BACKGROUND: Caves are special natural laboratories for most biota and the cave communities are unique. Establishing population in cave is accompanied with modifications in adaptability for most animals. To date, little is known about the survival mechanisms of soil animals in cave environments, albeit they play vital roles in most terrestrial ecosystems. Here, we investigated whether and how gut microbes would contribute to the adaptation of earthworms by comparing the gut microbiome of two earthworm species from the surface and caves. RESULTS: Two dominant earthworm species inhabited caves, i.e., Allolobophora chlorotica and Aporrectodea rosea. Compared with the counterparts on the surface, A. rosea significantly decreased population in the cave, while A. chlorotica didn't change. Microbial taxonomic and phylogenetic diversities between the earthworm gut and soil environment were asynchronic with functional diversity, with functional gene diversity been always higher in earthworm gut than in soil, but species richness and phylogenetic diversity lower. In addition, earthworm gut microbiome were characterized by higher rrn operon numbers and lower network complexity than soil microbiota. CONCLUSIONS: Different fitness of the two earthworm species in cave is likely to coincide with gut microbiota, suggesting interactions between host and gut microbiome are essential for soil animals in adapting to new environments. The functional gene diversity provided by gut microbiome is more important than taxonomic or phylogenetic diversity in regulating host adaptability. A stable and high-efficient gut microbiome, including microbiota and metabolism genes, encoded potential functions required by the animal hosts during the processes of adapting to and establishing in the cave environments. Our study also demonstrates how the applications of microbial functional traits analysis may advance our understanding of animal-microbe interactions that may aid animals to survive in extreme ecosystems.

Institute of Soil Biology Biology Centre Czech Academy of Sciences Na Sádkách 7 37005 České Budějovice Czech Republic

Soil Ecology Lab College of Resources and Environmental Sciences Nanjing Agricultural University Nanjing 210095 China

See more in PubMed

Poulson TL, White WB. The cave environment. Science. 1969;165:971–981. doi: 10.1126/science.165.3897.971. PubMed DOI

Christman MC, Culver DC, Madden MK, White D. Patterns of endemism of the eastern North American cave fauna. J Biogeogr. 2005;32:1441–1452. doi: 10.1111/j.1365-2699.2005.01263.x. DOI

Christman MC, Culver DC. The relationship between cave biodiversity and available habitat. J Biogeogr. 2001;28:367–380. doi: 10.1046/j.1365-2699.2001.00549.x. DOI

Mammola S. Finding answers in the dark: caves as models in ecology fifty years after Poulson and White. Ecography (Cop) 2019;42:1331–1351. doi: 10.1111/ecog.03905. DOI

Smrž J, Kováč L, Mikeš J, Šustr V, Lukešová A, Tajovský K, et al. Food sources of selected terrestrial cave arthropods. Subterr Biol. 2015;16:37–46. doi: 10.3897/subtbiol.16.8609. DOI

Phillips HRP, Guerra CA, Bartz MLC, Briones MJI, Brown G, Crowther TW, et al. Global distribution of earthworm diversity. Science. 2019;366:480–485. doi: 10.1126/science.aax4851. PubMed DOI PMC

Reeves WK, Reynolds JW. New records of cave-dwelling earthworms (Oligochaeta: Lumbricidae, Megascolecidae and Naididae) and other annelids (Aeolosomatida, Branchiobdellida and Hirudinea) in the Southeastern United States, with notes on their ecology. Megadrilogica. 1999;7:65–71.

Reynolds JW. Note on some cave earthworms (Oligochaeta: Lumbricidae) from the Isle of Man, U.K. Megadrilogica. 1996;6:89–90.

Protas ME, Trontelj P, Patel NH. Genetic basis of eye and pigment loss in the cave crustacean, Asellus aquaticus. Proc Natl Acad Sci U S A. 2011;108:5702–5707. doi: 10.1073/pnas.1013850108. PubMed DOI PMC

Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI. Worlds within worlds: evolution of the vertebrate gut microbiota. Nat Rev Microbiol. 2008;6:776–788. doi: 10.1038/nrmicro1978. PubMed DOI PMC

Shapira M. Gut microbiotas and host evolution: scaling up symbiosis. Trends Ecol Evol. 2016;31:539–549. doi: 10.1016/j.tree.2016.03.006. PubMed DOI

Ankrah NYD, Douglas AE. Nutrient factories: metabolic function of beneficial microorganisms associated with insects. Environ Microbiol. 2018;20:2002–2011. doi: 10.1111/1462-2920.14097. PubMed DOI

Chu CC, Spencer JL, Curzi MJ, Zavala JA, Seufferheld MJ. Gut bacteria facilitate adaptation to crop rotation in the western corn rootworm. Proc Natl Acad Sci U S A. 2013;110:11917–11922. doi: 10.1073/pnas.1301886110. PubMed DOI PMC

Yun JH, Roh SW, Whon TW, Jung MJ, Kim MS, Park DS, et al. Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host. Appl Environ Microbiol. 2014;80:5254–5264. doi: 10.1128/AEM.01226-14. PubMed DOI PMC

Thakuria D, Schmidt O, Finan D, Egan D, Doohan FM. Gut wall bacteria of earthworms: a natural selection process. ISME J. 2010;4:357–366. doi: 10.1038/ismej.2009.124. PubMed DOI

Rossmassler K, Dietrich C, Thompson C, Mikaelyan A, Nonoh JO, Scheffrahn RH, et al. Metagenomic analysis of the microbiota in the highly compartmented hindguts of six wood- or soil-feeding higher termites. Microbiome. 2015;:1–6. PubMed PMC

Ding J, Zhu D, Li H, Ding K, Chen QL, Lassen SB, et al. The gut microbiota of soil organisms show species-specific responses to liming. Sci Total Environ. 2019;659:715–723. doi: 10.1016/j.scitotenv.2018.12.445. PubMed DOI

Klappenbach JA, Dunbar JM, Schmidt TM. rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol. 2000;66:1328–1333. doi: 10.1128/AEM.66.4.1328-1333.2000. PubMed DOI PMC

Layeghifard M, Hwang DM, Guttman DS. Disentangling interactions in the microbiome: a network perspective. Trends Microbiol. 2017;25:217–228. doi: 10.1016/j.tim.2016.11.008. PubMed DOI PMC

Růžička V, Šmilauer P, Mlejnek R. Colonization of subterranean habitats by spiders in Central Europe. Int J Speleol. 2013;42:133–140. doi: 10.5038/1827-806X.42.2.5. DOI

Rutgers M, Orgiazzi A, Gardi C, Römbke J, Jänsch S, Keith AM, et al. Mapping earthworm communities in Europe. Appl Soil Ecol. 2016;97:98–111. doi: 10.1016/j.apsoil.2015.08.015. DOI

Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–336. doi: 10.1038/nmeth.f.303. PubMed DOI PMC

Langille MGI, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol. 2013;31:814–821. doi: 10.1038/nbt.2676. PubMed DOI PMC

R Core Team. R: a language and environment for statistical computing. R foundation for statistical computing, Vienna, Austria. URL http://www.R-project.org/. 2021.

Kembel SW, Cowan PD, Helmus MR, Cornwell WK, Morlon H, Ackerly DD, et al. Picante: R tools for integrating phylogenies and ecology. Bioinformatics. 2010;26:1463–1464. doi: 10.1093/bioinformatics/btq166. PubMed DOI

Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009;75:7537–7541. doi: 10.1128/AEM.01541-09. PubMed DOI PMC

Csardi G, Nepusz T. The igraph software package for complex network research. InterJournal. 2006; Complex Systems:1695.

Bredon M, Dittmer J, Noël C, Moumen B, Bouchon D. Lignocellulose degradation at the holobiont level: teamwork in a keystone soil invertebrate. Microbiome. 2018;6:1–19. doi: 10.1186/s40168-018-0536-y. PubMed DOI PMC

O’brien PA, Webster NS, Miller DJ, Bourne DG. Host-microbe coevolution: Applying evidence from model systems to complex marine invertebrate holobionts. MBio. 2019;10:1–14. PubMed PMC

Macke E, Tasiemski A, Massol F, Callens M, Decaestecker E. Life history and eco-evolutionary dynamics in light of the gut microbiota. Oikos. 2017;126:508–531. doi: 10.1111/oik.03900. DOI

Bon D, Gilard V, Massou S, Pérès G, Malet-Martino M, Martino R, et al. In vivo 31P and 1H HR-MAS NMR spectroscopy analysis of the unstarved Aporrectodea caliginosa (Lumbricidae) Biol Fertil Soils. 2006;43:191–198. doi: 10.1007/s00374-006-0092-7. DOI

Xiang Q, Zhu D, Chen QL, Delgado-Baquerizo M, Su JQ, Qiao M, et al. Effects of diet on gut microbiota of soil collembolans. Sci Total Environ. 2019;676:197–205. doi: 10.1016/j.scitotenv.2019.04.104. PubMed DOI

Mathipi V, de Mandal S, Chawngthu Z, Lalfelpuii R, Kumar NS, Lalthanzara H. Diversity and metabolic potential of earthworm gut microbiota in Indo-Myanmar biodiversity hotspot. J Pure Appl Microbiol. 2020;14:1503–1511. doi: 10.22207/JPAM.14.2.48. DOI

Sampedro L, Whalen JK. Changes in the fatty acid profiles through the digestive tract of the earthworm Lumbricus terrestris L. Appl Soil Ecol. 2007;35:226–236. doi: 10.1016/j.apsoil.2006.04.007. DOI

Drake HL, Horn MA. As the Worm Turns: The earthworm gut as a transient habitat for soil microbial biomes. Annu Rev Microbiol. 2007;61:169–189. doi: 10.1146/annurev.micro.61.080706.093139. PubMed DOI

Trigo D, Lavelle P. Changes in respiration rate and some physicochemical properties of soil during gut transit through Allolobophora molleri (Lumbricidae, Oligochaeta) Biol Fertil Soils. 1993;15:185–188. doi: 10.1007/BF00361609. DOI

Knapp BA, Seeber J, Podmirseg SM, Meyer E, Insam H. Application of denaturing gradient gel electrophoresis for analysing the gut microflora of Lumbricus rubellus Hoffmeister under different feeding conditions. Bull Entomol Res. 2008;98:271–279. doi: 10.1017/S0007485308006056. PubMed DOI

Knapp BA, Podmirseg SM, Seeber J, Meyer E, Insam H. Diet-related composition of the gut microbiota of Lumbricus rubellus as revealed by a molecular fingerprinting technique and cloning. Soil Biol Biochem. 2009;41:2299–2307. doi: 10.1016/j.soilbio.2009.08.011. DOI

Rudi K, Ødegård K, Løkken TT, Wilson R. A feeding induced switch from a variable to a homogenous state of the earthworm gut microbiota within a host population. PLoS ONE. 2009;4:e7528. doi: 10.1371/journal.pone.0007528. PubMed DOI PMC

Egert M, Marhan S, Wagner B, Scheu S, Friedrich MW. Molecular profiling of 16S rRNA genes reveals diet-related differences of microbial communities in soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta: Lumbricidae). FEMS Microbiol Ecol. 2004;48:187–97. PubMed

Gong X, Chen TW, Zieger SL, Bluhm C, Heidemann K, Schaefer I, et al. Phylogenetic and trophic determinants of gut microbiota in soil oribatid mites. Soil Biol Biochem. 2018;123:155–164. doi: 10.1016/j.soilbio.2018.05.011. DOI

Condon C, Liveris D, Squires C, Schwartz I, Squires CL. rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation. J Bacteriol. 1995;177:4152–4156. doi: 10.1128/jb.177.14.4152-4156.1995. PubMed DOI PMC

Klappenbach JA, Saxman PR, Cole JR, Schmidt TM. rrndb: The ribosomal RNA operon copy number database. Nucleic Acids Res. 2001;29:181–184. doi: 10.1093/nar/29.1.181. PubMed DOI PMC

Roller BRK, Stoddard SF, Schmidt TM. Exploiting rRNA operon copy number to investigate bacterial reproductive strategies. Nat Microbiol. 2016;1 November:1–7. PubMed PMC

Valdivia-Anistro JA, Eguiarte-Fruns LE, Delgado-Sapién G, Márquez-Zacarías P, Gasca-Pineda J, Learned J, et al. Variability of rRNA operon copy number and growth rate dynamics of bacillus isolated from an extremely oligotrophic aquatic ecosystem. Front Microbiol. 2016;6:1–15. doi: 10.3389/fmicb.2015.01486. PubMed DOI PMC

Food origin influences microbiota and stable isotope enrichment profiles of cold-adapted Collembola (Desoria ruseki)