Accepted Manuscript Hsp90, Hsp60 and sHsp families of heat shock protein genes in channel catfish and their expression after bacterial infections Yangjie Xie, Lin Song, Zhaohong Weng, Shikai Liu, Zhanjiang Liu PII:

S1050-4648(15)00126-6

DOI:

10.1016/j.fsi.2015.03.027

Reference:

YFSIM 3381

To appear in:

Fish and Shellfish Immunology

Received Date: 26 November 2014 Revised Date:

25 February 2015

Accepted Date: 20 March 2015

Please cite this article as: Xie Y, Song L, Weng Z, Liu S, Liu Z, Hsp90, Hsp60 and sHsp families of heat shock protein genes in channel catfish and their expression after bacterial infections, Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.03.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Hsp90, Hsp60 and sHsp families of heat shock protein genes in channel catfish and their

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expression after bacterial infections

3 Yangjie Xie a,b,1, Lin Song a,1, Zhaohong Weng a,b, Shikai Liu a, Zhanjiang Liu a*

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a

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of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA

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The Fish Molecular Genetics and Biotechnology Laboratory, Aquatic Genomics Unit, School

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Fisheries College, Jimei University, Xiamen 361021, P R China

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These authors contributed equally

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*Corresponding author. Tel.: +1 334-844-4784; fax: +1 334 844 9208. E-mail address:

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[email protected].

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Abstract

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Heat shock proteins (Hsps) are a suite of highly conserved proteins whose expressions are

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generally induced by elevated temperature. However, many Hsps play important roles in both

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innate and adaptive immunity. On the basis of our previous work on Hsp40 and Hsp70 gene

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families in channel catfish (Ictalurus punctatus), the objective of this study was to characterize

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Hsp90, Hsp60, Hsp10, and small Hsp genes, and to investigate their expression profiles after

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bacterial infections. A total of 20 Hsp genes were identified and annotated in the channel catfish

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genome, including five Hsp90 genes, one Hsp60 gene, one Hsp10 gene, and 13 sHsp genes. Six

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Hsp genes were differentially expressed after Edwardsiella ictaluri infection, and 12 were

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differentially expressed after Flavobacterium columnare infection. Although expression of these

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genes exhibited both temporal and spatial regulation, the induction of Hsp genes was observed

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soon after bacterial infection, while the suppression of Hsp genes was observed at later time-

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points, suggesting their distinct roles in immune responses and disease defenses. A pathogen-

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specific expression pattern of Hsp90 was observed. After F. columnare infection, all Hsp90

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genes were found up-regulated except Hsp90ab1, which was not significantly regulated.

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However, after E. ictaluri infection, only one Hsp90 gene was found significantly down-

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regulated. Both pathogen-specific and tissue-specific pattern of expression were observed with

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small Hsps after E. ictaluri and F. columnare bacterial infections. These results suggested that

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most of Hsp genes may play important roles in immune response and/or disease defense in

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channel catfish.

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Keywords: Channel catfish; Heat shock protein; Gene expression; Bacterial infection; immune

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response

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1 Introduction

41 Heat shock proteins (Hsps) are a suite of highly conserved proteins whose expressions are

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mostly increased when cells are exposed to elevated temperature or other stresses such as disease

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and hypoxia [1-3]. They are classified into five families based on molecular weight as well as

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domain structures and functions: Hsp90, Hsp70/Hsp110, Hsp60/Hsp10, Hsp40, and small heat

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shock protein (sHsp) families [4]. We previously identified and characterized two Hsp families

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of Hsp40 and Hsp70 in channel catfish [5, 6], but the remaining three families of Hsp genes:

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Hsp90, Hsp60/Hsp10, and small Hsps remain unexplored in catfish.

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Hsp90s not only function as molecular chaperones but also play roles in many other

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processes such as involvement in intracellular transport [7-9], protein degradation [10-12], and

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cell signaling [13, 14]. Hsp90s stabilize a number of proteins required for tumor growth, and

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therefore Hsp90 inhibitors are investigated as anti-cancer drugs [15-17]. In most species

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previously studied, Hsp90 genes are abundantly expressed, accounting for 1–2% cellular proteins

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[15]. They are found in the cytosol, nucleoplasm, endoplasmic reticulum (ER), mitochondria,

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and chloroplasts [15, 18, 19]. In human, 17 Hsp90 genes are found, including six functional

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genes (HSP90AA1, HSP90AA2, HSP90AB1, HSP90B1, TRAP1 and HSP90N), and 11

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pseudogenes [20]. Functional Hsp90s are dimeric with each monomer consisting of four

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structural domains: a highly conserved N-terminal domain (NTD) that has a high-affinity ATP-

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binding site, a "charged linker" region that connects the N-terminus with the middle domain, a

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protein-binding middle domain (MD) and a C-terminal domain (CTD) that interacts with co-

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chaperones [21-25].

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Hsp60/Hsp10 act as chaperones in mitochondria and they are also been found in the

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cytoplasm. In addition, they play important roles in the transportation of mitochondrial proteins

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into the mitochondrial matrix from the cytoplasm [26]. Moreover, studies have suggested that

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Hsp60 plays a key role in preventing apoptosis in the cytoplasm by forming a complex with

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proteins responsible for apoptosis and regulates the activity of these proteins [27]. The

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cytoplasmic Hsp60 is also involved in immune response [28] and cancer [27, 29, 30]. Hsp10 aids

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Hsp60 in protein folding by acting as a dome-like cover on the ATP active form of Hsp60 [28].

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Small heat shock proteins display in vitro chaperone-like activity [31, 32]. In vivo, sHsps

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have been implicated in an astounding variety of processes, such as enhancing cellular stress

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resistance [33], regulating actin and intermediate filament dynamics [34, 35], inhibiting

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apoptosis [36], modulating membrane fluidity [37], and regulating vasorelaxation [38].

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Mutations of sHsps in humans are responsible for various forms of hereditary cataract [39, 40],

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muscular diseases [41, 42] and neuropathies [43, 44]. sHsps are probably the most diverse in

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structure and function amongst the various families of stress proteins [45, 46]. Functional sHsps

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are multimeric complexes composed of one or more sHsp monomers [45, 47]. Each sHsp

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monomer contains an amino-terminal region, an α-crystallin domain (ACD) and a carboxyl-

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extension [45, 47-52].

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Hsps play important roles in both innate and adaptive immune responses [3, 53]. For innate

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immunity, Hsps are reported to mediate both humoral and cellular innate immune responses [54].

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The presence of Hsps in the extracellular environment served as a danger signal to activate

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innate immune such as dendritic cells (DCs) and macrophages [55-57]. Several cytokines can be

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induced by Hsps, such as tumour-necrosis-α (TNF-α), interleukin-1β (IL-1β), interleukin-12 (IL-

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12), nitric oxide and some chemokines [58-61]. For adaptive immunity, Hsps can stimulate

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adaptive immune responses as potent antigen carriers. For instance, Hsp60, Hsp70, Hsp90 have

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been proposed to interact with immune cells as a ligand for a variety of cell-surface receptors

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such as Toll-like receptors and a number of clusters of differentiation (CDs) such as CD14 and

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CD91 [62-65]. Elevated expression of Hsps after pathogen infection has been demonstrated in

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aquatic organisms [54, 66-69]. Increasing Hsps in aquatic organisms by heat shock, chemical

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application and feeding exogenous Hsps also enhanced resistance against infection [54, 70], and

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the level of tolerance correlated with the amount of accumulated Hsps [54].

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Channel catfish (Ictalurus punctatus) is an important aquaculture species in the United

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States. However, in recent years, catfish industry has encountered great challenges including

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devastating diseases which cause the largest economic losses. Of the serious disease problems,

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bacterial diseases are the major threats to the catfish industry. Enteric septicaemia of catfish

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(ESC), caused by Gram-negative bacterium E. ictaluri, is the most significant disease affecting

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the catfish industry [71]. Columnaris, caused by Gram-negative bacterium F. columnare, is the

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most frequently occurring disease in fish including catfish, causing huge economic losses

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worldwide [72].

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In this study, we identified and annotated Hsp90, Hsp60/Hsp10 and sHsp gene families in

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channel catfish, and determined their expression profiles after ESC and Columnaris disease

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infections, to provide insights into their roles in immune response and disease defense.

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2 Materials and Methods

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2.1 Gene identification and sequence analysis

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To identify the Hsp genes, the channel catfish transcriptome database [73-75] and the whole

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genome database of channel catfish (unpublished data) were searched using available Hsp90,

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Hsp60/Hsp10 and sHsp from teleosts (zebrafish (Danio rerio), stickleback (Gasterosteus

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aculeatus), medaka (Oryzias latipes), tilapia (Oreochromis niloticus), fugu (Takifugu rubripes))

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and other vertebrate species from amphibian to mammals (xenopus (Xenopus laevis or X.

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tropicalis), turtle (Pelodiscus sinensis), lizard (Anolis carolinensis), bird (chicken (Gallus gallus)

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or turkey (Meleagris gallopavo)), platypus (Ornithorhynchus anatinus), mouse (Mus musculus),

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human (Homo sapiens)) as query sequences, including 90 kDa heat shock proteins

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(HSP90AA1/Hsp90aa1.1, HSP90AA2/Hsp90aa1.2, Hsp90ab1, Hsp90b1, Trap1), 60 kDa heat

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shock proteins (Hspd1), 10 kDa heat shock proteins (Hspe1) and small heat shock proteins

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(Hspb1, Hspb2, Hspb3, Cryaa, Cryab/(Cryaba, Cryabb), Hspb6, Hspb7, Hspb8, Hspb9, Hspb10,

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Hspb11, Hspb12, Hspb15) (Supplementary Table 1). The e-value was set at an intermediately

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stringent level of e-10 for collecting as many as potential Hsps.

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The retrieved sequences from transcriptome database were translated into amino acid

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sequences using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The predicted ORFs

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were verified by BLASTP against NCBI non-redundant protein sequence database. The simple

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modular architecture research tool (SMART 7) [76] was used to predict the conserved domains

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based on sequence homology and further confirmed by conserved domain prediction from

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BLASTP.

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The identified catfish Hsp transcript sequences were verified by aligning with the draft

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catfish genome sequences (unpublished data). The genomic scaffold containing the catfish Hsps

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genes were retrieved, and the genes within genomic scaffolds were predicted by Fgenesh of

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Molquest software (Softberry Int.) [77]. The catfish Hsp genes were named following the ZFIN

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(Zebrafish Nomenclature Guidelines) and the Guidelines for the nomenclature of the human heat

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shock proteins [78]. The genes were named after the zebrafish or mammalian orthologues

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whenever possible. When zebrafish or mammalian orthologues were known, the same name and

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abbreviation were used, except all letters were italicized and in lower case. When a gene is

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homologous to a zebrafish gene or human gene, but orthology was ambiguous, the gene was

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named after the closest zebrafish or mammalian homologue with the word 'like' appended to the

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name and the letter 'L' appended to the gene symbol. The orthology was determined through

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syntenic analysis. The protein symbol was same as gene symbol, but non-italic and the first letter

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was uppercase.

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138 2.2 Phylogenetic analysis

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The amino acid sequences of Hsps from other vertebrate organisms were retrieved from

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Ensembl genome databases or NCBI database for phylogenetic analysis. Multiple protein

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sequences alignments were conducted using the Clustal W2 program [79], Muscle v3.8 [80] and

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the Auto (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i; depends on data size), L-INS-i, E-INS-i

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and G-INS-i strategies from MAFFT v7.01 [81] with default parameters. The program MUMSA

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[82] was employed to select the best-scoring multiple alignment.

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To find a best-fit model of Hsps’ evolution, the ProtTest program was used [83] according to

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the Bayesian information criterion. The best-fit model was the LG+G model for Hsp90 family,

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which used LG amino acid model [84] and the gamma distribution for modeling rate

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heterogeneity (+G); while the JTT+G model for sHsp family and Hspe family, which used Jones-

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Taylor-Thornton (JTT) matrix and the gamma distribution for modeling rate heterogeneity (+G);

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and the JTT+G+I model for Hspd family, which used JTT matrix and the gamma distribution

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with invariant sites for modeling rate heterogeneity (+G+I). The phylogenetic and molecular

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evolutionary analyses were conducted using MEGA 6 [85]. Using parameters of Bootstrap test of

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1000 replicates and 95% partial deletion method, the Maximum Likelihood trees, Minimum

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Evolution trees and the Neighbor Joining Trees were generated. Separate phylogenetic analyses

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were constructed per family with other representative vertebrate species including zebrafish,

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medaka, fugu, stickleback, tilapia, Xenopus, turtle, lizard, chicken/turkey, platypus, mouse and

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human (Supplementary Table 1).

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159 2.3 Syntenic analysis

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Syntenic analysis was conducted by analyzing syntenic regions harboring Hsp genes from

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several vertebrates based on genome information from Ensembl (Release 77) and NCBI, to

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provide additional evidence for gene identification and orthology. Briefly, the catfish Hsps

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sequences were used as queries to search against the draft channel catfish genome sequences to

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obtain the genomic scaffolds containing the catfish Hsps genes. The neighboring genes were

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identified by Fgenesh program [77] and BLASTP as above. The orders of these neighboring

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genes were compared with those from zebrafish and human using the software Genomicus [86].

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2.4 Expression analysis using available RNA-Seq datasets

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The RNA-Seq datasets used for expression analysis were obtained from our previous studies

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in channel catfish in response to E. ictaluri infection (SRP009069) [87] and in response to F.

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columnare (SRP012586) [88]. In brief, intestine tissues were collected at 3 h, 24 h and 3 d time-

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points after E. ictaluri challenge. At each time-point, 30 fish from both control and treatment

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were randomly selected and divided into three replicate pools (10 fish each), respectively. Total

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RNA was extracted from each tissue pool. For each time-point, equal amounts of RNA from

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three treatment replicates were pooled for RNA-Seq. For the control samples, the replicate pools

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spanned each of three time-points (3 h, 24 h and 3 d). A master pool composed of equal amounts

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of each replicate control pool was formed for RNA-Seq (See details in [87]). Similarly, after F.

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columnare challenge, gill tissues were collected at 4 h, 24 h, and 48 h time-points. At each time

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point, 18 fish from both control and treatment were randomly selected and divided into 3

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replicate pools (6 fish each), respectively. Total RNA was extracted from each tissue pool. For

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each time-point, equal amounts of RNA from three treatment replicates were pooled for RNA-

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Seq. For the control samples, the replicate pools spanned each of the three time-points (4 h, 24 h,

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and 48 h). A master pool composed of equal amounts of each replicate control pool was formed

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for RNA-Seq (See details in [88]).

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The gene expression analysis was performed using CLC Genomics Workbench software

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(V5.5.2). The trimmed high-quality reads were first mapped onto channel catfish Hsp transcript

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sequences. Mapping parameters were set as ≥ 95% of the reads in perfect allignment and ≤ 2

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mismatches. The total mapped reads number for each transcript was then determined and RPK

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(Reads Per Kilobase of exon model) was calculated. Beta actin gene (actb) was used as internal

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control for calculating relative expression value. In brief, actb transcript was identified from

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channel catfish transcriptome database and was used as reference for mapping along with Hsp

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transcripts. The expression fold-change of each Hsp gene was determined based on the ratio of

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its RPK to that of actb in the same sample. Transcrirps with absolute expression fold change

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value ≥ 1.5 and total gene reads ≥ 5 were included in the analyses as significantly differentially

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expressed genes.

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3 Results

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3.1 Identification of Hsp90s in channel catfish Five Hsp90 genes were identified in the channel catfish genome, including hsp90aa1.1,

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hsp90aa1.2, hsp90ab1, hsp90b1 and trap1L. Full-length coding sequences were obtained for all

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five channel catfish hsp90 genes (Table 1). Of the five Hsp90 genes, hsp90aa1.1 and hsp90aa1.2

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were arranged in the genome as a head-to-tail tandem. All Hsp90 proteins are comprised of

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HATPase_c and HSP90 domain in structure (Table 1). Phylogenetic analysis supported the

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annotation of catfish hsp90 genes. They were well placed into distinct clades, grouped with other

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fishes and then grouped together with tetrapods (Fig. 1).

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3.2 Identification of Hsp60/Hsp10 genes in channel catfish

One Hsp60 gene (hspd1) and one Hsp10 gene (hspe1) were identified in the channel catfish

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genome. Full-length coding sequences of both genes were obtained (Table 1). hspd1 and hspe1

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are present in the genome as a head-to-head gene pair. The Hspd1 protein was comprised of

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GroEL/cpn60 domain while the Hspe1 protein was comprised of GroES/cpn10 domain (Table

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1). Phylogenetic analysis supported the annotation of catfish hspd1 and hspe1 genes. In both

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phylogenetic trees, the channel catfish gene was clustered with its counterpart in zebrafish (Fig. 2

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and 3).

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3.3 Identification of sHsp genes in channel catfish

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A total of 13 sHsp genes were identified in the channel catfish genome. Full-length coding

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sequences were obtained for all of them (Table 1). All channel catfish sHsps have Alpha

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crystallin domain (ACD) (Table 1). Hspb2 and cryabb form a head-to-head gene pair on the

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same scaffold. The phylogenetic analysis supported the annotation of catfish sHsp genes, each

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catfish sHsp gene clustered with its respective counterpart of other species, and most of them

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clustered with their respective counterparts of zebrafish (Fig. 4). Of the small Hsps, hspb10 was

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not found in the channel catfish genome.

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3.4 Syntenic analysis

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In most cases, phylogenetic analysis provided strong evidence for the identities of Hsp genes,

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with exception of trap1, hsp90aa1.1, hspb9 and hspb12. To provide additional evidence for the

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identities of these Hsp genes, syntenic analysis was conducted. As shown in Figure 5-7,

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conserved syntenies were found between channel catfish and other species for hsp90aa1.1, hspb9

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and hspb12. For trap1, the neighboring genes were not conserved between catfish and zebrafish

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(Fig. 8). Due to the lack of evidence for conserved synteny, the catfish trap1 gene was named as

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“trap1-like (trap1L)”.

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3.5 Expression of Hsp genes after bacterial infections Expression of the Hsp genes after bacterial infections was determined using available RNA-

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Seq datasets (Supplementary Table 2). Of the 20 Hsp genes, 15 were differentially expressed

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after bacterial infections, with 12 genes differentially expressed after columnaris disease

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infection (Fig. 9), and six genes differentially expressed after ESC disease infection (Fig. 10).

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After F. columnare infection, six Hsp genes (hsp90aa1.2, hsp90b1, trap1L, hspd1, hspe1

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and hspb11) were up-regulated, while five Hsp genes (hspb1, hspb2, hspb3, cryabb and hspb9)

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were down-regulated, and one Hsp gene (hsp90aa1.1) was up-regulated at 4 hours and down-

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regulated at 48 hours (Fig. 9). However, the patterns of up-regulated or down-regulated

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expression of these genes were quite different. In general, the up-regulated genes were induced

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quite rapidly after bacterial infection with significant up-regulation was observed at 4 hours or 24

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hours after infection, whereas significant down-regulation was not observed until at least 24

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hours after infection (Fig. 9). Among these significantly regulated genes, hsp90b1 was most

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significantly up-regulated, approximately 5.7 times up at 24 h after challenge. Among down-

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regulated genes, cryabb was most down-regulated, approximately 3.6 times down at 24 h after

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challenge. The extent of induction or suppression for all other differentially expressed genes

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were modest, 1.5-4 folds up or down after the bacterial infection (Fig. 9). It should be noted that

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of the 20 genes under analysis, eight did not exhibit differential regulation after the bacterial

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infection, of which four (hsp90ab1, haspb6, haspb7 and hspb8) were not differentially

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expression after the bacterial infection, while the remaining four (cryaa, cryaba, hspb12, hspb15)

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had low levels of expression that were excluded from analysis.

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After E. ictaluri infection, three Hsp genes (hsp90ab1, hspd1 and hspe1) were down-

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regulated, while three Hsp genes (cryabb, hspb7 and hspb8) were up-regulated (Fig. 10). The

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level of induction or suppression was modest for all six genes, within 1.5-2 folds. Similar to the

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situation of columnaris infection, the up-regulation appeared to be more rapid than down

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regulation, with the significant up-regulation being detectable as early as 3 hours after challenge.

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However, down-regulation was much slower, with significant down-regulation being detectable

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three days after the bacterial challenge (Fig. 10).

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4 Discussion

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4.1 Hsp genes in channel catfish

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In this work, systematic analysis of Hsp90, Hsp60, Hsp10 and sHsps genes was conducted.

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A total of 20 Hsp genes were identified and annotated in the channel catfish genome, and their

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expressions after bacterial infections were examined to determine their involvement in defense

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responses. The 20 Hsp genes included five Hsp90 genes, one Hsp60 gene, one Hsp10 gene, and

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13 sHsp genes (Table 1). In most cases, their identities were readily determined by phylogenetic

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analysis (Fig. 1-4), but with four Hsp genes, syntenic analysis was conducted to provide

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additional evidence to properly annotate these genes. Of the four genes, three were found to be

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within well conserved syntenic region in the genomes (Fig. 5-7), thus their identities were

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determined. However, with one gene, trap1, phylogenetic analysis alone did not provide concrete

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answer to its identity, and the genomic region was not conserved between catfish and zebrafish

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(Fig. 8). Therefore, it was annotated as trap1-like (trap1L).

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4.2. Copy numbers of Hsp genes

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The copy numbers of the analyzed Hsp genes are generally conserved among many

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organisms across a broad evolutionary spectrum (Table 2). However, several differences were

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observed. First, the teleost and amphibian have two copies of hsp90aa1, while mammal, bird

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and reptile possess only one copy, suggesting that one copy of this gene was lost during

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evolution between amphibian and reptile. Second, Hspb10/Odf1 was not found in teleost and

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amphibian genomes, but was found in the genomes of reptiles, birds and mammals, suggesting

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that the gene was gained in higher vertebrate during evolution [89, 90]. Third, catfish as well as

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zebrafish have two copies of cryab gene, cryaba and cryabb, while other species have only one

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cryab genes (Table 2).

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4.3 Hsp genes were involved in disease defense Although reports were available for the involvement of the Hsp genes in disease defenses

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[53, 54, 66], there was no previous studies conducted systematically examining the expression of

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the Hsp genes after bacterial infection. In this study, we took advantage of the existing RNA-Seq

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datasets obtained after ESC and columnaris challenges [87, 88]. Among 20 Hsp genes being

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studied, five Hsp90 genes (hsp90aa1.1, hsp90aa1.2, hsp90ab1, hsp90b1, trap1L), one Hsp60

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gene (hspd1), one Hsp10 gene (hspe1), and eight sHsp genes (hspb1, hspb2, hspb3, cryabb,

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hspb7, hspb8, hspb9, hspb11) were found to be differentially expressed after bacterial challenges

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(Fig. 9-10). Although their roles and the mechanisms of regulation are not known at present, the

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findings that they are significantly regulated after bacterial challenges suggest that they are

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involved in disease responses, and perhaps also in disease defenses against infectious bacteria.

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Hsp90 proteins were known to be abundantly expressed in most cells under normal

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conditions. In terrestrial animal species, there are four Hsp90 gene paralogues: two (Hsp90aa and

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Hsp90ab) are cytosolic, Hsp90b1 is distributed in the endoplasmic reticulum (ER), and Trap1 is

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mitochondrial. Hsp90s are chaperone proteins playing a number of important roles including

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interacting with immune cells as a ligand for a variety of cell-surface receptors such as Toll-like

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receptors and CDs [62, 91]. In this study, hsp90aa1.1 was up-regulated in gill at first (4 h) and

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then down-regulated (48 h) after F. columnare infection (Fig. 9). However, it was not expressed

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in intestine after E. ictaluri infection (Fig. 10). Both hsp90aa1.2 and hsp90b1 were significantly

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up-regulated in all three time-points after F. columnare infection (Fig. 9), but they did not show

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significant fold change in intestine after E. ictaluri infection (Fig. 10). Due to the constitutive

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expression, hsp90ab1 had the highest expression values in both experiments, but it did not show

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significant fold change with the exception of being down-regulated at 3 d in intestine after E.

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ictaluri infection (Fig. 9-10). trap1L was significantly up-regulated in gill at 24 h after F.

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columnare infection (Fig. 9), but was not differentially expressed in intestine after E. ictaluri

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infection (Fig. 10). These results demonstrated the different expression patterns between

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Hsp90aa and Hsp90ab genes: the former is inducible but the latter is constitutively expressed

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[20]. Additionally, hsp90aa1.1 and hsp90aa1.2 had different expression profile though they were

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tandem repeated genes. Furthermore, four Hsp90 genes showed up-regulation in gill after F.

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columnare infection (Fig. 9), but they were not significantly regulated in intestine after E.

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ictaluri infection challenge at all time-points after infection (Fig. 10), suggesting pathogen-

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specific gene regulation.

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Hsp60 is an important chaperonin, and was also proposed to interact with immune cells as a

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ligand for a variety of cell-surface receptors such as Toll-like receptors and CDs [63-65]. Hsp60

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requires interacting with Hsp10 for proper function [92]. In this study, both hspd1 and hspe1

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showed up-regulation in gill after F. columnare infection (Fig. 9), however both were down-

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regulated in intestine at 3 d after E. ictaluri infection (Fig. 10), indicating that their expression

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was regulated temporally as well as by the functions of pathogenesis with different diseases.

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sHsps are the most diverse family amongst the heat shock proteins. Amongst 13 sHsp genes

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identified in channel catfish, eight sHsp genes exhibited differential expression after E. ictaluri

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or F. columnare infections (Fig. 9-10). hspb1 was down-regulated in gill at 24 h and 48 h after F.

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columnare infection (Fig. 9), but was not significantly regulated in intestine after E. ictaluri

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infection (Fig. 10). hspb2, hspb3 and hspb9 were down-regulated expression in gill after F.

332

columnare infection (Fig. 9) but their expression was low or not detected in intestine after E.

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ictaluri infection (Supplementary Table 2), suggesting tissue-specific expression. In this study,

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RNA-Seq datasets were from intestine (ESC) and gill (columnaris). These findings are consistent

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with previous reports that expression of hspb2 and hspb3 was restricted to heart and skeletal

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muscle cells in mammals [93, 94] because gill has skeletal muscle cells while intestine has not.

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cryabb was significantly down-regulated at 24 h in gill after F. columnare infection but was

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significantly up-regulated at 3 h and 24 h in intestine after E. ictaluri infection (Fig. 9-10). As the

339

major structural protein of eye lens, Cryaa (αA-crystallin) is abundantly expressed together with

340

Cryab (αB-crystallin) in the eye lens. In spite of their high degree of homology, Cryab is

341

expressed in many non-lenticular tissues; it has the greater chaperone-like activity than Cryaa

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and its heat-induced conformational change and aggregation is more susceptible than that of

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Cryaa [95-99]. This could explain why cryaa was not expressed in gill or intestine while cryabb

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was expressed in both tissues.

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One interesting observation was that all up-regulated Hsp genes tended to be differentially

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expressed at early time-points after infection, whereas the down-regulated Hsp genes exhibited

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differential expression at much later stages after infection. The exact reason of this observation is

348

unknown at present, but it is possible that higher levels of Hsp gene expression at early stages of

349

pathogenesis could be beneficial to the host because chaperone activities are demanded to fight

350

against the bacterial infection. However, as the pathogenesis progressed, additional high levels of

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Hsp expression could be detrimental to the host. Alternatively, the regulated Hsp gene

352

expression could also be a consequences of pathogenesis rather than proactive or active defense

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mechanisms of the host because it is well known that Hsps can be induced by many stresses

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including bacterial infections [54, 66-69].

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5. Conclusions

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A total of 20 Hsp genes were identified and annotated in the channel catfish genome

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including five Hsp90 genes, one Hsp60 gene, one Hsp10 gene, and 13 sHsp genes. Differential

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expression was observed from the majority of the Hsps after bacterial infections. Different

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temporal and spatial expression patterns were observed for these genes. In general, induced

361

expression was observed soon after bacterial infection, but suppressed Hsp gene expression was

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observed at later stages after bacterial infection. The observed differential expression of the Hsp

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genes indicated that they may be involved as a part of disease response genes or it is possible that

364

some of these Hsp genes may actually be a part of disease defense genes against the bacterial

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pathogens. Further studies are warranted to explore the mechanisms of regulation and to

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understand the roles of the Hsps in host defenses against infectious diseases.

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367 Acknowledgements

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This project was supported by USDA Aquaculture Research Program Competitive Grant (No.

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2014-70007-22395), and by Agriculture and Food Research Initiative Competitive Grants (Nos.

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2015-67015-22907 and 2015-67015-22975) from the USDA National Institute of Food and

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Agriculture (NIFA). Yangjie Xie was supported by the scholarship awarded by Education

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Department of Fujian Province for Visiting Scholars Abroad. Zhaohong Weng was sponsored by

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Fujian Fumin Foundation.

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Supporting Information

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Supplementary Table 1 Gene names and accessions of reference Hsps used in the study. (XLS)

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Supplementary Table 2 The channel catfish Hsp genes significantly expressed after E. ictaluri or

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F. columnare infection. (XLS)

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[96] Iwaki T, Kume-Iwaki A, Liem RK, Goldman JE. αB-crystallin is expressed in non-

620

lenticular tissues and accumulates in Alexander's disease brain. Cell. 1989 57:71-8.

621

[97] Sun T-X, Das BK, Liang JJ-N. Conformational and functional differences between

622

recombinant human lens αA-and αB-crystallin. Journal of Biological Chemistry. 1997 272:6220-

623

5.

624

[98] Horwitz J, Bova M, Huang Q-L, Ding L, Yaron O, Lowman S. Mutation of αB-crystallin:

625

effects on chaperone-like activity. International Journal of Biological Macromolecules. 1998

626

22:263-9.

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[99] Liang JJ, Sun TX, Akhtar NJ. Heat-induced conformational change of human lens

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recombinant alphaA- and alphaB-crystallins. Mol Vis. 2000 6:10-4.

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Tables:

631 632

Table 1. Identification of Hsp90, Hsp60/Hsp10 and small Hsp genes in channel catfish Gene

Accession Numbers

Domain

hsp90aa1.1

JT341208.1

HATPase_c--HSP90

hsp90aa1.2

JT411683.1

HATPase_c--HSP90

hsp90ab1

KP126796

HATPase_c--HSP90

hsp90b1

JT417742.1

HATPase_c--HSP90

trap1L

JT406894.1

SC

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Hsp90

HATPase_c--HSP90

Hsp60/Hsp10 JT407863.1

hspe1

JT408710.1

GroES/cpn10

hspb1

JT419201.1

ACD_HspB1_like

hspb2

JT416075.1

Crystallin--ACD_HspB2_like

hspb3

JT413022.1

ACD_HspB3_Like

cryaa

JT408754.1

Crystallin--ACD_alphaA-crystallin_HspB4

cryaba

KP126797

Crystallin--ACD_HspB4-5-6

JT407340.1

Crystallin--ACD_HspB4-5-6

JT410786.1

metazoan_ACD

JT408248.1

ACD_HspB7_like

JT417241.1

ACD_HspB8_like

hspb7 hspb8 hspb9 hspb11

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hspb12 hspb15

633 634

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hspb6

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sHsps

cryabb

GroEL/cpn60

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hspd1

JT412125.1

metazoan_ACD

JT473518.1

ACD_HspB9_like

JT471744.1

ACD_HspB7_like

JT426179.1

ACD_HspB1_like

635

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636

Table 2. Comparison of copy numbers of Hsp90, Hsp60/Hsp10 and sHsp genes among selected

637

vertebrate genomes Bird

Amphibian

Tilapia

Fugu

Medaka

Zebrafish

Catfish

Hsp90aa1 Hsp90ab1 Hsp90b1 Trap1

1 1 1 1

1 1 1 1

2 1 1 1

2 1 1 1

2 1 1 1

2 1 1 1

2 1 1 2

2 1 1 1

Total of Hsp90 family Hspd1 Hspe1 Total of Hsp60/Hsp10 family Hspb1 Hspb2 Hspb3 Cryaa Cryab Hspb6 Hspb7 Hspb8 Hspb9 Hspb10/Odf1 Hspb11 Hspb12 Hspb15

4 1 1

4 1 1

5 1 1

5 1 1

5 1 1

5 1 1

6 1 1

5 1 1

2 1 1 1 1 1 1 1 1 1 1 1 0 0

2 1 1 1 1 1 0 1 1 0 1 0 0 0

2 1 1 1 1 1 1 1 1 0 0 1 1 0

2 1 0 1 1 1 1 0 1 1 0 1 1 1

11 17

8 14

10 17

10 17

639 640

SC 2 1 0 1 1 1 1 1 1 0 0 1 1 1

2 1 0 1 1 1 0 0 1 0 0 1 0 1

2 1 1 1 1 2 1 1 1 1 0 1 1 1

2 1 1 1 1 2 1 1 1 1 0 1 1 1

10 17

7 14

13 21

13 20

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Total of sHsp family Total

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Gene Name

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Figures:

642 643 644

Fig. 1. Phylogenetic analysis of channel catfish Hsp90 genes. The phylogenetic tree was constructed using the

645

maximum likelihood method with LG+G model and 95% partial deletion method in MEGA6. The statistical

646

robustness of the tree was estimated by bootstrapping with 1000 replicates. Bootstrap values were indicated by

647

numbers at the nodes.

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648 649 650

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651 652

Fig. 2. Phylogenetic analysis of channel catfish hspd1. The phylogenetic trees were constructed using the

654

maximum likelihood method with JTT+G+I model and 95% partial deletion method in MEGA6. The statistical

655

robustness of the tree was estimated by bootstrapping with 1000 replicates. Bootstrap values were indicated by

656

numbers at the nodes.

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658

659

Fig. 3. Phylogenetic analysis of channel catfish hspe1. The phylogenetic trees were constructed using the

661

maximum likelihood method with JTT+G model and 95% partial deletion method in MEGA6. The statistical

662

robustness of the tree was estimated by bootstrapping with 1000 replicates. Bootstrap values were indicated by

663

numbers at the nodes.

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665 666

Fig. 4. Phylogenetic analysis of channel catfish sHsp genes. The phylogenetic tree was constructed using the

668

Minimum Evolution method with JTT+G model and 95% partial deletion method in MEGA6. The statistical

669

robustness of the tree was estimated by bootstrapping with 1000 replicates. Bootstrap values are indicated by

670

numbers at the nodes.

671

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672 Fig. 5. Syntenic analysis of channel catfish hsp90aa1.1 gene with zebrafish and human.

674

hsp90aa1.1 in channel catfish has common neighbored genes as that of zebrafish and human.

675

Abbreviations are Chr: chromosome; CINP: cyclin-dependent kinase 2 interacting protein; dio3:

676

iodothyronine deiodinase type 3; gimap8: GTPase IMAP family member 8; gtf2ird2: general

677

transcription factor II-I repeat domain-containing protein 2; HSP90AA1: heat shock protein

678

90kDa alpha (cytosolic), class A member 1; hsp90aa1.1: heat shock protein 90, alpha (cytosolic),

679

class A member 1, tandem duplicate 1; hsp90aa1.2: heat shock protein 90, alpha (cytosolic),

680

class A member 1, tandem duplicate 2; klhl10: Kelch-like protein 10; mgat2: mannosyl (alpha-

681

1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; MOK: MOK protein kinase; myh6:

682

myosin, heavy polypeptide 6, cardiac muscle, alpha; pak6: serine/threonine-protein kinase PAK

683

6; pak6b: p21 protein (Cdc42/Rac)-activated kinase 6b; PPP2R5C: protein phosphatase 2,

684

regulatory subunit B', gamma; ppp2r5cb: protein phosphatase 2, regulatory subunit B', gamma b;

685

rps29: ribosomal protein S29; slc25a29: solute carrier family 25, member 29; SLC25A47: solute

686

carrier family 25, member 47; slc25a47a: solute carrier family 25, member 47a; WDR20: WD

687

repeat domain 20; wdr20b: WD repeat domain 20b.

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689

690

Fig. 6. Syntenic analysis of channel catfish hspb9 gene with zebrafish and human. hspb9 in

692

channel catfish has one common neighbored gene as that of zebrafish and human. There are only

693

four identified genes in scaffold jcf7180003668739. Abbreviations are Chr: chromosome;

694

ccdc103: coiled-coil domain containing 103; DHX58: DEXH (Asp-Glu-X-His) box polypeptide

695

58; G6PC3: glucose-6-phosphatase 3, catalytic; g6pcb: glucose-6-phosphatase b, catalytic;

696

hspb9: heat shock protein, alpha-crystallin-related, 9; KAT2A: K(lysine) acetyltransferase 2A;

697

kat7: K(lysine) acetyltransferase 7; MPP2: MAGUK p55 subfamily member 2; mpp2a:

698

membrane protein, palmitoylated 2a; nbr1: neighbor of BRCA1 gene 1; PYY: peptide YY; pyya:

699

peptide YYa; racgap1: Rac GTPase-activating protein 1; rarab: retinoic acid receptor, alpha b;

700

rpl23: ribosomal protein L23; stxbp4: syntaxin-binding protein 4; ZNF385C: zinc finger protein

701

385C.

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703

704

Fig. 7. Syntenic analysis of channel catfish hspb12 gene with zebrafish and cave fish (Astyanax

706

mexicanus). Hspb12 in channel catfish has some common neighbored genes as that of cave fish

707

and has one common neighbored gene as that of zebrafish which only two neighbor genes were

708

known so far. Abbreviations are Chr: chromosome; alp: alkaline phosphatase, tissue-nonspecific

709

isozyme precursor; alp3: alkaline phosphatase 3; CABZ01071642.2: Uncharacterized protein

710

(ENSDARG00000087003); cep57: centrosomal protein 57kDa; chd: Chordin; clcn2: chloride

711

channel protein 2; ecel1: endothelin-converting enzyme-like 1; eif4g1: eukaryotic translation

712

initiation factor 4 gamma 1; hspb12: heat shock protein, alpha-crystallin-related, b12; psmd2:

713

26S proteasome non-ATPase regulatory subunit 2.

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716

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Fig. 8. Syntenic analysis of channel catfish trap1L gene with zebrafish and human. trap1 in

721

channel catfish has no common neighbored genes as that of zebrafish and human. Abbreviations

722

are Chr: chromosome; adcy9: adenylate cyclase 9; btbd8: BTB (POZ) domain containing 8,

723

isoform CRA_c; clcn2L: chloride channel protein 2-like; crebbp: CREB-binding protein;

724

crebbpb: CREB binding protein b; DNASE1: deoxyribonuclease I; ehbp1L: EH domain-binding

725

protein 1-like; kiaa1841L: uncharacterized protein KIAA1841-like; prdm9: histone-lysine N-

726

methyltransferase PRDM9; psmd2: 26S proteasome non-ATPase regulatory subunit 2; rnf213:

727

ring finger protein 213; rnps1: RNA binding protein S1, serine-rich domain; SLX4: SLX4

728

structure-specific endonuclease subunit; srl: sarcalumenin; tfap4: transcription factor AP-4;

729

trap1: TNF receptor-associated protein 1; trap1L: TNF receptor-associated protein 1-like; trim35:

730

tripartite motif containing 35; xpo1: exportin-1.

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718 719 720

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733

SC

734

Fig. 9. Expression of channel catfish Hsp90, Hsp60/Hsp10 and sHsp genes in gill after F.

736

columnare infection. Gene expressions were presented as fold-change relative to control samples

737

(only absolute fold change value≥1.5 was shown).

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741

SC

742

Fig. 10. Expression of channel catfish Hsp90, Hsp60/Hsp10 and sHsp genes in intestine after E.

744

ictaluri infection. Gene expressions were presented as fold-change relative to control samples

745

(only absolute fold change value≥1.5 was shown).

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Highlights:

2




Hsp90, Hsp60, Hsp10 and sHsp genes were identified in channel catfish genome.

3




Most Hsp genes were significantly regulated after bacterial infections.

4




Hsp genes were regulated in a time-dependent fashion after bacterial infections.

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Supplementary Table 1
 
 Gene names and accessions of reference Hsps used in the study

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Description Transcript ID Location Transcripts Name Transcript Protein ID Length (aa) organism heat shock protein 90kDa alpha Chromosome (cytosolic), 14: 102,547,075-102,606,036 class A member HSP90AA1-002 1 [Source:HGNC reverseENST00000216281 strand. Symbol;Acc:5253] ENSP00000216281 732 human heat shock protein 90, alpha Chromosome (cytosolic), 12: 110,690,605-110,702,728 class A member Hsp90aa1-001 1 [Source:MGI reverse Symbol;Acc:MGI:96250] ENSMUST00000094361 strand. ENSMUSP00000091921 733 mouse heat shock protein 90kDa alpha UltraContig (cytosolic), Ultra378: class7,237,297-7,242,966 A member HSP90AA1-201 1 [Source:HGNC reverse ENSOANT00000009133 Symbol;Acc:5253] strand. ENSOANP00000009131 733 platypus heat shock protein 90kDa alpha Chromosome: (cytosolic), 5; NC_006092.3 class A member (49388166..49392564, heat 1 shock protein NM_001109785.1 complement) HSP 90-alpha NP_001103255.1 567 chicken heat shock protein 90kDa alpha NW_005854351.1 (cytosolic), (1762380..1770681) class A member heat 1 shock protein XM_006119990.1 90kDa alpha (cytosolic), XP_006120052.1 class A member728 1 turtle Uncharacterized protein Source: Scaffold UniProtKB/TrEMBL GL343232.1: 1,912,641-1,921,714 H9G3B6 Novel forward ENSACAT00000000159 strand. ENSACAP00000000156 728 lizard heat shock protein 90kDa alpha NW_004668241.1 (cytosolic), (75835485..75847813, class A member heat 1, shock gene complement) 1protein NM_001016282.2 90kDa alpha (cytosolic), NP_001016282.1 class A member729 1, genexenopus 1 heat shock protein 90, alpha scaffold_16: (cytosolic), 1,109,271-1,112,372 class A member hsp90aa1.1-201 1, forward tandem duplicate strand. ENSTRUT00000031532 1 [Source:ZFIN;Acc:ZDB-GENE-990415-94] ENSTRUP00000031409 724 fugu heat shock protein 90, alpha Scaffold (cytosolic), GL831143.1: class 3,039,921-3,043,080 A member hsp90aa1.1-201 1, tandem duplicate reverse ENSONIT00000001041 strand. 1 [Source:ZFIN;Acc:ZDB-GENE-990415-94] ENSONIP00000001042 724 tilapia heat shock protein 90, alpha groupXVIII: (cytosolic), 14,758,907-14,763,271 class A member hsp90aa1.1-201 1, tandem forwardduplicate strand. ENSGACT00000017054 1 [Source:ZFIN;Acc:ZDB-GENE-990415-94] ENSGACP00000017020 723 stickleback heat shock protein 90, alpha Chromosome (cytosolic), 24: 17,509,256-17,513,475 class A member hsp90aa1.1-201 1, tandem forward duplicate strand. ENSORLT00000021929 1 [Source:ZFIN;Acc:ZDB-GENE-990415-94] ENSORLP00000021928 724 medaka heat shock protein 90, alpha Chromosome (cytosolic), 20: 54,337,468-54,367,194 class A member hsp90aa1.1-001 1, tandem reverse duplicate strand. ENSDART00000004756 1 [Source:ZFIN;Acc:ZDB-GENE-990415-94] ENSDARP00000022302 725 zebrafish heat shock protein 90kDa alpha NW_004668241.1 (cytosolic), (75856713..75864604, class A member heat 1, shock gene complement) 2protein NM_001079297.1 90kDa alpha (cytosolic), NP_001072765.1 class A member716 1, genexenopus 2 heat shock protein 90, alpha scaffold_16: (cytosolic), 1,114,145-1,117,181 class A member hsp90aa1.2-201 1, forward tandem duplicate strand. ENSTRUT00000031659 2 [Source:ZFIN;Acc:ZDB-GENE-031001-3] ENSTRUP00000031536 727 fugu heat shock protein 90, alpha Scaffold (cytosolic), GL831143.1: class 3,033,202-3,039,387 A member hsp90aa1.2-201 1, tandem duplicate reverse ENSONIT00000001023 strand. 2 [Source:ZFIN;Acc:ZDB-GENE-031001-3] ENSONIP00000001024 729 tilapia heat shock protein 90, alpha groupXVIII: (cytosolic), 14,763,666-14,768,466 class A member hsp90aa1.2-201 1, tandem forwardduplicate strand. ENSGACT00000017081 2 [Source:ZFIN;Acc:ZDB-GENE-031001-3] ENSGACP00000017047 713 stickleback heat shock protein 90, alpha Chromosome (cytosolic), 24: 17,515,059-17,518,400 class A member hsp90aa1.2-201 1, tandem forward duplicate strand. ENSORLT00000021940 2 [Source:ZFIN;Acc:ZDB-GENE-031001-3] ENSORLP00000021939 715 medaka heat shock protein 90, alpha Chromosome (cytosolic), 20: 54,322,501-54,336,777 class A member hsp90aa1.2-001 1, tandem reverse duplicate strand. ENSDART00000023550 2 [Source:ZFIN;Acc:ZDB-GENE-031001-3] ENSDARP00000026065 734 zebrafish heat shock protein 90kDa alpha Chromosome (cytosolic), 6: 44,214,824-44,221,620 class B member HSP90AB1-003 1 [Source:HGNC forward strand. ENST00000371646 Symbol;Acc:5258] ENSP00000360709 724 human heat shock protein 90 alphaChromosome (cytosolic), 17: class 45,567,775-45,573,271 B member 1Hsp90ab1-001 [Source:MGI reverseSymbol;Acc:MGI:96247] strand. ENSMUST00000024739 ENSMUSP00000024739 724 mouse heat shock protein 90kDa alpha Chromosome (cytosolic), 3: 29,630,922-29,636,756 class B member HSP90AB1-201 1 (HSP90AB1), reverse strand. ENSGALT00000016542 mRNA. [Source:RefSeq ENSGALP00000016523 mRNA;Acc:NM_206959] 725 chicken heat shock protein 90kDa alpha Chromosome (cytosolic), 1: 227,915,054-227,933,305 class B member HSP90AB1-201 1 [Source:HGNC forwardENSACAT00000014893 strand. Symbol;Acc:5258] ENSACAP00000014598 727 lizard heat shock protein 90kDa alpha NW_004668895.1 (cytosolic), (15744..22064) class B member heat 1 shock protein NM_001030484.1 90kDa alpha (cytosolic), NP_001025655.1 class B member723 1 xenopus heat shock protein 90, alpha Scaffold (cytosolic), GL831178.1: class 3,475,799-3,482,638 B member hsp90ab1-201 1 [Source:ZFIN;Acc:ZDB-GENE-990415-95] forward ENSONIT00000007477 strand. ENSONIP00000007472 725 tilapia heat shock protein 90, alpha groupXVIII: (cytosolic), 15,800,515-15,805,626 class B member hsp90ab1-201 1 [Source:ZFIN;Acc:ZDB-GENE-990415-95] reverse strand. ENSGACT00000017921 ENSGACP00000017886 728 stickleback heat shock protein 90, alpha Chromosome (cytosolic), 24: 5,421,972-5,426,821 class B member hsp90ab1-201 1 [Source:ZFIN;Acc:ZDB-GENE-990415-95] forward strand. ENSORLT00000014901 ENSORLP00000014900 679 medaka heat shock protein 90, alpha Chromosome (cytosolic), 20: 51,532,469-51,546,551 class B member hsp90ab1-001 1 [Source:ZFIN;Acc:ZDB-GENE-990415-95] forward strand. ENSDART00000020084 ENSDARP00000014978 725 zebrafish heat shock protein HSP 90-beta-like Chromosome: 16; NC_018905.1 (7612387..7617109, heat shock protein XM_003971742.1 complement) HSP 90-beta-likeXP_003971791.1 723 fugu heat shock protein 90kDa beta Chromosome (Grp94),12: member 104,323,885-104,347,423 1 [Source:HGNC HSP90B1-001 Symbol;Acc:12028] forwardENST00000299767 strand. ENSP00000299767 803 human heat shock protein 90, betaChromosome (Grp94), member 10: 86,690,209-86,705,509 1 [Source:MGIHsp90b1-001 Symbol;Acc:MGI:98817] reverse strand. ENSMUST00000020238 ENSMUSP00000020238 802 mouse heat shock protein 90kDa beta UltraContig (Grp94), Ultra443: member 1 [Source:HGNC 3,217,815-3,257,138 HSP90B1-201 Symbol;Acc:12028] forward ENSOANT00000008832 strand. ENSOANP00000008830 739 platypus heat shock protein 90kDa beta Chromosome (Grp94),1:member 54,869,423-54,879,269 1 (HSP90B1), HSP90B1-201 mRNA. reverse [Source:RefSeq strand. ENSGALT00000020773 mRNA;Acc:NM_204289] ENSGALP00000020744 795 chicken heat shock protein 90kDa beta NW_005871013.1 (Grp94), member (4771495..4791028, 1 heatcomplement) shock protein XM_006138017.1 90kDa beta (Grp94), XP_006138079.1 member 1 728 turtle heat shock protein 90kDa beta Chromosome (Grp94),5:member 18,171,869-18,181,959 1 [Source:HGNC HSP90B1-201 Symbol;Acc:12028] forward strand. ENSACAT00000016310 ENSACAP00000015990 795 lizard heat shock protein 90kDa beta (Grp94), member 1 heat shock protein NM_001090811.1 90kDa beta (Grp94), NP_001084280.1 member 1 745 xenopus heat shock protein 90, betascaffold_2: (grp94), member 3,905,705-3,910,334 1 [Source:ZFIN;Acc:ZDB-GENE-031002-1] hsp90b1-201 forward strand. ENSTRUT00000043644 ENSTRUP00000043499 803 fugu heat shock protein 90, betaScaffold (grp94),GL831160.1: member 1 [Source:ZFIN;Acc:ZDB-GENE-031002-1] 1,996,934-2,003,129 hsp90b1-201reverse ENSONIT00000010731 strand. ENSONIP00000010722 797 tilapia heat shock protein 90, betagroupXIX: (grp94), 11,751,314-11,756,060 member 1 [Source:ZFIN;Acc:ZDB-GENE-031002-1] hsp90b1-201 reverse strand. ENSGACT00000013325 ENSGACP00000013300 771 stickleback heat shock protein 90, betaChromosome (grp94), member 6: 8,851,521-8,856,114 1 [Source:ZFIN;Acc:ZDB-GENE-031002-1] hsp90b1-201 forward strand. ENSORLT00000005468 ENSORLP00000005467 759 medaka heat shock protein 90, betaChromosome (grp94), member 4: 8,898,701-8,906,072 1 [Source:ZFIN;Acc:ZDB-GENE-031002-1] hsp90b1-001 forward strand. ENSDART00000004879 ENSDARP00000013441 793 zebrafish TNF receptor-associated protein Chromosome 1 [Source:HGNC 16: 3,701,640-3,767,598 Symbol;Acc:16264] TRAP1-001 reverse strand. ENST00000246957 ENSP00000246957 704 human TNF receptor-associated protein Chromosome 1 [Source:MGI 16: 4,039,971-4,077,827 Symbol;Acc:MGI:1915265] Trap1-001 reverse strand. ENSMUST00000006137 ENSMUSP00000006137 706 mouse TNF receptor-associated protein Chromosome 1 (TRAP1), 14: 12,473,314-12,491,461 nuclear gene encoding TRAP1-201 reverse mitochondrial strand. ENSGALT00000012459 protein, mRNA. ENSGALP00000012445 [Source:RefSeq mRNA;Acc:NM_001006175] 697 chicken TNF receptor-associated protein Scaffold 1 [Source:HGNC JH210909.1: Symbol;Acc:16264] 456,153-502,881 TRAP1-202 reverse strand. ENSPSIT00000006698 ENSPSIP00000006661 707 turtle TNF receptor-associated protein Scaffold 1 [Source:HGNC GL343507.1: Symbol;Acc:16264] 520,668-529,369 TRAP1-201 reverse strand. ENSACAT00000012896 ENSACAP00000012640 674 lizard TNF receptor-associated protein Scaffold 1 [Source:HGNC GL172663.1: Symbol;Acc:16264] 1,682,329-1,691,724 TRAP1-201 reverse ENSXETT00000066219 strand. ENSXETP00000058756 659 xenopus TNF receptor-associated protein scaffold_115: 1 [Source:ZFIN;Acc:ZDB-GENE-030131-4257] 83,690-87,162 reverse trap1-201 strand. ENSTRUT00000010513 ENSTRUP00000010455 655 fugu TNF receptor-associated protein Scaffold 1 [Source:ZFIN;Acc:ZDB-GENE-030131-4257] GL831217.1: 675,285-688,542 trap1-201 reverse strand. ENSONIT00000006506 ENSONIP00000006501 719 tilapia TNF receptor-associated protein groupXI: 1 [Source:ZFIN;Acc:ZDB-GENE-030131-4257] 10,044,151-10,050,273 trap1-201 forward strand.ENSGACT00000015034 ENSGACP00000015006 671 stickleback TNF receptor-associated protein Chromosome 1 [Source:ZFIN;Acc:ZDB-GENE-030131-4257] 8: 11,408,875-11,415,031 trap1-201 forward strand. ENSORLT00000010500 ENSORLP00000010499 684 medaka TNF receptor-associated protein Chromosome 1 [Source:ZFIN;Acc:ZDB-GENE-030131-4257] 3: 9,814,733-9,851,981 trap1-202 forward strand. ENSDART00000124450 ENSDARP00000107323 719 zebrafish

TE D

Gene ID ENSG00000080824 ENSMUSG00000021270 ENSOANG00000005734 423463 102443811 ENSACAG00000000151 549036 ENSTRUG00000012406 ENSONIG00000000819 ENSGACG00000012875 ENSORLG00000017525 ENSDARG00000010478 780222 ENSTRUG00000012449 ENSONIG00000000805 ENSGACG00000012888 ENSORLG00000017533 ENSDARG00000024746 ENSG00000096384 ENSMUSG00000023944 ENSGALG00000010175 ENSACAG00000014791 595047 ENSONIG00000005936 ENSGACG00000013530 ENSORLG00000011892 ENSDARG00000029150 101076976 ENSG00000166598 ENSMUSG00000020048 ENSOANG00000005554 ENSGALG00000012726 102453638 ENSACAG00000016190 399408 ENSTRUG00000016985 ENSONIG00000008526 ENSGACG00000010067 ENSORLG00000004360 ENSDARG00000003570 ENSG00000126602 ENSMUSG00000005981 ENSGALG00000007686 ENSPSIG00000006020 ENSACAG00000012701 ENSXETG00000033239 ENSTRUG00000004400 ENSONIG00000005173 ENSGACG00000011350 ENSORLG00000008358 ENSDARG00000089242

EP

Gene Symbol

HSP90AA1 Hsp90aa1 Hsp90aa1 Hsp90aa1 Hsp90aa1 Hsp90aa1-like Hsp90aa1.1 hsp90aa1.1 hsp90aa1.1 hsp90aa1.1 hsp90aa1.1 hsp90aa1.1 Hsp90aa1.2 hsp90aa1.2 hsp90aa1.2 hsp90aa1.2 hsp90aa1.2 hsp90aa1.2 HSP90AB1 Hsp90ab1 Hsp90ab1 Hsp90ab1 Hsp90ab1 hsp90ab1 hsp90ab1 hsp90ab1 hsp90ab1 hsp90ab1 HSP90B1 Hsp90b1 Hsp90b1 Hsp90b1 Hsp90b1 Hsp90b1 Hsp90b1 hsp90b1 hsp90b1 hsp90b1 hsp90b1 hsp90b1 TRAP1 Trap1 Trap1 Trap1 Trap1 Trap1 trap1 trap1 trap1 trap1 trap1

AC C

No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

Scientific Name

HGNC

Homo sapiens HGNC:5253 Mus musculus Ornithorhynchus anatinus Gallus gallus Pelodiscus sinensis Anolis carolinensis Xenopus (Silurana) tropicalis Takifugu rubripes Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio Xenopus (Silurana) tropicalis Takifugu rubripes Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio Homo sapiens HGNC:5258 Mus musculus Gallus gallus Anolis carolinensis Xenopus (Silurana) tropicalis Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio Takifugu rubripes Homo sapiens HGNC:12028 Mus musculus Ornithorhynchus anatinus Gallus gallus Pelodiscus sinensis Anolis carolinensis Xenopus laevis Takifugu rubripes Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio Homo sapiens HGNC:16264 Mus musculus Gallus gallus Pelodiscus sinensis Anolis carolinensis Xenopus laevis Takifugu rubripes Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

heat shock 60kDa protein 1 Chromosome (chaperonin) 2: [Source:HGNC 198,351,305-198,381,461 Symbol;Acc:5261] HSPD1-001 reverseENST00000388968 strand. ENSP00000373620 573 human Homo sapiens HGNC:5261 heat shock protein 1 (chaperonin) Chromosome [Source:MGI 1: 55,077,835-55,088,243 Symbol;Acc:MGI:96242] Hspd1-001 reverse strand. ENSMUST00000027123 ENSMUSP00000027123 573 mouse Mus musculus heat shock 60kDa protein 1 SuperContig (chaperonin)Contig9065: [Source:HGNC 485-12,776 Symbol;Acc:5261] HSPD1-201 reverse strand. ENSOANT00000016517 ENSOANP00000016514 468 platypus Ornithorhynchus anatinus heat shock 60kDa protein 1 Chromosome (chaperonin) 7: (HSPD1), 9,720,880-9,729,711 nuclear HSPD1-201 genereverse encoding strand. mitochondrial ENSGALT00000013137 protein, ENSGALP00000013122 mRNA. [Source:RefSeq 573 mRNA;Acc:NM_001012916] chicken Gallus gallus heat shock 60kDa protein 1 NW_005853697.1 (chaperonin) (4681774..4700127, heatcomplement) shock 60kDa XM_006117918.1 protein 1 (chaperonin) XP_006117980.1 570 turtle Pelodiscus sinensis heat shock 60kDa protein 1 Scaffold (chaperonin) GL343436.1: [Source:HGNC 690,520-708,494 Symbol;Acc:5261] HSPD1-201 forward strand. ENSACAT00000009572 ENSACAP00000009377 677 lizard Anolis carolinensis heat shock 60kDa protein 1 (chaperonin) heat shock 60kDa NM_001090501.1 protein 1 (chaperonin) NP_001083970.1 579 xenopus Xenopus laevis 60 kDa heat shock protein, Chromosome: mitochondrial-like 1; NC_018890.1 (10583674..10587326) 60 kDa heat shock XM_003961632.1 protein, mitochondrial-like XP_003961681.1 575 fugu Takifugu rubripes heat shock 60kD protein 1 (chaperonin) Scaffold GL831337.1: [Source:ZFIN;Acc:ZDB-GENE-021206-1] 573,991-582,082 hspd1-201 reverse strand. ENSONIT00000017327 ENSONIP00000017312 575 tilapia Oreochromis niloticus heat shock 60kD protein 1 (chaperonin) groupXVI: 17,515,531-17,520,899 [Source:ZFIN;Acc:ZDB-GENE-021206-1] hspd1-202 reverse strand. ENSGACT00000011894 ENSGACP00000011870 582 stickleback Gasterosteus aculeatus heat shock 60kD protein 1 (chaperonin) ultracontig244: [Source:ZFIN;Acc:ZDB-GENE-021206-1] 4,508-9,291 reverse hspd1-202 strand. ENSORLT00000025854 ENSORLP00000025853 575 medaka Oryzias latipes heat shock 60kD protein 1 (chaperonin) Chromosome 9:[Source:ZFIN;Acc:ZDB-GENE-021206-1] 33,319,913-33,334,091 hspd1-001 reverse strand. ENSDART00000078596 ENSDARP00000073057 575 zebrafish Danio rerio heat shock 10kDa protein 1 Chromosome [Source:HGNC 2: Symbol;Acc:5269] 198,364,718-198,368,181 HSPE1-001 forwardENST00000233893 strand. ENSP00000233893 102 human Homo sapiens HGNC:5269 heat shock protein 1 (chaperonin Chromosome 10) [Source:MGI 1: 55,088,132-55,091,307 Symbol;Acc:MGI:104680] Hspe1-001 forward strand. ENSMUST00000075242 ENSMUSP00000074724 102 mouse Mus musculus heat shock 10kDa protein 1 SuperContig [Source:HGNCContig9065: Symbol;Acc:5269] 19,008-25,187 HSPE1-201 forwardENSOANT00000016519 strand. ENSOANP00000016516 102 platypus Ornithorhynchus anatinus heat shock 10kDa protein 1 Chromosome (chaperonin7:10) 9,732,777-9,734,998 (HSPE1), nuclear HSPE1-201 gene forward encoding strand. ENSGALT00000014762 mitochondrial protein, ENSGALP00000014746 mRNA. [Source:RefSeq 111 mRNA;Acc:NM_205067] chicken Gallus gallus heat shock 10kDa protein 1 Scaffold [Source:HGNC JH207332.1: Symbol;Acc:5269] 4,704,339-4,707,984 HSPE1-201 forward ENSPSIT00000020390 strand. ENSPSIP00000020294 102 turtle Pelodiscus sinensis heat shock 10kDa protein 1 Scaffold [Source:HGNC GL343436.1: Symbol;Acc:5269] 676,964-685,290 HSPE1-201 reverse strand. ENSACAT00000009479 ENSACAP00000009286 102 lizard Anolis carolinensis heat shock 10kDa protein 1 Scaffold (chaperonin GL172638.1: 10) [Source:Jamboree;Acc:XB-GENE-963412] 923,093-929,868 hspe1-201 forward strand. ENSXETT00000016357 ENSXETP00000016357 102 xenopus Xenopus laevis heat shock 10 protein 1 (chaperonin scaffold_31: 10) 1,504,416-1,506,338 [Source:ZFIN;Acc:ZDB-GENE-000906-2] hspe1-202 forward strand. ENSTRUT00000040106 ENSTRUP00000039965 102 fugu Takifugu rubripes heat shock 10 protein 1 (chaperonin Scaffold GL831337.1: 10) [Source:ZFIN;Acc:ZDB-GENE-000906-2] 584,659-586,100 hspe1-201 forward strand. ENSONIT00000017335 ENSONIP00000017320 99 tilapia Oreochromis niloticus heat shock 10 protein 1 (chaperonin groupXVI: 10) 17,523,127-17,523,660 [Source:ZFIN;Acc:ZDB-GENE-000906-2] hspe1-201 forward strand. ENSGACT00000011913 ENSGACP00000011889 99 stickleback Gasterosteus aculeatus heat shock protein 10 (hspe1), ultracontig244: nuclear gene11,102-12,333 encoding mitochondrial forward hspe1-201 strand. protein, ENSORLT00000025856 mRNA. [Source:RefSeq ENSORLP00000025855 mRNA;Acc:NM_001104762] 100 medaka Oryzias latipes heat shock 10 protein 1 (chaperonin Chromosome10) 9: [Source:ZFIN;Acc:ZDB-GENE-000906-2] 33,334,325-33,338,315 hspe1-001 forward strand. ENSDART00000143103 ENSDARP00000118521 100 zebrafish Danio rerio heat shock 27kDa protein 1 Chromosome [Source:HGNC 7: Symbol;Acc:5246] 75,931,861-75,933,612 HSPB1-001 forward strand. ENST00000248553 ENSP00000248553 205 human Homo sapiens HGNC:5246 heat shock protein 1 [Source:MGI Chromosome Symbol;Acc:MGI:96240] 5: 135,887,919-135,889,563 Hspb1-001 forwardENSMUST00000005077 strand. ENSMUSP00000005077 209 mouse Mus musculus heat shock 27kDa protein 1 Chromosome [Source:HGNC 21:Symbol;Acc:5246] 4,620,805-4,622,737 HSPB1-201 reverse strand. ENSMGAT00000004104 ENSMGAP00000003402 195 turkey Meleagris gallopavo heat shock 27kDa protein 1 Scaffold [Source:HGNC JH207163.1: Symbol;Acc:5246] 1,133,762-1,139,013 HSPB1-201 forward ENSPSIT00000010890 strand. ENSPSIP00000010835 216 turtle Pelodiscus sinensis heat shock 27kDa protein 1 Scaffold [Source:Jamboree;Acc:XB-GENE-480320] GL172708.1: 387,572-393,961 hspb1-201 reverse strand. ENSXETT00000019657 ENSXETP00000019657 212 xenopus Xenopus laevis heat shock protein, alpha-crystallin-related, scaffold_29: 162,409-165,242 1 [Source:ZFIN;Acc:ZDB-GENE-030326-4] forward hspb1-201 strand. ENSTRUT00000010704 ENSTRUP00000010646 202 fugu Takifugu rubripes heat shock protein, alpha-crystallin-related, groupVII: 14,587,963-14,593,757 1 [Source:ZFIN;Acc:ZDB-GENE-030326-4] hspb1-203 forward strand. ENSGACT00000026786 ENSGACP00000026734 217 stickleback Gasterosteus aculeatus heat shock protein, alpha-crystallin-related, scaffold1485: 5,245-9,176 1 [Source:ZFIN;Acc:ZDB-GENE-030326-4] forward hspb1-201 strand. ENSORLT00000025315 ENSORLP00000025314 199 medaka Oryzias latipes heat shock protein, alpha-crystallin-related, Chromosome 5: 3,584,621-3,601,420 1 [Source:ZFIN;Acc:ZDB-GENE-030326-4] hspb1-001 reverse strand. ENSDART00000060162 ENSDARP00000060161 199 zebrafish Danio rerio heat shock 27kDa protein 2 Chromosome (HSPB2), mRNA. 11: 111,782,966-111,789,574 [Source:RefSeq HSPB2-001 mRNA;Acc:NM_001541] forwardENST00000304298 strand. ENSP00000302476 182 human Homo sapiens heat shock protein 2 [Source:MGI Chromosome Symbol;Acc:MGI:1916503] 9: 50,751,072-50,752,354 Hspb2-201 reverse strand. ENSMUST00000042790 ENSMUSP00000042374 182 mouse Mus musculus heat shock 27kDa protein 2 Scaffold [Source:Jamboree;Acc:XB-GENE-940436] GL172675.1: 3,602,391-3,606,078 hspb2-201 forward ENSXETT00000022445 strand. ENSXETP00000022445 174 xenopus Xenopus laevis heat shock protein, alpha-crystallin-related, Chromosome 5: 58,979,568-58,990,471 b2 [Source:ZFIN;Acc:ZDB-GENE-050417-260] hspb2-001 reverse strand. ENSDART00000074306 ENSDARP00000068795 169 zebrafish Danio rerio heat shock 27kDa protein 3 Chromosome [Source:HGNC 5: Symbol;Acc:5248] 53,751,445-53,752,207 HSPB3-001 forward strand. ENST00000302005 ENSP00000303394 150 human Homo sapiens HGNC:5248 heat shock protein 3 [Source:MGI Chromosome Symbol;Acc:MGI:1928479] 13: 113,662,896-113,663,676 Hspb3-201reverseENSMUST00000054650 strand. ENSMUSP00000054193 154 mouse Mus musculus heat shock 27kDa protein 3 SuperContig [Source:HGNCContig4470: Symbol;Acc:5248] 27,522-27,962 HSPB3-201 reverseENSOANT00000003207 strand. ENSOANP00000003206 147 platypus Ornithorhynchus anatinus heat shock 27kDa protein 3 Scaffold [Source:HGNC JH224682.1: Symbol;Acc:5248] 8,432,803-8,433,246 HSPB3-201 forward ENSPSIT00000001656 strand. ENSPSIP00000001651 147 turtle Pelodiscus sinensis heat shock 27kDa protein 3 Chromosome [Source:HGNC 2: Symbol;Acc:5248] 2,909,694-2,910,128 HSPB3-201 reverse strand. ENSACAT00000015552 ENSACAP00000015243 144 lizard Anolis carolinensis heat shock 27kDa protein 3 Scaffold [Source:Jamboree;Acc:XB-GENE-969539] GL173336.1: 27,316-27,753 hspb3-201 reverse strand. ENSXETT00000050520 ENSXETP00000050520 146 xenopus Xenopus laevis heat shock protein, alpha-crystallin-related, Chromosome 5: 41,921,681-41,922,133 b3 [Source:ZFIN;Acc:ZDB-GENE-070705-338] hspb3-001 reverse strand. ENSDART00000097526 ENSDARP00000088297 150 zebrafish Danio rerio crystallin, alpha A [Source:HGNC Chromosome Symbol;Acc:2388] 21: 44,589,118-44,592,915 CRYAA-001 forward strand. ENST00000291554 ENSP00000291554 173 human Homo sapiens HGNC:2388 crystallin, alpha A [Source:MGI Chromosome Symbol;Acc:MGI:88515] 17: 31,677,933-31,681,722 Cryaa-201 forward strand. ENSMUST00000019192 ENSMUSP00000019192 196 mouse Mus musculus crystallin, alpha A [Source:HGNC Chromosome Symbol;Acc:2388] 1: 115,580,404-115,582,975 CRYAA-201 forwardENSMGAT00000016408 strand. ENSMGAP00000015452 131 turkey Meleagris gallopavo crystallin, alpha A [Source:HGNC Scaffold Symbol;Acc:2388] JH224647.1: 6,331,531-6,335,684 CRYAA-201 forward ENSPSIT00000007970 strand. ENSPSIP00000007929 173 turtle Pelodiscus sinensis crystallin, alpha A [Source:HGNC Chromosome Symbol;Acc:2388] 3: 137,146,449-137,151,667 CRYAA-201 reverseENSACAT00000017909 strand. ENSACAP00000017563 172 lizard Anolis carolinensis crystallin, alpha A [Source:Jamboree;Acc:XB-GENE-5940571] Scaffold GL177754.1: 206-2,610 cryaa-201 reverse strand.ENSXETT00000044922 ENSXETP00000044922 174 xenopus Xenopus laevis crystallin, alpha A [Source:ZFIN;Acc:ZDB-GENE-020508-1] scaffold_151: 21,420-22,810 reverse cryaa-201 strand. ENSTRUT00000006713 ENSTRUP00000006669 176 fugu Takifugu rubripes crystallin, alpha A [Source:ZFIN;Acc:ZDB-GENE-020508-1] Scaffold GL831293.1: 1,212,404-1,213,817 cryaa-201 reverse ENSONIT00000004731 strand. ENSONIP00000004728 176 tilapia Oreochromis niloticus crystallin, alpha A [Source:ZFIN;Acc:ZDB-GENE-020508-1] groupI: 27,017,070-27,018,422 reverse cryaa-201 strand.ENSGACT00000020268 ENSGACP00000020229 176 stickleback Gasterosteus aculeatus crystallin, alpha A [Source:ZFIN;Acc:ZDB-GENE-020508-1] ultracontig26: 68,806-71,329 reverse cryaa-201 strand. ENSORLT00000025614 ENSORLP00000025613 176 medaka Oryzias latipes crystallin, alpha A [Source:ZFIN;Acc:ZDB-GENE-020508-1] Chromosome 1: 28,027,491-28,031,533 cryaa-001 forward strand. ENSDART00000075539 ENSDARP00000070021 173 zebrafish Danio rerio

TE D

ENSG00000144381 ENSMUSG00000025980 ENSOANG00000010414 ENSGALG00000008094 102449187 ENSACAG00000009505 399217 101078140 ENSONIG00000013762 ENSGACG00000008860 ENSORLG00000020874 ENSDARG00000056160 ENSG00000115541 ENSMUSG00000073676 ENSOANG00000010415 ENSGALG00000009070 ENSPSIG00000017974 ENSACAG00000009482 ENSXETG00000007510 ENSTRUG00000015632 ENSONIG00000013770 ENSGACG00000008995 ENSORLG00000020879 ENSDARG00000056167 ENSG00000106211 ENSMUSG00000004951 ENSMGAG00000003680 ENSPSIG00000009825 ENSXETG00000008972 ENSTRUG00000004477 ENSGACG00000020233 ENSORLG00000020428 ENSDARG00000041065 ENSG00000170276 ENSMUSG00000038086 ENSXETG00000010175 ENSDARG00000052450 ENSG00000169271 ENSMUSG00000051456 ENSOANG00000002023 ENSPSIG00000001656 ENSACAG00000015513 ENSXETG00000023377 ENSDARG00000067714 ENSG00000160202 ENSMUSG00000024041 ENSMGAG00000014588 ENSPSIG00000007237 ENSACAG00000017837 ENSXETG00000020802 ENSTRUG00000002869 ENSONIG00000003758 ENSGACG00000015336 ENSORLG00000020678 ENSDARG00000053502

EP

HSPD1 Hspd1 Hspd1 Hspd1 Hspd1 Hspd1 Hspd1 hspd1 hspd1 hspd1 hspd1 hspd1 HSPE1 Hspe1 Hspe1 Hspe1 Hspe1 Hspe1 Hspe1 hspe1 hspe1 hspe1 hspe1 hspe1 HSPB1 Hspb1 Hspb1 Hspb1 Hspb1 hspb1 hspb1 hspb1 hspb1 HSPB2 Hspb2 Hspb2 hspb2 HSPB3 Hspb3 Hspb3 Hspb3 Hspb3 Hspb3 hspb3 CRYAA Cryaa Cryaa Cryaa Cryaa Cryaa cryaa cryaa cryaa cryaa cryaa

AC C

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

crystallin, alpha B [Source:HGNC Chromosome Symbol;Acc:2389] 11: 111,779,289-111,794,446 CRYAB-003reverseENST00000227251 strand. ENSP00000227251 crystallin, alpha B [Source:MGI Chromosome Symbol;Acc:MGI:88516] 9: 50,752,758-50,756,633 Cryab-201 forward strand. ENSMUST00000034562 ENSMUSP00000034562 crystallin, alpha B NW_001749496.1 (3231..5793, complement) alpha-crystallin NM_001242752.1 B chain NP_001229681.1 crystallin, alpha B [Source:HGNC Chromosome Symbol;Acc:2389] 26: 6,649,582-6,653,066 CRYAB-201 forward strand. ENSMGAT00000005255 ENSMGAP00000004537 crystallin, alpha B [Source:HGNC Scaffold Symbol;Acc:2389] JH205382.1: 1,033,776-1,043,832 CRYAB-201 reverse ENSPSIT00000017923 strand. ENSPSIP00000017841 crystallin, alpha B [Source:HGNC Scaffold Symbol;Acc:2389] GL343521.1: 32,522-33,000 CRYAB-201 forward strand. ENSACAT00000006481 ENSACAP00000006339 crystallin, alpha B [Source:Jamboree;Acc:XB-GENE-971379] Scaffold GL172675.1: 3,591,942-3,598,480 cryab-201 reverse ENSXETT00000022441 strand. ENSXETP00000022441 crystallin, alpha B, a [Source:ZFIN;Acc:ZDB-GENE-991119-2] scaffold_6: 3,188,735-3,189,997cryaba-201 forward strand. ENSTRUT00000043839 ENSTRUP00000043693 crystallin, alpha B, a [Source:ZFIN;Acc:ZDB-GENE-991119-2] Scaffold GL831183.1: 2,041,477-2,045,451 cryaba-201 reverse ENSONIT00000006226 strand. ENSONIP00000006222 crystallin, alpha B, a [Source:ZFIN;Acc:ZDB-GENE-991119-2] groupVII: 21,680,381-21,681,616cryaba-201 reverse strand. ENSGACT00000027357 ENSGACP00000027305 crystallin, alpha B, a [Source:ZFIN;Acc:ZDB-GENE-991119-2] Chromosome 14: 13,383,070-13,383,970 cryaba-201 reverse strand. ENSORLT00000007410 ENSORLP00000007409 crystallin, alpha B, a [Source:ZFIN;Acc:ZDB-GENE-991119-2] Chromosome 15: 17,063,601-17,068,283 cryaba-001 reverse strand. ENSDART00000062523 ENSDARP00000062522 crystallin, alpha B, b [Source:ZFIN;Acc:ZDB-GENE-040718-419] Chromosome 5: 58,990,767-59,000,186 cryabb-003 forward strand. ENSDART00000148665 ENSDARP00000124531 heat shock protein, alpha-crystallin-related, Chromosome 19: 36,245,469-36,248,980 B6 [Source:HGNC HSPB6-201 Symbol;Acc:26511] reverse strand. ENST00000004982 ENSP00000004982 heat shock protein, alpha-crystallin-related, Chromosome 7: 30,552,178-30,555,443 B6 [Source:MGI Hspb6-001 Symbol;Acc:MGI:2685325] forward strand. ENSMUST00000044048 ENSMUSP00000039172 heat shock protein, alpha-crystallin-related, Scaffold JH207379.1:B680,010-85,651 [Source:HGNC HSPB6-201 forward Symbol;Acc:26511] strand. ENSPSIT00000009034 ENSPSIP00000008988 heat shock protein, alpha-crystallin-related, Scaffold GL343276.1:B61,462,581-1,471,512 [Source:HGNC HSPB6-201 Symbol;Acc:26511] reverse ENSACAT00000016703 strand. ENSACAP00000016379 heat shock protein, alpha-crystallin-related, Scaffold GL173286.1:B6493,653-495,668 [Source:Jamboree;Acc:XB-GENE-876273] hspb6-201 reverse strand. ENSXETT00000038123 ENSXETP00000038123 heat shock protein, alpha-crystallin-related, Scaffold GL831286.1:b6172,505-174,914 [Source:ZFIN;Acc:ZDB-GENE-080214-7] hspb6-202 reverse strand. ENSONIT00000003448 ENSONIP00000003447 heat shock protein, alpha-crystallin-related, Chromosome 15: 36,257,981-36,260,993 b6 [Source:ZFIN;Acc:ZDB-GENE-080214-7] hspb6-001 forward strand. ENSDART00000085522 ENSDARP00000079957 heat shock 27kDa protein family, Chromosome member 1: 716,340,523-16,346,089 (cardiovascular) HSPB7-001 [Source:HGNC reverse strand. ENST00000375718 Symbol;Acc:5249] ENSP00000364870 heat shock protein family, Chromosome member 7 (cardiovascular) 4: 141,420,779-141,425,311 [Source:MGI Hspb7-001 forward Symbol;Acc:MGI:1352494] ENSMUST00000102486 strand. ENSMUSP00000099544 heat shock 27kDa protein family, Chromosome member 23:7 4,525,573-4,527,283 (cardiovascular) HSPB7-201 [Source:HGNC forward strand. ENSMGAT00000005732 Symbol;Acc:5249] ENSMGAP00000005006 heat shock 27kDa protein family, Scaffold member JH204940.1: 7 (cardiovascular) 343,112-351,742 HSPB7-201 [Source:HGNC reverse strand. ENSPSIT00000013121 Symbol;Acc:5249] ENSPSIP00000013058 heat shock 27kDa protein family, Scaffold member GL343609.1: 7 (cardiovascular) 348,103-351,257 HSPB7-201 [Source:HGNC reverse strand. ENSACAT00000026032 Symbol;Acc:5249] ENSACAP00000019633 heat shock 27kDa protein family, member 7 (cardiovascular) cardiovascular NM_001093089.1 heat shock protein NP_001086558.1 heat shock protein family, scaffold_147: alpha-crystallin-related, 276,085-278,423 b7 [Source:ZFIN;Acc:ZDB-GENE-041010-136] forward hspb7-201 strand.ENSTRUT00000007393 ENSTRUP00000007348 heat shock protein family, groupXII: alpha-crystallin-related, 5,795,327-5,797,360 b7 [Source:ZFIN;Acc:ZDB-GENE-041010-136] reverse hspb7-201 strand.ENSGACT00000007019 ENSGACP00000007001 heat shock protein family, Chromosome alpha-crystallin-related, 23: 24,543,282-24,550,320 b7 [Source:ZFIN;Acc:ZDB-GENE-041010-136] hspb7-201 reverse strand. ENSDART00000023984 ENSDARP00000006705 heat shock 22kDa protein 8 Chromosome [Source:HGNC 12:Symbol;Acc:30171] 119,616,447-119,658,936 HSPB8-001forwardENST00000281938 strand. ENSP00000281938 heat shock protein 8 [Source:MGI Chromosome Symbol;Acc:MGI:2135756] 5: 116,408,491-116,422,864 Hspb8-001 reverseENSMUST00000036991 strand. ENSMUSP00000037007 heat shock 22kDa protein 8 Chromosome [Source:HGNC 17:Symbol;Acc:30171] 10,279,516-10,281,446 HSPB8-201 reverse strand. ENSMGAT00000010170 ENSMGAP00000009350 heat shock 22kDa protein 8 Scaffold [Source:HGNC JH211961.1: Symbol;Acc:30171] 156,688-171,419 HSPB8-201 reverse strand. ENSPSIT00000015034 ENSPSIP00000014963 heat shock 22kDa protein 8 Scaffold [Source:HGNC GL343338.1: Symbol;Acc:30171] 513,698-527,697 HSPB8-201 reverse strand. ENSACAT00000004096 ENSACAP00000004004 heat shock 22kDa protein 8 Scaffold [Source:Jamboree;Acc:XB-GENE-945643] GL172976.1: 1,009,006-1,033,442 hspb8-201 forward ENSXETT00000054022 strand. ENSXETP00000054022 heat shock protein, alpha-crystallin-related, scaffold_4: 2,328,783-2,333,736 b8 [Source:ZFIN;Acc:ZDB-GENE-030131-2480] hspb8-201 reverse strand. ENSTRUT00000041582 ENSTRUP00000041438 heat shock protein, alpha-crystallin-related, Scaffold GL831281.1:b8276,193-288,229 [Source:ZFIN;Acc:ZDB-GENE-030131-2480] hspb8-201 reverse strand. ENSONIT00000013274 ENSONIP00000013264 heat shock protein, alpha-crystallin-related, groupXIII: 18,434,585-18,440,058 b8 [Source:ZFIN;Acc:ZDB-GENE-030131-2480] hspb8-201 forward strand. ENSGACT00000018865 ENSGACP00000018827 heat shock protein, alpha-crystallin-related, Chromosome 9: 2,753,704-2,762,119 b8 [Source:ZFIN;Acc:ZDB-GENE-030131-2480] hspb8-201 forward strand. ENSORLT00000001458 ENSORLP00000001457 heat shock protein, alpha-crystallin-related, Chromosome 5: 17,593,474-17,614,308 b8 [Source:ZFIN;Acc:ZDB-GENE-030131-2480] hspb8-001 forward strand. ENSDART00000134206 ENSDARP00000113545 heat shock protein, alpha-crystallin-related, Chromosome 17: 40,274,756-40,275,371 B9 [Source:HGNC HSPB9-001 Symbol;Acc:30589] forward strand. ENST00000355067 ENSP00000347178 heat shock protein, alpha-crystallin-related, Chromosome 11: 100,713,850-100,714,575 B9 [Source:MGI Hspb9-201 Symbol;Acc:MGI:1922732] forwardENSMUST00000169833 strand. ENSMUSP00000130551 heat shock protein, alpha-crystallin-related, Scaffold GL831179.1:9 1,302,379-1,307,731 [Source:ZFIN;Acc:ZDB-GENE-080214-6] hspb9-202 reverse ENSONIT00000007960 strand. ENSONIP00000007955 heat shock protein, alpha-crystallin-related, Chromosome 3: 33,437,532-33,438,196 9 [Source:ZFIN;Acc:ZDB-GENE-080214-6] hspb9-201 forward strand. ENSDART00000114023 ENSDARP00000103607 outer dense fiber of sperm Chromosome tails 1 [Source:HGNC 8: 103,563,800-103,573,245 Symbol;Acc:8113] ODF1-001forwardENST00000285402 strand. ENSP00000285402 outer dense fiber of sperm Chromosome tails 1 [Source:MGI 15: 38,219,203-38,226,735 Symbol;Acc:MGI:97424] Odf1-001 forward strand. ENSMUST00000081966 ENSMUSP00000080632 outer dense fiber protein 1-like NW_001611530.1 (17361..17869, complement) outer dense fiber XM_001516398.2 protein 1-like XP_001516448.1 outer dense fiber of sperm Chromosome tails 1 [Source:HGNC 3: 79,986,286-79,987,616 Symbol;Acc:8113] ODF1-201 forward strand. ENSMGAT00000013866 ENSMGAP00000012965 outer dense fiber of sperm Scaffold tails 1 [Source:HGNC JH208674.1: 1,940,955-1,941,269 Symbol;Acc:8113] ODF1-201 reverse ENSPSIT00000000031 strand. ENSPSIP00000000031 outer dense fiber of sperm Chromosome tails 1 [Source:HGNC 4: 13,907,726-13,917,938 Symbol;Acc:8113] ODF1-201 reverse strand. ENSACAT00000009157 ENSACAP00000008965 heat shock protein beta-11-like Chromosome: 15; NC_018904.1 (3919935..3920567, heat shock protein XM_003970887.1 complement) beta-11-like XP_003970936.1 heat shock protein, alpha-crystallin-related, Scaffold GL831363.1:b11 1,020,855-1,021,508 [Source:ZFIN;Acc:ZDB-GENE-030131-5148] hspb11-201 reverse ENSONIT00000026544 strand. ENSONIP00000026520 heat shock protein, alpha-crystallin-related, Chromosome 21: 19,211,712-19,212,753 b11 [Source:ZFIN;Acc:ZDB-GENE-030131-5148] hspb11-001 forward strand. ENSDART00000026430 ENSDARP00000026207 heat shock protein, alpha-crystallin-related, scaffold_123: 814,130-815,183 b12 [Source:ZFIN;Acc:ZDB-GENE-030131-2741] forward hspb12-201 strand.ENSTRUT00000031237 ENSTRUP00000031119 heat shock protein, alpha-crystallin-related, Scaffold GL831338.1:b12 140,160-146,780 [Source:ZFIN;Acc:ZDB-GENE-030131-2741] hspb12-201 forward strand. ENSONIT00000013589 ENSONIP00000013579

EP

CRYAB ENSG00000109846 Cryab ENSMUSG00000032060 Cryab 791109 Cryab ENSMGAG00000004705 Cryab ENSPSIG00000015818 Cryab ENSACAG00000006473 Cryab ENSXETG00000010174 cryaba ENSTRUG00000017054 cryaba ENSONIG00000004946 cryaba ENSGACG00000020643 cryaba ENSORLG00000005891 cryaba ENSDARG00000042621 cryabb ENSDARG00000052447 HSPB6 ENSG00000004776 Hspb6 ENSMUSG00000036854 Hspb6 ENSPSIG00000008165 Hspb6 ENSACAG00000016645 Hspb6 ENSXETG00000017563 hspb6 ENSONIG00000002750 hspb6 ENSDARG00000077236 HSPB7 ENSG00000173641 Hspb7 ENSMUSG00000006221 Hspb7 ENSMGAG00000005124 Hspb7 ENSPSIG00000011763 Hspb7 ENSACAG00000002673 Hspb7 446393 hspb7 ENSTRUG00000003137 hspb7 ENSGACG00000005290 hspb7 ENSDARG00000011538 HSPB8 ENSG00000152137 Hspb8 ENSMUSG00000041548 Hspb8 ENSMGAG00000009076 Hspb8 ENSPSIG00000013376 Hspb8 ENSACAG00000004110 Hspb8 ENSXETG00000025255 hspb8 ENSTRUG00000016209 hspb8 ENSONIG00000010551 hspb8 ENSGACG00000014260 hspb8 ENSORLG00000001186 hspb8 ENSDARG00000058365 HSPB9 ENSG00000197723 Hspb9 ENSMUSG00000017832 hspb9 ENSONIG00000006308 hspb9 ENSDARG00000078674 HSPB10/ODF1 ENSG00000155087 Hspb10/Odf1 ENSMUSG00000061923 Hspb10/Odf1-like 100086304 Hspb10/Odf1 ENSMGAG00000012316 Hspb10/Odf1 ENSPSIG00000000031 Hspb10/Odf1 ENSACAG00000009176 hspb11 101064707 hspb11 ENSONIG00000021218 hspb11 ENSDARG00000002204 hspb12 ENSTRUG00000012285 hspb12 ENSONIG00000010800

AC C

107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161

175 175 182 174 195 111 174 167 142 164 167 168 180 160 162 171 173 110 148 142 245 169 168 162 139 162 165 156 161 196 196 196 203 205 202 220 206 224 221 216 159 199 223 204 250 248 107 115 105 205 210 217 205 198 199

human mouse platypus turkey turtle lizard xenopus fugu tilapia stickleback medaka zebrafish zebrafish human mouse turtle lizard xenopus tilapia zebrafish human mouse turkey turtle lizard xenopus fugu stickleback zebrafish human mouse turkey turtle lizard xenopus fugu tilapia stickleback medaka zebrafish human mouse tilapia zebrafish human mouse platypus turkey turtle lizard fugu tilapia zebrafish fugu tilapia

Homo sapiens HGNC:2389 Mus musculus Ornithorhynchus anatinus Meleagris gallopavo Pelodiscus sinensis Anolis carolinensis Xenopus laevis Takifugu rubripes Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio Danio rerio Homo sapiens HGNC:26511 Mus musculus Pelodiscus sinensis Anolis carolinensis Xenopus laevis Oreochromis niloticus Danio rerio Homo sapiens HGNC:5249 Mus musculus Meleagris gallopavo Pelodiscus sinensis Anolis carolinensis Xenopus laevis Takifugu rubripes Gasterosteus aculeatus Danio rerio Homo sapiens HGNC:30171 Mus musculus Meleagris gallopavo Pelodiscus sinensis Anolis carolinensis Xenopus laevis Takifugu rubripes Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio Homo sapiens HGNC:30589 Mus musculus Oreochromis niloticus Danio rerio Homo sapiens HGNC:8113 Mus musculus Ornithorhynchus anatinus Meleagris gallopavo Pelodiscus sinensis Anolis carolinensis Takifugu rubripes Oreochromis niloticus Danio rerio Takifugu rubripes Oreochromis niloticus

ACCEPTED MANUSCRIPT

alpha-crystallin-related, Scaffold Zv9_NA428: b12 3,864-16,021 [Source:ZFIN;Acc:ZDB-GENE-030131-2741] hspb12-201 forward strand. ENSDART00000128824 alpha-crystallin-related, Scaffold GL831275.1:b15 1,057,316-1,061,428 [Source:ZFIN;Acc:ZDB-GENE-080214-5] hspb15-201 forward ENSONIT00000017555 strand. alpha-crystallin-related, groupXIV: 6,508,337-6,510,005 b15 [Source:ZFIN;Acc:ZDB-GENE-080214-5] reverse hspb15-201 strand.ENSGACT00000022430 alpha-crystallin-related, Chromosome 12: 13,237,928-13,238,990 b15 [Source:ZFIN;Acc:ZDB-GENE-080214-5] hspb15-201 reverse strand. ENSORLT00000010252 alpha-crystallin-related, Chromosome 5: 33,958,926-33,960,751 b15 [Source:ZFIN;Acc:ZDB-GENE-080214-5] hspb15-201 reverse strand. ENSDART00000110804

ENSDARP00000111674 ENSONIP00000017540 ENSGACP00000022388 ENSORLP00000010251 ENSDARP00000104334

RI PT

protein, protein, protein, protein, protein,

SC

shock shock shock shock shock

M AN U

heat heat heat heat heat

TE D

ENSDARG00000087665 ENSONIG00000013955 ENSGACG00000016949 ENSORLG00000008163 ENSDARG00000078411

EP

hspb12 hspb15 hspb15 hspb15 hspb15

AC C

162 163 164 165 166

205 202 206 198 154

zebrafish tilapia stickleback medaka zebrafish

Danio rerio Oreochromis niloticus Gasterosteus aculeatus Oryzias latipes Danio rerio

ACCEPTED MANUSCRIPT

RI PT

8 6

SC

2

M AN U

0

EP

TE D

-2

AC C

Fold Change

4

Columnaris_4h Columnaris_24h Columnaris_48h

ACCEPTED MANUSCRIPT

3

ESC_3h

2 ESC_3d

RI PT

0 -1

SC

-2 -3

M AN U

-4

EP

TE D

-5

AC C

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ESC_24h