Skip to main content
SpringerLink
Log in

Heterogeneity of the Tissue-specific Mucosal Microbiome of Normal Grass Carp (Ctenopharyngodon idella)

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

Microbiome plays key roles in the digestion, metabolism, and immunity of the grass carp (Ctenopharyngodon idella). Here, we characterized the normal microbiome of the intestinal contents (IC), skin mucus (SM), oral mucosa (OM), and gill mucosa (GM) of the grass carp, as well as the microbiome of the sidewall (SW) of the raising pool, using full-length 16S rRNA sequencing based on the PacBio platform in this specie for the first time. Twenty phyla, 38 classes, 130 families, 219 genera, and 291 species were classified. One hundred four common classified species might be core microbiota of grass carp. Proteobacteria, Bacteroides, and Cyanobacteria were the dominant phyla in the niche of grass carp. Proteobacteria and Bacteroides dominated the taxonomic composition in the SM, GM, and OM, while Proteobacteria, Planctomycetota, and Cyanobacteria preponderated in the IC and SW groups. Microbiota of IC exhibited higher alpha diversity indices. The microbial communities clustered either in SW or the niche from grass carp, significantly tighter in the SW, based on Bray–Curtis distances (P < 0.05). SM, GM, and OM were similar in microbial composition but were significantly different from IC and SW, while IC had similarity with SW due to their common Cyanobacteria (P < 0.05). Differences were also reflected by niche-specific and differentially abundant microorganisms such as Noviherbaspirillum in the SM and Rhodopseudomonas palustris, Mycobacterium fortuitum, and Acinetobacter schindleri in GM. Significantly raised gene expression was found in IC and SM associated with cell cycle control, cell division, chromosome, coenzyme transport and metabolism, replication, recombination and repair, cell motility, post-translational modification, signal transduction mechanisms, intracellular trafficking, secretion, and vesicles by PICRUSt. This work may be of great value for understanding of fish-microbial co-workshops, especially in different niche of grass carp.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

The 16S rRNA gene sequences were deposited to the NCBI Sequence Read Archive (SRA) database with the accession number PRJNA748818. The data set supporting the conclusions of this article is included within the article.

References

  • Abou Chacra L , Fenollar F (2021) Exploring the global vaginal microbiome and its impact on human health. Microb Pathog 160:105172

  • Anh HTH, Shahsavari E, Bott NJ, Ball AS (2021a) Application of co-culture technology to enhance protease production by two halophilic bacteria. Marinirhabdus Sp and Marinobacter Hydrocarbonoclasticus Molecules 26:3141

    CAS  PubMed  Google Scholar 

  • Anh HTH, Shahsavari E, Bott NJ Ball AS (2021b) The application of Marinobacter hydrocarbonoclasticus as a bioaugmentation agent for the enhanced treatment of non-sterile fish wastewater. J Environ Manag 291:112658

  • Astrofsky KM, Schrenzel MD, Bullis RA, Smolowitz RM, Fox JG (2000) Diagnosis and management of atypical Mycobacterium spp. infections in established laboratory zebrafish (Brachydanio rerio) facilities. Comp Med 50(6):666–672

  • Behra PRK, Pettersson BMF, Ramesh M, Dasgupta S, Kirsebom LA (2019) Insight into the biology of Mycobacterium mucogenicum and Mycobacterium neoaurum Glade members. Sci Rep 9:19259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brooks JP, Edwards DJ, Harwich MD, Rivera MC, Fettweis JM, Serrano MG, Reris RA, Sheth NU, Huang B, Girerd P et al (2015) The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiol 15:66

    Article  PubMed  PubMed Central  Google Scholar 

  • Cao SL, Guo JJ, Zhao WP, Yang WF, Zhang SL, Tao HZ Li JN (2021) Impacts of oral Vibrio mimicus double-targeted DNA vaccine on the gut microbiota in grass carps (Ctenopharyngodon idella) and correlations with intestinal mucosa innate immunity. Aquaculture 533

  • Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chung GT, Yoo JS, Oh HB, Lee YS, Cha SH, Kim SJ, Yoo CK (2008) Complete genome sequence of Neisseria gonorrhoeae NCCP11945. J Bacteriol 190:6035–6036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Coetzer WG, Coetzee LM, Cason ED, Grobler JP, Schneider SR, Boucher CE (2021) A preliminary assessment of skin microbiome diversity of zebrafish (Danio rerio): South African Pet Shop Fish. Indian J Microbiol 61:81–84

    Article  CAS  PubMed  Google Scholar 

  • Collado L, Cleenwerck I, Van Trappen S, De Vos P, Figueras MJ (2009) Arcobacter mytili sp. nov., an indoxyl acetate-hydrolysis-negative bacterium isolated from mussels. Int J Syst Evol Microbiol 59(Pt 6):1391–1396

  • Cui CY, Chen C, Liu BT, He Q, Wu XT, Sun RY, Zhang Y, Cui ZH, Guo WY, Jia QL et al (2020) Co-occurrence of plasmid-mediated tigecycline and carbapenem resistance in Acinetobacter spp. from waterfowls and their neighboring environment. Antimicrob Agents Chemother 64(5):e02502–19

  • Delghandi MR, El-Matbouli M, Menanteau-Ledouble S (2020) Mycobacteriosis and infections with non-tuberculous mycobacteria in aquatic organisms: a review. Microorganisms 8:1368

    Article  CAS  PubMed Central  Google Scholar 

  • Dortet L, Legrand P, Soussy CJ, Cattoir V (2006) Bacterial identification, clinical significance, and antimicrobial susceptibilities of Acinetobacter ursingii and Acinetobacter schindleri, two frequently misidentified opportunistic pathogens. J Clin Microbiol 44:4471–4478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Earl JP, Adappa ND, Krol J, Bhat AS, Balashov S, Ehrlich RL, Palmer JN, Workman AD, Blasetti M, Sen B et al (2018) Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes. Microbiome 6:190

    Article  PubMed  PubMed Central  Google Scholar 

  • Food and Agriculture Organization (FAO) (2018) The State of World Fisheries and Aquaculture 

  • Gloor GB, Hummelen R, Macklaim JM, Dickson RJ, Fernandes AD, MacPhee R, Reid G (2010) Microbiome profiling by illumina sequencing of combinatorial sequence-tagged PCR products. PLoS One 5(10)

  • Gomez D, Sunyer JO, Salinas I (2013) The mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens. Fish Shellfish Immunol 35:1729–1739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Groussin M, Mazel F, Alm EJ (2020) Co-evolution and co-speciation of host-gut bacteria systems. Cell Host Microbe 28:12–22

    Article  CAS  PubMed  Google Scholar 

  • Han QF, Zhang XR, Xu XY, Wang XL, Yuan XZ, Ding ZJ, Zhao S, Wang SG (2021) Antibiotics in marine aquaculture farms surrounding Laizhou Bay, Bohai Sea: distribution characteristics considering various culture modes and organism species. Sci Total Environ 760:143863

  • Hu A, He J, Chu KH, Yu CP (2011) Genome sequence of the 17beta-estradiol-utilizing bacterium Sphingomonas strain KC8. J Bacteriol 193:4266–4267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang S, Sheng P, Zhang H (2012) Isolation and identification of cellulolytic bacteria from the gut of Holotrichia parallela larvae (Coleoptera: Scarabaeidae). Int J Mol Sci 13:2563–2577

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huttenhower C, Gevers D, Knight R, Abubucker S, Badger JH, Chinwalla AT, Creasy HH, Earl AM, FitzGerald MG, Fulton RS et al (2012) Structure, function and diversity of the healthy human microbiome. Nature 486:207–214

    Article  CAS  Google Scholar 

  • Ijaz MU, Ahmed MI, Zou X, Hussain M, Zhang M, Zhao F, Xu X, Zhou G, Li C (2018) Beef, casein, and soy proteins differentially affect lipid metabolism, triglycerides accumulation and gut microbiota of high-fat diet-fed C57BL/6J mice. Front Microbiol 9:2200

    Article  PubMed  PubMed Central  Google Scholar 

  • Jamieson WD, Pehl MJ, Gregory GA, Orwin PM (2009) Coordinated surface activities in Variovorax paradoxus EPS. BMC Microbiol 9:124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI (2011) Human nutrition, the gut microbiome and the immune system. Nature 474:327–336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ke FM, Gao YP, Liu L, Zhang C, Wang Q, Gao XW (2020) Comparative analysis of the gut microbiota of grass carp fed with chicken faeces. Environ Sci Pollut Res 27:32888–32898

    Article  CAS  Google Scholar 

  • Kelly C, Salinas I (2017) Under pressure: interactions between commensal microbiota and the teleost immune system. Front Immunol 8:559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klemetsen T, NP Willassen and CR Karlsen (2019) Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis. Microbiologyopen 8(10)

  • Li G, Brun YV, Tang JX (2013) Holdfast spreading and thickening during Caulobacter crescentus attachment to surfaces. BMC Microbiol 13:139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li XH, Yu YH, Li C, Yan QY (2018) Comparative study on the gut microbiotas of four economically important Asian carp species. Science China-life Sciences 61:696–705

    Article  PubMed  Google Scholar 

  • Lin T, Chen T, Liu J, Tu XM (2021) Extending the Mann-Whitney-Wilcoxon rank sum test to survey data for comparing mean ranks. Stat Med 40(7):1705–1717

  • Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R (2011) UniFrac: an effective distance metric for microbial community comparison. ISME J 5:169–172

    Article  PubMed  Google Scholar 

  • Miller WG, Yee E, Bono JL (2018) Complete genome sequence of the Arcobacter mytili type strain LMG 24559. Microbiol Resour Announc 7:e01078-e1118

    PubMed  PubMed Central  Google Scholar 

  • Mosher JJ, Bowman B, Bernberg EL, Shevchenko O, Kan J, Korlach J, Kaplan LA (2014) Improved performance of the PacBio SMRT technology for 16S rDNA sequencing. J Microbiol Methods 104:59–60

    Article  CAS  PubMed  Google Scholar 

  • Mugetti D, Tomasoni M, Pastorino P, Esposito G, Menconi V, Dondo A, Prearo M (2021) Gene sequencing and phylogenetic analysis: powerful tools for an improved diagnosis of fish mycobacteriosis caused by Mycobacterium fortuitum group members. Microorganisms 9:797

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakatsuji T, Cheng JY, Gallo RL (2021) Mechanisms for control of skin immune function by the microbiome. Curr Opin Immunol 72:324–330

    Article  CAS  PubMed  Google Scholar 

  • Ni JJ, Yan QY, Yu YH, Zhang TL (2014) Factors influencing the grass carp gut microbiome and its effect on metabolism. FEMS Microbiol Ecol 87:704–714

    Article  CAS  PubMed  Google Scholar 

  • Nunkaew T, Kantachote D, Nitoda T, Kanzaki H, Ritchie RJ (2015) Characterization of exopolymeric substances from selected Rhodopseudomonas palustris strains and their ability to adsorb sodium ions. Carbohydr Polym 115:334–341

    Article  CAS  PubMed  Google Scholar 

  • Nyerges G, Han SK, Stein LY (2010) Effects of ammonium and nitrite on growth and competitive fitness of cultivated methanotrophic bacteria. Appl Environ Microbiol 76:5648–5651

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pan HJ, Li ZF, Xie J, Liu D, Wang HJ, Yu DG, Zhang Q, Hu ZY, Shi CB (2019) Berberine influences blood glucose via modulating the gut microbiome in grass carp. Front Microbiol 10:1066

    Article  PubMed  PubMed Central  Google Scholar 

  • Pootakham W, Mhuantong W, Yoocha T, Putchim L, Sonthirod C, Naktang C, Thongtham N, Tangphatsornruang S (2017) High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system. Sci Rep 7:2774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig WG, Peplies J, Glockner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35:7188–7196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Quivey RG, O’Connor TG, Gill SR, Kopycka-Kedzierawski DT (2021) Prediction of early childhood caries onset and oral microbiota. Mol Oral Microbiol 36:255–257

    Article  PubMed  Google Scholar 

  • Reeve W, Nandasena K, Yates R, Tiwari R, O’Hara G, Ninawi M, Gu W, Goodwin L, Detter C, Tapia R et al (2013) Complete genome sequence of Mesorhizobium australicum type strain (WSM2073(T)). Stand Genomic Sci 9:410–419

    Article  PubMed  PubMed Central  Google Scholar 

  • Roh H, Chu KH (2011) Effects of solids retention time on the performance of bioreactors bioaugmented with a 17beta-estradiol-utilizing bacterium, Sphingomonas strain KC8. Chemosphere 84:227–233

    Article  CAS  PubMed  Google Scholar 

  • Rosado D, Perez-Losada M, Pereira A, Severino R, Xavier R (2021) Effects of aging on the skin and gill microbiota of farmed seabass and seabream. Anim Microbiome 3:10

    Article  PubMed  PubMed Central  Google Scholar 

  • Ryan MP, Pembroke JT, Adley CC (2006) Ralstonia pickettii: a persistent gram-negative nosocomial infectious organism. J Hosp Infect 62:278–284

    Article  CAS  PubMed  Google Scholar 

  • Salipante SJ, Kawashima T, Rosenthal C, Hoogestraat DR, Cummings LA, Sengupta DJ, Harkins TT, Cookson BT, Hoffman NG (2014) Performance comparison of Illumina and ion torrent next-generation sequencing platforms for 16S rRNA-based bacterial community profiling. Appl Environ Microbiol 80:7583–7591

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C (2011) Metagenomic biomarker discovery and explanation. Genome Biol 12:R60

    Article  PubMed  PubMed Central  Google Scholar 

  • Sehnal L, Brammer-Robbins E, Wormington AM, Blaha L, Bisesi J, Larkin I, Martyniuk CJ, Simonin M, Adamovsky O (2021) Microbiome composition and function in aquatic vertebrates: small organisms making big impacts on aquatic animal health. Front Microbiol 12:567408

  • Shao S, Hu Y, Cheng J, Chen Y (2018) Research progress on distribution, migration, transformation of antibiotics and antibiotic resistance genes (ARGs) in aquatic environment. Crit Rev Biotechnol 38:1195–1208

    Article  CAS  PubMed  Google Scholar 

  • Shen YB, Wang L, Fu JJ, Xu XY, Yue GH, Li JL (2019) Population structure, demographic history and local adaptation of the grass carp. BMC Genomics 20:467

    Article  PubMed  PubMed Central  Google Scholar 

  • Sullam KE, Essinger SD, Lozupone CA, O’Connor MP, Rosen GL, Knight R, Kilham SS, Russell JA (2012) Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis. Mol Ecol 21:3363–3378

    Article  PubMed  Google Scholar 

  • Sylvain FE, Holland A, Bouslama S, Audet-Gilbert E, Lavoie C, Val AL, Derome N (2020) Fish skin and gut microbiomes show contrasting signatures of host species and habitat. Appl Environ Microbiol 86:e00789-e820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tan KY, Dutta A, Tan TK, Hari R, Othman RY Choo SW (2020) Comprehensive genome analysis of a pangolin-associated Paraburkholderia fungorum provides new insights into its secretion systems and virulence. Peerj 8:e9733

  • Tran NT, Zhang J, Xiong F, Wang GT, Li WX, Wu SG (2018) Altered gut microbiota associated with intestinal disease in grass carp (Ctenopharyngodon idellus). World J Microbiol Biotechnol 34:71

    Article  CAS  PubMed  Google Scholar 

  • Uslan DZ, Kowalski TJ, Wengenack NL, Virk A, Wilson JW (2006) Skin and soft tissue infections due to rapidly growing mycobacteria - comparison of clinical features, treatment, and susceptibility. Arch Dermatol 142:1287–1292

    Article  PubMed  Google Scholar 

  • Wang ST, Meng XZ, Dai YF, Zhang JH, Shen YB, Xu XY, Wang RQ Li JL (2021) Characterization of the intestinal digesta and mucosal microbiome of the grass carp (Ctenopharyngodon idella). Comp Biochem Physiol Part D: Genomics & Proteomics 37:100789

  • Wang ST, Meng XZ, Zhang JH, Dai YF, Shen YB, Xu XY, Wang RQ Li JL (2020) 16S rRNA sequencing analysis of the correlation between the intestinal microbiota and body-mass of grass carp (Ctenopharyngodon idella). Comp Biochem Physiol Part D: Genomics & Proteomics 35:100699

  • Wong SD, Rawls JF (2012) Intestinal microbiota composition in fishes is influenced by host ecology and environment. Mol Ecol 21:3100–3102

    Article  PubMed  PubMed Central  Google Scholar 

  • Wu SG, Wang gt, Angert ER, Wang WW, Li WX, Zou H (2012) Composition, diversity, and origin of the bacterial community in grass carp intestine. PloS One 7(2): e30440

  • Wu ZB, Zhang QQ, Lin YY, Hao JW, Wang SY, Zhang JY, Li AH (2021) Taxonomic and functional characteristics of the gill and gastrointestinal microbiota and its correlation with intestinal metabolites in New Gift strain of farmed adult nile tilapia (Oreochromis niloticus). Microorganisms 9:617

    Article  PubMed  PubMed Central  Google Scholar 

  • Xiao L, Zhao Z, Ma ZJ, Chen J, Song ZF (2020) Immobilization of Rhodopseudomonas palustris P1 on glass pumice to improve the removal of NH4+-N and NO2–N from aquaculture pond water. Biotechnol Appl Biochem 67:323–329

    CAS  PubMed  Google Scholar 

  • Xiong JB, Nie L, Chen J (2019) Current understanding on the roles of gut microbiota in fish disease and immunity. Zool Res 40:70–76

    Article  PubMed  Google Scholar 

  • Xu Z, Parra D, Gomez D, Salinas I, Zhang YA, Jorgensen LV, Heinecke RD, Buchmann K, LaPatra S, Sunyer JO (2013) Teleost skin, an ancient mucosal surface that elicits gut-like immune responses. Proc Natl Acad Sci USA 110:13097–13102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zelezniak A, Andrejev S, Ponomarova O, Mende DR, Bork P, Patil KR (2015) Metabolic dependencies drive species co-occurrence in diverse microbial communities. Proc Natl Acad Sci USA 112:6449–6454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao YM, Zhang L, Wang CF, Xie CX (2020) Biology and ecology of grass carp in China: a review and synthesis. North Am J Fish Manag 40:1379–1399

    Article  Google Scholar 

  • Zhou L, Lin KT, Gan L, Sun JJ, Guo CJ, Liu L, Huang XD (2019) Intestinal microbiota of grass carp fed faba beans: a comparative study. Microorganisms 7:465

    Article  CAS  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by the China Agriculture Research System (CARS-45–03) and the Project of Shanghai Engineering and Technology Center for Promoting Ability (19DZ2284300). China Agriculture Research System,CARS-45–03,the Project of Shanghai Engineering and Technology Center for Promoting Ability,19DZ2284300,Jiale Li

Author information

Authors and Affiliations

  1. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai, 201306, China

    Na Zhao, Jiamin Guo, Yuting Liu, Yubang Shen & Jiale Li

  2. College of Fisheries and Life Science, Shanghai Engineering Research Centre of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China

    Yubang Shen & Jiale Li

  3. Hangzhou Academy of Agricultural Sciences, Hangzhou, 310024, China

    Kai Liu

  4. Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Zhanjiang, China

    Na Zhao & Bo Zhang

Authors
  1. Na Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiamin Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bo Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kai Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuting Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yubang Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jiale Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yubang Shen or Jiale Li.

Ethics declarations

Research Involving Human and Animal Participants

All handling of fishes was conducted in accordance with the guidelines on the care and use of animals for scientific purposes set up by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Ocean University, Shanghai, China.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, N., Guo, J., Zhang, B. et al. Heterogeneity of the Tissue-specific Mucosal Microbiome of Normal Grass Carp (Ctenopharyngodon idella). Mar Biotechnol 24, 366–379 (2022). https://doi.org/10.1007/s10126-022-10113-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-022-10113-3

Keywords

Search

Navigation