Fish and Shellsh Immunology 134 (2028) 108639, Contents lists available nt ScienceDirect Fish and Shellfish Immunology ELSEVIER journal homepage: www elsevier comfocatelis) Full length article Omics analysis revealed the possible mechanism of streptococcus disease |S. outbreak in tilapia under high temperature Zaoya Zhao“ ', Qianxing Zou "*', Shuyu Han‘, Jingu Shi °, Haijun Yan“, Dasheng Hu’, Yi Yi” * Guanes Laborer of Gren rc of Saar Resure Calle of Bll al Chel gine Gap Unborn of sec end Tel ath £45006, China Depo of Reroute Medina Pepe's Hato 54500, Cine * Gaon Faery Tcl Exon Son, Ning. 530022. Cina Suey Lara of Ben De of Agate Ecos Anral on Curae Proine eyLabrary fr Aquat Fone Anal, Sut ha Se ‘Bio Roce Boat and Colairate Dain Gover, Sol of Lif Sen, Sun Ya Set Unt, Cur, S000, Cina Ties Seneca place ign omperaare "High temperate isa mnin cms to result inthe omtbrenk of tilapia steptocoeal dense, However, the wn etving mechansns ale not well understood. ln this sudy, we fst coated that tapi infected with Srpuocaceus aol (Saga) had a higher moctalty ae high temperature (35°C) than that at aoc reniperatue (26°C). Subsequently, the effects of high empearute on gene expression pater ofS agalactiae nd incestinal mirobiora of tilapia were respectively derected by RNA Seq and 16S 1DNA sequencing. RNA seq ented 357 difeusally expessed genes (DEGS) ln S. golactiaeeultuied at 28°C and 35 °C. GO and KEG alse showed that these DEGs were highly involved ia metabolle protests, ncuding scot, lipid and amino ‘eid metbolisns, which indicates that S.aglatae have stronger vitals ad are litely to be move infec liner high temperature. Microblota analysis revened that high temperature onld intense the bacteria ‘community conspsiion of api nestine, accompanied by changes in intestinal structure. Computed feed a 28 °C the total bacteal species as well as pathogens, suchas noraak F Rhizoblaleslnceriae Seis, Pseudo ‘hodoplanes, Ateylobacter, in apa latestine were significantly intense at 25°C, which aay weaken the “nme resiance of api. Taken together our rests gest tha high tempat evoked lain susceptible ‘oS qglacoe should be the combined effet of enhanced S.aglactoe netabolis and dysregulated dala lneestnal leobioa 1. Introduetion below 29°C (1. More diteetly, high temperature can inerease the sus ceptbility of tilapia to S. agalactiae and leed to lager numbers of tilapia Sweprococcus agalactiae (S. agalactiae), one of the most important — death [0] pathogens associated with disease outbreaks in farm-raised ilapia (Oreochromis mossambicus) in south of China, which eauses significant economic losses annually (1,2. The pathogen causes septicemia, rmeningoencephalits, exophthalmia, anorexia and ascites in many fish species and commonly affects adult fs (J. Noticeably the outbreaks of tilapia streptococcal disease usally accu i sumer, sggesting that high temperature should be a high risk factor for strepracaceal infections [1 tn fae, studies showed that the death of tlapin resulting from '.qgulactoe infection i not usually occurred when water tenperature * conesponding author Contesponding acon There have several reports attempt to explain the reasons for igh, temperarire enusing the outbreaks of streptococcal disease in tipi For example, Wang etal. reported tha high temperacure (82 °C) cou reduce the oxygen levels in water compared to 22 °C an impair the defense of lapia against S. agalactiae infection [7]. A GC/MS (Gas Chromatography Mass Spectroneter) based nuetabolomies study showed that high temperarure (30 °C) could decrease the abundance of amino aeids and increase the abundance of exsbobydeates in tilapia compared to 25 °C, and further investigation revealed that injecting Ema atreses: raya 20126 com (2. Zo, 20 ining 168.com (Q, Zou), hesy07702168.com (, Ham), 35847804340q9.com (J. Shi), 113127751568 ‘9q.com (H. Yan), hdshengettom.com (D. Hu), 1000005226hexust eden (¥. YD, * conmibured equally ap://doL.ong/10.1016/}.4.2023.108699 Received 19 November 2022, Received in evised form 5 February 2023; Asepred 22 Febuary 2023 Avail online 24 Febnany 2023 1050 4648/2028 The Authos. Published by Elsevet Ld. This an open aces article under the CC BY-NC ND Heese //eeavecommons og ienses/ nend/400.‘proline cond rescue amino acid metabolism and thereby reduce apn mortality [6]. Additionally, high temperature also affeets the tran scriptome and proteome of 5. agalactiae, particularly the expression of ‘genes involved in metabolism, virulence factors, and rdgptaton [6]. However, how to combine altered gene expression of S. agalactiae with disease resistance in tilapia under high temperature is an unsolved problem, thas been revealed that S. agalactiae adhered at the apical portion of Intestinal folds and invaded through transepithelial ronte is @ major pattern to infect tilapia (9), Colonization of intestinal mucosal surfaces ‘with « normal microbiota has a positive effect on immune regulatory netlons of the intestinal, Disturbing tls microbiota ean affect ites tinal immnne resistance and contribute tothe occurrence of wariot sliseases in ish [10,11]. Based on the important role ofthe intestine and intestinal mierobiota in fish immunity, we hypothesized chat high Temperature would change tilapia’ intestinal microbiota, and thereby feance che susceptibility of tilapia to S. agalactiae Since the advent of new sequencing technologes, many studies have ‘been conducted on fish fatestinal microbiota and global gene expression, In this study, in order to uncover the presumable mechanisms of high Temperature inducing tilapia streptococcal disease, we respectively tected the effects of high temperature on S, agalactiae’s whole genes ‘expression and tlapia's intestinal mierobios using RNA seq and 168 FDNA-seq, and found that upregulation of metabolism related genes ‘expression in S. agalactice and dysregulation of normal enirobfota in Ulapia intestine respond to high temperature stress via bioinformatics ‘analyses. Our results shed light on the understanding of the mechani ‘of streptococcal disease ombresk in farm-raised tilapia under high Temperature conditions, which is ertiel to minimize the danger of ‘aquaculture practices and capture fisheries succumbing tothe effects of slobal warming, 2. Materials and methods 21. 8. agolactae culture and treatment The S. qglactiae strain (YZF005) was isolated from a sick tilapia by ‘our group, and identified by PCR based on CAMP factor pore-forming toxin (fd) and 165 rDNA genes. The sequence was submitted to NCBI (National Center for Biotechnology Information) (GenBank ID: KF2I2401). S. agalactiae were cultured in BHI mes. at 28 °C for ‘overnight and then diluted (1-100) int fresh BHT medium at 28 °C oF 89°C (n=). The gronsth curves were draivn according tothe vlies of ‘OD600 at different time points, The bacteria were emimersted by ‘counting colony forming units (cfu) on BHT agar media, For RNAseq, ‘experiment, 5. agalactiae were cultured at 28 °C until ODGOO = 1.0, followed by trestment at 28 °C or 35 °C for 2h, then the eututes were harvested for RNA extraction (1 = 3). 22. RNASeq ofS. agoluctiae oval RNAS of S. aglactiae were extracted using TRIzol Reagent Cavitrogen, USA) sccording to the manufacturer's instructions, then ‘quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Teh nologies, USA), NanoDrop (Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis. One jig total RNAS with RIN value above 7 were used for following next generation sequencing library preparation. Libraries were constructed using NEBNext Ultea™ RNA library Prep Kit for lumina (New England Biolabs, USA) and then sequenced on an ‘lumina Hiseq instrument (lumina, USA). The sequencing was pro: ‘cessed and analyzed by Beling, Genomies Instioute Co., Led Being, china). 2.3. RNASeq data analysis aw reds were processed to remove adapters primers and bases less ‘et and Seti ero 196 (2023) 108699 than 15 nt by SOAPnuike (v1.5.2) 0 obtain high quality clean data {12} All clean reads were mapped tothe reference genome downloaded from ENSEMBL —(hitps:/bacterin.ensembl.org/Streptococcus agalactine 26013 3% 000007265/InforIndex/) vik software Hist2 (92.0.1) (15). Clean reads were aligned t0 reference gene sequences using Bowed software (v2.2.5), and then the gene expression levels were calculated by RSEM software (V1.2.8) [1,15], DESeq sofware was used to analyze differential expression, in which genes with P-value <0.05 wore regarded as significantly different (16,171. Subsequently, GO (Gene Ontology) and KEGG (Kyoto Eneyelopedia of Genes and Ge- homes) analysis, and heatmap drawing were performed by Dr. Tom sytem (hits://tepor.bg.com/ps/logn/logn.m}, a sequencing and ualysis service provided by Beljng Genomics Intinite Co, Li. 24, Realsime quantitative PCR ‘Ten of candidate dtferenilly expressed genes (DEGS) were selected ‘o validate the fidelity of RNA.seq by ral-tinie quantitative PCR (qPCR) using the remaining sonpls for sequencing. The sequences of candidate fenes were obtained ftom NCBI, and che primers were designed using online toot Primer3 (hitps//primerS.utee/). QPCR reactions were prepared using SYBR Green qPCR Mix (Guangzhou Dongsheng Biotech, China) nnd rin on a LightCyeler96 system (Roche, Switzerland) ne cording tothe wser miantals.16S RNA was used as the reference gene, tnd the expression ofeach gene was normalized to 165 RNA. All of the primers were listed in Table SI 25. Tlpia reament ‘Tilapia (25.3 4 2.2 g) were obtained from Liwzhou Fishery Teel: nology Extension Station (Liuzhou, Guangxi, China) and randomly divided into wo groups. All apia were fed tice with commercial extruded feed (Tonge, Foshan, China) daily, and the water temper ture was kept at 28 °C and water quality was controlled with dissolved oxygen >5 mg/L, pH 7.4-8.1, total ammonia <0.05 mg/L, and ntete ‘<1 4M, Alter domesticated fr two weeks, the water temperature of ene group was controlled at 24°C and the ther group at 35 -C. Two weeks lace, lapl’s intestines and srestinal eontents Were sampled for sub sequent experiment. For mortality test, 1 106 ef S- agalactiae were Injected into each tilapia after they farmed at 28 °C and 35 °C for two weeks, aud the numberof dead tlapia was consecutively recorded forl4 days. All animal experiments were caried out in accordance with the felines snd approval from the Experimental Animal Ethies Com micee of Guangai University of Science and Technology. 26, Hisolagy ‘The sections of tilapia Kntestinal tissue Were made according to the previous report [18]. Briefly, the tissues were fixed with 49% pare formaldehyde for overnight at 4 °C, chen embedded in paraffin after ‘dehydration through a series of inteasing concentrations of ethanol ‘Three setions (5 ym thick) were cut from each sample and then stained with hematoxylin and eosin (HE) fr histology observation [1] 27. High trougipur 168 rDNA Mumina Miseq sequencing ‘The DNA of intestinal contents wes extracted tsing E-Z.N.A.® Sol DNA Kit (Omega Blotek, USA), and the purity and eoneentration of genomic DNA were detected by 1% agarose gel electrophoresis and Nanobrop2000 spectrophiotomieter, respectively. 16S 1DNA’s V3-V4 region was anuplifed by PCR sing primer 338F (ACTCCTACGG: GAGGCAGCAG) and 806R (GGAGTACHYGGGTWTCTAAT). PCR prod: ucts were separated by 2% agarose gel electrophoresis and recovered from gels us AxyPrepDNA Gel Recovery Kit (Aaygen, USA), The I brary was constructed using TruSeqTM DNA Sample Prep Kit lumina, 'USA) and subsequently sequenced on lumina Hiseq2500. Anslyssequencing data were performed by Shanghai Majorbio Bio-pharm ‘Technology (Shanghai, Chins), 2.8, 168 rDNA sequencing data analysis After sample splitting of PE reads obtained by MiSeq sequencing, ‘quality contol and filtering of double-ended reads were performed based on sequencing quality. Then, splicing was carried out according to ‘overlap relationship between double-ended reads to obtain the opt tized data, The optimized data were processed by sequence noise ‘eduction method (DADA2), and the representative sequences and abundance statistics of ASV (Amplicon Sequence Variant) of each sa ple were obtained. Based on ASV, the species texonomy, community diversity, species differences and functional prediction of the samples were analyzed using Majorbio Cloud Platform (Wi: anajrbio.con 091 29, Statistical analysis Data were expressed as the means = SEM unless otherwise stared, ‘Seatstical significance was aseseed by two-taled inpaited Student's T test, Statistical significance was defined as p < 0.05, ad p < 0.03 was noted with *, p< 0.01 with *, and p < 0.001 with >. 3. Results 1. High temperature alters gene expression of S. agalactiae Firly, the morality of tilapia under S. aguactiae exposure at different temperatures was calculated, and the results shoved that high Aw . mon ime ie : = ae moe B ce. . 1. PA ad GO analysis of DEGS in ferent rermperataes eated S ‘et and Seti ero 196 (2023) 108699 temperature increased the mortality of tilapia (Mig. SA). As expected, the growth rate of S, agilactiae at 95°C was faster than at 28 °C (ig S1B). We speculated that high temperature ean increase the ability ‘ofS. agalactiae to inet lpia, but the underlying mechanism stil known. Therefore, a RNA-Seq ofS. agalactiae ner 28°C and 39 °C was performed, We identified a total of 1767 transcripts in S. agalactiae, in Which 97.989 with clean reads and corresponding 10 82.28% of the predicted genome of the strain (Table S2). PCA analysis showed that fata from 28 °C to 35 °C conld be discriminated (Pi. 1A), sggesting ‘har transeripromic data hd saul variations among repens In oral, 357 differentially expressed genes (DEGS) were identified in, ‘agalactiae der 28°C and 35°C, with 229 DEGs up regulated ad 128 EGS down-regulated (1s. 1B, Table’). The aeeutaey of RNA.seq was validated by quantifying 10 exndidate genes miRNA expression levels ising qPCR. Pearsons correlation coofieent berwcen q PCR and RNA- sea, was 0:809 (p < 0.01, Pip, SIC), suggesting that our RNA.seq data land high reliability. These results indicated that high temperature could alter the global gene expression ofS. agalactiae, which may regulate the nfectivity and metabolism of. agalactiae to inerease mortality of ilapia ‘under infection, 2. GO and KEGG anabsis of DEGS GO analysis showed that large of DEGs belong to celular process and metabolic process in terms of biological process, cellular anatomical entity in terms of cellular component, and binding and catalytic ctvity in terms of molecular funetion (Fig. 1C, Table $4. GO enriched bubble chart showed that DEGS were mainly enriched in carbohydrate and ‘nino acid metabotisms, such as glycogen metabolic process, pyruvate rmetabolie process, and nucleoside diphosphate metabolic process matte ‘galaesice. (A) PCA aay, (B) Numbet of DEG, (C-D) GO analysis of DEC,(ig. 1D). The changed carbohydrate and amino acid metabolism induced by high temperature might affect the pathogenicity. of '. agalactiae to tilapia. Inorder to further explote the possible mechanism, KEGG analysis of DDEGs was conducted. Rests showed thar metabolism pathotays, esp- ally the related pathways of lucose, lipid and amino acid metabolisms ‘were strongly enriched (Pig. 24-B), suggesting that high temperature could alter the metabolism ofS. agalactiae. As the effects of up-regulated DDEGs and doven regulated DEG on S. aulaciae may be diferent, the ‘enriched pathways of total DEGS, vp-regulated DEGS and. dow: regulated DEGS were separately analyzed and listed in Table 1, The ‘ess showed dat up regulated DEGs enricued pathways were simular to (ota DEGs, in which carbohydrate metabolism (ko00010, Ko00500), ‘amino acid metabolism (K000350, ko01230, ko00250), and environ ‘ment interaction (ko04626, ko00710, ko01 120) weve strongly enriched (rable 1). Dovwn-regulated DEGS were mainly enriched in pyrimidine ‘metabolism and RNA degradation (Table 1). To directly observe the effet of high temperatute on genes involved in carbohydrate and amino ‘acid metabolism in. agalactiae, dhe heat-maps were drawn (ig. 26D). ‘The hent-muap shoved that genes related to glucose and lipid metabo lisms were up-regulated (Py, 2C), while genes related co pyrimidine ‘metabolism were down-regulated (ig. 2D). S. agalactiae can produce a wide variety of virulence factors, Inchiding toxins and proteins which facilitate adhesion, colonization ‘and invasion of host cells [20]. We summarized the probable virulence actors existed in DEG in Inble SS, Among these virulenee factors, the vec Patmay Ee ‘et and Seti ero 196 (2023) 108699 expression of alpC efo and al were significantly upregulated, while others’ expression were downregulated under high temperature (Table $5), GO analysis showed that these virulence factors mainly belong 1 biological regulation (G0:0065008, —GO:0019725, {60:0009987, GO:0050789, GO:0065007 and GO:0050794), response ro stress (G0:0050896, G0:0006950 and GO:0006979), oxidoreductase activity (G0:0016491, GO:0016668, G00016667 and GO:0015036) and binding activity (G0:0000166, G0:0036094, Go:0043168, {60:0005488, and GO:1901363) (Table S4, Table SS). KEGG analysis shovted saldase family protein belongs to metabolism pachneay (001100, 000600 and Ko00S11) (Fabe $5). Taken together, gh temperate changed the expression of virulence faetors, whieh may relate the pathogenicity of S. agalactiae by affecting the cellular pro cess and metabolie processes, 3. Bets of high remperanare on intestinal structure of tapia In addition to affecting the pathogenicity ofS. agalactiae, high te perature may also alter the tolerance of tilapia to S. agalactiae. Since ‘agalactiae can colonize in the intestinal tise of tilapia, we examined the Intestinal structure of tapi uncer high temperature. As expected, the length sad with of intestinal villus were decreased at high te erature, and the museular layer was aso thinner (Pig. 9). 1Cis worth noting that high temperate led to an increase in the number of goblet cells in the intestine, whieh may secret more mucus o resist pathogens. During experiment procedures, we observed a phenomenon that high ic Fig. 2. KEGG anass and heat map of DGS diferent remperanues rested S. agate. (A-B) KEGG anal (C) Heat nap of DEGS In ghucoipd-metabolsn rele signaling pathways, () Heat-np of DEG in amino aed an pytinidine metabolisns elated signaling padays‘Table 1 “The envched pathways in tol DEGs, up teguated DEG and down-regulated DEGS by KEGG analysis Paiway Pavey Name Rakai Peale ‘e0n550_ Bwanoce meabaisn ieee an01701 oonat0 Pyridine etal, ban antour 000710 Caton aston phous saete2 —aaner71 000525 Nepthniene degtatin 1 ‘1038001 004625 Pleatpuhogen itercton 1 ‘035001 o00010 Geo Cleconeapenens 3s OHI368 000570. AmtaaeRNA Hoyas bs else Upreguleted Des 200710" Caton fasten photoset saets2 —o0onas7 Sson010 Ges lucomoneness 036 oootses o00350 Tyron metal r S008 oor129 Microbial metab a dere asses antes ooar26 Roti formation ceric col GSS 2A son525 Naphthalene degdatin 1 ooisene 004625 Plan pathogen intercon 1 botse02 o00053 Snot and aldsrnte metiem GENS o00240. Frimidine etal sow 4ane05 fe0t212— Langeniyvepulting pthvay worm Of. 00377 oonss0Pepogiensbosyatess zzz 0009576 oor3s0 Bynes of cota isrias — anete9 200570 AminnarARNA Northen oz ‘.aasi04 Temperature inereased food intake in capa, which should be the reason to result in increased goblet cells numbers and intestinal secretion fonction 3.4. Bijects of high remperarure on ieetinal microbiota diversity of tilapia Dysregulation of intestinal microbiota is the main cause for fish lsease outbreak, To detect the feet of high temperature on intestinal tmicrobiocs composition of tilapia, a high-throughput 168 +DNA Tina MMiseq sequencing was performed, The summary sttistes of 165 rDNA sequencing reads were Hsted in Table Se. Eaeh sample's rarefaction ‘curve tending flat indicated that current sequencing quantity could reflect the species versity of the specimens (Pig. 1D). The shlarity ‘and difference of merobial commnity compestions of 12 stestinal content samples isolated from rlapia at 28 °C and 35 °C were showed in PCA plot with PCI accounting fr 36.65% ofthe total variation and PC2 ‘accounting for 11.9496, Asa result, samples from 35 °C (F35) formed a dlistnct cluster and clearly separated from 28 °Csamuples (T28) (Fe. A). Notably, microbial communities of T35 exhibited greater Individual ‘et and Seti ero 196 (2023) 108699 variation then 128 (Fig. 4A). Common and unique ASVs analyses were conducted to reveal intestinal microbial community in two groups though a Venn diagram. A total of 235 ASV were identified in group “728 nnd 416 ASVS in group T35, of whieh 168 ASVs overlapped in these [v0 groups, 67 unique ASYs in group T2S and 248 unique ASVs in group "735 (Fig. 4B). The Sobs index and Shannon index of ASV also increased a¢ 39 °C compared to at 28 °C (Fig, 4C_D). These results suggested that high temperature resulted in the ineease of intestinal microbiota di versity, whieh may account for tilapia susceptible to streptococcal Aisense 55, Effet of hightemperature on iestinl microbiota compostion of tiapia Species composition analysis showed that che domtant bacteria of ntestinal microbiota in group T28 were Actinobacteria (56.54%) and Proteobacteria (40.8%) at phylum level, andthe proportion of Actin: Dnacteria (43.2%) was decreased and’ Proteobacteria (48.5%) was Increased in group T35 (Fig. 5A). Statistically, the abundance of the Atinobmetera was signfieandy different berween group T28 ne group "735 (p < 0105), whereas Proteobacteria exhibited no significant differ. fence (Fg. SB). In addition tothe dominant phylum, Verricomicrobiota, Firmiewtes and Desulfobacterota were also significantly diferent be tnecen group T28 and group T35 (p < 0.05) [At gents love, the dominant bacteria were Delfin (22.9%), Rho ocoecus (20.39%), Aurantinierobium (17.7%) and Mycobacterinm (8.7%) in group 28, while Delftia (14.79), Rhodococeus (11.7%), Mycobacterium (8.9%), Mierobacrerium (11.2%) and nor Rhizobiales corte Sedis (a family of Rhzobiales without si fentile genus name) (11.7%) in group 735 (ig. SC). Moreover, compared with group T28, the abundance of Auraatinerobiam (p = 0.01) was significantly lower in group T35, whereas nor Rhizobiales Incertae Seas (p < 0.01), Pseudorhodoplanes (p < 10.05), Ancylobacter (p < 0.01), norank £_norsnk.o_PeAM15 (p < 0.01), Candidamns Protochlamydia (p < 0.01), and Hyphomicrobium (p< 0.01) were significantly increase (ig. 9D). These results suggested that high temperature could cause intestinal iicrobiota disorder in tilep 6, Functional prediction of differential ASVs In order to further sméy whether the diferent bacterial microbiots tthe two groups affected the disease resistance of lap, we performed functional analysis of significantly differenced ASV. Microbial func dons were predicted by PICRUSW2 as show in ig. 6A, The dominant ‘microbial fanetion of eifferent bacterial mrabiota wns involved in tistabolism processes, such as amino acid metabolism, fatty. acid metabolism, glyeolysis/gluconeogeness. Subsequently, phenotypic prediction of differential ASVs was conducted by Bugase, The results shovved that nine porentlal mierobial phenotypes including Aerobie, Anaerobic, Containing Mobile Element, Facultatively Anaerobic, Forms Biofilms, Gram negative, Gram positive, Potentially Pathogenic, and Seress Tolerant were decected (Fig, OB), The relative abundance of Potentially Pathogenic, Facultatively Anaerobic, Contains Mobile Ele ments and Stress Tolerant in group 89 were significantly decreased compared with that in group 728, while Forms Bios was inereased (ig. B.D), Inverestingly, although the relative abundance of Potentially Pathogenie was decreased in group T35, i contained genera (Anox- ybacillus, Xanthobacter, Kista, Aquicella) was increased compared With that in group 728 (Fi. CC). Additionally, Pseutorhodoplan (29.236, Aquicella (19.190) and Mesorhizobium (26.190) were the major genera contained in Stress Tolerant in group 128, while the major genera were Psendorhodoplanes (14.33), Nocardia (8.0%) and unclas sified Xanthobacteracese (6.3%) in group TSS (Fig. GD). Taken together, high temperature altered the composition and function of in testinel nirobiots, which thereby woul affect elapie immunity.2 toe ‘et and Seti ero 196 (2023) 108699 Fig. 9. High temperance affects the intestinal struct of tilapia Intestinal suuctges of tapi under 28°C (A-C) and 95 °C (D-E). The length and wide of Intestinal villus). double sided arow, the length of ltestinal vies saight line, the width of intestinal villus; Hack tangle, gle cells seis, ed blood cells pro, scala layers. A PCA on ASV level B c D soo 34s — 3 Bs ais B35 % 23 2 S25 $ so ee $ 200 Bus ai ee g4 gm dos o ol Te Ts 18 735 Sample Group Sample Group ig 4. Community diversity as ysis of teent temperatures Heated apla (A) PCA analysis of ASUS, () Venn diagram. (C-D) Sbs and Shannon indexes of ASS. 4. Diseussion Group B Streptococcus (GBS) at 22 °C and 31 °C showed 10% and 80% mortality respectively [21]. Another study reported that the cumulative 4.1. High remperanre increases the pathogenicity of 8. agalactiae 10 mortality was 70%, 5035, snd OM in tilapia under infection at 30 siapia 25 °G, and 20°C, respectively [6]. Our results confined that high temperarire could increase the mortality of tilapia wnder S. agelactiae 1 Js well known that high temperature can increase the pathoge- infection and accelerate the growth ofS. agalactiae, Reports showed that icity of S. agalactiae to tilapia. Ia Broil, the tilapia challenged with temperature variations ca affect the inmuunty of is species, and highA cones apt ae ‘et and Seti ero 196 (2023) 108699 Beene eb ot ont D oon ran et ton gr Se acca coe ah Fig. 5. Community composition analysis of intestinal microbioca in ifevent temperate treated lpia (A-B) Analysis of community composition and community iference at phy level. (C-D) Annysesofconmnity competion and commit eferene at gen evel Temperature usually has inmunosuppressive effects via inhibiting the ‘generation of carrier specific helper cellsand immune factors [722]. We Ihave known that 8. agarariae can feet fish st low temperate (21) ‘Thus, when water temperature gots warmer, the growth and hemolysis activity ofS. agalcatae will can be increased, which cnuses extensive tissue damage with a massive inflammatory responses, thereby dis rupting fish's defense system and ineveasing mortality (25). 4.2. High temperature changes transcriptome of S. agalactiae In our transcriptome analysis ofS. agalacia, cellu metabolisn related pathvinys were highly enriched under high temperarare (ig. 2A), Among those pathways up-regulated nt 35 °C, the pathways oF slyeolysis/gluconeogenesis (ko00010), starch and sucrose metabolism (4000500) and carbon metabolism (ko0 200) pathways were involved in glacose homeostasis. For pathogente bacteria, glucose ean regulate the vinulence of vations bacteria, such as Pseudomonas aeruginosa (24, Group A Streptococcus (GAS) [25], and Salmonella enterea [20]. A previous study demonstrated that glucose transport related genes were "upregulated In GBS at 32 °C compared to at 22 °C [6]. Moreover, _lucose can influence the produetion oF CAMP factor in S. agalactiae [07]. For the host, scase homeostasis fs essential for the prevention ‘and tolerance of ‘pathogen infection [28]. in addition, ensichment ‘analysis of amino acid and pyrimidine metabolism pathways under high temperature suggested an Increase in amino and pyrimidine synthesis (ig 20). 1 has been showed that pyrimidine biosynthetie pathway is ‘essential for the growth and of virulence maintenance of Cryptococcus neoformans a¢ high temperature 2, In the present study, augment of pyrimidine might be associated with an adaptive condition, in which S. agalactiae inereases the requirements of thymine, uracil and eytosine for growth at high temperacure. The increased pyrimidine could be used for DNA synthesis and replication, andthe reduction of RNA degrade. tion indicated that there had more RNAs for translating proteins (lable D, which could promote the growth of S. agalactiae and the expression of virulence fietors, Taken together, high teniperatre loads to large amounts of glucose produced from glycolipid metabolism, which flones into the riearboxylfe acid eyele to produce ghitamine for amino sugar and nucleoside sugar metabolisms, and finally for prot eration ofS. agalactiae. 4.3. High temperarure compromises intestinal suture of tilapia Fluctuations in water temperature ean result in several physiologion bnormalities i fish. Good intestinal nstcasal morphology isthe fot dation for nutrient absorption, animal growth and disease resistance (Our results showed that high temperature treated for 14 days decreased the length and width of tapia intestine villas (Fig. 9). A proviows study in tlapia gostrointestinal tract revenled that inimunostinslants eon Increase villus eight [90], which implies the comrelation becween villus height and fish inumunity. The atenuated intestinal architecture under high temperature indicated smaller surface area for absorption as well is reduced imaumologieal protection agnist pathogens [92]. th add tion, high temperature increased the numberof intestinal goblet cells where synthesize and secrete mci to form mucosal barter. although rucosal barrier i a part of innate immune system to protect fish from pathogens [321 increased mucus in this study may be de to infeetion ar lover feeding, As we observed during experiment, tlapin’s food intake twas significantly increased at 35 “C compared to at 28 °C (data not shown), which could explain the increased number of intestinal goblet cells at high temperature. Collectively, it can be concluded that high temperature reduces the height and width of intestinal villi, whiel may lend to the decline of tilapia immunity‘et and Seti ero 196 (2023) 108699 Wilcoxon rank-sum test on phenatype r= —oeee col Fig. 6. Fuetinal preieson analysis of ferential ASV in diferent resperates ceed pia (A) Heatnap of KEGG abdance with dfeental ASUS, (B) Phenotype dtfeseces in T28 and 735, (C-D) Species phenotype contiution analysis, 44. High temperature disrapts intestinal mnirobior of ilapia Intestinal microbiota participate in carbohydrate and protein me: tabolisms and the syuthesis of many vitamins aad nonessential amino ‘acids, and promote the absorption of mineral elements such as iou and magnestum [35]. Thus, the colonization aad homeostasis of intestinal tmjerobiows have far-reaching. impacts on the nutrition, phystology, and immunity of hos. For aquatic animals, uctuations in water environ- ‘ment may cause changes in the composition of ish intestinal microbiota, In this study, owo predominant bacterial phyla, Actinobacteria and Proteobacteria, were identified in tilapia intestine. Zhu et al charac teri2ed microbial communities in the intestine of genetically insproved farmed tilapia (GIFT, Oreochromis niloticus) fed with low-protein diet ‘and found that che dominant bacterial phyla were Fusobacteria, Bac teroldetes, Proteobacteria, and Flrmieutes [5]. Shullrly, Fan et al ‘eported tha the dominant bacterial phyla in intensively cultured elspis ‘wore Proteobacteria, Actinobacteria snd Firmiewtes [5]. I has been revealed chat the abundance of phyla was related 10 diet, age, sex, ‘growth environment, and even the method of sampling and analysis bo According 1 the index of bacterial diversity, high temperature cold Increase intestinal mierobial diversity of elapia. Similarly, a previous ‘study shoved dramatic dectease in microbial diversity (Shannon index Hand richness when tilapia were exposed to cold temperature (12°C) [67], Parthermore, dhe microbial communities in the fish that were ‘subjected to cold temperatures exhibited lower variability in richness [07], which was consistent with or results, Low-temiperatre is 8s lective condition fr constraining microbial communities and decreasing ther inter individual variation. At the same time, high temperature led to dramatic ehanges in the composition ofthe intestinal merobfome by decreasing several genera of Actinobacteria (Aurantnnirobium, and Rhodococcus) and increasing Proteobacteria (nor ‘ank [_Rhizobiales Incertae Sedis, sewdorhodoplanes and AAneylobacten). Its well known thet Actinobacterions, especialy Bf ddobacterium, isa typieal probiote dha ean acidity intestinal environ: tent and protect intestinal barrier [si]. Nevertheless, miost Proteobacteria are pathogenic bacteria, such as scerichia cli, Salno- rela phimurium, and Vibrio cholera. ta particular, Rhodococcus can hance fish survival dhrough decteasing the population ofthe pathogen Flavobacterium psyehrophilum [59]. Rhodococcus is characterized by its probiotic properties in intestine, which has been reported to have beneficial effets on enhancing the intestinal immune function [40,1], Furthermore, we found that high temperature caused damage t0 the iestinal mucosa of tilapia (Fig. 9. Therefore, it ean be inferred that adverse impacts on intestinal meosa and imma ineced by high temperature may be related to the decrease in the abundance of Rho- docoecus, Meanwhile, Rhizobiales is pathogenic bacterium that maybe a souree of transmission for Stony Coral Tissue Loss Disease [12] “Therefore high emperatre many enlace the susceptibility of tilapia to ‘agalactiae vin decreasing the peoportion of probioties and increasing pathogens ‘The microbial phenotype prediction of differential ASVs showed that, high temperscure could change the composition ad function of tuicrobiots i Gaia intestine, 1 8 worth noting that although the abundance of Potentially Pathogenic was decreased wnder high te perature, the genera of pathogenicity related microorganisms were Increased, such as Kaistia and Anoxyhacillus. The interactions among Dathogeate mieroorganisns can sigalficantly affect host disease ress lance. For example, co-infection i fish ean aggravate the course and severity of many diseases, by synergistic and antagonistic interactions (09), Additionally, we observed a decrease in the abundance of sess Tolerance, such #5 Pseudorhiodoplanes, Mesorhizobium. and Aquicell ‘These mieroorganisms ean regulate hosts immunologiesl and metsbole (olerance, which is erucial 0 host against diseases [14]. Therefor, the lteration of intestinal microbiota incnding the augment of Potentially Pathogeaie diversity and the abatement of Stress Tolerance abundance,‘may be an important reason for high temperature exacerbating tilapia septocoecal disease 4.5. Conjoint analysis Under high temperature exposure, we found a significant change (enrichment or depletion) in several pathways in intestinal micro biomes, amino acid metabolism, faty acid metabolism and glycolysis’ shiconeogenesis. Changes in metabolic levels are normal adaptive ‘response o high temperatures in fsh microbiomes. in Shaoxing ducks (Anas plaryrhynchos}, the abundance of starch and sucrose metabolism ‘and energy metabolism pathways were inereased after heat creatment (05). The point is tha changes in metabolie levels and their metabolites ‘nay affect the hosts ability to resist disease. Notably, ou analysis fom transcriptome and 16S DNA sequencing revealed that pathways related to glucose aud lipid metabolisa were up-tegulated under high temper ‘ature, suggesting that S. agalactiae make extensive use of glucose ob tained from tilapia for growth and proliferation. Glucose level ean affect the imianity of hos, in both disease resistance snd tolerance [517]. ‘metabolomie study showed that the glucose level was significantly decreased in tlapia after Edwardsella tarda infection (48, ku pati: lla, exogenous administration of glacose greatly enhanced the sirvival ‘of api. der Eswardsellarada infetion by reprogramnsing meta folomies [49]. These studies indicate that low level of glucose may attenuate host resistance to pathogens. Meanwhile, glucose homeostasis plays an important role in maintaining tolerance to sepsis, in whieh jockdown of ferritin disrupts hepatie G6P enzyme expression and glucose production, and thereby reduces host tolerance (0 multiple ‘mleroblal infections [50]. Altogether, high tempereture may affect the ‘susceptibility of tilapia to 8 agalactiae through metabolites, especially slucose. 15. Conclusions High temperature can increase the susceptibility of tilapia to 'S agolactae infection. In this study, we found that high temperature promoted the growth and altered global genes expression of S. agalactiae. GO and KEG analysis of DEGs showed thatthe metabolic related pathways were mainly enriched. In addition, the length and ‘width of intestinal villus of tilapia were decreased under high temper ature, suggesting that high temperature might affect intestinal structure ‘and microbiota. Indeed, 165 rDNA seq showed that high temperature ‘can affect intestinal microecology, leading to nn imbalance of the ‘composition of intestinal microbiota, in rota, high temperature cas accelerate the growth ofS. agalactiae by modulating transcriptome, and dlismpt the structure and mietobiots of tpl Intestine, whe nally leads to increase susceptibility of api to S. agalactiae, ‘CRedit authorship conteibution statement Zaoya Zhao: Conceptialization, Methixiology, Dats curaton, Investigation, Writing ~ origi on, Funding acquis. tion. Qianxing Zou: Concepmalization, Data curation, investigation, Haljun Yan: Resources, Daca curation. Dasheng Hi tration, Data curation, Funding acquiscon. YE Project administration, Funding scaision, Declaration of competing interest ‘The authors declare there are no confit of interest. Das wil be misde available on request ‘et and Seti ero 196 (2023) 108699 Acknowledgements This work was supported by the Guangxi Science and Technology Base sud Special Talent Foundation (AD22080018), Livzhou Science ‘and Technology Plan (2020NACBO802), the Initial Seientifc Research Fund of Higherlevel ‘Talents in Liuzhott People’s Hospital (LRYGCC202208), the open fund of Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture (GXKEYLA2019 06), the Young and Middle-aged Teachers Ability Promotion Projet of Guangxi Autonomous Region (2022KY0332) and the Special Fund for Guangxi University of Science and Technology (No, 21245), Appendix A. 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