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Cloning and Characterization of IL-1β, IL-8, IL-10,and TNFα from Golden Tilefish (Lopholatiluschamaeleonticeps) and Red Snapper (Lutjanuscampechanus)Kristina L. DeakUniversity of South Florida, [email protected]

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Scholar Commons CitationDeak, Kristina L., "Cloning and Characterization of IL-1β, IL-8, IL-10, and TNFα from Golden Tilefish (Lopholatiluschamaeleonticeps) and Red Snapper (Lutjanus campechanus)" (2014). Graduate Theses and Dissertations.https://scholarcommons.usf.edu/etd/5416

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Cloning and Characterization of IL-‐1β, IL-‐8, IL-‐10, and TNFα from Golden Tilefish (Lopholatilus

chamaeleonticeps) and Red Snapper (Lutjanus campechanus) by

Kristina L. Deak

A thesis submitted in partial fulfillment of the requirements for the degree of

Master of Science with a concentration in Biological Oceanography

Department of Marine Science College of Marine Science University of South Florida

Major Professor: Steve A. Murawski, Ph.D. Cathy J. Walsh, Ph.D. Dana L. Wetzel, Ph.D.

Date of Approval: November 4, 2014

Keywords: Genetics, Immunology, Oil Spill, Deepwater Horizon

Copyright © 2014, Kristina L. Deak

DEDICATION

This thesis is dedicated to all those affected by the Deepwater Horizon explosion of April 20,

2010, including the 11 men who lost their lives that day. I hope that we will continue to learn from this

tragedy and work towards efforts to mitigate the ecological impact of future spills.

ACKNOWLEDGMENTS

Funding for this work was provided by a grant from the BP/Gulf of Mexico Research Initiative, C-‐

IMAGE, the Fish Florida scholarship, and the Guy Harvey Ocean Foundation scholarship.

I would like to express my most sincere gratitude to my advisor and mentor, Dr. Steve

Murawski. Working with Dr. Murawski has been an incredible experience, and has taught me a great

deal about fish, fishing, professional development, and how to effectively communicate science. I look

forward to many more summers at sea, especially catching that obligatory last set tiger.

Thank you to many members of Mote Marine Laboratory, including my committee members Dr.

Cathy Walsh and Dr. Dana Wetzel. Dr. Walsh’s years of marine immunology and biochemistry

experience were an incredible asset to my research, and I appreciate her assistance with

troubleshooting and data analysis. Dr. Wetzel provided endless amounts of lab space, reagents, and

flexibility as I juggled my commitments as a chemist in her lab and a student at USF. Dr. Tracy

Sherwood was a valuable resource for method optimization and data interpretation. Finally, I thank Dr.

Carl Luer, for use of his laboratory instrumentation throughout this project.

A world of credit is due to (doctoral candidate!) Erin Pulster. She has been absolutely

instrumental to my development as a scientist, and working with her, first as an intern, then as a co-‐

worker at Mote, has opened so many doors. Her daily assistance has significantly and positively laid the

foundation for my career. Thank you, for the thousands of pieces of advice, millions of questions

answered, and countless laughs over the years. I am excited for all of our future collaborations.

Finally, I am grateful for the unwavering support from Jesse Hoemann, Amy Wallace, the ladies

of the Murawski lab, and my parents. I truly appreciate all of your assistance throughout this thesis…

now let’s rally for the PhD!

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TABLE OF CONTENTS List of Tables iii List of Figures iv Abstract viii Chapter One: Introduction 1

1.1 Deepwater Horizon, PAH immunotoxcity, and traditional biomarkers for PAH exposure 1

1.2 Teleost cytokines and the immune response 2 1.3 Characteristics of vertebrate IL-‐1β, IL-‐8, IL-‐10, and TNFα 4 1.4 Objectives 7 Chapter Two: Methods 10 2.1 Isolation of total RNA 10 2.2 cDNA production 11 2.3 Initial identification and cloning of IL-‐1β, IL-‐8, IL-‐10, and TNFα fragments 11 2.4 5’-‐ and 3’-‐RACE 13 2.5 Sequence confirmation 14 2.6 Analysis of confirmed protein coding sequence 14 Chapter Three: Results and Discussion 16 3.1 Cloning and sequence analysis of golden tilefish IL-‐1β 16 3.2 Cloning and sequence analysis of red snapper IL-‐1β 19 3.3 Cloning and sequence analysis of golden tilefish IL-‐8 19 3.4 Cloning and sequence analysis of red snapper IL-‐8 21 3.5 Cloning and sequence analysis of golden tilefish IL-‐10 22 3.6 Cloning and sequence analysis of red snapper IL-‐10 23 3.7 Cloning and sequence analysis of golden tilefish TNFα 25 3.8 Cloning and sequence analysis of red snapper TNFα 26 Chapter Four: Conclusions and Future Work 51 4.1 Conclusions 51 4.2 Future work 52 References 53 Appendix A: Map of 2013 Tissue Sampling Stations 63 Appendix B: 2013 Tissue Sampling Location Data 64

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Appendix C: Golden Tilefish and Red Snapper Biometrics Data 65 Appendix D: Extended PCR Parameters 66 Appendix E: Experimentally Obtained Nucleotide Sequences 67 Appendix F: Experimentally Derived Cytokine Amino Acid Sequences 73 Appendix G: Sequence Confirmation 74 Appendix H: Table of Common Names and Their Associated Scientific Names 87

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LIST OF TABLES Table 1: Oligonucleotide primers used throughout this study, including their purpose, 15 annealing temperature, expected fragment band size in number of base pairs (bp), and primer source. Table 2: Percent amino acid identity between confirmed, mature amino acid sequence and 31 the top five most homologous fish species, according to BLASTp analysis. Table 3: Percent amino acid identity among vertebrate groups. 32 Table B1: 2013 longline tissue sampling location data. 64 Table C1: Golden tilefish and red snapper biometrics data, specific to fish whose spleens were 65 used in this study. Table D1: Extended PCR parameters for primers used. 66 Table F1: Amino acid sequences for each cytokine, translated from the experimentally 73 obtained nucleotide sequence by the ExPASy Translate tool. Table H1: List of all common names used in this thesis with the associated scientific name. 87

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LIST OF FIGURES

Figure 1: Schematic of PCR results including locations of the coding region, 5’ UTR, 3’ UTR, 28 locations at which primers bound, and the size and position of the resulting fragments. Figure 2: Clustal W multiple sequence alignment of representative vertebrate IL-‐1β, shaded 29 with BOXSHADE. Figure 3: Multiple sequence alignment of golden tilefish IL-‐1β with the top five most 33 homologous species, according to BLASTP. Figure 4: Rooted phylogenetic tree of vertebrate IL-‐1β proteins, produced in MEGA 6 with a 34 NJ method. Figure 5: Clustal Omega alignment between golden tilefish and red snapper IL-‐1β. 35 Figure 6: Clustal W multiple sequence alignment of representative vertebrate IL-‐8, shaded 36 with BOXSHADE. Figure 7: Multiple sequence alignment of golden tilefish IL-‐8 with the top five most 37 homologous species, according to BLASTP. Figure 8: Rooted phylogenetic tree of vertebrate IL-‐8 proteins, produced in MEGA 6 with 38 a NJ method. Figure 9: Multiple sequence alignment of red snapper IL-‐8 with the top five most 39 homologous species, according to BLASTP. Figure 10: Clustal Omega alignment between golden tilefish and red snapper IL-‐8. 40 Figure 11: Clustal W multiple sequence alignment of representative vertebrate IL-‐10, shaded 41 with BOXSHADE. Figure 12: Clustal Omega alignment between red snapper and golden tilefish IL-‐10. 43 Figure 13: Multiple sequence alignment of red snapper IL-‐10 with the top five most 44 homologous species, according to BLASTP. Figure 14: Rooted phylogenetic tree of vertebrate IL-‐10 proteins, produced in MEGA 6 with a 45 NJ method.

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Figure 15: Multiple sequence alignment of representative vertebrate TNFα, shaded with 46 BOXSHADE. Figure 16: Clustal Omega alignment between red snapper and golden tilefish TNFα 48 Figure 17: Multiple sequence alignment of red snapper TNFα with the top five most 49 homologous species, according to BLASTP. Figure 18: Rooted phylogenetic tree of vertebrate TNFα proteins, produced in MEGA 6 with 50 a NJ method. Figure A1: 2013 longline stations aboard the R/V Weatherbird II yielding tissue samples. 63 Figure E1: Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish IL-‐1β, 67 derived from the assembled 5’-‐ and 3’-‐RACE products. Figure E2: Confirmed 5’ to 3’ nucleotide sequence for the coding region of golden tilefish 68 IL-‐1β, obtained via cloning from two different fish. Figure E3: Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper IL-‐1β, 68 derived from the partial cloned fragment. Figure E4: Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish IL-‐8, 68 derived from the 3’-‐RACE product. Figure E5: Confirmed 5’ to 3’ nucleotide sequence for the coding region of golden tilefish IL-‐8, 69 obtained via cloning from two different fish. Figure E6: Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper IL-‐8, derived 69 from the assembled 5’-‐ and 3’-‐RACE products. Figure E7: Confirmed 5’ to 3’ nucleotide sequence for the coding region of red snapper IL-‐8, 69 obtained via cloning from two different fish. Figure E8: Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish IL-‐10, 70 derived from the assembled 5’-‐RACE and initial fragment products. Figure E9: Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper IL-‐10, derived 70 from the assembled 5’-‐ and 3’-‐RACE products. Figure E10: Confirmed 5’ to 3’ nucleotide sequence for the coding region of red snapper IL-‐10, 71 obtained via cloning from two different fish. Figure E11: Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish TNFα, 71 derived from the assembled 3’-‐RACE and initial fragment products.

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Figure E12: Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper TNFα, 72 derived from the assembled 5’-‐ and 3’-‐RACE products. Figure E13: Confirmed 5’ to 3’ nucleotide sequence for the coding region of red snapper TNFα, 72 obtained via cloning from two different fish. Figure G1: 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐1β, 75 cloned from fish F. Figure G2: 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐1β, 75 cloned from fish H. Figure G3: Clustal Omega alignment of the nucleotide sequences for golden tilefish IL-‐1β 76 from fish F and H. Figure G4: Clustal Omega alignment of the protein sequences for golden tilefish IL-‐1β from 77 fish F and H. Figure G5: Clustal Omega alignment of golden tilefish IL-‐1β from fishes F and H and the top 77 five homologous species, according to BLASTp. Figure G6: 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐8, 78 obtained from fish F. Figure G7: 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐8, 78 obtained from fish H. Figure G8: Clustal Omega alignment of the nucleotide sequences for golden tilefish IL-‐8 from 79 fish F and H. Figure G9: Clustal Omega alignment of the protein sequences for golden tilefish IL-‐8 from fish 79 F and H. Figure G10: 5’ to 3’ nucleotide sequence for red snapper IL-‐8, obtained from fish C. 80 Figure G11: 5’ to 3’ nucleotide sequence for red snapper IL-‐8, obtained from fish E. 80 Figure G12: Clustal Omega alignment of the nucleotide sequences for red snapper IL-‐8 from 80 fish C and E. Figure G13: Clustal Omega alignment of the protein sequences for red snapper IL-‐8 from fish 81 C and E. Figure G14: 5’ to 3’ nucleotide sequence for the translated region of snapper IL-‐10, cloned 81 from fish C. Figure G15: 5’ to 3’ nucleotide sequence for the translated region of snapper IL-‐10, cloned 81 from fish E.

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Figure G16: Clustal Omega alignment of the nucleotide sequences for red snapper IL-‐10 82 from fish C and E. Figure G17: Clustal Omega alignment of the protein sequences for red snapper IL-‐10 from 83 fish C and E.

Figure G18: Clustal Omega alignment of red snapper IL-‐10 from fish C and E and the top 83 five homologous species, according to BLASTp. Figure G19: 5’ to 3’ nucleotide sequence for the translated region of red snapper TNFα, 84 cloned from fish C. Figure G20: 5’ to 3’ nucleotide sequence for the translated region of red snapper TNFα, 84 cloned from fish E. Figure G21: Clustal Omega alignment of the nucleotide sequences for red snapper TNFα 85 from fish C and E. Figure G22: Clustal Omega alignment of the protein sequences for red snapper TNFα from 86 fish C and E.

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ABSTRACT

Cytokines are pleiotropic and redundant signaling molecules that govern the inflammatory

response and immunity, a critical ecological parameter for organism success and population growth.

Produced at the site of injury or pathogen intrusion by a variety of cell types, cytokines mediate cell-‐

signaling in either an autocrine or paracrine manner. The type and magnitude of the cytokine milieu

produced subsequently dictates the strength and form of immune response. As the most diverse

vertebrate group, with a high sensitivity to contaminants, fish represent an important foci for the

evaluation of immune system evolution, function, and alteration upon toxicant exposure. While many

cytokines have been identified in teleosts, primary study has been limited to model species (e.g.

zebrafish and fugu). However, evidence exists for several variations of cytokine genes within taxa,

underscoring the need for species-‐specific evaluation.

In this study, two pro-‐inflammatory cytokines (IL-‐1β and TNFα), one chemokine (IL-‐8), and one

anti-‐inflammatory cytokine (IL-‐10) were cloned, sequenced, and characterized for the first time in two

commercially relevant Perciformes in the Gulf of Mexico, golden tilefish (Lopholatilus chamaeleonticeps)

and red snapper (Lutjanus campechanus). The complete amino acid sequence was obtained and

confirmed for IL-‐1β and IL-‐8 from golden tilefish and for IL-‐8, IL-‐10, and TNFα from red snapper, with

partial sequences obtained for the remaining proteins. The results indicate high homology among

Perciformes for all cytokines studied, but divergence with other teleost orders, and low conservation

when compared to birds, amphibians, and mammals.

The sequences will be used to create a multi-‐plexed antibody-‐based assay for the routine

detection of cytokines in teleost serum. This would allow the biochemical response to fish health

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challenges, such as oil spills and other contamination events, to be monitored at the protein level,

building upon the current regime of genetic biomarkers. Thus, this work will aid in the understanding of

how oil spills and other contamination events may alter the immune response in fishes.

1

CHAPTER ONE:

INTRODUCTION

1.1 Deepwater Horizon, PAH immunotoxicity, and traditional biomarkers for PAH exposure

The Deepwater Horizon (DWH) explosion on April 20th, 2010 and subsequent oil spill resulted in

the second largest environmental disaster and response effort in human history [1, 2]. Before final

cessation on July 15, 2010, 780 million liters (L) of crude oil (3.9% polycyclic aromatic hydrocarbons

(PAHs) by weight) were released into one of the most productive fisheries in the United States, resulting

in a 36.6% offshore fishery closure [2-‐4]. In addition to a surface sheen, four temporally persistent

subsurface plumes were noted at 25 meters (m), 265 m, 875 m, and 1175 m [2]. A total of 7 million L of

dispersant were utilized in an attempt to mitigate oil reaching the shoreline, including 4 million L applied

at the surface and 2.9 million L applied at the wellhead [1]. This application of dispersant at depth had

never before been attempted, and its resulting longevity and effect in the water column were uncertain,

though recent sampling indicates dispersal of petroleum far beyond the scope of the 1175 m plume [2].

In addition, there is evidence of dispersant-‐enhanced extraction of toxic compounds, like benzene and

PAHs, to the entire span of the water column, increasing overall aquatic toxicity [2, 4]. These soluble

enhanced constituents were bioavailable and free to diffuse across biological membranes, linking

dispersant application to an increased PAH uptake by fish [5].

Metabolism of PAHs induces their immunotoxic effects and triggers cytokine expression in

characteristic ways. When PAHs cross biological membranes they activate the aryl hydrocarbon

receptor (AhR) pathway, causing AhR to dissociate from cofactors, including heat shock protein 90 [6, 7].

The AhR and PAH complex then enters the nucleus and upregulates cytochrome P450 (CYP1A)

2

expression, a hydroxylase that aids in PAH metabolism [6, 8, 9]. This induction of CYP1A generally

increases the intracellular concentration of reactive oxygen species [10]. ROS are produced naturally, as

a product of anaerobic respiration, and cells contain antioxidant systems to neutralize them [11, 12].

However, if these antioxidant systems are overwhelmed, the NF-‐кB pathway can be initiated, a stress

response nuclear factor network that triggers the release of many pro-‐inflammatory cytokines [10]. This

release of pro-‐inflammatory cytokines is a clear indication of the immune response.

Traditional biomarkers utilized to understand the effect of oil contamination on marine

organisms focus on the earlier portion of the PAH biochemical pathway, particularly through

measurement of AhR and Cyp1A gene expression [13, 14]. While these targets have utility, they

primarily indicate the exposure to and metabolism of oil, not necessarily the resulting biological impact

[15, 16]. A need exists for a novel suite of biomarkers that can quantify the alterations in the

biochemistry of marine organisms that lead to physiological response and aberrant pathologies, such as

the noted increase in lesions in fishes found in contaminated waters [7, 17, 18]. This includes the

identification and quantification of a link between PAH exposure and subsequent alterations of immune

system function [19-‐21].

1.2 Teleost cytokines and the immune response

Immunity is an ecologically critical biological function that allows an organism to fight pathogens

while continuing to proliferate, thus aiding in population growth [22]. Cytokines are pleiotropic

signaling peptides, proteins, and glycoproteins that mediate inflammation and influence both innate and

adaptive immunity [23-‐25]. Secreted by a variety of cell types in response to a stressor, cytokines have

pro-‐ and anti-‐inflammatory roles and control the ratio of various T helper (Th) cell subsets, which in turn

helps determine the type of immune response and maintains homeostasis in vertebrates [26, 27].

Cytokines can regulate the ability of phagocytes to destroy foreign pathogens, as well as control antigen

3

presentation on T cells, and stimulate lymphocyte migration throughout the body [25]. They can

function through either autocrine or paracrine signaling and their expression can either be altered in

quick bursts, such as during infection, or their aberrant expression can be prolonged, as is the case in

well-‐studied human diseases like rheumatoid arthritis [28-‐30].

Fish are one of the earliest vertebrate groups to possess hallmarks of both innate and adaptive

immunity and are frequently used as models in evolutionary immunological studies [31, 32]. While

some of the main immune organs are different (in addition to the thymus and spleen, teleost immunity

is also dependent upon the head kidney) many of the cell types involved in the immune response are

conserved between fish and mammals, including monocytes/macrophages, non-‐specific cytotoxic cells

(NCC), natural killer cells (NK), and granulocytes [33]. In addition, critical complexes and receptors to

the mammalian immune system are conserved in teleosts, including immunoglobulins, T cell receptors

(TCR), and major histocompatibility complexes (MHC) [33-‐38]. Given their continual dermal exposure to

aquatic viruses, bacteria, contaminants, and other threats, it is not surprising that fish would have also

developed unique immune strategies, including the presence of specialized toll-‐like receptors (TLR)-‐22

and TLR3 for the detection of viral RNA on the cell surface and intracellularly, respectively [34]

The major secretory cells of cytokines, Th cells, are produced upon naive CD4+ cell recognition

of a foreign peptide and subsequent differentiation [25, 35]. Genomic work in teleosts confirmed the

presence of critical T cell function and signaling-‐related molecules indicative of all four major Th subsets,

including Th1, Th2, Th17, and Treg cells, and functional studies suggest similar lymphocyte activity to

mammals [25, 31, 35]. However, the definitive presence of these Th cell subsets cannot yet be

substantiated, given the lack of teleost-‐specific antibodies [25, 34]. In addition, unique T cell molecules

have been identified in fish. For example, teleost cells appear to have relevant Th1 cytokines, including

IFNγ, although they also have a teleost-‐specific molecule, IFNγ-‐rel [35].

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Many important mammalian cytokines have been identified in fish, including at least 19

interleukins, and members of the tumor necrosis factor, chemokine, and interferon families. However,

teleost functional studies are significantly lagging behind mammalian studies and gene examination is

limited to a few model species [32, 36-‐38]. Teleost cytokine identification is complicated by the fish

specific genome duplication event (3R or FSGD), which lead to fish-‐specific copies of several known

cytokines (potentially partitioning functions of ancestral genes among them), along with a likely fourth

duplication event in Salmoniformes [32, 39, 40]. In addition to possible differential function and

expression between isoforms of the same signaling protein, certain species of fish may have multiple

variations of a single gene, while other species do not, underscoring the need for species-‐specific

examination [24, 25, 40-‐42].

1.3 Characteristics of vertebrate IL-‐1β , IL-‐8, IL-‐10, and TNFα

In this study, four cytokines were explored in two important fishery species in the Gulf of

Mexico, golden tilefish (Lopholatilus chamaeleonticeps) and red snapper (Lutjanus campechanus). The

proteins of interest include two proinflammatory cytokines (interleukin (IL)-‐1β and tumor necrosis factor

alpha (TNFα)), one chemokine (IL-‐8), and one anti-‐inflammatory cytokine (IL-‐10). These targets were

selected given their importance in the inflammatory and immune response as well as their extensive

prior evaluation in model fish species, primarily in relation to toxicant exposure and with regards to

parasite infection in an aquaculture setting [19, 21, 37, 38, 43].

Interleukin-‐1β is one of the earliest expressed pro-‐inflammatory cytokines, induced by tissue

injury, microbial invasion, and autoimmune disease [37, 44]. Produced by a variety of cell types,

primarily macrophages and neutrophilic granulocytes in teleosts, IL-‐1β stimulates T cells and initiates a

cascade of potent effects by subsequent up-‐ or down-‐regulation of other cytokines, resulting in

inflammation [39, 45]. The IL-‐1β gene has been identified in a number of teleost species, including

5

Cypriniformes, Perciformes, Salmoniformes, and Gadiformes, and similarities to its mammalian

counterpart are observed, though the teleost gene clusters independently of birds, mammals and

amphibians on phylogenetic trees [38, 44, 46-‐51].

Differential phylogenetic clustering could, in part, be due to fundamental sequence

discrepancies between the mammalian and fish orthologues. For example, while mammalian IL-‐1β

exhibits a seven exon structure, the Perciform orthologue has four to five exons, and Salmoniformes

have six, though the classic β trefoil shape with 12 β-‐sheet structure is conserved among all studied

fishes [38, 47]. In addition, mammalian IL-‐1β is produced as an inactive peptide that must be

proteolytically cleaved by caspase-‐1 at the IL-‐1β converting enzyme (ICE) cut site [47, 52, 53].

Interestingly, teleosts typically lack this ICE cut site, though experimental evidence suggests the fish

orthologue is also produced in an inactive form and later activated via a cleavage mechanism [47].

Multiple isoforms of the gene exist in several species, including carp, trout, salmon, catfish, and

platyfish, possibly due to duplication of the IL-‐1 locus during the FSGD event [47, 53].

Despite these differences, functional studies in rainbow trout, haddock, and olive flounder,

suggest that teleost IL-‐1β is involved with regulation of immune genes, lymphocyte activation, migration

of leucocytes, phagocytosis, and bactericidal activity [37, 54]. Recombinant IL-‐1β from sea bass and

rainbow trout can induce the release of calcium (Ca2+) in teleost leukocytes, an activity not clearly

demonstrated in mammalian species [53]. An increase in intracellular [Ca2+] has also been noted in fish

cells upon exposure to benzo(a)pyrene and other PAH’s, potentially linking the activities of this cytokine

to PAH exposure [7]. Upregulation of the IL-‐1β gene has been noted after exposure to heavy crude oil in

rockfish liver [19]. Suggested functions from mammalian studies include response to tissue injury [44].

Chemokines, like IL-‐8, are chemoattractant cytokines that primarily assist in cell migration,

principally by invoking leukocyte mobilization to sites of infection or injury [55]. Specifically, IL-‐8

belongs to the CXC family of chemokines and attracts neutrophils, T lymphocytes, and basophils to

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inflamed regions [37]. While current functional assays of IL-‐8 in teleosts display similar activity and

basal levels of expression as the mammalian orthologue, teleost sequences have only 30-‐40% amino

acid identity with the mammalian protein [55, 56].

Recent evidence suggests that teleost CXC is a novel lineage from other vertebrates, with up to

three distinct strains evolving within fish families [25, 42, 56, 57]. The hallmark of IL-‐8 proteins in

mammals is the presence of an ELR motif before the first cysteine, which most fish homologues (except

Gadoids) lack. This motif is necessary for neutrophil chemotaxis, therefore, the function of IL-‐8 in

teleosts may be slightly different from that in mammals [42, 48, 56]. However, a recent study in

flounder (which has a SLH motif, rather than the mammalian ELR motif) designated a leucine residue at

the N-‐terminus of the IL-‐8 gene as responsible for induction of chemotaxis in the fish [57]. In addition,

in vitro functional studies in rainbow trout demonstrate the ability of IL-‐8 to recruit monocyte cells, as

well as induce expression of pro-‐inflammatory cytokines, such as IL-‐1β and TNFα [42, 49]. Upon

exposure to benzo(a)pyrene, a toxic PAH, IL-‐8 was up-‐regulated in flounder macrophage cells [21, 58]

One of the most well studied pro-‐inflammatory cytokines in fish, TNFα has been shown to have

numerous, pleiotropic functions [38, 39]. Some of these functions include upregulation of adhesion

molecules, induction of chemokine release, induction of the NF-‐кb pathway, regulation of apoptosis,

and stimulation of IL-‐6 production. TNFα has also been shown to upregulate the production of IL-‐1β and

IL-‐8 in rainbow trout [37, 59, 60]. Therefore, TNFα is critical in antimicrobial defense and response to

injury, and helps potentiate the inflammatory response [38, 61]. Its function in fish mirrors that noted in

mammals, although multiple forms of the gene have been observed in teleosts, including two variants in

bluefin tuna [38, 39, 62]. Structural information for TNFα among cloned species has indicated high

levels of divergence, indicating a need for species-‐specific evaluation of the molecule [62]. However,

the majority of the TNFα family signature and two cysteine residues are conserved among all studied

vertebrates [60].

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Finally, IL-‐10 is one of the most important and potent anti-‐inflammatory cytokines [41].

Through induction of Treg cells, IL-‐10 inhibits production of pro-‐inflammatory cytokines by macrophages

and dendritic cells, including the regulation of TNFα [41, 63, 64]. Produced after proinflammatory

mediators, IL-‐10 can inhibit Th1 and Th17 immunity, partially through presentation of antigen

presenting cells, and helps prevent a run-‐away immune response. Essentially, IL-‐10 is responsible for

limiting the ability of monocytes/macrophages to partake positively in the immune response while

simultaneously aiding in the production of anti-‐inflammatory mediators [41]. In addition, in mammalian

systems, IL-‐10 has been implicated in the down-‐regulation of ROS, which can be produced as a by-‐

product of PAH metabolism [10, 65]. This interleukin can regulate the function of many leukocyte

classes, including the activation of B cells, the proliferation of NK cells, and the differentiation of

cytotoxic T cells [66, 67].

To date, IL-‐10 has been identified in several teleosts, including European seabass, rainbow trout,

cod, fugu, common carp, silver carp, zebrafish, and goldfish, and functional studies suggests analogous

function to that of its mammalian counterpart [25, 64, 66, 68-‐70]. For example, recombinant goldfish IL-‐

10 can regulate multiple isoforms of TNFα, IL-‐1β1, and IL-‐8 in fish monocytes [64]. Furthermore, the IL-‐

10 receptor subunit 1 has been identified in grass carp, indicating at least partially conserved signaling

pathways for the molecule throughout evolution [67]. To date, rainbow trout are the only teleost

species identified with more than one gene encoding IL-‐10, though both trout genes have 92% identity

in the translated region [25, 71]. In addition to four disulfide bond-‐forming cysteine residues conserved

in all vertebrates, fish IL-‐10 contains two cysteine residues that appear to be teleost-‐specific [70].

1.4 Objectives

In this study, the above-‐mentioned cytokines (IL-‐1β, IL-‐8, IL-‐10, and TNFα) were isolated, cloned,

sequenced, and characterized for the first time from two important fishery species in the Gulf of Mexico,

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red snapper and golden tilefish. Red snapper are reef-‐associated, offshore Perciformes with high

fecundity [72, 73]. They represent the most economically important snapper species to the Gulf of

Mexico commercial fishery, whose 3.24 million pound yield in 2011 produced $13.8 million in revenue

[72, 73]. They are also one of the most targeted fishes for the enormous Gulf of Mexico recreational

fishery, with their annual landings almost always exceeding the Annual Catch Limit set by NOAA,

sometimes exceeding the limit by as much as 49.6% [74]. Golden tilefish represent a smaller

commercial fishery, but their habitat selection method of burrow formation in deep sediments may

enhance their exposure to settled hydrocarbons and other contaminants, making them valuable for

environmental health assessment studies [75, 76].

It was hypothesized that field-‐caught golden tilefish and red snapper cytokine sequences would

share high homology with the corresponding Perciform genes. Anomalies were anticipated as

comparisons across other evolutionary lineages ensued, including comparisons between other

Actinopterygii, as well as between cartilaginous fish, amphibians, birds, and mammals. The results of

this study will aid in the body of literature identifying conserved genetic regions within fishes and among

vertebrate taxa and strengthen the phylogenetic analysis of cytokine structure, potentially aiding in

predictions concerning the evolution of the immune system.

The resulting characterized sequences and phylogenetic correlations from this study will

eventually be used to create antibodies for the generation of a highly cross-‐reactive magnetic bead-‐

based assay for detection of cytokines at the protein level in Perciform serum, plasma, and cell culture

supernatant. The kit would likely be validated utilizing a dosing study and correlated with an evaluation

of gene expression patterns in vivo and in vitro in response to a water accommodated fraction (WAF) of

oil (DWH crude oil) and a chemically enhanced WAF (CEWAF; DWH crude oil + Corexit 9500). In

addition, gene expression patterns and protein production levels will likely be compared to those found

in fish samples with known PAH body burdens collected from the Gulf of Mexico in the vicinity of the

9

DWH explosion from 2012-‐2014. This work will form the basis of the PhD dissertation to be prepared by

the author from 2015 through 2018 and will be the first to produce a thoroughly validated, Perciform-‐

specific antibody-‐based kit for cytokine detection. Given the amount of variability in cytokine genes

across teleosts, such a thorough investigation first required an analysis of the target genes in each

species of interest.

10

CHAPTER TWO:

METHODS

2.1 Isolation of total RNA

Golden tilefish (n=39) and red snapper (n=38) spleen samples were obtained from freshly caught

fish from the Gulf of Mexico in August 2013 aboard the R/V Weatherbird II (a map of sampling locations

can be found in Appendix A, with location coordinates and depths in Appendix B). For this study, four

golden tilefish spleen samples were used, from fish weighing 2.630-‐4.054 kg, caught at stations SL11-‐

150 (labeled F and G) and SL14-‐100 (labeled T8 and T9; all biometrics data in Appendix C). In addition,

two red snapper spleen samples were used, labeled C and E, from fish weighing 4.508-‐5.274 kg, caught

at station SL4-‐40. Fish were caught via demersal longline fishing from 35 to 225 m depth for red

snapper and from 156 to 508 m depth for golden tilefish, depending upon the site, using a 5 mile line

with approximately 500 baited hooks and an average soak time of two hours.

Fish were landed on deck, bled, and tissue samples were immediately taken. All spleen tissue

was subsampled into 5 mm x 5 mm x 5 mm cubes, immediately stored in 1 mL RNAlater (Life

Technologies, Grand Island, New York), and placed in a -‐20oC freezer, until transport to a final storage

location at -‐80oC. Samples were thawed at room temperature and subsampled if necessary to 50-‐100

mg pieces. Total RNA was isolated using TRI Reagent (Molecular Bioproducts, San Diego, California)

according to manufacturer’s protocol and dissolved in 25 µL nuclease-‐free water (Life Technologies,

Grand Island, New York). Samples were DNAse treated using the Turbo DNA-‐free kit, according to

manufacturer’s instructions (Ambion, Austin, Texas). RNA was quantified using a Nanodrop

11

spectrophotometer (Nanodrop Technologies, Wilmington, Delaware) and sample concentration was

adjusted to 1 µg/µL with nuclease-‐free water. Samples were stored at -‐80oC.

2.2 cDNA production

Total RNA (1 µL) was reverse transcribed to cDNA using the Reverse Transcription System, per

manufacturer’s instructions (Promega, Madison, Wisconsin). Proper performance of the reverse

transcription reaction was assessed via sufficient amplification of β-‐actin, using reverse-‐transcription

polymerase chain reaction (RT-‐PCR), GoTaq Green Master Mix (Promega, Madison, Wisconsin) and

European seabass β-‐actin primers and parameters [50]. Each PCR product was visualized on a 1.2%

Ultrapure agarose (Life Technologies, Grand Island, New York) gel with 1x TAE buffer (Life Technologies,

Grand Island, New York), run at 70 V for 45 minutes and viewed on a Chemidoc gel imager (BioRad,

Hercules, California). Rapid Amplification of cDNA Ends (RACE)-‐ready cDNA was also produced using 1

µL Total RNA, but with the SMARTer RACE 5’/3’ kit according to the manufacturer’s protocol (CloneTech,

Mountain View, California).

2.3 Initial identification and cloning of IL-‐1β , IL-‐8, IL-‐10, and TNFα fragments

Degenerate primers were taken from the teleost literature or designed from available teleost

FASTA sequences in GenBank, using PriFi (cgi-‐www.daimi.au.dk/cgi-‐chili/PRiFi/main; Table 1). Primer

dimer plausibility was screened for using the Multiple Primer Analyzer Tool

(www.thermoscientificbio.com/webtools/multipleprimer; Thermo Scientific). All oligonucleotides were

purchased from Invitrogen and adjusted to 10 µM with nuclease-‐free water (Life Technologies, Grand

Island, New York). Gradient RT-‐PCR experiments were utilized to obtain fragments of the expected base

pair (bp) size as predicted in the literature or using PriFi (Table 1). All RT-‐PCR experiments began with a

94oC initialization for five minutes, followed by 35 cycles of denaturation at 94oC for 30-‐45 seconds,

12

primer annealing at 48-‐60oC for 30-‐45 seconds, and primer extension at 72oC for 45-‐60 seconds, ending

with a final extension at 72oC for 5-‐10 minutes (for extended PCR parameter information, please see

Appendix D). Initial 25 µL reactions were performed and products were viewed on a 1.2% Ultrapure

agarose gel (Life Technologies, Grand Island, New York), as previously described.

Reaction parameters producing a single band of the expected size (as predicted in the literature

or primer design software), were repeated to produce 100 µL of PCR product, and subsequently run on a

1.2% Ultrapure agarose gel (Grand Island, New York) for isolation. Bands were excised from the gel and

DNA fragments were purified using the Wizard SV Gel and PCR Clean-‐Up System by centrifugation,

according to the manufacturer’s protocol (Promega, Madison, Wisconsin). Purified DNA was quantified

using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, Delaware) and cloned using

the pGEM T Easy Vector System, per manufacturer’s instructions (Promega, Madison, Wisconsin).

Ligation reactions (10 µL) were prepared with a 1:3 insert:vector ratio. Following an overnight

incubation at 4oC, the reaction was transformed into JM109 High Efficiency Competent Cells (Promega,

Madison, Wisconsin) and grown with SOC medium (Fisher, Waltham, Massachusetts) for 90 minutes at

37oC on an orbital shaker plate, rotating at 550 rpm. Following incubation, 100 µL aliquots of the

transformation reactions were transferred to two agar plates (Sigma-‐Aldrich, St. Louis, Missouri), one

undiluted and one diluted 1:10 in SOC medium (Fisher, Waltham, Massachusetts). Colonies were grown

at 37oC for 18 hours.

Ten colonies were selected from each plate and grown in 2 mL LB broth (5 g Tryptone (EMD,

Rockland, Massachusetts), 2.5 g yeast (EMD, Rockland, Massachusetts), 2.5 g NaCl (Fisher, Waltham,

Massachusetts), 500 µL 100 mg/mL Ampicillin (Fisher, Waltham, Massachusetts), to 500 mL DI H2O) in

15 mL conical vials. Conical vials were placed on an orbital shaker at 550 rpm at 37oC for 20 hours.

Plasmid purification was performed using the Manual FastPlasmid Mini Kit (5Prime, Hamburg,

Germany). Colonies were screened for insert using RT-‐PCR with universal T7 and SP6 primers, ordered

13

from Life Technologies (Grand Island, New York; Table 1). Purified plasmid DNA was quantified as

before, adjusted to 50-‐100 ng/uL and two samples with sufficient 260/280 values and concentrations

were sent to Eurofin Genomics for sequencing (Eurofins MWG Operon, Huntsville, Alabama). The vector

was removed from sequencing results using VecScreen (www.ncbi.nlm.nih.gov/tools/vecscreen) and the

remaining fragment was input to the nucleotide Blast Local Alignment Tool (BLASTn;

www.ncbi.nlm.nih.gov/blast).

2.4 5’-‐ and 3’-‐RACE

If sufficient homology existed between the obtained fragment and other available teleost

nucleotide sequences for the same cytokine, 5’/3’-‐RACE was performed using the SMARTer RACE 5’/3’

Kit and the Advantage 2 Polymerase Mix (Clonetech, Mountain View, California). Gene-‐specific

oligonucleotide primers designed for 5’/3’-‐RACE (GSP1 for 3’-‐RACE and GSP2 for 5’-‐RACE) using

Primer3Plus (www.bioinformatics.nl/cgi-‐bin/primer3plus) and the parameters dictated in the SMARTer

5’/3’-‐RACE protocol. Primer dimer plausibility between designed primers and kit provided long and

short universal primers (Clonetech, Mountain View, California) was again screened using the Multiple

Primer Analyzer Tool.

Gradient RT-‐PCR was performed to optimize the annealing temperature using RACE-‐ready cDNA

and PCR products were visualized on a 1.2% Ultrapure agarose gel (Life Technologies, Grand Island, New

York). When a bright, single band was obtained, a 100 µL reaction was repeated under the same

conditions, extracted and purified, as above. Products were screened for inserts using via RT-‐PCR with

the respective GSP1 or GSP2 primer. After quantification, samples were sent for sequencing, as

previously described. The resulting 5’/3’ RACE sequences were assembled and translated into amino

acids using the ExPASy translate tool (web.expasy.org/translate). Homology with teleost proteins was

confirmed using BLASTp (www.ncbi.nlm.nih.gov/blast).

14

2.5 Sequence confirmation

Gene-‐specific primers were designed with Primer3Plus to isolate the coding sequence for the

protein of interest. They were applied to the cDNA of two different fish from each species, in order to

validate the sequence of the coding region and eliminate the possibility of single nucleotide

polymorphisms skewing sequence data. The isolation, amplifying, cloning, and sequencing were

performed according to previously described methods. The resulting sequences were compared using

Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo), BLASTn, and BLASTp and any discrepancies were

resolved by comparison to other teleost sequences and experimental data.

2.6 Analysis of confirmed protein coding sequence

All confirmed protein sequences were screened for the presence of the family signature,

according to PROSITE (prosite.expasy.org) and the relevant teleost literature [38, 44, 48, 66, 68]. N

glycosylation sites were identified by the NetNGlyc 1.0 server (www.cbs.dtu.dk/services/NetNGlyc),

putative signal peptide cut sites were identified using SignalP 4.1 (www.cbs.dtu.dk/services/SignalP),

and transmembrane domain regions were identified using TMPred

(www.ch.embnet.org/software/TMPred_form.html). Percent identity was calculated by comparing

amino acid sequences using BLASTp. Phylogenetic trees were generated using complete sequences by

the neighbor-‐joining (NJ) method in MEGA6 (www.megasoftware.net), validated by bootstrap analysis

with 10,000 repetitions using the p-‐distance method with partial deletions and an 80% cutoff [48, 77].

15

Table 1: Oligonucleotide primers used throughout this study, including their purpose, annealing temperature, expected fragment band size in number of base pairs (bp), and primer source.

Name Nucleotide,Sequence,(5'33>,3') Use

Annealing,temp.,(C)

Fragment,size,(bp)

SourceBA_F ATCGTGGGGCGCCCCAGGCACA

BA_R CTCCTTAATGTCACGCACGATTTC

gtIL-1b_F GGGAAAGAATCTRTACCTGTCYTG

gtIL-1b_R GTGCTGATGAACCAGT

gtIL-8_F GCAGCTGAACMAAGAAAAGGAAAG

gtIL-8_R CAGCGGCAGTGCAKCTCCAYTCCCA

gtIL-10_F TGCTGCTCCTTCGTGGAGGGCTTCCC

gtIL-10_R CCAGCTCCCCCATGGCTTTATA

gtTNFa_F AGGCGTCSTTCAGAGTCTCC

gtTNFa_R GGSCTACTACAATCTCAGGTCA

rsIL-1b_F GCTGGAGAGCGTAGTYGAAGA

rsIL-1b_R CGGAGTCCTGTTTGTAGAAGAGAR

rsIL-8_F GCAGCTGAACMAAGAAAAGGAAAG

rsIL-8_R CAGCGGCAGTGCAKCTCCAYTCCCA

rsIL-10_F TGCTGCTCCTTCGTGGAGGGCTTCCC

rsIL-10_R CCAGCTCCCCCATGGCTTTATA

rsTNFa_F ATCAGCAGCAAAGCCAAGGCAGC

rsTNFa_R GAAGGTCTTGCCCTCGTCTGTCTCCAG

T7 TAATACGACTCACTAAGGG

SP6 ATTTAGGTGACACTATAGAA

gtIL-1b_GSP1 CCTGCCACAAGGATGGTGAAGAGCC 64 775

gtIL-1b_GSP2 CCAGTCACTGTAGGGGACGGACACG 64 750

gtIL-8_GSP1 TGAACAGCAGAGTCTTCGTCGCCACT 70 634

gtIL-10_GSP2 GGCTTGAGGTCTCTGGTGTCCTCCGT 62 513

gtTNFa_GSP1 AAACCGGCCTCTACTTCGTCTAC 60 779

rsIL-8_GSP1 GAGGAGAGAGGGAACTGAGCAAAAGA 68 784

rsIL-8_GSP2 CACCACAATAGAGGCGATGA 66 279

rsIL-10_GSP1 GAAGCAAACGACGACTTGGACAGAGC 68 711

rsIL-10_GSP2 TCCATGTGAGGCTTGAGGTCTCTGGT 68 519

rsTNFa_GSP1 TCTACAGCCAGGCGTCGTTCAGG 70 777

rsTNFa_GSP2 TACCAGCCGTGTCCGTCTCTGTAGC 70 744

gtf_IL-1b_F TGGGGAGAACAACACTGAGA

gtf_IL-1b_R TCCACACTCCACTTTGGTTG

gtf_IL-8_F CACAGTGCTTCACCAGAAGG

gtf_IL-8_R AAGGACATTAAGCCGTCCTCT

rsf_IL-8_F TTCAGGCATCTTTTTGAAGGA

rsf_IL-8_R GGCCTCAGTTGGATGAATGT

rsf_IL-10_F GCCAGCAACATCCTCTTCAT

rsf_IL-10_R TCTTCAAGCAGAGGCCACAT

rsf_TNFa_F GCCAAGTGCTCGACTGGTAG

rsf_TNFa_R GCAAACACGCCAAAGAAAGT56

48

54

54

54

48

48

50

55

50

54

Scapigliati,H2001RTHqualityH

control

Scapigliati,H2001

Buonocore,H2007

Xie,H2008

Lu,H2008

55

48

50

54

500

199

279

441

572

474 Covello,H2009

UniversalIsolatingH

fromHvector

430

277

406

Fragment

+H160Hbp

Deak,HPriFi

Buonocore,H2007

ObtainingH

intitialH

fragments

Deak,HPriFi

ObtainingH

finalH

proteinH

sequence

Deak,HHHHHHHHHH

Primer3Plus

881

513

453

647

884

5'-HandH3'-H

RACEH

Deak,H

Primer3Plus

16

CHAPTER THREE:

RESULTS AND DISCUSSION

3.1 Cloning and sequence analysis of golden tilefish IL-‐1β

Degenerate primers gtIL-‐1b_F and gtIL-‐1b_R were used to amplify, via PCR, an initial 199 base

pair (bp) fragment from cDNA isolated from golden tilefish spleen F (Table 1). The fragment was cloned,

sequenced, and utilized for the design of gene-‐specific oligonucleotides to perform 5’-‐ and 3’-‐RACE

(Table 1). The 5’-‐RACE reaction yielded a 750 bp product and 3’-‐RACE resulted in a 775 bp product, with

a 153 bp overlap between the two fragments. This resulted in an assembled RACE product of 1,396 bp

with a 756 bp translated region (Figure 1a). The 547 bp 3’ untranslated region (UTR) contained three

instability motifs (ATTTA), five endotoxin responsive motifs (TTATTTAT), two of which were overlapping,

and a polyadenylation signal (AATAAA) ending 14 bp upstream of the poly-‐A tail. The 5’ UTR was 93 bp

in length and likely incomplete (all experimentally determined nucleotide sequences may be found in

Appendix E). The assembled RACE product was utilized to design gene-‐specific primers to isolate the

coding sequence for subsequent cloning and sequence confirmation through analysis of clones from

different fish (Table 1).

The resulting sequences, cloned from separate fish, shared high consensus, yielding a final,

confirmed 756 bp coding region, which was translated into 251 amino acids and contained the family

signature, [FCL]-‐x-‐S-‐[ASLV]-‐x(2)-‐[PRS]-‐x(2)-‐[FYLIV]-‐[LI]-‐[SCAT]-‐T-‐x(7]-‐[LIVMK], as

FvSVpySdwYISTagddnkpV (Figure 2; all experimentally derived amino acid sequences may be found in

Appendix F, all confirmed sequence comparisons may be found in Appendix G). No conservation was

observed at the aspartic acid residue marking the ICE cleavage site in mammals (Figure 2). This residue is

17

typically absent in non-‐mammalian vertebrates, though processing by other proteases has been

postulated [78, 79]. No signal peptide was predicted by SignalP, which suggests secretion by a non-‐

canonical pathway, avoiding the golgi-‐endoplasmic reticulum excretion mechanism, another vertebrate

characteristic for IL-‐1β [78, 80].

Five N-‐glycosylation sites were predicted using the NetNGlyc 1.0 server, at golden tilefish (gt)N8,

gtN48, gtN103, gtN132, and gtN209 (Figure 3). The gtN8 motif was shared with other Perciformes and

Pleuronectiformes and the gtN132 motif was shared with all other screened type II IL-‐1β proteins in

teleosts (except red seabream). The gtN48 and gtN209 motifs, however, were shared exclusively with red

seabream (Figures 3). Two cysteine residues were observed, at gtC166 and gtC235 (Figure 2). The first

was conserved among all vertebrates, while the latter was conserved among several teleosts (Figure 2;

Figure 3). There was high conservation among the 12 β-‐sheets across all vertebrates screened in the

multiple alignment, particularly in sheets 3-‐11 (Figure 2). This might indicate that similar folding

patterns and tertiary structure have been upheld throughout evolutionary lineages [38]. Interestingly,

there appears to be a golden tilefish-‐specific sequence addition between sheets 5 and 6, not found in

any other screened vertebrate (Figure 2).

However, of the seven residues cited as being essential for binding to the IL-‐1 receptor in

humans, tilefish shared only two with humans, residues human (h)R4 and hK103, numbered according to

the location of the mature human peptide [81]. The hK103 residue was highly conserved in many

Perciformes, chicken, and the mammals screened in the multiple alignment, but substituted with an

arginine in the Salmoniformes, Gadiformes, and Cypriniformes (Figure 2). Two specific sites were

previously characterized as being critical for human IL-‐10 ligand-‐receptor binding, Site A and Site B [82].

Of the 25 residues involved with Van der Waals force-‐dependent Site A, golden tilefish demonstrated

conservation with 11 of them, including identity with 7 amino acids (hS13, hL31, hQ32, hG33, hN129, hP131,

and hQ142; Figure 2). Of the 25 residues important for Site A binding, four are critical, hR11, hQ15, hH30,

18

and hQ32 [80]. Golden tilefish IL-‐1β had a similar amino acid substitution (asparagine) at hQ15, and

shared identity with hQ32 (Figure 2). Golden tilefish IL-‐1β showed weaker conservation at site B,

demonstrating identity with four of the important hydrophilic residues (hR4, hK103, hN108, hS152), but no

conservation with the remaining 17 residues (Figure 2).

The lack of conservation among the ligand-‐receptor binding sites may indicate high diversity in

binding mechanisms and sequences and limits the likelihood of cross-‐reactivity between non-‐placental

mammals and other vertebrates [38, 83]. In human mutagenic studies, even conserved amino acid

substitutions were sufficient to interrupt 30-‐50% of receptor binding activity, with a complete loss of

activity when the critical Site A hQ15 residue was mutated [80]. Therefore, even though the hQ15 residue

was substituted with the structurally similar arginine in tilefish, this might be enough to disrupt similar

receptor binding mechanisms between mammalian and teleost IL-‐1β molecules.

Golden tilefish IL-‐1β shared high amino acid percent identity with other Perciformes studied,

including 70% amino acid identity with European seabass, 70% with Mandarin fish, and 68% identity

with barred knifejaw (Table 2; all common names used and their associated scientific names may be

found in Appendix H). Identity was significantly less well-‐correlated with Cypriniformes (33%),

Elasmobranchs (28%), amphibians (33%), birds (31%), and mammals (29-‐32%; Table 3). Low identity

with the smaller spotted catshark was anticipated, as it is one of the most ancestral IL-‐1β molecules

identified [78].

On the constructed phylogenetic tree, teleost fishes clustered separately from remaining

vertebrates, with additional subdivisions between Cypriniformes, Gadiformes, Salmoniformes, and the

remaining Perciformes, which clustered with Plueronectiformes (Figure 4). These branchings could be a

result of the Type I and Type II division of IL-‐1β molecules. Perciformes typically contain Type II IL-‐1β,

while Cypriniformes, Elasmobranchs, amphibians, birds, and mammals contain Type I and

Salmoniformes and Gadiformes display both types [78]. Type II IL-‐1β molecules tend to be shorter,

19

owing to their lack of an additional exon at the 5’ end of the protein, and generally share higher

homology among one another as compared to their identity with known Type I molecules [76]. There

was an apparent split in the teleost portion of the tree, dividing the Perciformes, Pleuronectiformes,

Gadiformes, and Salmoniformes Type II IL-‐1 β from the zebrafish, amphibian, bird, and mammal Type I

IL-‐1 β [49]. Golden tilefish IL-‐1β exhibited stronger homology with Type II molecules, is short, at only

251 amino acids in length, and therefore, likely belonged to the Type II group.

3.2 Cloning and sequence analysis of red snapper IL-‐1β

Degenerate primers rsIL-‐1b_F and rsIL-‐1b_R were utilized to amplify an initial 430 bp fragment

from red snapper spleen cDNA, according to PCR conditions given (Table 1). This fragment was cloned,

sequenced, and its 297 bp coding region was translated into a 99 amino acid sequence. The partial

amino acid sequence for red snapper IL-‐1β had good homology with other teleost IL-‐1β proteins,

including European seabass (67%) and southern Bluefin tuna (67%; Table 3). It shared 64% identity with

the 43% of golden tilefish IL-‐1β it covered, although it did not share any N-‐glycosylation sites (Table 3;

Figure 5). The partial sequence did not include the family signature, however, it did include β-‐sheets 3-‐

9, the majority of which showed significant levels of conservation with other vertebrate species (Figure

2). The final sequence was not cloned and confirmed from multiple fish, as there was sufficient

information to provide a comparison between golden tilefish and red snapper IL-‐1β for the purpose of

antibody production (Figure 5).

3.3 Cloning and sequence analysis of golden tilefish IL-‐8

Degenerate primers gtIL-‐8_F and gtIL-‐8_R were designed with PriFi and used to amplify, clone,

and sequence an initial 279 fragment from golden tilefish spleen cDNA (Table 1). This sequence was

then used to design primers for 5’-‐ and 3’-‐RACE, gtIL-‐8_GSP2 and gtIL-‐8_GSP1, respectively (Table 1).

20

The resulting 3’-‐RACE product was 634 bp in length and provided sufficient sequence information for

analysis, without attaching a 5’-‐RACE product (Figures 1b). Instead, the 3’-‐RACE product was assembled

with the initial fragment. This assembled sequence contained a 172 bp 5’ UTR, 297 bp translated region,

and 381 bp 3’ UTR, which contained 2 instability motifs (ATTTA), two endotoxin motifs (one TTATTTAT

and one ATATTTAT), and a polyadenylation signal (ATTAAA), ending 18 bp upstream of the poly-‐A tail.

Gene-‐specific primers were designed (gtfIL-‐8_F and gtfIL-‐8_R) to clone and confirm the IL-‐8 coding

sequence from spleen cDNA of two different golden tilefish fish according to the PCR parameters given

(Table 1). This resulted in a 479 bp product with a 297 bp coding region, which was translated into 98

amino acids.

The translated portion included the IL-‐8 family signature, CXC-‐[LIVMS]-‐x(4,6)-‐[LIVMFY]-‐x(2)-‐

[RKSEQNA]-‐x-‐[LIVM]-‐x(2)-‐[LIVMA]-‐x(5)-‐[STAG]-‐x(2)-‐C-‐x(3)-‐[EQ]-‐[LIVM](2)-‐x(9,10)-‐C-‐[LV]-‐[DN], as

CrCIqteskpigRhIakVeliipnshCeetEIIatlkktgqevCLD (Figure 6). A putative cut site for the signal peptide

was predicted between gtG22 and gtM23 (Figure 6). This site aligned with the end of the signal peptide

and start of mature protein among all other vertebrates as well [48, 49]. No N glycosylation sites were

found.

The sequence contained the CXC motif but lacked the ELR motif, the latter of which is critical for

neutrophil chemotaxis and angiogenesis in mammals [45]. The lack of a complete ELR sequence is a

characteristic pattern shared by many fish homologues [42, 57, 84]. Golden tilefish had ELH in place of

ELR, as did striped trumpeter, European seabass, hapuku, and many other fishes (Figure 7). A study

utilizing recombinant proteins for the ELR region in black seabream concluded that the ELR motif could

not be the only residues stimulating chemotaxis and that fish species lacking this motif may express an

ancestral version of the chemokine [84]. Given the high conservation of the SLGV motif in fishes prior

to the ELR-‐CXC motif, L6 has been proposed as the responsible residue for inducing chemotaxis in

21

flounder [57]. This residue was conserved in golden tilefish, so the L6 functional hypothesis may hold for

this species as well (Figure 7).

The mature golden tilefish IL-‐8 sequence shared high homology with other Perciformes,

including striped trumpeter (86% amino acid identity), Mandarin fish (84%), and European seabass (84%;

Table 2). High amino acid identity was seen with other teleosts, however, lower identities were shared

with Elasmobranchs (43%), rodents (36%) and humans (41%; Table 3). Teleosts grouped separately from

other vertebrates in the phylogenetic tree, with a division noted between the two predicted types of IL-‐

8 protein in teleosts, designated by branches separating Salmoniformes and Cypriniformes from other

bony fishes (Figure 8). As anticipated, golden tilefish clustered with other Perciformes, with high

bootstrapping statistical support (Figure 9).

3.4 Cloning and sequence analysis of red snapper IL-‐8

PriFi-‐designed degenerate primers rsIL-‐8_F and rsIL-‐8_R were used to amplify, clone, and

sequence an initial 277 bp fragment from red snapper spleen cDNA according to the PCR conditions

listed (Table 1). Gene-‐specific primers, rsIL-‐8_GSP2 and rsIL-‐8_GSP1, were designed for 5’-‐ and 3’-‐RACE

based upon this fragment yielding a 5’-‐RACE product of 279 bp and a 784 bp 3’-‐RACE product with a 127

bp overlap between the two sequences (Table 1; Figure 1c). When aligned, this produced a 937 bp

sequence with a 249 bp 5’ UTR, 300 bp translated region, and a 388 bp 3’ UTR containing 3 instability

motifs (ATTTA), two endotoxin responsive motifs (ATATTTAT) and one polyadenylation signal, ending 11

bp upstream of the poly-‐A tail. Gene-‐specific primers rsfIL-‐8_F and rsfIL-‐8_R were designed to amplify,

clone, and confirm the coding region from two different red snapper fish samples (Table 1). This

confirmed the sequence for mature red snapper IL-‐8, with a translated region of 300 bp, encoding 99

amino acids.

22

The protein sequence contained the IL-‐8 family signature in the form:

CrCIqtesrpigRhIgkVelippnshCeetEIIatlkktgqevCLD (Figure 6). The sequence contained the CXC motif but

again lacked the ELR motif, which was substituted with ELH (Figure 9). Signal P 4.1 predicted a cut site

for the signal peptide at red snapper (rs)G22 and rsM23, which aligned well with the cut site in other

vertebrates (Figures 6). As with the tilefish sequence, no N glycosylation sites were predicted.

The mature red snapper IL-‐8 protein shared the highest homology with humphead snapper (92%

amino acid identity), European seabass (88%), and Mandarin fish (88%; Table 2). It was also 84%

identical to the mature golden tilefish IL-‐8 sequence, though golden tilefish was slightly more

homologous to mammals than red snapper (Table 3; Figure 10). The red snapper partial sequence

grouped with other Perciform IL-‐8 proteins on the rooted phylogenetic tree, and branched before the

European seabass and red seabream node (Figure 8).

3.5 Cloning and sequence analysis of golden tilefish IL-‐10

Degenerate primers gtIL-‐10_F and gtIL-‐10_R were used to amplify a 441 bp fragment from

golden tilefish spleen cDNA using the PCR conditions listed (Table 1). This product was cloned,

sequenced, and used to design primers for 5’-‐ and 3’-‐RACE, though 3’-‐RACE did not yield sufficient

sequence data, and therefore, was omitted from the table (Table 1). 5’-‐RACE resulted in 513 bp

sequence, which had a 197 bp overlap with the originally cloned fragment (Figure 1d). The assembled

sequence consisted of 714 bp and a 493 bp coding region that translated into 164 amino acids.

The first teleost IL-‐10 family signature motif was conserved as follows: L-‐[FILMV]-‐x(3)-‐[ILV]-‐x(3)-‐

[FILMV]-‐x(5)-‐C-‐x(5)-‐[LIMV]-‐[LIMV]-‐x(3)-‐L-‐x(2)-‐[IV]-‐[FILMV] as LLdqtIehsLktpfaChainsILgfyLgtVL (Figure

11). Only a portion of the second teleost IL-‐10 family signature was present (Figure 12). The partial

sequence shared high homology with other teleost IL-‐10, including European seabass (76%) amino acid

identity, gilthead seabream (76%), and green spotted puffer (66%; Table 3). The final sequence was

23

confirmed via sequencing of the IL-‐10 PCR fragment from two additional golden tilefish spleen samples

(Figure 12).

3.6 Cloning and sequence analysis of red snapper IL-‐10

An initial 406 bp fragment was amplified from red snapper spleen cDNA by degenerate primers

rsIL-‐10_F and rsIL-‐10_R under the PCR conditions listed (Table 1). The fragment was cloned, sequenced,

and utilized to design gene-‐specific primers for 5’-‐ and 3’-‐RACE (Table 1). 5’-‐RACE yielded a 519 bp

fragment and 3’-‐RACE yielded a 711 bp fragment, with a 71 bp overlap between the two products

(Figure 1d). The assembled RACE sequence contained 1108 bp with a 564 bp translated region, 180 bp

5’ UTR, and a 364 bp 3’ UTR. The 3’ UTR contained three instability motifs (ATTTA), one endotoxin

responsive motif (ATATTTAT), and one polyadenylation motif (AATAAA), ending 15 bp upstream of the

poly-‐A tail. Gene-‐specific primers were designed to confirm the IL-‐10 coding sequence from two

different red snapper spleen cDNA samples, using the PCR conditions given (Table 1). The amplified

sequence was 647 bp in length with a 564 bp coding region that translated into 187 amino acids.

The translated red snapper IL-‐10 sequence contained two teleost IL-‐10 family signatures in the

following forms: L-‐[FILMV]-‐x(3)-‐[ILV]-‐x(3)-‐[FILMV]-‐x(5)-‐C-‐x(5)-‐[LIMV]-‐[LIMV]-‐x(3)-‐L-‐x(2)-‐[IV]-‐[FILMV] as

LLdqtIehsLktpfaChainsILgfyLgtVL and KA-‐x(2)-‐E-‐x-‐D-‐[ILV]-‐[FLY]-‐[FILMV]-‐x(2)-‐[ILMV]-‐[EKQZ] as

KAmgElnLLFnyIE [48, 66, 68]. Two potential N-‐glysoylation sites were predicted using the NetNGlyc 1.0

sever at rsN13 and rsN146, the latter of which was shared among all Perciformes analyzed (Figure 13). Of

note, however, is rsN146 as it was within alpha helix E, and therefore, unlikely to be functional. Mouse

and human IL-‐10 have an N glycosylation site between the D and E chains, on the surface of the

molecule, where it may be readily available for use [85].

A putative cut site for the signal peptide was predicted between residues rsC22 and rsT23 using

SignalP 4.1, which occurred after the human cut site in the multiple alignment (Figure 11). Six conserved

24

cysteine residues were observed, including two teleost-‐specific cysteines (rsC26 and rsC31) and four

vertebrate conserved cysteines responsible for forming intra-‐chain disulfide bonds in humans (rsC30,

rsC80, rsC130, and rsC136; Figure 11; Figure 13). Within fish there was good conservation among all six

alpha helical chains, however, discrepancies arose, particularly in chain D and E, when the comparison

was extended to chicken, frog, and mammals (Figure 11; Figure 13).

There are 28 conserved hydrophobic residues in humans, critical for stabilizing the molecule

[86]. Of these, 10 were identical in red snapper IL-‐10 and 16 were substituted for other hydrophobic

amino acids (Figure 11). There are six conserved amino acids between viral and human IL-‐10, which

implies their importance, four of which were identical in red snapper and one of which was similarly

substituted by another amino acid [86]. In addition, red snapper IL-‐10 contained all five amino acid

residues conserved between human IL-‐10 and IFNγ, residues also implicated in core stabilization [87].

The critical core-‐stabilizing surface ion pair hR27-‐hE151 was perfectly conserved in red snapper [85]. The

hI69 residue, responsible for the molecules immunostimulatory properties in humans, was not conserved

directly in the multiple sequence alignment, however, all teleosts, including red snapper, had an

isoleucine residue preceding hI69 by one amino acid [88].

In humans, IL-‐10 cellular action requires two receptor chain assemblies to occur sequentially on

the cell surface, at Site 1 and Site 2 [89]. There are nine crucial residues for binding to IL-‐10R1 in the Site

1 region [88]. Red snapper shared four of them, hP20, hR27, hK138, and hE151 [89]. However, of the ten

important residues for binding IL-‐10/IL-‐10R2 at Site 2, only hH90 was conserved in red snapper [89]. The

hR24 residue has a shared importance in both Site 1 and Site 2 binding [89]. While snapper did not have

this residue, it contained an arginine one amino acid further along the sequence (Figure 11).

Importantly, IL-‐10R1 has been confirmed in several fish species, including grass carp, goldfish, and

zebrafish, definitive teleost orthologues to human IL-‐10R2 have not been confirmed [67]

25

Red snapper IL-‐10 shared the highest amino acid identity with European seabass (77%), gilthead

seabream (76%), and cod (66%; Table 2). Lower homology was observed with Cypriniformes (45-‐50%),

frog (37%), chicken (33%), and mammals (29%; Table 3). Red snapper IL-‐10 clustered with European

seabass after phylogenetic analysis and there was a clear division between Cypriniformes and other

teleosts, as well as between fishes, chicken, and mammals (Figure 14). There was high homology

between red snapper and golden tilefish IL-‐10 molecules, with 99% identity over the sequenced portion

of the protein (Table 3; Figure 12). The high identity is unusual, given the typical range of amino acid

homology in fishes, however, the nucleotide sequences for golden tilefish and red snapper were

confirmed from at least two fish, each. The high identity could be an artifact of the Fish Genome

Duplication event, where similar gene copies have been conserved in these fish, or, it is possible the

molecule is highly conserved among Perciformes, which may become more apparent as more fish IL-‐10

proteins are sequenced.

3.7 Cloning and sequence analysis of golden tilefish TNFα

Degenerate primers were used to obtain a 572 bp fragment from golden tilefish spleen cDNA

isolated from spleen under the PCR conditions given (Table 1). This fragment was cloned, sequenced,

and used to create gene-‐specific primers for 5’-‐/3’-‐RACE, although the 5’-‐RACE primer failed to provide

sufficient data for inclusion (Table 1). The 3’-‐RACE product was 779 bp in length, and contained the

entirety of the initially isolated fragment. The assembled product was 795 bp in length with a 290 bp

coding region that was translated into 96 amino acids. The resulting amino acid sequence corresponded

with the C terminal of the TNFα protein, and while it was not cloned and confirmed, sufficient data was

obtained to indicate significant homology with a portion of red snapper TNFα and that of other

Perciformes (Table 3; Figure 15; Figure 16).

26

3.8 Cloning and sequence analysis of red snapper TNFα

An initial 474 bp fragment was obtained from red snapper spleen cDNA using degenerate

primers rsTNFa_F and TNFa_R under the PCR conditions given (Table 1). This fragment was cloned,

sequenced, and used to design primers for 5’-‐/3’-‐RACE (Table 1). The 5’-‐RACE reaction resulted in a 744

bp product, the 3’-‐RACE reaction yielded a 777 bp product, and there was a 91 bp overlap between the

two fragments (Figure 1e). The assembled product was 1431 bp in length, with a 764 bp translated

region, 156 bp 5’ UTR, and a 510 bp 3’ UTR. The 3’ UTR contained 3 instability motifs (ATTTA), two

endotoxin responsive motifs (TTATTTAT and ATATTTAT) and one polyadenylation signal (ATTAAA)

ending 4 bp upstream from the poly-‐A tail. This product was used to design primers to amplify, clone,

and confirm the coding sequence of red snapper TNFα sequence from the spleen cDNA from two

different fish (Table 1). The resulting fragment was 884 bp in length with a 768 bp coding region, which

translated into a 256 amino acid product.

The red snapper TNFα sequence contained the TNFα family signature, [LV]-‐x-‐[LIVM]-‐x(3)-‐G-‐

[LIVMF]-‐Y-‐[LIVMFY](2)-‐x(2)-‐[QEKHL], as ivIpqtGLYFVysQ (Figure 15). Two cysteine residues were

observed, at rsC156 and rsC196 (Figure 17). These appear to correspond to the two human cysteine

residues responsible for proper folding of mature TNFα. In the multiple sequence alignment, red

snapper C156 appeared one residue before the mammalian cysteine, while the position of rsC196 was

conserved among all vertebrates examined (Figure 15). A transmembrane domain was predicted by

TMPred from amino acid rsV35 through rsW54 (Figure 15). This corresponded perfectly with the

predicted transmembrane domain in other Perciformes, suggesting that red snapper TNFα may be

membrane-‐bound (Figures 17). The TACE cleavage site was identified at rsT86-‐rsL87, a few residues later

than where it occurs in Cypriniformes and mammals (Figure 17). In having both a predicted

transmembrane domain and TACE cleavage site, it is possible that red snapper TNFα is secreted in a

manner similar to its mammalian orthologue, with the mature molecule freely secreted from its

27

membrane-‐bound state after cleavage at the TACE site [38]. No glycosylation sites were predicted. High

conservation in the C terminal region of the protein was observed among all analyzed vertebrates,

common to the TNF homology domain (THD; Figure 15; Figure 17).

Red snapper TNFα shared the highest homology with barred knifejaw (81% amino acid identity),

striped trumpeter (80%), and orange-‐spotted grouper (78%; Table 2). Homology was reduced between

red snapper and Salmoniformes (40%), Cypriniformes (38-‐46%), frog (27%), chicken (42%, over a partial

sequence), and mammals (35-‐36%; Table 3). This was not surprising, given the high degree of genetic

divergence noted in TNFα genes [62]. Carp, rainbow trout, and salmon all exhibit multiple isoforms of

the gene, likely as a result of a tetraploidation event which occurred along these lineages, while the two

forms discovered in bluefin tuna are indicative of gene duplication events [60, 62, 90, 91]. Phylogenetic

analysis suggested clustering of red snapper with red seabream, and this pair clustered with other

Perciformes (Figure 18). While the analysis displayed vague distinctions among Perciformes, there was

clear clustering between the Perciform and Pleuronectiform node, and Tetraodontiformes,

Salmoniformes, Cypriniformes, and tetrapods (Figure 18). Red snapper TNFα shared 80% amino acid

identity with the partial TNFα sequence from golden tilefish, as may be expected as much of this

covered the THD region (Table 3; Figure 16).

28

Figure 1: Schematic of PCR results including the locations of the coding region, 5’ UTR, 3’ UTR, locations at which primers bound, and the size and position of the resulting fragments. Genes shown are: a) golden tilefish IL-‐1β, b) golden tilefish IL-‐8, c) red snapper IL-‐8, d) red snapper IL-‐10, and e) red snapper TNFα.

30

Figure 2. Clustal W multiple sequence alignment of representative vertebrate IL-‐1β, shaded with BOXSHADE. The family signature is boxed, the mammalian ICE cleavage site is marked with an arrow, and the vertebrate conserved cysteine is marked with an asterisk (*). The 12 β-‐sheets are underlined and numbered. Residues involved with ligand-‐receptor binding at Site A are labeled with an “a” above the alignment and those involved with binding at Site B are labeled with a “b”. The GenBank accession codes used were: European seabass (CAC41006.1), red seabream (AAP33156.1), southern bluefin tuna (AGB24759.1), olive flounder (BAB8682.1), Atlantic salmon (NP_001117054.1), rainbow trout (CAA11684.1), Atlantic cod (ABV59377.1), haddock (CAJ58294.1), zebrafish (AAH98597.1), smaller spotted catshark (CAC09453.1), African clawed frog (CAB53499.1), chicken (CAA75239.1), mouse (AAA39276.1), and human (AAA59135.1).

31

Table 2. Percent amino acid identity between confirmed, mature amino acid sequence and the top five most homologous fish species, according to BLASTp analysis.

Target Species Highest.scoring.teleosts Order Query.cover E.valueAmino.acid.identity.(%)

European)seabass)|)CAC41006.1 Perciformes 98% 5.00E=119 70%

Mandarin)fish)|)AAV65041.1 Perciformes 100% 1.00E=117 70%

Barred)knifejaw)|)ACH87392.1 Perciformes 100% 3.00E=117 68%

Striped)trumpeter)|)ACQ99510.1 Perciformes 98% 2.00E=116 70%

Red)seabream)|)AAP33156.1 Perciformes 97% 1.00E=108 67%

European)seabass)|)CAM32186.1 Perciformes 100% 8.00E=53 84%


Mandarin)fish)|)AFK65606.1 Perciformes 98% 7.00E=52 84%

Humphead)snapper)|)AGV99968.1 Perciformes 96% 7.00E=51 83%

Red)seabream)|)ADK35756.1 Perciformes 100% 8.00E=51 84%

European)seabass)|)CAM32186.1 Perciformes 98% 8.00E=53 88%

Humphead)snapper)|)AGV99968.1 Perciformes 95% 2.00E=55 92%

Mandarin)fish)|)AFK65606.1 Perciformes 97% 4.00E=55 88%

Red)seabream)|)ADK35756.1 Perciformes 100% 5.00E=55 87%


European)seabass)|)ABH09454.1 Perciformes 99% 2.00E=96 77%

Gilthead)seabream)|)AGS55345.1 Perciformes 92% 8.00E=93 76%

Atlantic)cod)|)ABV64720.1 Gadiformes 84% 7.00E=74 66%

Green)spotted)puffer)|)CAD67773.1 Tetraodontiformes 87% 2.00E=73 65%

Rainbow)trout)|)NP_001232028.1 Salmoniformes 83% 2.00E=57 57%

Barred)knifejaw)|)ACM69339.1 Perciformes 98% 2.00E=158 81%


Orange=spotted)grouper)|)AEH59794.1 Perciformes 98% 1.00E=153 78%

Large)yellow)croaker)|)ABK62876.1 Perciformes 98% 2.00E=153 79%

European)seabass)|)AAZ20770.1 Perciformes 98% 8.00E=152 77%

IL=1β

IL=10)

TNFα

IL=8

Golden)tilefish)

Golden)tilefish)

Red)snapper

Red)snapper

Red)snapper

32

Table 3: Percent amino acid identity among vertebrate groups. Asterisks (*) denote partial sequences.

Snapper TilefishGolden tilefish (F) Bony fishes Perciformes 64 xRed snapper (C) * Bony fishes Perciformes x 64

European seabass | CAC80553.1 Bony fishes Perciformes 67 70Red seabream | AAP33156.1 Bony fishes Perciformes 66 67

Southern bluefin tuna | AGB24759.1 Bony fishes Perciformes 67 63Olive flounder | BAB8682.1 Bony fishes Pleuronectiformes 63 56

Atlantic salmon | NP_001117054.1 Bony fishes Salmoniformes 61 51Rainbow trout | CAA1164.1 Bony fishes Salmoniformes 59 51Atlantic cod | ABV59377.1 Bony fishes Gadiformes 61 51Haddock | CAJ58294.1 Bony fishes Gadiformes 61 51Zebrafish | AAH98597.1 Bony fishes Cypriniformes 45 33

Smaller spotted catshark | CAC09435.1 Cartilaginous fishes Carcharhiniformes 40 28African clawed frog | CAB53499.1 Amphibians Anura 38 33

Chicken | CAA75239.1 Birds Galiformes 38 31Mouse | AAA39276.1 Mammals Rodentia 42 32Human | AAA59135.1 Mammals Primates 47 29Golden tilefish (H) Bony fishes Perciformes 84 xRed snapper (C) Bony fishes Perciformes x 84

European seabass | CAM32186.1 Bony fishes Perciformes 88 84Red seabream | ADK35756.1 Bony fishes Perciformes 87 84Olive flounder | AAL05442.1 Bony fishes Pleuronectiformes 69 70Atlantic salmon | ADK11046.1 Bony fishes Salmoniformes 58 57Rainbow trout | AAO25646.1 Bony fishes Salmoniformes 61 59

Haddock | CAD97422.2 Bony fishes Gadiformes 68 74Silver carp | AEX32919.1 Bony fishes Cypriniformes 63 59

Zebrafish | XP_001342606.2 Bony fishes Cypriniformes 59 58Smaller spotted catshark | CAD91126.3 Cartilaginous fishes Carcharhiniformes 45 43

African clawed frog | AEB96252.1 Amphibians Anura 54 46Chicken | ABD49203.2 Birds Galiformes 47 49Marmot | ABY67262.1 Mammals Rodentia 35 36Human | AAH13615.1 Mammals Primates 40 41Golden tilefish (T8)* Bony fishes Perciformes 99 xRed snapper (C) Bony fishes Perciformes x 99

European seabass | CAK29522.1 Bony fishes Perciformes 77 78Gilthead seabream | AGS55345.1 Bony fishes Perciformes 76 76Green spotted puffer | CAD67773.1 Bony fishes Tetraodontiformes 65 66Atlantic salmon | ABM46994.1* Bony fishes Salmoniformes 50 50

Atlantic cod | ABV64720.1 Bony fishes Gadiformes 66 66Grass carp | AEA50953.1 Bony fishes Cypriniformes 45 43Zebrafish | AAW78362.1 Bony fishes Cypriniformes 50 47

African clawed frog | CAE92388* Amphibians Anura 37 35Chicken | CAF18432.1 Birds Galiformes 33 32Rat | AAA41425.1 Mammals Rodentia 29 28

Human | AAI04254.1 Mammals Primates 29 28Golden tilefish (H)* Bony fishes Perciformes 80 xRed snapper (C) Bony fishes Perciformes x 80

European seabass | AAZ20770.1 Bony fishes Perciformes 77 90Red seabream | AAP76392.1 Bony fishes Perciformes 77 79

Pacific bluefin tuna | BAG72141.1 Bony fishes Perciformes 68 85Fugu | NP_001033074.1 Bony fishes Tetraodontiformes 64 69

Olive flounder | BAA94970.1 Bony fishes Pleuronectiformes 65 72Rainbow trout | CAB92316.1 Bony fishes Salmoniformes 48 61Grass carp | ADY80577.1 Bony fishes Cypriniformes 46 45Zebrafish | NP_998024.2 Bony fishes Cypriniformes 38 45

African clawed frog | BAF95747.1 Amphibians Anura 27 26Chicken | AEW10516.1* Birds Galiformes 42 36Mouse | BAA19513.1 Mammals Rodentia 35 43Human | NP_000585.2 Mammals Primates 36 43

IL-‐10

TNFα

Cytokine Species Amino acid identity (%)

IL-‐1β

IL-‐8

OrderVertebrate group

33

Figure 3. Multiple sequence alignment of golden tilefish IL-‐1β with the top five most homologous species, according to BLASTP. Identical (*) or similar (: or .) amino acid sequences were identified by Clustal Omega. Predicted N-‐glycosylation sites are boxed and both the family signature motif and conserved cysteine residues are shaded in grey. The locations corresponding to the 12 β-‐sheets are underlined, with the corresponding human sequence below along with the identifying sheet number. The mammalian ICE cleavage site is marked with an arrow. The GenBank accession codes used were: European seabass (CAC41006.1), mandarin fish (AAV65041.1), barred knifejaw (ACH87392.1), striped trumpeter (ACQ99510.1), and red seabream (AAP33156.1).

34

Figure 4. Rooted phylogenetic tree of vertebrate IL-‐1β proteins, produced in MEGA 6 with a NJ method. The p-‐distance method was used, bootstrapped 10,000 times, with results reflected at the branch nodes. Partial sequences were excluded and human IFNγ was used as an outgroup. The Genbank Accession codes used were: European seabass (CAC80553.1), red seabream (AAP33156.1), southern bluefin tuna (AGB24759.1), olive flounder (BAB8682.1), Atlantic salmon (NP_01117054.1), rainbow trout (CAA11684.1), Atlantic cod (ABV59377.1), haddock (CAJ58294.1), zebrafish (AAH98597.1), smaller spotted catshark (CAC09435.1), African clawed frog (CAB53499.1), chicken (CAA75239.1), mouse (AAA39276.1), human (AAA59135.1) and human IFNγ (AAB59534.1).

35

Figure 5. Clustal Omega alignment between golden tilefish and red snapper IL-‐1β. Identical (*) or similar (: or .) amino acid sequences were identified and the locations of the β-‐sheets covered by both sequences are underlined and numerated, corresponding to their sheet number in the complete protein (1-‐12).

36

Figure 6. Clustal W multiple sequence alignment of representative vertebrate IL-‐8, shaded with BOXSHADE. The family signature is boxed, the ELR motif is marked by carrots (^) and the CXC motif is marked using asterisks (*). The putative cut site for the signal peptide is marked with an arrow above the sequence. The GenBank accession codes used were: European seabass (CAM32186.1), red seabream (ADK35756.1), olive flounder (AAL05442.1), Atlantic salmon (ADK11046.1), rainbow trout (AAO25646.1), haddock (CAD97422.2), silver carp (AEX32919.1), zebrafish (XP_001342606.2), smaller spotted catshark (CAD91126.3), African clawed frog (AEB896252.1), chicken (ABD49203.2), marmot (ABY67262.1), and human (AAH13615.1).

37

Figure 7. Multiple sequence alignment of golden tilefish IL-‐8 with the top five most homologous species, according to BLASTP. Identical (*) or similar (: or .) amino acid sequences were identified by Clustal Omega. The ELR motif is boxed, the CXC motif is underlined, the putative cut site for the signal peptide is marked with an arrow, and the family signature is shaded in grey. The GenBank accession codes used were: European seabass (CAM32186.1), Mandarin fish (AFK65606.1), humphead snapper (AGV99968.1), red seabream (ADK35756.1), and striped trumpeter (ACQ99511.1).

38

Figure 8. Rooted phylogenetic tree of vertebrate IL-‐8 proteins, produced in MEGA 6 with a NJ method. The p-‐distance method was used, bootstrapped 10,000 times, with results reflected at the branch nodes. Partial sequences were excluded and human IFNγ was used as an outgroup. The Genbank accession codes used were: European seabass (CAM32186.1), red seabream (ADK35756.1),olive flounder (AAL05442.1), Atlantic salmon (ADK11046.1), rainbow trout (AAO25646.1), haddock (CAD97422.2), silver carp (AEX32919.1), zebrafish (XP_001342606.2), smaller spotted catshark (CAD91126.3), African clawed frog (AEB96252.1), chicken (ABD49203.2), marmot (ABY67262.1), human (AAH13615.1) and human IFNγ (AAB59534.1).

39

Figure 9. Multiple sequence alignment of red snapper IL-‐8 with the top five most homologous species, according to BLASTP. Identical (*) or similar (: or .) amino acid sequences were identified by Clustal Omega. The ELR motif is boxed, the CXC motif is underlined, the putative cut site for the signal peptide is marked with an arrow, and the family signature is shaded in grey. The GenBank accession codes used were: European seabass (CAM32186.1), Mandarin fish (AFK65606.1), humphead snapper (AGV99968.1), red seabream (ADK35756.1), and striped trumpeter (ACQ99511.1).

40

Figure 10. Clustal Omega alignment between golden tilefish and red snapper IL-‐8. Identical (*) or similar (: or .) amino acid sequences were identified and the position of the ELR and CRC motifs are underlined.

41

Figure 11. Clustal W multiple sequence alignment of representative vertebrate IL-‐10, shaded with BOXSHADE. The family signature is boxed, the four vertebrate conserved cysteines are marked with asterisks (*), and the alpha helical domains are underlined and lettered. The putative cut site for the human signal peptide is marked with an arrow below the alignment and the putative cut site for the snapper signal peptide is marked with an arrow above the alignment. Below the alignment, residues critical for ligand-‐receptor binding at Site 1 are marked with a “1” and those critical for ligand-‐receptor binding at Site 2 are labeled with a “2.” The residue responsible for hIL-‐10 immunostimulatory properties is marked with an “I” below the sequence. Above the sequence, hydrophobic residues are

42

marked with an “h,” critical core-‐stabilizing amino acids for IL-‐10 and IFNγ are marked with a “c” and the viral conserved amino acids are marked with a “v.” The GenBank accession codes used were: European seabass (CAK29522.1), gilthead seabream (AGS55345.1), green spotted puffer (CAD67773.1), Atlantic salmon (ABM46994.1), Atlantic cod (ABV64720.1), grass carp (AEA50953.1), zebrafish (AAW78362.1), African clawed frog (CAE92388.1), chicken (CAF18432.1), rat (AAA41425.1) and human (AAI04254.1).

43

Figure 12. Clustal Omega alignment between red snapper and golden tilefish IL-‐10. Identical (*) or similar (: or .) amino acid sequences were identified and the positions of the six alpha helical domains are underlined and labeled.

44

Figure 13. Multiple sequence alignment of red snapper IL-‐10 with the top five most homologous species, according to BLASTP. Identical (*) or similar (: or .) amino acid sequences were identified by Clustal Omega. Two predicted N-‐glycosylation site are boxed and the two IL-‐10 family signatures are highlighted in grey. Six conserved cysteine residues are highlighted in grey, including two fish-‐specific residues at rsC26 and rsC31. The six alpha helical domains are underlined and lettered “a-‐f.” The putative cut site for the signal peptide is marked with an arrow. The GenBank accession codes used were: European seabass (CAK29522.1), gilthead seabream (AGS55345.1), green spotted puffer (CAD67773.1), Atlantic cod (ABV64720.1), and rainbow trout (NP_001232028.1).

45

Figure 14. Rooted phylogenetic tree of vertebrate IL-‐10 proteins, produced in MEGA 6 with a NJ method. The p-‐distance method was used, bootstrapped 10,000 times, with results reflected at the branch nodes. Partial sequences were excluded and human IFNγ was used as an outgroup. The GenBank accession codes used were: European seabass (CAK29522.1), gilthead seabream (AGS55345.1), green spotted puffer (CAD67773.1), Atlantic cod (ABV64720.1), grass carp (AEA50953.1), zebrafish (AAW78362.1), chicken (CAF18432.1), rat (AAA41425.1), human (AAI04254.1), and human IFNγ (AAB59534.1).

47

Figure 15. Clustal W multiple sequence alignment of representative vertebrate TNFα, shaded with BOXSHADE. The family signature is boxed, the predicted Perciform transmembrane domain is underlined, the snapper TACE cleavage site is marked with an arrow above the alignment, and the human TACE cleavage site is marked with an arrow below the alignment. The partially conserved cysteine residue is notated with a colon (:) and the fully conserved cysteine is marked with an asterisk (*). The GenBank accession codes used were: European seabass (AAZ20770.1) , red seabream (AAP76392.1), Pacific bluefin tuna (BAG72141.1), fugu (NP_001033074.1), olive flounder (BAA94970.1), rainbow trout (CAB92316.1), grass carp (ADY80577.1), zebrafish (NP_998024.2), African clawed frog (BAF95747.1), chicken (AEW10516.1), mouse (BAA19513.1), and human (NP_00585.2).

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Figure 16. Clustal Omega alignment between red snapper and golden tilefish TNFα. Identical (*) or similar (: or .) amino acid sequences were identified.

49

Figure 17. Multiple sequence alignment of red snapper TNFα with the top five most homologous species, according to BLASTP. Identical (*) or similar (: or .) amino acid sequences were identified by Clustal Omega. The predicted transmembrane domain is underlined, the TACE cleavage site is boxed, and the family signature and two conserved cysteine residues are highlighted in grey. The GenBank accession codes used were: barred knifejaw (ACM69339.1), striped trumpeter (ACQ99509.1), orange-‐spotted grouper (AEH59794.1), large yellow croaker (ABK62876.1), and European seabass (AAZ20770.1).

50

Figure 18. Rooted phylogenetic tree of vertebrate TNFα proteins, produced in MEGA 6 with a NJ method. The p-‐distance method was used, bootstrapped 10,000 times, with results reflected at the branch nodes. Partial sequences were excluded and human IFNγ was used as an outgroup. The GenBank accession codes used were: European seabass (AAZ20770.1) , red seabream (AAP76392.1), Pacific bluefin tuna (BAG72141.1), fugu (NP_001033074.1), olive flounder (BAA94970.1), rainbow trout (CAB92316.1), grass carp (ADY80577.1), zebrafish (NP_998024.2), African clawed frog (BAF95747.1), mouse (BAA19513.1), human (NP_00585.2), and human IFNγ (AAB59534.1).

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CHAPTER FOUR:

CONCLUSIONS AND FUTURE WORK

4.1 Conclusions

In this study, IL-‐1β, IL-‐8, IL-‐10, and TNFα were, for the first time, isolated, cloned, sequenced,

and characterized from golden tilefish and red snapper. The full amino acid sequences were obtained

for golden tilefish IL-‐1β and IL-‐8, as well as for red snapper IL-‐8, IL-‐10 and TNFα. Partial sequences were

obtained for golden tilefish IL-‐10 and TNFα, as well as red snapper IL-‐1β. In comparing these sequences,

it was evident that there was a high level of conservation across all four genes in Perciformes, however,

there was divergence among other teleost lineages, particularly when Perciformes were compared to

Salmoniformes and Cypriniformes. Homology was low when golden tilefish and red snapper cytokine

sequence were compared with other vertebrate groups, including cartilaginous fishes, amphibians,

birds, and mammals.

There was conservation among secondary structural elements across vertebrates, evidenced in

both the β-‐sheets of IL-‐1β and the alpha helices of IL-‐10. There also appeared to be similar cleavage

sites present in red snapper and golden tilefish for the mammalian orthologues known to be

proteolytically cleaved prior to generation of the mature peptide, as predicted in the IL-‐8, IL-‐10, and

TNFα teleost sequences. However, there was low conservation at known receptor binding sites

between mammals and teleosts, particularly in the IL-‐1β and IL-‐10 sequences presented here. This

would suggest diversity among receptor sites and binding mechanisms among vertebrate groups.

Coupled with the low overall amino acid identity observed between mammals and the teleosts, the

52

likelihood that cross-‐reactivity would occur between mammalian cytokine antibodies and red snapper or

golden tilefish is limited. Importantly, this suggests that commercially available detection kits for

mammalian cytokines probably would not work well for accurately detecting and quantifying cytokine

expression in Perciformes. The results of this study unequivocally substantiate the need for species-‐

specific evaluation of these critical immune system regulators.

4.2 Future work

Building upon the information gathered here, antibodies can be designed for Perciform-‐specific

IL-‐1β, IL-‐8, IL-‐10, and TNFα. These antibodies can then be multiplexed into a magnetic bead-‐based

assay for detection of the above-‐mentioned cytokines in fish serum, plasma, and cell culture

supernatant. Given the high level of nucleotide and amino acid similarity between red snapper and

golden tilefish, as well as among the available Percifromes, it is possible that this kit will be cross-‐

reactive with many families in this large teleost order.

A suite of in vivo and in vitro gene expression studies would aid in our understanding of cytokine

expression by evaluating the timing and magnitude of cytokine release upon exposure to contaminants,

such as DWH crude and Corexit 9500. Correlations could be investigated between gene expression in

field collected golden tilefish and red snapper samples caught in the vicinity of the DWH explosion from

2013 and 2014, and the resulting circulating serum cytokine concentration. Additional study goals could

include an analysis of the timing and magnitude of the expression of different markers along the PAH

metabolic pathway leading to the induction of an immune response.

The thoroughly validated kit would provide a novel tool for the rapid assessment of immune

system status in Perciformes. Not only would this be useful to aid in fishery management decisions

during future contamination events, but also for daily use in the burgeoning aquaculture field. This

work will form the basis of the author’s PhD dissertation, beginning January 2015.

53

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63

APPENDIX A:

MAP OF 2013 TISSUE SAMPLING STATIONS

Figure A1: 2013 longline stations aboard the R/V Weatherbird II yielding tissue samples. Yellow-‐circled stations indicate the locations from which golden tilefish spleen samples were used in this study. Red-‐circled stations indicate the locations from which red snapper spleen samples were used in this study.

64

APPENDIX B:

2013 TISSUE SAMPLING LOCATION DATA

Table B1: 2013 longline tissue sampling location data. Latitude and longitude data are provided in degrees, minutes, decimal minutes format from shipboard GPS data at the start and end of setting out gear. The mean depth is the average of the start and end shipboard depth data obtained while setting out the gear.

Station ID Sampling dateLatitude

at Set StartLongitude at Set Start

Latitude at Set End

Longitude at Set End

Mean depth (m)

PCB03-‐100 8/15/2013 29o 41.360 86o 24.080 29o 44.101 86o 19.080 101

SL7-‐150 8/15/2013 29o 29.226 86o 40.371 29o 26.437 86o 44.838 329

SL14-‐60 8/16/2013 29o 27.619 87o 26.448 29o 30.645 87o 21.768 235

SL14-‐100 8/16/2013 29o 24.292 87o 29.536 29o 19.455 87o 29.499 362

SL8-‐40 8/19/2013 29o 53.263 87o 17.663 29o 49.051 87o 20.614 54

SL8-‐100 8/19/2013 29o 44.666 87o 11.552 29o 42.253 87o 07.648 211

SL9-‐80 8/20/2013 29o 17.746 88o 00.589 29o 16.038 87o 56.303 191

SL9-‐150 8/20/2013 29o 14.984 87o 59.256 29o 13.356 87o 54.006 328

SL10-‐40 8/21/2013 29o 12.305 88o 52.125 29o 08.116 88o 53.573 59

HE-‐365 8/25/2013 28o 22.609 90o 30.839 28o 18.876 90o 27.504 49

SL16-‐150 8/26/2013 28o 37.780 90o 00.397 28o 34.138 89o 56.721 204

SL12-‐100 8/26/2013 28o 30.962 89o 36.251 28o 33.833 89o 33.017 381

SL11-‐150 8/27/2013 29o 02.685 88o 36.926 29o 03.926 88o 32.267 328

SL5-‐200 8/28/2013 28o 28.897 86o 07.870 28o 29.553 86o 02.774 371

SL5-‐100 8/29/2013 28o 33.130 85o 22.370 28o 32.686 85o 27.755 179

SL4-‐40 8/29/2013 28o 05.265 84o 26.300 28o 07.896 84o 22.086 61

65

APPENDIX C:

GOLDEN TILEFISH AND RED SNAPPER BIOMETRICS DATA

Table C1: Golden tilefish and red snapper biometrics data, specific to fish whose spleens were used in this study.

Species cDNA Sample ID Station ID Sex Standard Length (cm) Fork Length (cm) Weight (kg)

F SL11-‐150 F 58 66 3.672

G SL11-‐150 M 58 68 4.054

T8 SL14-‐100 U 57 63 2.630

T9 SL14-‐100 U 56 63 2.974

C SL4-‐40 M 57 65 4.508

E SL4-‐40 F 62 70 5.274

Golden tilefish

Red snapper

66

APPENDIX D:

EXTENDED PCR PARAMETERS

Table D1. Extended PCR parameters for primers used.

Temp%(C)% Time%(min) Temp%(C)% Time%(min) Temp%(C)% Time%(min) Temp%(C)% Time%(min) Temp%(C)% Time%(min)

BA 94 5 94 0.75 55 0.75 72 0.75 72 5 35

rs_IL:1b 94 5 94 0.75 48 0.75 72 0.75 72 10 35

rs_IL:8 94 5 94 0.5 50 0.5 72 1 72 10 35

rs_IL:10 94 5 94 0.75 55 0.75 72 0.75 72 10 35

rs_TNFa 94 5 94 0.5 50 0.5 72 1 72 10 35

gtIL:1b 94 5 94 0.75 48 0.75 72 0.75 72 10 35

gtIL:8 94 5 94 0.5 50 0.5 72 1 72 10 35

gtIL_10 94 5 94 0.75 54 0.75 72 0.75 72 10 35

gt_TNFa 94 5 94 0.5 48 0.5 72 1 72 10 35

T7/SP6 94 5 94 0.5 48 0.5 72 0.75 72 5 35

rsIL:8_GSP1 94 5 94 0.5 68 0.5 72 1 72 10 35

rsIL:8_GSP2 94 5 94 0.5 66 0.5 72 1 72 10 35

rsIL:10_GSP1 94 5 94 0.5 68 0.5 72 1 72 10 35

rsIL:10_GSP2 94 5 94 0.5 68 0.5 72 1 72 10 35

rsTNFa_GSP1 94 5 94 0.5 70 0.5 72 1 72 10 35

rsTNFa_GSP2 94 5 94 0.5 70 0.5 72 1 72 10 35

gtIL:1b_GSP1 94 5 94 0.5 64 0.5 72 1 72 10 35

gtIL:1b_GSP2 94 5 94 0.5 64 0.5 72 1 72 10 35

gtIL:8_GSP1 94 5 94 0.5 70 0.5 72 1 72 10 35

gtIL:10_GSP2 94 5 94 0.5 62 0.5 72 1 72 10 35

gtTNFa_GSP1 94 5 94 0.5 60 0.5 72 1 72 10 35

rsf_IL:8_F 94 5 94 0.5 54 0.5 72 1 72 10 35

rsf_IL:8_R 94 5 94 0.5 54 0.5 72 1 72 10 35

rsf_IL:10_F 94 5 94 0.5 54 0.5 72 1 72 10 35

rsf_IL:10_R 94 5 94 0.5 54 0.5 72 1 72 10 35

rsf_TNFa_F 94 5 94 0.5 56 0.5 72 1 72 10 35

rsf_TNFa_R 94 5 94 0.5 56 0.5 72 1 72 10 35

gtf_IL:1b_F 94 5 94 0.5 54 0.5 72 1 72 10 35

gtf_IL:1b_R 94 5 94 0.5 54 0.5 72 1 72 10 35

gtf_IL:8_F 94 5 94 0.5 54 0.5 72 1 72 10 35

gtf_IL:8_R 94 5 94 0.5 54 0.5 72 1 72 10 35

#"of"CyclesReaction

Initital"Denaturation" Denaturation Annealing Extension Final"Extension"

67

APPENDIX E:

EXPERIMENTALLY OBTAINED CYTOKINE NUCLEOTIDE SEQUENCES

Figure E1. Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish IL-‐1β, derived from the assembled 5’-‐ and 3’-‐RACE products. Black text reflects the 5’ and 3’ UTR, the red text indicates the start (ATG) and stop (TAA) codons, the continuous green and blue text indicate the coding region for the golden tilefish IL-‐1β protein, and the blue text represents the overlap between the 5’ and 3’-‐RACE products.

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Figure E2. Confirmed 5’ to 3’ nucleotide sequence for the coding region of golden tilefish IL-‐1β, obtained via cloning from two different fish. Black text represents the 5’ and 3’ UTR, red text indicates the start (ATG) and stop (TAA) codon, and green text designates the coding region.

Figure E3. Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper IL-‐1β, derived from the partial cloned fragment. Black text designates the 5’ UTR, red text reflects the start codon (ATG) and green text indicates the coding region for the red snapper IL-‐1β protein.

Figure E4. Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish IL-‐8, derived from the 3’ RACE product. Black text reflects the 5’ and 3’ UTR, the red text indicates the start (ATG) and stop (TGA) codons, the continuous green text indicates the coding region for the golden tilefish IL-‐8 protein.

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Figure E5. Confirmed 5’ to 3’ nucleotide sequence for the coding region of golden tilefish IL-‐8, obtained via cloning from two different fish. Black text represents the 3’ UTR, red text indicates the start (ATG) and stop (TGA) codon, and green text designates the coding region.

Figure E6. Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper IL-‐8, derived from the assembled 5’-‐ and 3’-‐RACE products. Black text reflects the 5’ and 3’ UTR, the red text indicates the start (ATG) and stop (TGA) codons, the green text marks the coding region for the red snapper IL-‐8 protein, and the blue text represents the overlap between the 5’-‐ and 3’-‐RACE products.

Figure E7. Confirmed 5’ to 3’ nucleotide sequence for the coding region of red snapper IL-‐8, obtained via cloning from two different fish. Black text represents the 5’ and 3’ UTR, red text indicates the start (ATG) and stop (TGA) codon, and green text designates the coding region.

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Figure E8. Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish IL-‐10, derived from the assembled 5’ RACE and initial fragment products. Black text reflects the 5’ UTR, the red text indicates the start (ATG) codon, the green text marks the coding region for the golden tilefish IL-‐10 protein, and the blue text represents the overlap between the 5’-‐RACE and initial fragment products. The initial fragment accounts for the 3’ portion of the gene.

Figure E9. Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper IL-‐10, derived from the assembled 5’ and 3’ RACE products. Black text reflects the 5’ and 3’ UTR, the red text indicates the start (ATG) and stop (TGA) codons, the continuous green and blue text indicate the coding region for the red snapper IL-‐10 protein, and the blue text represents the overlap between the 5’-‐ and 3’-‐RACE products.

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Figure E10. Confirmed 5’ to 3’ nucleotide sequence for the coding region of red snapper IL-‐10, obtained via cloning from two different fish. Black text represents the 5’ and 3’ UTR, red text indicates the start (ATG) and stop (TGA) codon, and green text designates the coding region.

Figure E11. Experimentally obtained 5’ to 3’ nucleotide sequence for golden tilefish TNFα, derived from the assembled 3’-‐RACE and initial fragment products. Red text indicates the start (ATG) codon, the green text marks the coding region for the golden tilefish TNFα, protein, and the blue text represents the overlap between the 3’-‐RACE and initial fragment products. The initial fragment accounts for the 5’ portion of the gene.

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Figure E12. Experimentally obtained 5’ to 3’ nucleotide sequence for red snapper TNFα, derived from the assembled 5’-‐ and 3’-‐RACE products. Black text reflects the 5’ and 3’ UTR, the red text indicates the start (ATG) and possible stop (AAA) codons, the continuous green and blue text indicate the coding region for the red snapper TNFα protein, and the blue text represents the overlap between the 5’-‐ and 3’-‐RACE products. The stop codon was not observed in the cloned and confirmed sequence and may be a sequencing artifact.

Figure E13. Confirmed 5’ to 3’ nucleotide sequence for the coding region of red snapper TNFα, obtained via cloning from two different fish. Black text represents the 5’ and 3’ UTR, red text indicates the start (ATG) codon, and green text designates the coding region. The stop codon is absent.

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APPENDIX F:

EXPERIMENTALLY DERIVED CYTOKINE AMINO ACID SEQUENCES

Table F1. Amino acid sequences for each cytokine, translated from the experimentally obtained nucleotide sequence by the ExPASy Translate tool.

Complete( Partial(

Golden'tilefish

MESKMKCNMTKMWSPKMPEGLDFEITHHPLTMRSVVNLIVAMEKFKGNRSESPLSTEFRDENLLNIMLDSMEEEETVFECFSAPSVQYSRTGEHQCRVTDSENRSLVLVPDSMELHAVMLQGGSENCKVHLNMSTYLDLTPSAGARTVALGITGKTLEDKNLYLSCHKDGEEPTLHLEAVANKDSLLRIGSSSDMERFLFYKHVTGLNNTTFVSVPYSDWYISTAGDDNKPVEMCLENARRHRTFNIQRQS

✓

Red''''''snapper

MELHAVRLQGGSEESNEVHLNMATYAHPTPTTDALPVALGIKGTNLYLSCHKEGDKPTLHLEAVENNSSLSNISSTSDMVRSLFYKQDSESNSRGRHGG

✓

Golden'tilefish

MNSRVFVATIVVLLAFSAISEGMTLRSLGVELHCRCIQTESKPIGRHIAKVELIIPNSHCEETEIIATLKVTGQEVCLDPEAPWVKRVINRIMSNRKR

✓

Red''''''snapper

MSSRVFIASIVVVLAFLATSEGMALRSLGVELHCRCIQTESRPIGRHIGKVELIPPNSHCEETEIIATLKKTGQEVCLDPEAPWVKKVIEKIMSNSSRR

✓

Golden'tilefish

MTPRSLLLCVLVNLSVLCTVWCTPLCNNDCCRFVERFPVRLERLRADYSQIRGYYEANDDLDRALLDQTIEHSLKTPFACHAINSILGFYLGTVLPEALAEVTEDTRDLKPHMESIQTIFDQLKTDVTRCKHYFSCKEQFDITTLNSTYTQMESKGLYKAMGEL

✓

Red''''''snapper

MTPRSLLLCVLVNLSVLRTVWCTPLCNNDCCRFVERFPVRLERLRADYSQIRGYYEANDDLDRALLDQTIEHSLKTPFACHAINSILGFYLGTVLPEALAEVTEDTRDLKPHMESIQTIFDQLKTDVTRCKHYFSCKEQFDITTLNSTYTQMESKGLYKAMGELNLLFNYIETYLASKRPKNHVASA

✓

Golden'tilefish

DGDREGAGKRLTPLSHRIWSYSDSVGNKVSLMSAVRSACQNTAQEDSYKVGQGWYNAIYLGAVFQLNRGDKLWTETNQLSELDTDEGKTFFGVFAL

✓

Red''''''snapper

MVAYTTAPGDVEMGVDKRTVVLVEKKSSTQRMWKVSAALLFVAICVGGVMLFAWYWTRRPEMQTQSGQTGAQTGREATEKTDPHYTLRRISSKAKAAIHLEGSYDEGENSNHQLEWKNGQGQAFAQGGFRLVNNEIIIPQTGLYFVYSQASFRVSCSDGDAEGAGKGLMPLSHRIWRLSDSIGSRASLMSAVRSACQNTAQDDSYRDGHGWYNAIYLGAVFQLNKGDRLWTETNQLSELETEDGKTFFGVFAIEFP

✓

Sequence(data(

ILC1β

ILC8

ILC10

TNFα

Cytokine Species Amino(acid(sequence

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APPENDIX G:

SEQUENCE CONFIRMATION

G.1 Golden tilefish IL-‐1β sequence confirmation To confirm the coding region sequence for golden tilefish IL-‐1β, the fragment was isolated from

two different fish, designated fish F and fish H, and compared (Figures D1-‐5). Discrepancies were

resolved by first confirming that the nucleotide was properly recognized by visual inspection of the

chromatogram file, using Chromas lite. Next, nucleotide sequences were compared to one another

using BLASTn (Figure D3). The sequences were translated into amino acids using the Expasy Translate

tool and compared using BLASTp, to determine whether or not single nucleotide polymorphisms altered

the final protein sequence (Figure D4).

There were two discrepancies between fish F and fish H, at amino acid positions 50 and 240.

Fish F has S50, encoded for by TCA, while fish H has P50, encoded for by CCA. The difference therefore

lies in the presence of a cysteine, rather than a threonine, though both chromatogram peaks were clear

at this point. A multiple sequence alignment was performed with the five most homologous fish and the

serine residue was most commonly found at position 50 (Figure D5).

The second discrepancy arose at R240 in fish F and K240 in fish H, both similar amino acids with

positively charged R groups. R240 is encoded for by AGA while K240 is encoded by AAA, therefore, the

difference between the two is the A versus G at nucleotide position 811. Arginine was more conserved

across the Perciformes screened, therefore, fish F’s sequence was selected for the final analysis.

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Figure G1. 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐1β, cloned from fish F. Red text indicates the stop and start codons, green text reflects the translated region, and black text designates the untranslated region. Underlined codons are those that had single nucleotide differences between fish F and fish H.

Figure G2. 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐1β, cloned from fish H. Red text indicates the stop and start codons, green text reflects the translated region, and black text designates the untranslated region. Underlined codons are those that had single nucleotide differences between fish F and fish H.

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Figure G3. Clustal Omega alignment of the nucleotide sequences for golden tilefish IL-‐1β from fish F and H. Identical nucleotides are indicated by an asterisk (*).

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Figure G4. Clustal omega alignment of the protein sequences for golden tilefish IL-‐1β from fish F and H. Identical (*) and similar (:) amino acids are indicated.

Figure G5. Clustal omega alignment of golden tilefish IL-‐1β from fish F and H and the top five homologous species, according to BLASTp. Identical (*) or similar (: or .) amino acid sequences were identified. The two amino acids of interest are shaded in grey. The GenBank accession codes used were:

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European seabass (CAC41006.1), mandarin fish (AAV65041.1), barred knifejaw (ACH87392.1), striped trumpeter (ACQ99510.1), and red seabream (AAP33156.1). G.2 Golden tilefish IL-‐8 sequence confirmation The mature protein was cloned and sequenced from two different fish, fish F and H, and the

resulting sequence data was identical between the two. Comparing direct PCR sequencing results from

golden tilefish F and fish H confirmed the complete protein, including the signal peptide sequence

(Figures D6, D7). There was a single nucleotide difference, as fish F had an adenine at position 102 and

fish H had a guanine (Figure D8). However, this did not change the translated protein sequence (Figure

D9).

Figure G6. 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐8, obtained from fish F. Black text indicates the untranslated regions, red indicates the start and stop codons, and green text shows the span of the translated region.

Figure G7. 5’ to 3’ nucleotide sequence for the translated region of golden tilefish IL-‐8, obtained from fish H. Black text indicates the untranslated regions, red indicates the start and stop codons, and green text shows the span of the translated region.

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Figure G8. Clustal Omega alignment of the nucleotide sequences for golden tilefish IL-‐8 from fish F and H. Identical nucleotides are indicated by an asterisk (*).

Figure G9. Clustal Omega alignment of the protein sequences for golden tilefish IL-‐8 from fish F and H. Identical (*) and amino acids are indicated. G.3 Red snapper IL-‐8 sequence confirmation The mature protein was cloned and sequenced from two different fish, fish C and E, and the

resulting sequence data was identical between the two. Comparing direct PCR sequencing results from

golden tilefish C and fish E confirmed complete protein, including the signal peptide sequence (Figures

D10, D11). There was one discrepancy, at nucleotide 141, where fish C had a cytosine and fish E had a

thymine, however, this did not change the overall protein sequence (Figures D12, D13).

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Figure G10. 5’ to 3’ nucleotide sequence for red snapper IL-‐8, obtained from fish C. Black text indicates the untranslated regions, red indicates the start and stop codons, and green text shows the span of the translated region.

Figure G11. 5’ to 3’ nucleotide sequence for red snapper IL-‐8, obtained from fish E. Black text indicates the untranslated regions, red indicates the start and stop codons, and green text shows the span of the translated region.

Figure G12. Clustal Omega alignment of the nucleotide sequences for red snapper IL-‐8 from fish C and E. Identical nucleotides are indicated by an asterisk (*).

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Figure G13. Clustal Omega alignment of the protein sequences for red snapper IL-‐8 from fish C and E. Identical (*) amino acids are indicated. G.4 Red snapper IL-‐10 sequence confirmation To confirm the coding region sequence for red snapper IL-‐10 the fragment was isolated from

two different fish, designated fish C and fish E, and compared (Figures D14-‐18). Sequences were

analyzed as described above. One discrepancy was noted. Fish C had residue M151, encoded for by

ATG, where fish E had residue V151, encoded by GTG. When compared against the top five most

homologous species, according to BLASTp, the methionine was more conserved (Figure D18).

Therefore, sequence from fish C was utilized.

Figure G14. 5’ to 3’ nucleotide sequence for the translated region of red snapper IL-‐10, cloned from fish C. Black text indicates the untranslated regions, red indicates the start and stop codons, and green text shows the span of the translated region. The underlined text highlights the codon that differs between fish C and fish E.

Figure G15. 5’ to 3’ nucleotide sequence for the translated region of red snapper IL-‐10, cloned from fish E. Black text indicates the untranslated regions, red indicates the start and stop codons, and green text shows the span of the translated region. The underlined text highlights the codon that differs between fish C and fish E.

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Figure G16. Clustal Omega alignment of the nucleotide sequences for red snapper IL-‐10 from fish C and E. Identical nucleotides are indicated by an asterisk (*).

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Figure G17. Clustal Omega alignment of the protein sequences for red snapper IL-‐10 from fish C and E. Identical (*) and similar (:) amino acids are indicated.

Figure G18. Clustal Omega alignment red snapper IL-‐10 from fish C and E and the top five homologous species, according to BLASTp. Identical (*) or similar (: or .) amino acid sequences were identified. The amino acids of interest is shaded in grey. The GenBank accession codes used were: European seabass (ABH09454.1), gilthead seabream (AGS55345.1), Atlantic cod (ABV64720.1), green spotted puffer (CAD67773.1), and rainbow trout (NP_001232028.1).

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G.5 Red snapper TNFα sequence confirmation To confirm the coding region sequence for red snapper TNFα the fragment was isolated from

two different fish, designated fish C and fish E, and compared as described above (Figures D19-‐D22). The

only difference occurred at nucleotide position 85. Fish C had a guanine residue and fish E had an

adenine residue at this location. However, as this was in the 5’ UTR, it was not considered a significant

difference (Figure D21). The protein sequences were identical (Figure D22).

Figure G19. 5’ to 3’ nucleotide sequence for the translated region of red snapper TNFα, cloned from fish C. Black text indicates the untranslated region, red indicates the stop codons, and green text shows the span of the translated region. The underlined text highlights the nucleotide that differs between fish C and fish E.

Figure G20. 5’ to 3’ nucleotide sequence for the translated region of red snapper TNFα, cloned from fish E. Black text indicates the untranslated regions, red indicates the start codons, and green text shows the span of the translated region. The underlined text highlights the nucleotide that differs between fish C and fish E.

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Figure G21. Clustal Omega alignment of the nucleotide sequences for red snapper TNFα from fish C and E. Identical nucleotides are indicated by an asterisk (*).

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Figure G22. Clustal Omega alignment of protein sequences for red snapper TNFα from fish C and E. Identical amino acids are indicated by an asterisk (*).

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APPENDIX H:

TABLE OF COMMON NAMES USED AND THEIR ASSOCIATED SCIENTIFIC NAMES

Table H1: List of all common names used in this thesis with the associated scientific name.

Common name Scientific name

African clawed frog Xenopus laevis

Atlantic cod Gadus morhua

Atlantic salmon Salmo salar

Barred knifejaw Oplegnathus fasciatus

Channel catfish Ictalurus punctatus

Chicken Gallus gallus

Common carp Cyprinus carpio

European seabass Dicentrarchus labrax

Fugu Takifugu rubripes

Gilthead seabream Sparus aurata

Golden tilefish Lopholatilus chamaeleonticeps

Goldfish Carassius auratus

Grass carp Ctenopharyngodon idella

Green spotted puffer Tetraodon nigroviridis

Haddock Melanogrammus aeglefinus

Hapuku Polyprion oxygeneios

Human Homo sapiens

Humphead snapper Lutjanus sanguineus

Mandarin fish Siniperca chuatsi

Marmot Marmota monax

Mouse Mus musculus

Olive flounder Paralichthyes olivaceus

Orange-‐spotted grouper Epinephalus coioides

Platyfish Xiphophorous maculatus

Rainbow trout Oncorhynchus mykiss

Rat Rattus norvegicus

Red seabream Pagrus major

Red snapper Lutjanus campechanus

Rockfish Sebastes schlegeli

Silver carp Hypophthalmichthys molitrix

Smaller spotted catshark Scyliorhinus canicula

Southern bluefin tuna Thunnus maccoyii

Striped trumpeyer Latris lineata

Zebrafish Danio rerio

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