Variations of the Intestinal Gut Microbiota of Farmed Rainbow Trout, Oncorhynchus Mykiss (Walbaum), Depending on the Infection Status of the Fish"

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This is the peer reviewed version of the following article: Parshukov, A.N., E.N. Kashinskaya, E.P. Simonov, O.V. Hlunov, G.I. Izvekova, K.B. Andree, and M.M. Solovyev. 2019. "Variations Of The Intestinal Gut Microbiota Of Farmed Rainbow Trout, Oncorhynchus Mykiss (Walbaum), Depending On The Infection Status Of The Fish". Journal Of Applied Microbiology. Wiley. doi:10.1111/jam.14302., which has been published in final form at https://doi.org/10.1111/jam.14302. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions http://www.wileyauthors.com/self- archiving.

This article isprotected rights by All copyright. reserved. 10.1111/jam.14302 doi: lead been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer 7 6 Nekouzskii 5 4 Siberian Branch of 3 Sciences, Novosibirsk, 2 Petrozavodsk, 1 K. A. Articletype : MISS ELENAKASHINSKAYA (Orcid ID : 0000 ALEKSEYDR. PARSHUKOV (Orcid ID : 0000 Tomsk State University,Tomsk State Tomsk InstitutePapanin Biology for Inland ofWaters, Academy Russian Sciences, Borok, of LLC“FishForel” Laboratory ResearchBiotechnology, forand Genomic Krasnoyarsk Science the Center of IRTA Institute ofSystematicsand Siberian Ecology Branch, ofAnimals, Ru Institute ofBiology Karelian ofthe Research ofthe Centre Academy Russian ofSc

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This article isprotected rights by All copyright. reserved. in species aquaculture main the of one is trout Rainbow Accepted 2016). (FAO, 2013 in value by decade recent in strongly increased has trade world the to According region. in aquaculture for fish important most the of one and production wh species aquaculture successful most the among are Salmonids decades recent disease, Keywords: developmentprograms. for ofnew control disease strategies of microbiota Articlefish. healthy with compared if groups unhealthy the in intestine and stomach the of communities stomachbetween of and intestine (p≤0.05). themicrobiota from bacteria as Renibacterium such taxa unique registered Serratia, Lactobacillus to belonging OTU's by made fish. unhealthy

Aquaculture has been the world’s fastest growing food producing industry during during industry producing food growing fastest world’s the been has Aquaculture I Study of Impact and Significance Conclusions. ntroduction freshwater cage a cagefreshwater Bacillus

atra cmuiy salmonids, community, bacterial . farmed rainbow trout, rainbow farmed . , irboa f nelh fs in fish unhealthy of Microbiota The greatest contribution to the gut microbial composition of healthy of composition microbial gut the to contribution greatest The

Analysis of similarities (ANOSIM test) revealed the significant dissimilarity significant the revealed test) (ANOSIM similarities of Analysis producing and ,

A s

ubsta Food and Agriculture Organization, the share of salmon and trout in trout and salmon of share the Organization, Agriculture and Food Pseudomonas quaculture

p o 60.4 to up ntial finding was the absence of differences of absence the was finding ntial Bac

O. mykiss O. lu, erta Pseudomonas Serratia, illus, . In microbiota of unhealthy fish fish unhealthy of microbiota In . b lin tons illion . These results demonstrated alterations of the gut gut the of alterations demonstrated results These

bacterial during co during most most s, becoming the largest single commodity commodity single largest the becoming s, f ih products fish of

the ae ws ersne by represented was cases kidney

infections and can be useful for the the for useful be can and infections

family

disease, en measured by measured en Mycoplasmataceae per year (FAO, 2010). 2010). (FAO, year per the ,

Cetobacterium bacterial

oten Europe northern between microbial microbial between there h genera the cold were growth of growth

fish was was fish - and ,

water water

also and and an

This article isprotected rights by All copyright. reserved. Accepted2007 in hatcheries Finnish from imported fry with Karelia in introduced was that Such before. pathogenic, with that known countries N and Krai Krasnodar Oblast, Arkhangelsk Oblast, Leningrad fingerlings and reproductive Karelia in lack the is salmonids of diseases with among disease are trout cage in diseases parasitic trout developed highly with scientific the in coverage Articleand literature wide a have not do sporadic, are Karelia in aquaculture cage (Bebak ( parasites and crustacean trematoda, cestoda, fungi, bacteria, viruses, as such agents pathological different products where 97% ofproduct Europe. northern the in located Karelia impor most the Federation Russian the million 250 exceeds production its Europe: ), - n o te ot motn polm i aquaculture in problems important most the of One many others ( others many Williams et al., 2002). al., et Williams

g uh as such yrodactylosis,

including different pathogens that h that pathogens different including purposes purposes was the was are re are be transport of transport At ntd Kingdom United bought from bought presented Evseyeva the present time, Karelia produces around 21 thousand thousand 21 around produces Karelia time, present the situation, for example, with triploid triploid with example, for situation, to north

support d gna teaoe infe trematode igenean -

farming by east fish species fish , ion etc.) that cause s cause that etc.) Unfortunately, studies regarding problems of fish diseases in diseases fish of problems regarding studies Unfortunately, Dzubuk other regions of of regions other only only the

part of Russia and has has and Russia of part is attributed to ichthyobodosis UA Belarus, USA, , local aquaculture industry,aquaculture local and with future broad investment prospects. The main The prospects. investment broad future with and few a , 2016; Ieshko et al., 2016). al., et Ieshko 2016; , at ein o cg auclue f amnd is salmonids of aquaculture cage for region tant

te organisms other

works tons ave never appeared in the local water bodies bodies water local the in appeared never ave ignificant damage and great economical loss economical great and damage ignificant

with a value close to 700 million million 700 to close value a with the rainbow trout. , which , ction of

local brood stocks of rai of stocks brood local Denmark, Russia ostiasis), ostiasis), ,

rsaen d crustacean

climate a clone n Federation n are introduced, some potentially some introduced, are under orth Ossetia orth i s netos disease infectious is chthyophthiriasis which necessitates which n Finland and

- yoatls s Gyrodactylus representation representation Aggravating sae ( iseases similar similar

(Murmansk Oblast, (Murmansk - Alania) and Alania) .

to e.g It is very well well very is It nbow trout for for trout nbow the situation situation the tons of countries . for alaris alaris

protozoan, protozoan, a . the

rgulosis), rgulosis), s euros

hite spot hite This has This a region region a

of fish fish of u to due spawn other other RBT . In .

This article isprotected rights by All copyright. reserved. Acceptedapproaches have 2009) al., et Navarrete 19 al., et Trust others (among salmonids of community microbial gut the of structure the several on focused Although have studies gut. the of dysbiosis resulting potential the of terms in evaluated been feeding parasites and bacteria by caused disorders the that known well very 2016, al., Inge 2014, al., et Geurden 2012, al., et Navarrete 2012, al., et Mansfield 2006, al., et (Heikkinen etc water, of composition chemical and the others among includes compare if restricted is factors of number the conditions nature in e ArticleAraújo 2000, al., et (Verschuere pathogens different against defense the in role key a play can and digestion of cageaquaculture inKarelia trout of introduction Yersinia are Karelia in salmonids for pathogens bac studied most The extent. lesser much a to studied been have pathologies bacterial including rainbow trout. problematic a become revealed The bacterial community of f of community bacterial The pathogen invasive to contrast In

eair and behavior ( Bruce et al., 2017 al., Bruceet aquaculture Under community. bacterial gut fish the affect to able potentially are R

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yfnto o dysfunction . 4 Ysiiu t l, 96 Chl, 1990, Cahill, 1976, al., et Yoshimizu 74, and and

from Finland, Denmark, etc Denmark, Finland, from ultur

proximate composition of diet, fish age, fish diet, of composition proximate , Lyons et al., 2017, Huyben et al., 2018 al., etHuyben 2017, Lyonsal., et , harmf 2007).

( e are There

Nechaeva - of dependent ul the T parasite species for both native and native both for species parasite the f e rgn f hs bceil ahgn ws intensive was pathogens bacterial these of origin he s gt osse dfeet ucin sc as such functions different possesses gut ish

microbial

c parasite ic s, described a plethora of biotic and abiotic factors that factors abiotic and biotic of plethora a described , 2014) system. digestive Pseudomonas, Aeromonas, Flavobacterium Flavobacterium Aeromonas, Pseudomonas, uh s NGS as such approach .

community ( community o ribw trout rainbow of s es .

during

are

nbe icvr of discovery enable well d hs cags ae o always not have changes These

rslev et al., 2014a, 2014a, al., et rslev a period of period a ih conditions with Amann et al., 1995) al., et Amann - developed for developed Ringø , Mente et. al., 2018 al., et.Mente , ambient ambient ,

in may

cultured cultured active t l, 95 2008, 1995, al., et the et al., 2010, Desai Desai 2010, al., et temperature, pH pH temperature,

ed to lead Karelia region region Karelia n natur in salmonids

develop Llewellyn et et Llewellyn unculturable salmonids while aiding in in aiding altered e ). terial terial ment ,

It is and and but but the ,

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Accepted1 Article and volume of 2500 m3. 2500 of volume and - 5 m 5 existent Fish M The t

aim he dominance of γ of dominance he a with average de average with aterials and methodsaterials and

bacterial infection and infection bacterial

1 between stomach and intestine

, water ,

first (node (Reveco et al., 2014; Ingerslev(Reveco 2014; et2014b). al., et al., groups of bacteria (Ring bacteria of groups 5.4±0.5 a t cmae h vraiiy f h studied the of variability the compare to was i o te rsn suy w study present the of aim - the

less nylon thread) nylon less and diets and , 15.3±0.4 , gut

pth notntl, studies Unfortunately, Hye e a. 2018 al., et Huyben , - bacteria , Proteobacteria

i of water open the in placed were Cages and uut n September and August n this part of the of part this n .

, and 1 and , Twenty nine Twenty Karelia (Russia, Karelia ,

, including a comparison with comparison a including , 05 ( 2015 Spirochaetae f the of l

omnt fo ribw trout rainbow from community 4 ø with diameter of 17.8 m, area of 2 of area m, 17.8 of diameter with .

2±0.04 six et al., 2016). 2016). al., et tmc and stomach

from among intestinal microbiota of rainb of microbiota intestinal among

fish) individuals (two years old) of rainbow trout (O trout rainbow of old) years (two individuals ( h ifune of influence the °C respectively. °C as fifteen lake lake

Wn e a. 2013 al., et (Wong 60°50′03″ N, 31°33′10″ E). 31°33′10″ N, 60°50′03″

healthyand unhealthy r Mne t a. 2018 al., et. Mente ,

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25 m. 25

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The fish farm is located on the on located is farm fish The uninfected different pathogens is almost almost is pathogens different The fish density was was density fish The of parameters , the composition composition nese e a. 2014a, al., et Ingerslev belongs ). ainbow trout. ainbow lake Moreover 50 rainbow trout rainbow

Fish were Fish m2, uig t during

at to s measured at at measured s a ow trout was trout ow the height of 15 of height

Tenericutes, depth depth , as well as as well as in others others in he acute acute he bacterial

that the that 2.7 kept

of of . were - The 3.5 up up in .

This article isprotected rights by All copyright. reserved. extraction. in ethanol 70% in stored was together. content gut and layer mucosa from performed was extraction difficult was content gut and layer mucosal of separation appropriate the spatula a using separately l mucosal month one After nitrogen. in put abdome afterwardsthe ethanol; 70% with disinfected wad cotton a using mucus of cleaned was skin fish dissection to Prior and 4 intestine) the occupy 1 than (less particles food of amount 0 of scale a on ranked The standard(c length additive from clams) of meat fermented the on based by treated cages. in trout rainbow of infections S Verones mm 6 size pellet kg/m3. Accepted Articleveterinarian local by applied were treatments pecial - 5

h dge o flns o ter ietv tat soah n intestina and (stomach tract digestive their of fullness of degree The sacrificed humanely were fish studied The a All fish were fed twice per twice fed were fish All – i Rybin based A(probiotic onBacillus sp.) , Ve ,

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antibiotic antibiotic rom zip ) .

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– Due to the mucosal the to Due ,

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oee by covered o h feig al poie by provided table feeding the to n was dissected using sterile instruments and the gut removedgutthe and instruments sterile using dissectedwas n a separate day n iud irgn te us were guts the nitrogen, liquid in

the anterior the ste following the included treatments The – ( 2% 6.6 lmnm foil aluminum sterile - of of 9.2 g per fish) per g 9.2 1st 1st layer the total possible volume of food that could could that food of volume possible total the to 14th of July and by MIDIVET (additive (additive MIDIVET by and July of 14th to diet

, mid , 0 to 11th of July of 11th to

microcentrifuge tube at at tube microcentrifuge during tw - by

1 with some distention of distention with some theintestine wall

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s being (feed manufacturer) (feed

pithing with a with pithing in order to prevent the annual bacterial bacterial annual the prevent to order in dle, gut is empty or containing a negligible negligible a containing or empty is gut pproximately filled at 50% capacity; 50% at filled pproximately ,

liquid in frozen immediately and

and damaged by damaged by the by o days (15 o days posterior ,

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– After that the mucosa the that After the

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probiotic is n the and h wer h with was was A e . . ,

This article isprotected rights by All copyright. reserved. ofmiddle August, 2015). midd the in collected fish to belonged kidney, and spleen ( of consistent and size tissues organ of operculum, of condition ectopa external to mg/dm sulphates mg/dm and mgO index color analyzed were parameters (www.clatikarelia.ru) Karelia hours few a within depth collected were samples Water community. water Acceptedhttps://www.necropsymanual.net/en/additional Article total 2 /dm was also analyzed also was aie o fungi or rasites of 3 3 Healthy / unhealthy fish unhealthy / Healthy At ) ) . iron total ,

m 5 3 nitrogen ), the (8.

nitrite the signs 4.±. degree) (45.7±4.6 41 mg/dm 34±1 Wtr ape wr kp i gas n pl and glass in kept were samples Water . time of time , healthy group were divided were group healthy haemorrhages

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as a possible factor that may affect the structure of structure the affect may that factor possible a as

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, ulcerations , i products oil l (BOD5) days 5 in demand oxygen biological , hr te ape wr analyzed were samples the where opacity, .

3 Fish were classified as “healthy” or “unhealthy” a “unhealthy” or “healthy” as classified were Fish ),

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This article isprotected rights by All copyright. reserved. AcceptedAG CTC TGG psychrophilum psychrophil AAC CAA 95 forward were: reaction amplification of round second 438 reverse of protocol PCR of identification with slow, KDM salmoninarum Renibacterium according Articleinspection made etc.) organs, and skin the on spots and ulcers appetite, of lack the behavior, in (changes signs Russia (Moscow, transported after observed Se of beginning the in and July of middle amplification - - - clear that June June C CAG 2 (Kidney Disease Medium 2) and SKDM (Selective Kidney Disease Medium) Disease Kidney (Selective SKDM and 2) Medium Disease (Kidney 2 T f Unhealthy - 3 axonomical ’ from the middle of June 2015 and fish mortality in cages progressively increased progressively cages in mortality fish and 2015 June of middle the from

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This article isprotected rights by All copyright. reserved. stomach (intestine, tissue of type i.e. variables, al. et (Hammer PAST in conducted was group using calculated were sample per index) Simpson and (Shannon estimates analysis. reference. al. et DeSantis ‘gg_13_8’; alignment(release reference Greengenescore open and reference closed both of steps several involves algorithm This 2010). al., et Price 2.1.3; (FastTree tree and 2010) al., et Caporaso 1.2.2; (PyNAST alignment sequence (UCLAST), open perform to used was options default with ‘pick_open_reference_otus.py’ script QIIME the filtering, ‘ option an with (‘identify_chimeric_seqs.py’ po identify to applied was 2010) (Edgar, 6.1 USEARCH using continued bp 500 than greater bp eight than more of homopolymers and sites ambiguous M with filtered quality and merged v2 (500 cycles). on USA

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2006) was used as a as used was2006) MiSeq Reag MiSeq QIIME ER w as MANOVA . To test for test To

- used for for used diversity building building ent Kit Kit ent ) on ) ) -

This article isprotected rights by All copyright. reserved. Acceptedgram 0 was (arbitrary scale kidney the of inflammation bladder) abnormality. signs without of observations, of period 4 was fish sacrificed of fullness gut of scale (arbitrary activity feeding ( group p=0.0 Sequence Archive(NCBI Read Articlesamplesby werecalculated PERMANO using obtained genes of quantity total from percentages to transformed were then genomes) in genes of group every of occurrences of (frequency cat The 3. and 2, 1, levels at categories gene different to functions categorized and genomes) and the genes of encyclopedia generated (Kyoto pathways KEGG We communities. microbial of content functional metagenome predict to performed usingphyloseq non samples of groups among differences UniFrac weighted communities bacterial of groupings the - 1 negative ) Unhealt from fish the of (±SD) weight mean The R numbers accession sequence Nucleotide analysis. Functional example h es igher ults

Flavobacterium psychrophilum Flavobacterium hy fish (group D (group fish hy (

627 iue 1 Figure

distan ±55.8 - 2). 2). with From ce matrix matrix ce )

g) could be characterized as fish with normal daily behavior and and behavior daily normal with fish as characterized be could - package The PICRUSt software package ( package software PICRUSt The transparent than in than espe - the kidney of kidney the ) SRA, BioProject PRJNA BioProject SRA, had fin rot and skin ulcers ( ulcers skin and rot fin had cially in the posterior part. part. posterior the in cially cluae i QIIME) in (calculated the (McMurdie, Holmes,2013). (McMurdie,

, two cornea, unhealthy group (396±48 group unhealthy - - fish way and one and way metri VA , the , .

the and viscera (h viscera and , at p≤0.05. in Nucleotide sequences were deposited in the in deposited were sequencesNucleotide

mliiesoa scaling multidimensional c causative agent of myxobacteriosis. From From myxobacteriosis. of agent causative ,

groupD healthy group was group healthy and the differences between groups of of groups between differences the and egorized functions for different levels levels different for functions egorized

482823 - way PERMANOVA way

there example were Langille et al. 2013) was used used was 2013) al. et Langille e h gut The ) art, kidney, liver, and swim swim and liver, kidney, art,

was isolated and identified identified and isolated was

g performed ). Fish from Fish ). ,

Figure significantly ( significantly - 5) during 5) was almost empty empty almost was

2a .

( T based on based ). PCo visualize o the

There was was There t he A healthy healthy t=2.72

) was was ) entire the ,

This article isprotected rights by All copyright. reserved. representedFile inSupplementary 1. of composition individual group by presented mostly was bacteria rainbow of stomach correspondingly) ( sequencing metagenomic twenty to belonged trout rainbow of intestine and stomach lacustri reached copepod parasitic Caligidae August to July pale aetiologic specific No fat and viscera muscles, in hemorrhages exophthalmia, by characterized were salmoninarum the Accepted37.9 Article ie ad ki and liver color , D of intestine and stomach of composition Microbiota trout Rainbow

s 59.7

were detected. by

( family from family % with % Figure and , Actinobacteria , the ,

(groups A, B, C, D) C, B, A, (groups dney dney ,

was 15.4% and 0.85 ind. host ind. 0.85 and 15.4% was 2 b and

an intensity an causative agent of agent causative

trout 30.8 ).

there al were infected by infected were

Fusobacteria

the

agent was identified by culture on selective media. media. selective on culture by identified was agent

. from the healthy groups (A, B and C) and B (A, groups healthy the from

Crustacea In regard regard In

, correspondingly), %, a ioae gram isolated was (17. the of 4 %) and for group E by E group for and %) atra cmuiy o eey ih t hlm ee is level phylum at fish every for community bacterial 2.6 ind. host ind. 2.6 Proteobacteria .

( to unhealthy fish groups (D groups fish unhealthy to

Caligus lacustr Caligus 17.7 subphylum. bacterial C aligus , 0.6 - - , and and , oiie bacteria positive

1 - kidney disease. Unhealthy fish from group E E group from fish Unhealthy disease. kidney lacustri 1 respectively. In August the prevalence the August In respectively. 1 ,

(77. whereas in September no individuals of individuals no September in whereas In July the prevalence and intensity and prevalence the July In Firmicutes 7.9 is 4

is a parasitic copepod that belongs to to belongs that copepod parasitic a is s %, and

Firmicutes (Steenstrup and Lütken, 1861) Lütken, and (Steenstrup

- correspondingly) to dominate t dominate to correspondingly)

two two 75.1

rainbow trout rainbow

phyla %, dniid as identified

( have and E), the composition of composition the E), and 41.6

correspondingly) and for for and correspondingly) ( 22.7

, of bacteria. Result bacteria. of shown 38.3 %) .

and , from OTUs All The l The (Figure

Proteobacteria Renibacterium Renibacterium ( Figure 2 b,c 2 Figure iver had had iver

3 58.7 of this this of ).

from from The The s of of s had had %, he he C. ) a .

This article isprotected rights by All copyright. reserved. by wasand(groups E) dominated D Pseudomonadaceae Bacillaceae of microbiota healt the to related that differences showed also trout SupplementaryFi of composition Pseudomonadaceae by fi dominated unhealthy was fish Pse healthy from trout (Fig stomach in phyla bacterial abundant most the of ratio the in reflected Tenericutes Firmicutes by presented C Bacteroidetes and were cases of majority the in intestine for groups dominant The C and B (A, groups fish healthy

Accepted Article. dmndca Fusobacteriaceae, udomonadacea, In ure 38.1

ead o the to regard On a lower taxonomic level, the microbial community from the intestine of rainbow rainbow of intestine the from community microbial the level, taxonomic lower a On taxonomic lower a On of composition taxonomical The 6 ) , correspondingly), %, .

(37. (31. h gop ad ) h mcoit ws oiae by dominated was microbiota the E) and D (group sh 4., 14 ad 03 %) 20.3 and 21.4, (40.0,

the the ( the 11.4 4 the 2 %) for group E (Figure E group for %) le 2. following dominants: dominants: following and netn fo te elh fs (rus , ad ) was C) and B A, (groups fish healthy the from intestine , , bacterial community for every fish at fish every for community bacterial

12.6 69 97 ad 10 %) 11.0 and 9.7, (6.9,

nelh fs gop (D groups fish unhealthy

Bacillaceae 8. , a , 1 , correspondingly), %, nd

level, the microbial community from the stomach of rainbow rainbow of stomach the from community microbial the level, 10.4 ) was sim was ) Firmicutes and , %, correspondingly) %, Enterobacteriaceae ,

and Proteobacteria Enterobacteriaceae Enterobacteriaceae 4 ilar to ilar the ). Micrococcaceae

The effect of the different health status was also was status health different the of effect The wees irboa rm h uhaty fish unhealthy the from microbiota whereas ; Lactobacillaceae atra community bacterial ( 50.1 the

Act n E, h cmoiin f atra was bacteria of composition the E), and , bacterial community from community bacterial inobacteria 33.5 h status of th of status h

and (30.8 and 28.2 %), (30.8 and 28.2 (49.

Bacillaceae and , the

5 Fusobacteria 1., 48 ad 84 %), 18.4 and 14.8, (12.1, (Figure (Figure

and family level is represented in in represented is level family hra i te tmc of stomach the in whereas

(9 Proteobacteria 33.5 58.9 9 ) o gop D group for %) .9 e groups (Figure groups e from

, (Figure 5) (Figure

, correspondingly), %, 7 %, correspondingly), correspondingly), %, Enterobacteriaceae Enterobacteriaceae ).

h intestin the (16.2%) for group group for (16.2%)

Bacillaceae h individual The their dominated by by dominated

and intestine intestine and

( 35.6 stomach. 8 from e , ). The The ).

(36.2 (36.2 50.1

and and and and , , , This article isprotected rights by All copyright. reserved. microbial community of indices Shannon and Chao1 to according C and B A, (groups of richness and to (31. changed had dominant the (September) autumn during collected stomach both in present was noted be to has It and 5.8%) Serratia as such genera by dominated was C) and B A, (groups the lowest level taxonomical represented is File inSupplementary 3. was %) 29.4 %) %) 27.6 and 29.5, (11.4, of E %) 5.7 and

(Figure

Accepted Article, healthy

2 and also detected also %) Diversity analysis of the intestinal microbiota of rainbow trout of rainbow microbiota intestinal the of analysis Diversity genera the fish unhealthy For microbi The t level, taxonomic lowest the On

Cetobacterium Cetobacterium

in the intestine ( (8.9, 9.5, and 13.9 %) 13.9 and 9.5, (8.9, Pseudomonas 8 , ). ano tot gop A B n C and B A, (groups trout rainbow and Pseudomonadaceae

Cetobacterium the . that for fish from group D collected during summer (July) summer during collected D group from fish for that The individual composition of composition individual The al bacterial com bacterial )

were omnt from community

(12.6%) (25.0 and 23.1 %) 23.1 and (25.0 , Figure the Serratia significant stomach between hea (17 (14.5 %) ,

10 for group A for (9.2 and 15.0 %), and %), 15.0 and (9.2 Pseudomonas .

munity in munity ). 0 (11.1, 26.6, and 4.3 %) 4.3 and 26.6, (11.1, %)

Serratia ly and intestine and

he microbi he for grfor

the high ,

Tee ee no were There . were detected were . For unhealthyfish For . the the , netn o h of intestine oup C oup rta rm unhealthy from than er

Bacillus and Bacillus

ws domin was ) (6.9, 9.6, and 11.0 %) 11.0 and 9.6, (6.9, intestine of rainbow of intestine lthy and 3 fish (Table unhealthy

al the .

community fromcommunity (9.6%)

bacterial community for every fish on fish every for community bacterial Mycoplasmataceae

, Bacillus

and Pseudomonas aty ( ealthy Pseudomonas hra o fis for whereas , td by ated significant significant Bacillus Bacillus the genera the

(37.4, 19.7, and 19.5 %) 19.5 and 19.7, (37.4, Figur

such such

, trout from healthy from trout fish the . Bacteroides

for group E group for The species diversityspecies The e

(11.0, 20.5, and 6.7 and 20.5, (11.0, differences for for differences Mycoplasmataceae

stomach stomach were also detected also were

genera 10 (31.2%) Serratia gop D (groups h from group E group from h ) rainbow trout trout rainbow )

Renibacterium ). as

(Figure

for (34.5 and and (34.5 (6.5, 7.0, 7.0, (6.5, (18.1 %) (18.1 Bacillus

and E) and

group group fish the 9 ) . ,

This article isprotected rights by All copyright. reserved. intestine are shown fish differ unhealthy significant The respectively. and healthy for 46.4±0.4% and 48.7±0.3 (mean±SE) value average with shown) not (data healt between found differences significant predicted trout rainbow the of metagenomes functional Predicted differences were significant these fish healthy for whereas (P=0.1077) fish unhealthy for intestine and stomach between respectively “tissue” the stomach and intestine fish unhealthy by formed cluster the showed fish P fish (group intestine Accepted ArticleER MANOVA, p≤0.05) eaeoe aa a aaye uig IRS t dtrie h microbiome the determine to PICRUSt using analyzed was data Metagenome that indicated and results these by supported (ANOSIM) similarities of analysis The The for significant was fish of was status health The maximum The of healthy fish (group (group fish healthy of functions E ) for )

PCoA grouping of samples of grouping ) where the Simp ) where the using PICRUSt. n “ and

. The most abundant predicted function predicted abundant most The . forming forming

using

in f u o haty n uhaty fish. unhealthy and healthy of gut of elh status health

(Figure the the ipo idx value index Simpson (Table 4 egtd nFa dsac matrix distance UniFrac weighted Figure 7 the

(P=0.

gut bacterial community (Table 5 (Table community bacterial gut 11 son index value was minimal

B ). ) in comparison with comparison in ) 00 ). into

. ences between healthy and unhealthy fish for stomach and stomach for fish unhealthy and healthy between ences wr sgiiat atr (P=0. factors significant were ”

two big clusters (healthy and unhealthy). and (healthy clusters big two ) . a no had

hy and unhealthy fish for stomach and intestine and stomach for fish unhealthy and hy s

gut were la differe clear irboa fr microbiota eetd in detected the Chao1

microbial co microbial at the at

(Table 3)

c bten irboa of microbiota between nce ewe haty n unhealthy and healthy between At the level 1 there were no no were there 1 level the At and Shannon index (two index Shannon and ) om the the . L2 level was “ was level L2 No differences were found found were differences No irba comm microbial . elh ad unhealthy and healthy

mmunity of mmunity 00 01

n P=0.0 and Interestingly, metabolism” metabolism” un unity healthy healthy - way 428 the the in ,

This article isprotected rights by All copyright. reserved. Accepted its Ringø microbial core the supplements 2008) fish freshwater belonging of members intestin and stomach between composition the approach NGS wereet by Wongal.(2013) obtained but phyla same bydominated Article diseasenutrition, resistance and immunity fish. unhealthyfish for and intestine38.0% stomach were respectively. 80.8 functions predicted different significantly (stomach (PERMANOVA, ability to ability intestine

he bacterial he et al., 1995, Kim et al., 2007) and wid and 2007) al., et Kim 1995, al., et According t According gut The D a were there 3 level the At aig oe ebr pce go cniae a poitc pce fr feed for species probiotic as candidates good species member some making iscussion –

3.4 and intestine intestine and 3.4 .

stimulate stimulate the genus the The genus The f ano trout. rainbow of Proteobacteria, Proteobacteria, the

that microbiota is a very important very a is microbiota Mne t l, 2018) al., et (Mente with the addition of addition the with p≤0.05

genus genus has fish of microflora o our results our o community of community the phyla the the host host the Cetobacterium ) Bacillus Cetobacteri for healthy ( healthy for Proteobacteria, Firmicutes Proteobacteria, – Firmicutes, Fusobacteria Firmicutes,

immune system, immune , 4.3%)

n low In the microbiota of the of microbiota the has been identified in many studies to be a common part of part common a be to studies many in identified been has the the

and Bacteroidetes. and and um

stomach fish collected in different time points time different in collected in er

Lyons al.(2016) et been associated with numerous functions, including functions, numerous with associated been

the the are are (

e sgiiaty ifrn peitd functions predicted different significantly few e of healthy fish are related to high to related are fish healthy of e taxonomi Austin 200 Austin gut (PERMANOVA, (PERMANOVA, e stomach and and stomach able to produce vitamin vitamin produce to able

iey itiue i te netnl rcs of tracts intestinal the in distributed widely ly applied ly

constituent –

produce (Austin and Al and (Austin

0 .0

Similar results Similar

a level cal and intestine and 2 stomach , Wang et

and in and

antimicrobial and as a probiotic a as

of any verteb any of Bacillus

who have demonstrated who have demonstrated s Bacteroidetes te ifrne i microbial in differences the ,

p≤0.05 the - of healthy rainbow trout was was trout rainbow healthy of arn 18,Chl, 1990, Cahill, 1988, Zahrani al., 2018

– intestine dominated intestine

for healthy rainbow trout trout rainbow healthy for 2.4%) and unhealthyand 2.4%) in ) compounds the the В between healthy and and healthy between in rate animal including animal rate . On other hand other On . 12 (Tsuchiya et al., al., et (Tsuchiya 12

aquaculture due to due aquaculture intestine. Bacteria intestine.

were abundance dominant , and other and using an an using by ,

fi the the the the sh of of in

This article isprotected rights by All copyright. reserved. further composition microbial the on influence greater of al. (2017 al. sho previously was as results the affect also may protocols extraction muco intestine middle the in (mucous+content), intestine extraction study) 9.4 was factors groups research different by obtained results of variability great Such work. Wong’s with genus dominant identical no share Bacillus, work, Clostridia Staphylococcus, Streptococcus, Enterococcus Lactococcus, Lactobacillus, level results Wang et al.,2008,Kamgar substances useful

Acceptedthe Article ( 2017)

d sa f genus of 2 different 2) ;

core microbial community of rainbow trout rainbow of community microbial core , the

only one identical dominant genus ( genus dominant identical one only At such as such and º (yn e a. 21) 12.5 2016), al., et (Lyons C ) rm neir mdl ad otro part posterior and middle anterior, from

. ; 5) life cycle stage Llewel stage cycle life 5) ; Serratia,

microbial h sm ti same the But ( e.g. in in e.g. those

ctneoatru, aelccu, tetccu, esel, Leuconostoc, Weissella, Streptococcus, Catellicoccus, Acetanaerobacterium, at . 1) yn e al et Lyons the same time same the different different obtained by Wong et al. (2013) and Lyons et al. (2016) (2016) al. et Lyons and (2013) al. et Wong by obtained such the Cetobacterium, ,

community of healthy fish was dominated by bacteria from bacteria by dominated was fish healthy of community whereas in the present study the study present the in whereas it ad premix and diets me Lyons et al. (2016) (2016) al. et Lyons s ietv ezms aio cd, etc acids, amino enzymes, digestive as ,

okn with working rearing water conditions conditions waterrearing we we et al.,

and the studyby the rsn te idns of findings the present

( the variability the 2016) 2016)

2014, Ol and Bacillus lyn et al. et lyn º (og e (Wong C identified

;

Pseudomonas the same fish species may be explained by several by explained be may species fish same the ) different 3) mos and Paniagua mos and

were found were Bacillus Wong et al. (2013) the genomic DNA genomic the (2013) al. et Wong

study study

found found whereas ( 2014)

. uh genera such

a. 21) n 14.2 and 2013) al., t To understand which of these of which understand To the ) with ) in all three studies threeall in of s could ; 6) ; genomic DNA source was a was source DNA genomic

. ape matrix/tissue sample

by Wong et al. (2013) al. et Wong by sourc Th considerable differences among our our among differences considerable

te genera other differen the those s te ih rm u study our from fish the us,

also - netgtos r needed. are investigations Michel, 2014). e of of e

intestine be related to related be as as . fish from Lyons’s work and and work Lyons’s from fish

ces over time time over ces (Newaj genomic DNA was distal distal was DNA genomic yolsa Brevinema, Mycoplasma, wn by Kashinskaya et et Kashinskaya by wn e.

) ; 4) different DNA DNA different 4) ; - g. water temperature temperature water g. like - 15.4 Fyzul et al., 2007, 2007, al., et Fyzul

at a used for DNA DNA for used º . (the C

great plasticity greatplasticity the taxonomic taxonomic the Lactobacillus, Lactobacillus, In the present the In Gonçalves features has features source the mixture of of mixture

present genera

have have The The was

et et

This article isprotected rights by All copyright. reserved. Acceptedpsychroph (Linares for shown was bacteria of behavior species/strains different that known well is dysbiosis. it and Indeed, balance microbial normal the of disruption to lead may factors virulence suppr otherwise be would arecomposition consequen causes or But, diseases. health, host impact to due fish of gastrointestinalmicrobiota of understanding improved like conditions specific culture a in form media Articlespecific gut the of parts different bacteria; specific of enrichment differen providing to analogous as seen be can pH This 2018). 2015, al., et (Solovyevetc digestiveenzymes, of as composition and activity values, such conditions different support gut the of parts different the digestion), of stage they were truly dominant Ring used that in Pseudomonas dominant ø

t l, 1995 al., et yboi o te u de o boi fcos a la t opruitc netos that infections opportunistic to lead can factors abiotic to due gut the of Dysbiosis that species (fish species fish gastric in that known well is It - Otoya et al., 2017). 2017). al., et Otoya and be abl be and ge ilum nera culture , and , unfortunately, it remains unclear whether changes in the intestinal microbiota microbiota intestinal the in changes whether unclear remains it unfortunately,

is a bacterial disease that affects a broad host broad aaffects that diseasebacterial a is uh eea as genera such absent absent ) Bacillus . which mayaidwhich - e dependent However

produce various compounds with antibacterial activity. antibacterial with compounds various produce in ,

is unknown dueis unknown to - se. n otat a agesv ptoe producing pathogen aggressive an contrast, In essed. ), studies by studies dependent approach for growing specific groups of bacteria. of groups specific growing for approach dependent but Myxobacteriosis or Bacterial Cold Bacterial or Myxobacteriosis ,

approaches h as the the abundance of these genera these of abundance the registered i registered

development of more of development yoatra Cytophagales Myxobacteria, ces of different fishdiseases.ces ofdifferent sociation between alterations in the diversity of of diversity the in alterations between sociation Wong et al. (2013) and Lyons et al. (2016) al. et Lyons and (2013) al. et Wong infectious infectious

( Nieto et al., 1984 al., et Nieto n the limitation the present study present the tps f eetv mda o clue and culture for media selective of types t diseases efficient strategies to prevent or treatpreventor to efficientstrategies

of culture - will illuminate will range of fish species that inhabit that species fish of range ;

Austin and Al and Austin and therefore whether or not or whether therefore and were -

water Disease caused Disease water

and certain certain and -

dependent earlier earlier have stomach and acid acid and stomach have may show predatory predatory show may

how gut microbes microbes guthow found - Zahra Flavobacteria approach This activity activity This

in a study a in ( ni, powerful Serratia,

1988 by es An F . ;

This article isprotected rights by All copyright. reserved. AcceptedPseudomonas Apparentl by represented were phyla etiology. different with differ the of composition and dysbiosis regarding microbiota made be to inferences some enable can andfish unhealthy healthysignificant theobserveddiversitybetween microbial of differences parameters quality communities pathogen of abundance low commensals are genera this of species among that species of level the to identification enable not does analysis library 16S The in seen hemat and excretory the disrupting tissue kidney the in Articlefound localize inflammation 2005). al., et (Toranzo Finland in trout rainbow including salmonids cultured and wild of variety by caused (BKD) intracellular facultative disease kidney Bacterial myxobacteriosis. and microbiota (Starliper, 2011). predominat occurs and waters fresh cold, n o te an eut o te rsn wr i te ifrne in difference the is work present the of results main the of One provides study present The s hs ies las o h dvlpet of development the to leads disease This Figure 9 and Figu and 9 Figure

in various organs and tissues, the ovarian fluid, but more more but fluid, ovarian the tissues, and organs various in y, .

in Further, the patt the Further, until conditionsuntil become for less favourable thehost. with between healthy and unhealthy fish unhealthy and healthy between , farmed rainbow trout rainbow farmed of Renibacterium

the

, which which , a

internal organs. internal ih ee o audne f uh eea as genera such of abundance of level high bacteria bacteria re 10 re ern of change in the gut microbial diversity can be expected to to expected be can diversity microbial gut the in change of ern remaine rtoatra Atnbcei, Firmicutes Actinobacteria, Proteobacteria, In the present work in the group of unhealthy fish the dominant the fish unhealthy of group the in work present the In , and bacteria from from bacteria and ,

Renibacterium salmoninarum Renibacterium species of species ,

O. mykiss O. a This bacter This rltvl cntn drn te td period study the during constant relatively d ecito of description Renibacterium l a wtr eprtrs f 16 of temperatures water at ely

during ia appears to have low tissue specificity and and specificity tissue low have to appears ia . Al . though no association existed to water to existed association no though an epizootic of epizootic an the alterations of of alterations the the exophthalm

were identified in identified were family

is a chronic disease affecting a a affecting disease chronic a is opoietic functions in fish. in functions opoietic

ia Mycoplasmataceae , anemia, ascites, and and ascites, anemia, , bacterial kidney disease disease kidney bacterial fe i clinical in often ic the the Serratia, the and ,

strains living as as living strains gastrointestinal gastrointestinal un C n below and °C , Gram

the but it can be be can it but healthy fish. fish. healthy Tenericutes Bacillus, - bacterial bacterial positive positive . , cases cases

The The the the As . This article isprotected rights by All copyright. reserved. the hypothesized and trout, rainbow healthy determined althou animals, of Mycoplasma status physiological normal the of medias.artificial on cultivation their of difficulties by explained be could studies early the in bacteria of group The 2017). al., et Villasante 2015, al. et Ozorio 2015; al. et Lowrey 2015; al. researchers intestina microbiome only trout rainbow f to up (classified colonization for space physical or is community reactions specific than ( KDM as such media nutrient dense on colonies fish, in disease the In intestine. the in also but kidneys, the in only e Balfry rainbow trout was from bacteria of occurrence Accepted ArticleMedium Disease Kidney Selective a latent carrier T

t al., 1996) al., t bacterial infection bacterial infection mcoit o sloi fish. salmonid of microbiota l he specific changes in changes specific he

to since then then since

eeiue ( Tenericutes

f amn a frt ecie in described first was salmon of every infection. infection. every was designated as a pathogen (Holben, 2002). Lyons et al. (2016) have have (2016) al. et Lyons 2002). (Holben, pathogen a as designated was direct competition between pathogens and host microbiota host and pathogens between competition direct the At early revealed and explained by horizontal transmission (Sakai transmission byexplained horizontal et early and revealed amily , in in state

the present time, one of the possible roles of roles possible the of one time, present the and a and ih ee of level high group E. group (Abid et al. 2013; Green et al. 2013; Zarkasi et al. 2014; Llewellyn et Llewellyn 2014; al. et Zarkasi 2013; al. et Green 2013; al. et (Abid .

genus ccording to Bullock et al. (1978) al. et Bullock to ccording Mycoplasmataceae affecting the O The prevalence of of prevalence The the the Mycoplasma) changing of ways possible the of ne genus Tu, a Thus, . bacterial gut microbial community community microbial gut )

the fish host and, as aresult, the fish and, host on the 8 the on Renibacterium T his crig o u rsls te phylum the results, our to ccording otmnto and contamination ) was was ) th e exists re

s dominant a as a day observation study the - 2 indicate dominant

phylotype present (

iny ies Mdu 2 Medium Disease Kidney

specific by in

obn t l (2002) al. et Holben

the the Renibacterium Renibacterium an open form of infe of form open an

h earlier gh a been has likely study, the study, n h itsie f unhealthy of intestine the in taxa taxa ity gut microbiota of unhealthy unhealthy of microbiota gut Mycoplasma Mycoplasma omto of formation a Mycoplasma Mycoplasma great of of depend depend 8.9) in (81.59%) this bacterial taxa for for taxa bacterial this

confirmed number host number clinical sign clinical

tde designated studies for either resources resources either for the may upon in the intestinal intestinal the in host microbial microbial host absenc is maintenanceis Renibacterium

be the the Tenericutes ction rather ction f the of

the

) found not found by many many by al., 1992; al., 1992; SKDM , - e species s of the the of s specific gut of of gut of this this of

core core -

This article isprotected rights by All copyright. reserved. Acceptedgroup between differences no stomach activity feeding of level Cyanobacteria low very had year fish unhealthy andhealthy group high not did activity feeding fish of marker 70.0% to up was group this of ratio the elimination we study our In 2017). al., et Dehler 2013; al., et (Wong analysis the from removed and Articlecontaminants phylum the to assigned OTU's the example, for via, pathogens of influence infectious agents. which intestine, distal the induced enteritis in shown As may prokaryotes by utilized are that acids rainbow

(July abundance abundance s s , this phylum was not the main source of significant differences obtained between between obtained differences significant of source main the not was phylum this , . nte psil wy f changing of way possible Another alter

of fish of trout - August) have

the main the . Atlantic

Holben et al. (2002) (2002) al. et Holben , n epan hi almost their explain and

of on the gut microbial community is also supported also is community microbial gut the on ees of levels and

eliminate also

Cyanobacteria in was simultaneously by accompanied a numberwas high of

be caused by their acti their by caused be Ladoga Lake (Voloshko et al., 2008). al., et (Voloshko Lake Ladoga findings salmon bacterial ad h cnoiat hne i hdohoi ai secretion acid hydrochloric in changes concomitant the and ,

. Acinetobacter d are able to produce antimicrobial peptides and proteins to control to proteins and peptides antimicrobial produce to able are

It is known that that known Itis

edn activity feeding Cyanobacteria

(Supplementary File 4) File (Supplementary 2014) al., et (Reveco when it was removed fr removed was it when communities communities

Cyanobacteria spp. reveal

among healthy among

alterations s abn sources. carbon as complete ed bloom

, which would have prevented ingestion of of ingestion prevented have would which , ve struggle against pathogenic microorganisms. microorganisms. pathogenic against struggle ve the

from from that that of

the ot irba cmuiy is community microbial host s w

Mycoplasma

absen of of fish feeding activity. activity. feeding fish of ere considered p considered ere the which , the , stomach and intestine and stomach . Cyanobacteria Moreover, the phylum the Moreover, trout om bioinformatics

ce inclusion inclusion

could

analys

in as h hg occurrence high The We assume that unhealthy fish unhealthy that assume We the the compare

is able to produce different different produce to able is be be e by the by w While s. gut. of soybean meal soybean of interpreted as interpreted otential plant chloroplast plant otential

microalgae occur every every occur microalgae

The influence of the the of influence The d ana

fact with L. lactis L. In lysis in unhealthy fish unhealthy in

e that Cyanobacteria several s several

the have found have

by u before but an indirect indirect an there were there of unhealthy unhealthy

subsp. in indirect indirect in feed feed in several tudies tudies

This article isprotected rights by All copyright. reserved. of Academy Russian the of Centre Research Karelian the of Facility Core the of equipment 116121410124 2013 for Programme Research Scientific Fundamental Federal The and was study The A the digestive tract. alo flora microbial of transmission on have can pH stomach the in changes that influence in the was study. further requires diseases fish different of consequences Bacteroidetes trout Actinobacteria Proteobacteria, trout rainbow in differences significant Summarizing status. health differing of species same the of cohorts distinct two with infection natural a on the affect can of recovery exposure for prognosis and outcome, severity, of route and dosage The process. infection normal a of part not are injection intraperitoneal and doses high The intraperitoneally. injected routinely are fish utilize experiments challenge Controlled sense. some in relevant more yet and rare, more even are individuals infected with contrasted be can individuals healthy pathology. microbial of area advance th required to additional approaches are and further studies and al.,(Kotob 2016) et cknowledgments

Acceptedthe Article unhealthy groupunhealthy among h qetos soitd ih atra co bacterial with associated questions The absence

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- Aquaculture Aquaculture & sáae C (07. irboe yai mdlto through modulation dynamic Microbiome (2017). C. Escárate, - ) in juvenile rainbow trout trout rainbow juvenile in ) Andersen, G.L. (2006). (2006). G.L. Andersen, & 5072. . ans AC (03. itr syen rti concentrate protein soybean Dietary (2013). A.C. Barnes,

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Otoya, L.,Linares Otoya, 1664. utnoe, . (2013). C. Huttenhower, H. ,

(Walbaum) tochallenge infections. in rainbow ntrout ( ntrout rainbow in - (Walbaum) using next generation sequencing (NGS). (NGS). sequencing generation next using (Walbaum) uaqi ML, Campos M.L., Yupanqui, -

. Jresn LV, tue ML, asn N, asar, I., Dalsgaard, N., Larsen, M.L., Strube, L.V., Jorgensen, C.,

diet type. the art in their characterization, their in art the

The

Frontiers in Microbiology Frontiers in

81, eeomn o te u mcoit i ribw ru i afce b first by affected is trout rainbow in microbiota gut the of development 15(10), Oncorhynchus mykissOncorhynchus tion of general 16S ribosomal RNA gene PCR primers for classical and and classical for primers PCR gene RNA ribosomal 16S general of tion

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100 Merrifield, D.L. (2016). Effect of dietary dietary of Effect (2016). D.L. Merrifield, 246, - Verstraete, W. (2000). Probiotic bacteria as as bacteria Probiotic (2000). W. Verstraete, 1063.

Renibacterium salmoninarum Renibacterium 2(2),

, .. Hrmn, . Hlitr E.B., Hollister, M., Hartmann, J.R., l, - 110.

& 37 wr fr eciig n comparing and describing for tware & in Russian

Gisbert, E. (2018). (2018). E. Gisbert, 97 - Glupov, V.V. (2015). pH values and values pH (2015). V.V. Glupov, 61.

- ad oeua Booy Reviews Biology Molecular and y (08. s o 1S RA gene rRNA 16S of Use (2008). . 108.

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Drabkova, Drabkova, Letters in in Letters problemy

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This article isprotected rights by All copyright. reserved. Accepted Article Taylor, C.,Hatje,Zarkasi, Neuman, R.S., E., Abell, G.C.J., K.Z., T., Kimura, M., Yoshimizu, T., Welch, P.B., Kenney, F., Barrows, J., Davidson, S., Summerfelt, T., Waldrop, S., Wong, ( I. Dalsgaard, S., M. Bruun, L., Madsen, T., Wiklund, Sun, C., Wang, Y. Wang, Journal Microbiology ofApplied of bacteria & of the Japanese 1. salmonids. 4984. density. rearing and diet in variation ( J.F., Rawls, K., Snekvik, G.D., Wiens, Applied Microbiol psychrophilum Oceanology andLimnology unhealthysalmon Atlantic Aquaculture nohnhs mykiss Oncorhynchus omn JP (04. Pyrosequencing (2014). J.P. Bowman,

- B., L B.,

tatc amn ( salmon Atlantic

281 i, J. i,

The intestinal microflora of fish reared fish of microflora intestinal The . L, . L, . & i, Y. Liu, & X., Li, S., Li, G.,

rm ih ise n wtr ape b PR amplification PCR by samples water and tissue fish from Society for the Science for Society ofFish , -

R., R., 1 ogy - 4. &

88, pses lre oe netnl microbiota intestinal core large a possess ) Lin, J. (2008). Probiotics in aquaculture: challenges and outlook. outlook. and challenges aquaculture: in Probiotics (2008). J. Lin,

299 Salmo salar Salmo & 36 (2), - am salar Salmo Sakai, M. (1976). Studies on the intestinal microflora of of microflora intestinal the on Studies (1976). M. Sakai, 307

117, .

414

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- L. in a recirculating aquac recirculatingL.a in 426. - & 27.

. wti a omril mariculture commercial a within L.) Good C. (2013). Aquacultured rainbow trout trout rainbow Aquacultured (2013). C. Good

(2018 -

ae caatrzto of characterization based and Environmental Microbiology Environmental and 42,

91 ).

netnl irboa f elh and healthy of microbiota Intestinal 99. 2000

in freshwater and seawater. seawater. and freshwater in

). Detection of Detection ).

Tamplin, M.L., Katouli, M., M.L., M., Tamplin, Katouli, ulture system. ulture

ht s eitn to resistant is that

Flavobacterium Flavobacterium gastrointestinal .

ora of Journal

Jou 79,

Bulletin Bulletin system. system. rnal of rnal

4974 - This article isprotected rights by All copyright. reserved. Pseudomonadaceae Fusobacteriaceae trout. rainbow E) and D (groups unhealthy Bacillaceae and C) and B A, (groups healthy of 8 Figure Streptococcaceae; Micrococcaceae Bacillaceae and C) and B A, (groups healthy of 7 Figure Figure 6 Figure 5 Fusobacteria 1 using (MiSeq). trout rainbow sequencing of E) and D (groups unhealthy and C) 4 Figure Fusobacteria metagenomic rDNA 16S using (MiSeq). trout rainbow sequencing of E) and D (groups unhealthy and C) 3 Figure lacustris E: Figure 2 Figure 1

Accepted Article b) petechiae in perivisceral fat (pt), pale (pt), fat perivisceral in petechiae b)

infection (cl),necrosis fin (fn). Ratio Ratio instomach. abundant bacterial oftheRatio phyla most Fishgroup from healthy(group C) Fish from unhealthyFish from Phylum composition of microbial community in intestine healthy (groups A, B and and B A, (groups healthy intestine in community microbial of composition Phylum Phylum composition of microbial community in stomach healthy (groups A, B and B A, (groups healthy stomach in community microbial of composition Phylum Top OTUs at the family taxonomic level of the bacterial community from intestine from community bacterial the of level taxonomic family the at OTUs Top Top OTUs at the family taxonomic level of the bacterial community from stomach stomach from community bacterial the of level taxonomic family the at OTUs Top ( ; ( ; ; ( ; ( of the most abundant bacterial phyla inintestine. abundant bacterialof themost phyla ; ( ; Enterobacteriaceae ) ) ) Bacteroidac ) ( ;

Prot Proteobacteria ( ; ) ( ) )

Moraxellaceae eobacteria

Other ) Micrococcaceae

( ( Others. ) ) s.

group D: group Actinobacteria Actinobacteria eae

; ( ; ( ( ; ) ) Others. ; ( ;

Tenericutes; ( ; )

( ; a) extensive deep ulcers (ul ulcers deep extensive a) ) nelh (rus ad ) ano trout. rainbow E) and D (groups unhealthy Comamonadaceae Pseudomonadaceae; )

- )

coloured liver (lv); c) exophthalmia; d) exophthalmia; c) (lv); liver coloured ( ; ( ; Fusobacteriaceae Moraxellaceae; ( ) ) ) Others. Bacteroidetes Bacteroidetes

( ;

( ; (

( ( ( ) ) )

) )

( ; ( ; ), fin necrosis (fn); fin(fn); necrosis ), Sphingomonadaceae; ) 6S rDNA metagenomic metagenomic rDNA 6S Healthy Enterobacteriaceae Healthy Mycoplasmataceae; Lactobacillaceae ) ) ; ( ; Firmicutes Firmicutes ; ( ) ) Unhealthy. Unhealthy. Caligus ( ; ( ; group group ( ; ( ;

( ( ( ( ) ) ) ) ) ) ) )

This article isprotected rights by All copyright. reserved. ( i denotes circle 11 Figure Unknown Enterobacteriaceae Pseudomonas; t rainbow 10 Figure Others. Lactobacillus trout. rainbow 9 Figure

Accepted ) Article

rout. Dominant groups of bacteria at the genus taxonomic level in stomach of studied of stomach in level taxonomic genus the at bacteria of groups Dominant PCoA based on weighted UniFrac distance matrix for all studied rainbow trout rainbow studied all for matrix distance UniFrac weighted on based PCoA Dominant groups of bacteria at the genus taxonomic level in intestine of studied of intestine in level taxonomic genus the at bacteria of groups Dominant ( ;

( ntestine ( ( ) ) ) )

Acinetobacter Lactococcus; Acinetobacter Renibacterium; ;

triangle denotes s denotes triangle ; ( ) ; ( ;

( ; ( Unknown Mycoplasmataceae; ) ) Bacillus )

( Bacillus ) Pseudomonas; )

Serratia; tomach. Group: tomach. ; ( ; ;

(

) ( Cetobacterium ) ( Bacteroides; )

)

nnw Comamonadaceae Unknown Renibacterium;

(

( ) A )

; ( ; )

( ; Others.

( ) )

Cetobacterium ) Fusobacterium B; (

) (

Serratia; ) C ; ( ; ( ; ( ; ; ( ;

) . (

D; ) ) ) )

This article isprotected rights by All copyright. reserved. ( ( Others. Micrococcaceae Aeromonadaceae ( Lactobacillaceae Comamonadaceae (Healthy): Pseudomonadaceae Bacteroida ( Leuconostocaceae Comamonadaceae studied Supplementation in everygroup; S (Unhealthy): Bacteroidetes ( Fusobacteria; ( Proteobacteria; ( studied 1 Supplementation ) ( Proteobacteria; ) Accepted) Others. ) ) Article

Vibrionaceae; Moraxellaceae Streptococcaceae;

fish groups. fish Group E (Unhealthy):E Group ih groups. fish

ceae



( ( ; ( -

( ; fish number ineverygroup;fish number S ) Firmicutes; ) ( ; ) ( ; ) Proteobacteria; ( Proteobacteria; ) ; - Firmicutes; ) ( ; ; ( ; (

Others. ) 2 ( ; stomach; I stomach; ; ( ( )

Group A (Healthy A Group Aeromonadaceae )

( ) ) Others. niiul aiblt i soah n itsie f atra fam bacterial of intestine and stomach in variability Individual Individual variability in stomach and intestine of bacterial phyla in in phyla bacterial of intestine and stomach in variability Individual Comamonadaceae Neisseriaceae ) ) ) ) ) Others. ) Porphyromonadaceae

) ) ru A (Healthy): A Group Pseudomonadaceae Enterobacteriaceae

Clostridiaceae Bacillaceae Streptococcaceae; Mycoplasmataceae; ( )

(

ru C (Healthy): C Group

– ( Group BGroup (Healthy):

Vibrionaceae

) intestine. (

Group B (Healthy): B Group ) Proteobacteria; Aeromonadaceae Fsbcei; ( Fusobacteria; ) ; ; ) Others. ) ; ( ): ( ( ; ) ( ; (

) ( Pseudomonadaceae

; ( ;

) Bacteroidetes; ( Bacteroidetes; ) ; ) Enterobacteriaceae ( )

; ; (

)

Enterobacteriaceae -

( ( Group D (Unhealthy): D Group ) stomach; Istomach; ) Enterobacteriaceae

( ) ( Unknown Rhizobiales Unknown Bacillaceae Fusobacteriaceae ) ) ( Sphingomonadaceae;

Others. ) ) ( Pseudomonadaceae Pseudomonadaceae

; ) (

( Bacillaceae Tenericutes ) Poebcei; ( Proteobacteria; ) ( Aeromonadaceae Bceodts ( Bacteroidetes; ) )

atriee; ( Bacteroidetes; )

Bacillaceae –

intestine. ( ;

; ) Firmicutes; ( Firmicutes; ) Group ( ; ( ( ; ( ; ; ) ; ( ( ; Bacteroidaceae ) ( Sphingomonadaceae ) ; ( ; )

)

Bacteroidaceae ) ( ) Others. ; Fusobacteriaceae ; ;

Fusobacteriaceae ) (Unhealthy): D

( Lactobacillaceae; ; ( ( ) ( ) ( Moraxellaceae Enterobacteriaceae ) Actinobacteria; Others. )

) Others. ) ) ) ) Streptococcaceae; )

) Firmicutes; )

Shewanellaceae Shewanellaceae Bacillaceae Firmicutes;

 ) Fusobacteria; )

fish number fish

ru E Group Group C C Group ( ; le in ilies ( ; ;

; ( ; ;

; (

( ( ( ( ( ( ( ( ) ) ) ) ) ) ) ) ) ) ) ) ; ; ;

This article isprotected rights by All copyright. reserved. Cyanobacteria; Supplementa group; S Enterobacteriaceae Pseudomonas; Enterobacteriaceae Renibacterium Cetobacterium Enterobacteriaceae Unknown Lactobacillus ( Enterobacteriaceae Unknown ( (Healthy): Unknown Pseudomonas; ( groups Supplementation

Accepted Article) ) )

Bacillus Streptococcus; Cetobacterium

t h oet aooia level taxonomical lowest the at -

stomach; Istomach; ( Enterobacteriaceae ;

( ; ) tion (

; ;

Acinetobacter (

( ( ( ( ) Bacteroides ) ) ) Firmicutes; ) ) ) 3 ; ; )

Serratia;

Clostridium; Clostridium; ( ; 4 ( ( Lactococcus; Individual variability of bacteria in stomach and intestine of studied fish studied of intestine and stomach in bacteria of variability Individual Serratia; Serratia; – ) ) ) Others. ) ( intestine.

tmc pya ih Cyanobacteria with phyla Stomach Unknown Mycoplasmataceae Unknown nnw Aeromonadaceae Unknown )

( ; Fusobacterium

; ; ;

( ( ;

)

( ( ( (

( Shewanella Group ) ) ( ( ) ) ) ) Bacillus

Sphingobium ) ( Fusobacteria; Cetobacterium Others. ) ) nnw Rhizobiales Unknown Streptococcus; Deefgea; nnw Leuconostocaceae Unknown )

. E (Unhealthy): E Pseudomonas;

Group A (Healthy): A Group ;

( ; ( ;

( ) Bac ) ( ru D (Unhealthy): D Group ) ;

) ) ( Unknown Aeromonadaceae Unknown ; (

Janthinobacterium ( teroides ) Lactobacillus ( ; )

Pro ; ) (

nnw Bacillaceae Unknown

) ( Clostridium; Clostridium; (

(

teobacteria; ) nnw Comamonadaceae Unknown

; ) Others. ) ) ) nnw Comamonadaceae Unknown

; ( Acinetobacter;

Serratia; (

( Others. ) . )

) Pseudomonas; ( ;

( Bacillus ;

 Actinobacteria ) ( ; (

( - (

( ) ) Others.

) fish number in every in number fish

Others. ) ru C (Healthy): C Group ) Fusobacterium ) (

Lactococcus;

Pseudomonas; ;

( Shewanella ) ( ; ;

) ( ) Bacteroides ) Bacillus (

Bacillus

(

) )

) Unknown Unknown Group B B Group

Serratia; ; ; ( ;

;

; ; ( ;

( ( ( ( ( ( ( ) ) ) ) ) ) ) ) ) ;

This article isprotected rights by All copyright. reserved. (A,Vitamins D3, E,C),traceelements, citricgallate astaxanthin, acid, propyl Sodium Phosphorus Calcium Ash Crude fiber Oils andfats Crude protein Ingredi Table Table Accepted ArticleGroup D А C B E

2 1

ents

Composition ofthe com Characteristics the investigatedtrout rainbow of Status offishStatus

Unhealthy Unhealthy

Healthy He Healthy

althy

individuals Number of 6 4 8 3 8 mercial diet

Body weight 499.5±32.9 240.9±37.7 850.7±70.1 342.0±22.4 510.8±25.7

(from thefeed(from manufacturer) (g)

Total 32.8±0.5 27.4±1.4 27.1±0.8 29.3 32.8±0.5 (c

m length ±0.8 )

09.09.2015 18.07.2015 16.08.2015 18.07.2015 17.07.2015 sampling

Date of

16.4 0.4 1.3 8.3 1.6

28 42 % 2

This article isprotected rights by All copyright. reserved. Table Stomach Intestine Type of of Type

Accepted Articletissue *

3 The groupsat healthy differencesp≤0.05 significant and between were unhealthy

Alpha diversityanalysis microbiotarainbow of of

Unhealthy Unhealth Healthy Healthy Fish

Group mean mean mean mean D C A D C A E B E B

633.0±77.7* 483.5±32.3 548.1±36.4 449.6±84.3 452.8±67.3 296.9±71.2 280.9±64.6 550.4±93.7 788.2±22.5 560.3±64.4 369.2±49.7 418.8±57.4 319.5±39.5 Mean±SE 288.9±8.0 Chao1

trout

ShannonH ShannonH Mean±SE 3.3±0.05 5.0±0.5* 5.1±0.3 5.7±0.5 4.5±0.8 5.0±0.8 3.2±0.7 3.3±0.3 4.6±0.9 5.9±0.8 4.4±0.5 3.9±0.1 4.0±0.4 3.8±0.6

Mean±SE 0.85±0.03 0.88±0.05 0.87±0.01 0.80±0.11 0.72±0.04 0.68±0.14 0.75±0.05 0.80±0.05 0.74±0.11 0.89±0.08 0.76±0.06 0.82±0.02 0.83±0.04 0.80±0.08 Simpson

This article isprotected rights by All copyright. reserved. Accepted Article microbiota of Number rainbow 10000. ofpermutations, trout. Table

eut o two of Results Health status*tissue Tissue (healthy/unhealthy) Health status Health status*tissue Tissue (healthy/unhealthy) He Health status*tissue Tissue (healthy/unhealthy) Health status Factor alth status -

way PERMANOVA PERMANOVA way

(intestine (intestine (intestine , , , / / / stomach) stomach) stomach)

Shannon Simpson Chao

o rcns ad iest estimates diversity and richness for

17.42 8.72 3.35 0.45 4.87 1.94 0.50 7.90 1.70 F

p 0.0001 0.003 - 0.49 0.49 0.69 0.16 0.48 0.99 0.18 value

of of This article isprotected rights by All copyright. reserved. Accepted Article test (with 10000permutations) onweighted UniFrac dissimilarity matrix Table

The im Two One One One One One GroupB, (A, C,D,E) Health status (healthy/unhealthy) Health status (healthy/unhealthy) Tissue (intestine/stomach) Tissue (intestine/stomach) Health status*tissue Tissue Health status (healthy/unhealthy) ------way PERMANOVA onall samples PERMANOVA way onstomach PERMANOVA samples way onintestine samples PERMANOVA way onunhealthy PERMANOVA way fish onhealthy PERMANOVA way fish way onall samples PERMANOVA pactgut ofdifferent factors onfish microbiota assessedPERMANOVA by (intestine/stomach) Test

2.108 4.531 2.521 10.65 - 2.35 8.15 4.05 0.77 F

p 0.1077 0.0041 0.0715 0.0428 0.0001 0.0 0.0003 0.0098

- value 034

This article isprotected rights by All copyright. reserved. Accepted Article