-2 and sulfate-reducing bacteria (SRB). No hybridization signals were observed for ANME-SRB consortia that were positively hybridized with the DSS probe when probed with ANME-1-targeting (Figure c) or ANME-3-targeting (Figure d) probes. Oligonucleotide probes DSS658-R and EUB338-R were employed to target SRB and bacteria, respectively, and were labeled red. Probes ARCH915-H, ANME-1-350-H, ANME-2-538-H, and ANME-3-1249-H were used to target archaea, ANME-1, ANME-2, and ANME-3, respectively, and were labeled green. DAPI staining was applied for nuclear staining and appeared blue.)

Tab.3 The ANME consortia number observed in the sediments at different depths

层位	细胞团数量/个
层位	ANME-1	ANME-2	ANME-3
0~2 cm	0	86	0
4~6 cm	0	71	0
8~10 cm	0	73	0
12~14 cm	0	82	0
16~18 cm	0	78	0
平均	0	78	0

Fig.3 The composition of the consortia size in the sediment samples of Formosa cold seep

Fig.4 Heatmap of Pearson’s correlation coefficients between environmental factors and consortia sizes

Fig.5 The HCR-FISH images of the consortia cluster (The microbial consortia exhibited positive hybridization with the SRB-targeting probe (DSS658-R, red), DAPI staining appeared blue. Figures a to c represent scanning results from different z-axes.)

References 38

[1]	KNITTEL K, BOETIUS A. Anaerobic oxidation of methane: Progress with an unknown process[J]. Annual Review of Microbiology, 2009, 63: 311-334. DOI PMID
[2]	PAULL C K, HECKER B, COMMEAU R, et al. Biological communities at the Florida escarpment resemble hydrothermal vent taxa[J]. Science, 1984, 226(4677): 965-967. PMID
[3]	FENG D, QIU J W, HU Y, et al. Cold seep systems in the South China Sea: An overview[J]. Journal of Asian Earth Sciences, 2018, 168: 3-16.
[4]	SCHREIBER L, HOLLER T, KNITTEL K, et al. Identi-fication of the dominant sulfate-reducing bacterial partner of anaerobic methanotrophs of the ANME-2 clade[J]. Environmental Microbiology, 2010, 12(8): 2327-2340.
[5]	SCHÖNHUBER W, FUCHS B, JURETSCHKO S, et al. Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification[J]. Applied and Environ-mental Microbiology, 1997, 63(8): 3268-3273.
[6]	VOLPI E V, BRIDGER J M. FISH glossary: An overview of the fluorescence in situ hybridization technique[J]. Bio-Techniques, 2008, 45(4): 385-409.
[7]	AMANN R, FUCHS B M. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques[J]. Nature Reviews Microbiology, 2008, 6(5): 339-348. DOI PMID
[8]	YAMAGUCHI T, KAWAKAMI S, HATAMOTO M, et al. In situ DNA-hybridization chain reaction (HCR): A facilitated in situ HCR system for the detection of environmental microorganisms[J]. Environmental Microbiology, 2015, 17(7): 2532-2541. DOI PMID
[9]	YAMAGUCHI T, FUCHS B M, AMANN R, et al. Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH)[J]. Systematic and Applied Microbiology, 2015, 38(6): 400-405. DOI PMID
[10]	BHATTARAI S, CASSARINI C, LENS P L. Physiology and distribution of archaeal methanotrophs that couple anaerobic oxidation of methane with sulfate reduction[J]. Microbiology and Molecular Biology Reviews, 2019, 83(3): e00074-18.
[11]	KNITTEL K, LÖSEKANN T, BOETIUS A, et al. Diversity and distribution of methanotrophic Archaea at cold seeps[J]. Applied and Environmental Microbiology, 2005, 71(1): 467-479. PMID
[12]	NAUHAUS K, ALBRECHT M, ELVERT M, et al. In vitro cell growth of marine archaeal-bacterial consortia during anaerobic oxidation of methane with sulfate[J]. Environ-mental Microbiology, 2007, 9(1): 187-196.
[13]	NIU M Y, FAN X B, ZHUANG G C, et al. Methane-metabolizing microbial communities in sediments of the Haima cold seep area, northwest slope of the South China Sea[J]. FEMS Microbiology Ecology, 2017, 93(9): 105-110.
[14]	RUFF S E, BIDDLE J F, TESKE A P, et al. Global dispersion and local diversification of the methane seep microbiome[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(13): 4015-4020. DOI PMID
[15]	孙瑜, 牛明杨, 刘俏, 等. 南海Formosa冷泉区沉积物微生物多样性与分布规律研究[J]. 微生物学报, 2022, 62(6):2001-2020.
	SUN Y, NIU M Y, LIU Q, et al. Diversity and distribution of microorganisms in the sediment of Formosa cold seep in South China Sea[J]. Acta Microbiologica Sinica, 2022, 62(6): 2001-2020.
[16]	JIA Z Y, DONG Y J, XU H, et al. Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for detection of microbes in sediments[J]. Marine Life Science & Technology, 2021, 3(4): 529-541.
[17]	BOLYEN E, RIDEOUT J R, DILLON M R, et al. Repro-ducible, interactive, scalable and extensible microbiome data science using QIIME 2[J]. Nature Biotechnology, 2019, 37(8): 852-857.
[18]	QUAST C, PRUESSE E, YILMAZ P, et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools[J]. Nucleic Acids Research, 2013, 41(Database issue): 590-596. DOI PMID
[19]	WEI T, SIMKO V, LEVY M, et al. corrplot: Visualization of a correlation matrix[CP]. (2017-10-16). https://CRAN.R-project.org/package=corrplot.
[20]	KOLDE R. pheatmap: Pretty Heatmaps[CP]. (2022-10-14). https://CRAN.R-project.org/package=pheatmap.
[21]	CHOI H M T, CHANG J Y, TRINH L A, et al. Programmable in situ amplification for multiplexed imaging of mRNA expression[J]. Nature Biotechnology, 2010, 28(11): 1208-1212. DOI PMID
[22]	MATSUBAYASHI M, SHIMADA Y, LI Y Y, et al. Phylogenetic diversity and in situ detection of eukaryotes in anaerobic sludge digesters[J]. PLoS One, 2017, 12(3): e0172888.
[23]	MOTER A, GÖBEL U B. Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms[J]. Journal of Microbiological Methods, 2000, 41(2): 85-112. DOI PMID
[24]	LEVSKY J M, SINGER R H. Fluorescence in situ hybridization: Past, present and future[J]. Journal of Cell Science, 2003, 116(14): 2833-2838.
[25]	BOETIUS A, RAVENSCHLAG K, SCHUBERT C J, et al. A marine microbial consortium apparently mediating anaerobic oxidation of methane[J]. Nature, 2000, 407(6804): 623-626.
[26]	PERNTHALER A, DEKAS A E, BROWN C T, et al. Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(19): 7052-7057. DOI PMID
[27]	LASO-PÉREZ R, WU F B, CRÉMIÈRE A, et al. Evolutionary diversification of methanotrophic ANME-1 archaea and their expansive virome[J]. Nature Microbiology, 2023, 8(2): 231-245.
[28]	MAIGNIEN L, PARKES R J, CRAGG B, et al. Anaerobic oxidation of methane in hypersaline cold seep sediments[J]. FEMS Microbiology Ecology, 2013, 83(1): 214-231. DOI PMID
[29]	STOKKE R, ROALKVAM I, LANZEN A, et al. Integrated metagenomic and metaproteomic analyses of an ANME-1-dominated community in marine cold seep sediments[J]. Environmental Microbiology, 2012, 14(5): 1333-1346. DOI PMID
[30]	KEVORKIAN R T, CALLAHAN S, WINSTEAD R, et al. ANME-1 archaea may drive methane accumulation and removal in estuarine sediments[J]. Environmental Microbiology Reports, 2021, 13(2): 185-194. DOI PMID
[31]	RISTOVA P P, WENZHÖFER F, RAMETTE A, et al. Spatial scales of bacterial community diversity at cold seeps (Eastern Mediterranean Sea)[J]. ISME Journal, 2015, 9(6): 1306-1318. DOI PMID
[32]	CHEN Y, XU C L, WU N Y, et al. Diversity of anaerobic methane oxidizers in the cold seep sediments of the Okinawa trough[J]. Frontiers in Microbiology, 2022, 13: 819187.
[33]	MCGLYNN S E, CHADWICK G L, KEMPES C P, et al. Single cell activity reveals direct electron transfer in methanotrophic consortia[J]. Nature, 2015, 526(7574): 531-535.
[34]	WANKEL S D, ADAMS M M, JOHNSTON D T, et al. Anaerobic methane oxidation in metalliferous hydrothermal sediments: Influence on carbon flux and decoupling from sulfate reduction[J]. Environmental Microbiology, 2012, 14(10): 2726-2740. DOI PMID
[35]	MCILROY S J, LEU A O, ZHANG X Q, et al. Anaerobic methanotroph ‘Candidatus Methanoperedens nitroreducens’ has a pleomorphic life cycle[J]. Nature Microbiology, 2023, 8(2): 321-331.
[36]	ORPHAN V J, HOUSE C H, HINRICHS K U, et al. Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(11): 7663-7668. DOI PMID
[37]	KRUKENBERG V, RIEDEL D, GRUBER-VODICKA H R, et al. Gene expression and ultrastructure of meso- and thermophilic methanotrophic consortia[J]. Environmental Microbiology, 2018, 20(5): 1651-1666. DOI PMID
[38]	KLEINDIENST S, RAMETTE A, AMANN R, et al. Distribution and in situ abundance of sulfate-reducing bacteria in diverse marine hydrocarbon seep sediments[J]. Environmental Microbiology, 2012, 14(10): 2689-2710. DOI PMID