Link to Google Drive Folder with Papers Supplementary - A single iPython notebook streamlining all analyses - Or minimally, put up multiple notebooks on Figshare OCTOBER 20 AGENDA ________________ TITLE Pulling apart the host response to sea star wasting disease Authors Lauren Fuess, Morgan Eisenlord, Collin Closek, Allison Tracy, Ruth Mauntz, Sarah Gignoux-Wolfsohn, Monica Moritsch, Reyn Yoshioka, Drew Harvell, Carolyn Friedman, Colleen Burge, Steven Roberts Echinoderms provide a powerful system to examine invertebrate immune function from both an organismal and evolutionary perspective. With a taxonomic position at the base of the deuterostomes, echinoderms are more similar to chordates and vertebrates than other invertebrate lineages (Blair and Hedges 2005). This is evident in part through their complex innate immune system with specialized phagocytic cells, signalling molecules that circulate in coelomic fluid, and a melanization response (Franco et al. 2011; Smith et al. 2010; Smith and Soderhall 1991). Laboratory based studies have established the existence of echinoderm recognition proteins (Bulgakov et al. 2013; Gowda et al. 2013), cytokine signaling molecules (Beck et al. 1996), and effector mechanisms, such as antimicrobial peptides (Li et al. 2010). Conserved immune response pathways include the complement system, Toll pathway, and cell adhesion regulation (Hibino et al. 2006; Smith et al. 2010; Dheilly et al. 2013). While the discovery of these major pathways highlights broad similarities across the phylogenetic tree, echinoderms also have regions of unusually high diversification as well as unique combinations of innate and adaptive immune capabilities. For example, it has been reported that the sea urchin Strongylocentrotus purpuratus possess over two hundred Toll Like Receptor (TLR) genes as well as genes that suggest overlap with vertebrate immunity (Hibino et al., 2006). Infectious diseases of marine echinoderms are on the rise (Burge et al. 2014), providing an opportunity to investigate the varied immune responses of these animals to multiple pathogens. This rise in disease epidemics further underscores the need for a thorough understanding of the echinoderm immune system. Sea star wasting disease (SSWD), a newly emerging infectious disease affecting echinoderms, has caused severe population declines in multiple sea star species along the North American Pacific coast since summer 2013 (MARINe 2013). Early symptoms of disease include abnormally twisted arms, a deflated appearance, and white lesions on the aboral body wall; this rapidly progresses to tissue degradation, structure loss, arm loss, and death (Eckert et al. 2000). Many basic details of SSWD are still unknown, including the causative pathogen, the mode of disease spread, and conditions influencing its severity (MARINe 2013). As with any keystone species, loss of asteroid populations to disease outbreaks has the potential to shift community composition of intertidal and subtidal ecosystems (Paine 1974; Duggins 1983). In this study, we characterize the sea star immune response in the sunflower star, Pycnopodia helianthoides, by injecting healthy individuals with homogenate from stars with signs of wasting disease and examining gene expression patterns of control and treated stars. The P. helianthoides transcriptome generated here is the first immune-related transcriptome in the class Asteroidea. Physiological responses to SSWD included activation of the the Toll and complement pathways, alterations in nervous and connective tissues, and Wnt signaling pathway disruption. This transcriptome not only provides an important resource for future evolutionary and organismal studies but provides the first evidence of how sea stars mount a response to this devastating disease. Understanding the response to this rapidly-progressing disease will certainly provide insight into dynamics of host-pathogen interactions in marine systems. Furthermore this detailed exploration of the sea star response to disease can enhance our understanding of the evolution of immune response. Methods Experimental Design Pycnopodia helianthoides (radius 160.5 +/- 30.9 mm) were collected from three sites in Washington State: Dabob Bay (DB), WA (47.813, -122.820); Port Hadlock Marina (PH), WA (48.030, -122.745); Friday Harbor (FH), WA (48.545, -123.012). Sites[a] had no reports of SSWD infection at time of collection and no animals collected showed clinical signs of the disease. An effort was made to collect animals within a similar size range to reduce variation based on body mass but otherwise selection was random. Experiments were conducted at Marrowstone USGS Laboratory, WA. P. helianthoides were transported to the lab on the day of collection in coolers, wrapped in cloth soaked in seawater from their respective sites. Upon arrival, animals were examined for signs of disease or trauma and then transferred to individual, 37.8 L aquariums with separate flow-through, sand-filtered, and UV-treated seawater kept at ~8.5°C. The acclimation period for each star ranged from 3 days to 2.5 months, depending on time of collection. During this period, animals were examined for signs of disease and fed live manila clams (Ruditapes philippinarum), every 3-4 days. Water flow was checked daily. An infection challenge was conducted with tissue homogenate prepared from the tube feet, dermal tissue, and coelomic fluid of three P. helianthoides with clinical signs of SSWD. The tissue was ground with a mortar and pestle, then placed in a Stomacher with 10 mL seawater, and centrifuged at 1000 x g for 5 min at 4°C. Organisms injected with the homogenate boiled for 7 minutes are considered the control group. The treated group were injected with homogenate that was syringe-filtered through a 0.22 μm polyethylsulfone filter. Specifically, one animal from each site was inoculated on 6 April 2014 with either boiled (controls) or filtered (treated) inocula (0.5 mL) by injection into the coelomic cavity, under the dermis on the aboral side of the arm, and at the arm base. Animals were checked twice daily and any physical or behavioral changes recorded. Animals were sacrificed on 15 April 2014, 9 days post injection once all three treatment animals showed small lesions. Stars were sampled while lesions were small and they still had turgor so the coelomic fluid could be extracted from an intact animal. Coelomic fluid was collected from the arm using a syringe. The samples were centrifuged for 5 minutes at 1200 rpm at 4°C to separate the coelomocytes. The fluid was then removed and the pellet containing the coelomocytes flash frozen in liquid nitrogen and stored at -80C. High Throughput Sequencing and Assembly Total RNA was extracted using Tri-Reagent per manufacturer’s instruction. Potential DNA carry-over was removed from extracted RNA using the Turbo DNA-free treatment according to the manufacturer's instructions (Ambion). RNA quality, library preparation, and sequencing was performed by the Cornell University Institute for Biotechnology. RNA quality was assessed using an Advanced Analytical Fragment Analyzer. Libraries were prepared using the Illumina TruSeq RNA Sample Preparation kit according to the manufacturer’s protocol (including bar-coding for multiplexing), except our sample concentration was between 99-1503 ngs per sample. Samples were multiplexed where six samples were run in one lane for Illumina Hiseq 100 bp paired end sequencing. Quality trimming of resulting sequencing reads was performed using CLC Genomics Workbench v. 7.0 (CLC Bio, Germany) with the following parameters: quality limit = 0.05, number of ambiguous nucleotides < 2 on ends, reads shorter than 20 bp were removed, and Illumina PCR primers were removed. De novo assembly was performed with Genomics Workbench 7.0 (CLC Bio, Germany) on quality trimmed sequences with the following parameters; automatic bubble size, automatic word size, auto-detect paired distances, perform scaffolding, and minimum contig size of 500 bp. In order to remove bacterial sequences from the transcriptome data, consensus sequences were compared to the NCBI nt database using the BLASTn algorithm. Consensus sequences with significant matches (evalue = 0) to bacterial sequences (n=1102) were removed and not considered in subsequent analyses. Transcriptome Characterization In order to characterize relative completeness of the transcriptome, it was compared to complete transcriptomes available for the bat star Patira miniata (http://echinobase.org) and purple sea urchin Strongylocentrotus purpuratus (http://spbase.org, Cameron et al. 2009). Specifically, gene sets of P. minata (29,805) and S. purpuratus (31,159) were used as query with blastn comparison to our P. helianthoides transcriptome. P. helianthoides transcriptomic sequences were subsequently annotated by comparing contiguous sequences to the UniProtKB/Swiss-Prot database. Comparisons were made using the BLASTx algorithm with a 1.0E-5 e-value threshold. Genes were classified according to Swiss-Prot Gene Ontology (GO) associations, as well as respective parent categories (GO Slim). Differential Expression Analysis Differential expression of contigs was calculated using a negative binomial GLM in the R package DESeq2[b] (Anders and Huber 2010). Read counts were determined by aligning reads with CLC v7.0 with the following parameters: mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.8, similarity fraction = 0.8, maximum number of hits for a read = 10. The read counts were first normalized using the size factors method and fit to a negative binomial distribution. Significantly differential contig expression (Benjamini-Hochberg adjusted p<0.05 ) between control and treated animals was determined using the Wald test for significance of GLM terms. Enrichment Analysis Enriched GO terms associated with differentially expressed genes were identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v. 6.7 (Huang et al. 2009). Specifically, UniProt accession numbers for differentially expressed genes were uploaded as a gene list, while UniProt accession numbers for all annotated contigs were used as a background. Significantly enriched GO terms were identified as those with Benjamini-Hochberg adjusted p<0.05. Results Inoculation Experiment During the acclimation period, no signs of disease were observed. All treated P. helianthoides developed signs of SSWD including curling and lesions (Table 1[c]). Lesions were noted at 8 to 9 days post injection in all three treatment animals. There was some individual variation in the onset and duration of clinical sign but signs were consistent between animals. Control animals did not show any clinical signs. Transcriptome This sequencing effort results in a combined 2.9x108 paired end reads among all six libraries. Sequencing reads are available in NCBI SRA Accession # (*). After quality trimming, reads were assembled into 29476 consensus sequences with an N50 value of 1757 bp (SUPPLEMENTAL FILE). Using Patria miniata and Stronglycentrotus purpuratus transcriptomes as for references we found that 52% and and 26% of the respective transcriptomes had match to the Pycnopodia helianthoides transcriptome using an 1.0E-5 e-value threshold. Comparisons of the Pycnopodia helianthoides transcriptome sequences to the UniProtKB/Swiss-Prot database resulted in annotation of 10513 contigs (SUPPLEMENTAL FILE). Differentially Expressed Genes[d] Of those contigs identified as differentially expressed (n=3773), 1629 were expressed lower and 2103 were expressed higher in treated individuals (Figure 1). A total of 1183 differentially expressed contigs (31.7%) were annotated based on comparison to Uniprot/SwissProt database. DESeq dots.png [e] (Needs to be redone when we get our transcriptome cleared of bacterial sequences) Figure 1. DESeq2 analysis of contigs resulted in 3773 differentially expressed genes (red) that were used in further analyses. Gene Ontology (GO) Enrichment Seventeen BP_FAT gene ontology (GO) terms were significantly enriched in the subset of annotated differentially expressed genes. These terms fell into three broad categories: immune response, regulation of cytokine production, and biological adhesion (Figure 2). Immune response was the largest of these categories, with thirteen associated enriched GO terms. Table S1 lists all significantly enriched GO terms. Regulation of cytokine biosynthetic process (GO:0042035) and activation of immune response (GO:0002253) were the two most enriched GO terms with fold enrichments of 5.881791 and 5.614436 respectively (Figure 3). In addition to enriched terms and categories, contigs annotated as part of the toll-like receptor-mediated pathway (Table 2) , complement cascade (Table 3), and clotting/ mutable connective tissue (Table 4) were noted. Furthermore a number of contigs involved in the WNT-signaling pathway (Table 5), neural/ mutable connective tissue processes (Table 6), and G-coupled proteins were also observed (Table 7) (Table S1). FoldEnrichmentGraph.png Figure 3 Fold Enrichment of each of the 18 significant (padj<.05) BP_FAT GO terms. immune_heatmaps.jpeg neural_heatmap.jpeg Neural- Growth /organization clottingheatmap.jpeg Clotting WNT_heatmap.jpeg Wnt Table 4 List of contigs involved in clotting/ MCT Contig ID Annotation log 2 FC padj Phel_clc_contig_1871, Phel_clc_contig_20104 Q91Y47 Coagulation factor XI (FXI) 7.75 5.37 0.00 0.00 Phel_clc_contig_1128 O15072 ADAM-TS 3 3.77 0.00 Phel_clc_contig_6569 Q9UKP5 ADAM-TS6 -2.33 0.02 Phel_clc_contig_7590 Q76LX8 ADAM-TS 13 (vWF-cleaving protease) 7.96 0.00 Phel_clc_contig_3408 P98139 Coagulation factor VII 2.68 0.02[f][g] Phel_clc_contig_1865 Q8JIY1 ADAM 10 2.77 0.00 Phel_clc_contig_1112 Q61824 ADAM 12 4.17 0.00 Phel_clc_contig_163, Phel_clc_contig_12224, Phel_clc_contig_1077, Phel_clc_contig_5796 A2AVA0 Sushi, von Willebrand factor 2.20 4.20 1.61 -1.91 0.00 0.00 0.00 0.03 Phel_clc_contig_1522 P11584 Integrin beta-PS 1.74 0.05 Phel_clc_contig_2142, Phel_clc_contig_13337 Q00651 CD antigen CD49d 3.07 3.63 0.00 0.00 Phel_clc_contig_9206 B0FYY4 CD antigen CD29 -1.40 0.01 Phel_clc_contig_6080 Q9JHI0 MMP-19 4.22 0.00 Phel_clc_contig_61 Q9R0S2 MMP-24 2.24 0.00 Phel_clc_contig_360 P28863 Stromelysin-1 3.97 0.00 Phel_clc_contig_1508 Q9ULZ9 MMP-17 4.08 0.00 Phel_clc_contig_202 Q9WTR0 MMP-16 3.43 0.00 Phel_clc_contig_25693 P57044 Integrin-linked protein kinase (ILK) 0.00 -3.37 Table 5 List of contigs involved in the wnt signaling pathway Contig SPID Annotation log2 FC padj Phel_clc_contig_1252 Q5REY6 Transforming protein RhoA 6.22 2.52E-11 Phel_clc_contig_25 Q9QYP1 Low-density lipoprotein receptor-related protein 4 4.48 1.79E-06 Phel_clc_contig_9741 Q90Y90 Kremen protein 1 3.52 4.98E-04 Phel_clc_contig_3608 Q99N43 Kremen protein 1 2.64 2.61E-10 Phel_clc_contig_2240 Q4JIM4 Presenilin-1 1.8 1.07E-03 Phel_clc_contig_7024 P35223 Catenin beta 1.47 4.02E-04 Phel_clc_contig_9770 Q8NCW0 Kremen protein 2 -1.42 6.13E-03 Phel_clc_contig_9571 Q6FHJ7 Secreted frizzled-related protein 4 -1.93 3.01E-03 Phel_clc_contig_12417 Q5BL72 Frizzled-7 -2.26 1.61E-03 Phel_clc_contig_13198 P33945 Protein Wnt-5b -2.92 1.35E-05 Phel_clc_contig_24757 Q2TJA6 Protein naked cuticle homolog 1 (Naked-1) -3.62 6.70E-05 Table 6: List of selected neurological/MCT contigs Contig SPID Annotation log2 FC padj Phel_clc_contig_296 O77656 Collagenase 3 / Matrix Metalloproteinase 13 10.84 1.01E-39 Phel_clc_contig_475 O77656 Collagenase 3 / Matrix Metalloproteinase 13 7.82 5.07E-16 Phel_clc_contig_235 O77656 Collagenase 3 / Matrix Metalloproteinase 13 3.39 0.000411 Phel_clc_contig_29418 Q80T41 Gamma-aminobutyric acid type B receptor subunit 2 -3.56 0.0168 Phel_clc_contig_1368 P23975 Sodium-dependent noradrenaline transporter / Norepinephrine transporter 4.64 1.51E-13 Phel_clc_contig_6750 P23975 Sodium-dependent noradrenaline transporter / Norepinephrine transporter -4.76 2.08E-21 Phel_clc_contig_7409 B3STU3 Botch (Blocks Notch protein) 4.25 7.63E-20 Phel_clc_contig_3774 Q07817 Bcl-2-like protein 1 4.31 6.23E-08 Phel_clc_contig_18669 P97799 Neurensin-1 3.85 4.40E-06 Phel_clc_contig_4949 Q8K1S2 Netrin Receptor UNC5D 6.05 4.27E-15 Phel_clc_contig_1127 Q8JGT4 Netrin Receptor UNC5B -1.19 0.0262 Phel_clc_contig_360 P28863 Stromelysin-1 3.97 5.91E-05 Phel_clc_contig_1066 P35624 Tissue Inhibitor of Metalloproteinases 1 6.58 8.50E-16 Phel_clc_contig_4458 Q9JHB3 Tissue Inhibitor of Metalloproteinases 4 3.27 0.000347 Phel_clc_contig_2994 P81556 Tissue Inhibitor of Metalloproteinases 4 -1.81 0.00149 Phel_clc_contig_27280 P26652 Tissue Inhibitor of Metalloproteinases 3 -4.17 2.33E-05 Phel_clc_contig_24134 Q86GC8 Acetylcholinesterase -2.34 0.0446 Phel_clc_contig_16508 P21836 Acetylcholinesterase -1.94 0.0411 Phel_clc_contig_17140 Q29499 Acetylcholinesterase -1.6 0.0214 Table 7: List of contigs involved in G-proteins Discussion Here we provide the first report of the immune response through enriched transcriptome of P. helianthoides in relation to (or “following”?) pathogen exposure. In an effort to gain a better understanding of the immune response of P. helianthoides to SSWD, Specifically, the comparison of gene expression patterns between treatment and control organisms was in an effort to gain provides a better understanding of the immune response of P. helianthoides to SSWD. As might be predicted from the dramatic mortality events that have been reported from Canada to …. (CITE), SSWD poses an existential threat to many seastar populations. [This could continue straight into the next paragraph] basics[h][i] The results indicate that P. helianthoides shows signs of SSWD and mounts a strong immune response when infected with homogenate from sea stars with signs of SSWD. This response includes increased expression of genes associated with the Toll pathway, complement cascade, melanization, and coagulation. Furthermore, we document a number of changes in gene expression that may contribute to the observed clinical signs of SSWD. These include contigs involved in G-coupled protein processes, WNT signaling, cell adhesion, and neural processes. A close examination of these genes enhances knowledge of host-pathogen dynamics in sea stars. A total of XXXX differentially expressed genes were identified ….[j][k] Toll Pathway The differential expression and enrichment of multiple genes involved in the Toll-like receptor-mediated pathway of the infection treatment sea stars suggests that this pathway plays an important role in P. helianthoides immunity. Toll-like receptors (TLRs) in both vertebrates and invertebrates recognize patterns to discriminate between self and non-self, inducing a response to bacterial and viral pathogens (Aderem and Ulevitch, 2000; Finberg et al. 2007; Zambon et al. 2005; Valanne et al. 2011). The sea urchin genome contains 222 TLRs (Rast et al. 2006[l]). It is not surprising, therefore, that we find differentially expressed TLRs in the sea star transcriptome (TLR1,TLR2,TLR8). The differential expression of TLR8 is especially interesting as this receptor has been linked to viral recognition (Akira et al. 2006). Following recognition, TLRs act through a network of signaling molecules to activate different immune and inflammatory cascades (Valanne et al. 2011). One potential pathway following TLR recognition in the treated sea stars is activation of Rac1, which initiates a pathway to activate NF-kB (Cuadrado et al. 2014), a transcription factor that triggers multiple immune effectors (Janeway et al. 2001). This response has been seen in immune-challenged Manila clams (Moreira et al. 2012). Additional enriched genes [m]could act through an alternate pathway of NF-kB activation: TOLLIP and Myd88 could be activated by TLR1 and TLR2, in turn activating IRAK1 (Takeda et al. 2004; Muzio et al. 1997). Evidence of the cascade from Myd88 to IRAK1 to TRAF6 has been found in both invertebrate and vertebrate systems (Wang et al. 2011), including Myd88 in both sea urchins and sea cucumbers and TRAF6 in sea cucumbers (Rauta et al. 2014). Downstream signaling in linked to the Myd88 pathway includes a suite of closely interacting genes that belong to enriched processes in the treatment sea stars: PINK1, Traf6, Sqstm-1, HSP60[n] and MALT1 (Sun et al. 2004, Bitto et al 2014, Muroi et al. 2008; Coscia et al. 2011; Into et al. 2010; Cohen-Sfady et al. 2005). Further downstream in the TLR-mediated pathways, the enriched gene BLC3 is could be involved in negatively regulating NF-kB (Wang et [o]al. 2009). NF-kB, NF-kB1 subunits[p][q][r][s][t], and NF-rKB are themselves enriched genes in treatment sea stars. The signaling cascade initiated by TLRs leads to the production of inflammatory cytokines including IL-6, which is differentially expressed in treatment sea stars and has been found in past research on immune-challenged sea stars (Beck & Habicht 1996). We also identified other enriched genes that have been linked with the TLR-mediated pathway including RASGEF1B (Andrade et al. 2010), TBK1, which activates IRF3 instead of NF-kB (Shin et al. 2013), and Cebp-b, another pro-inflammatory transcription factor (Rojo et al. 2007; Grohmann et al. 2010). The histamine H2 receptor (Hrh2) is also differentially expressed and has been linked to the regulation of IL-10 and IL-12 regulation in mammals (Elenkov et al. 1998). Differentially expressed genes in enriched processes appear in multiple branches of the TLR-mediated pathway, serving as evidence that, as in other echinoderms, Toll pathways play an important role in P. helianthoides immunity[u]. Complement Cascades The presence of multiple differentially expressed components of the complement cascade suggests that sea stars utilize this pathway, in addition to Toll-like receptors, to combat infection by SSWD. The complement cascade oposonizes pathogens leading to inactivation and phagocytosis by immune cells and also damages pathogens directly through the formation of the membrane attack complex (Janeway 2001). Although a previous study into the sea star coelomocyte proteome did not show homologous complement proteins, this study identifies four complement cascade proteins that are differentially expressed: Ficolin-2 (FCN2; contig_7739, contig_3867, contig_107, contig_1813, contig_6139), Complement C3 (C3; contig_3127, contig_632), Properdin (CFP; contig_312), and Complement C2 (C2; contig_1529) (Franco et al 2011). For all of four protein annotations, at least one corresponding contig was more highly expressed in infected stars than in control samples. However, contigs annotated as FCN2 had opposing log fold changes, suggesting either gene duplication and differential function or alternative splicing[v]. The complement cascade has been well documented in Echinoderms previously and contains C3, which functions in opsonization and phagocytosis, and factor B/C2, which increases C3 production (Smith et al. 1999[w]). Here we document the presence of two additional complement cascade proteins, FCN2 and CFP. Both FCN2 and CFP induce the complement cascade: Ficolin through association with mannan-binding lectin-associated serine proteases (Matsushita et al 2000), and CFP by binding to apoptotic cells resulting in complement activation (Kemper et al, 2008). Our results suggest that the complement cascade is an important part of sea star response to wasting disease, and furthermore, that echinoderms may have a more complex complement cascade system than previously thought. Melanization The melanin synthesis cascade in invertebrates serves roles in wound healing and immune response by creating chemical/physical barriers and encapsulating pathogens for phagocytosis (Cerenius & Söderhäll 2004). One of the contigs with the largest log2 fold change was annotated as a quinone oxidoreductase (CRYZ). Similar oxidoreductases (NQO1) in humans have been described as increasing melanin synthesis by increasing tyrosinase catalytic activity (Yamaguchi et al, 2010). Various aspects of the melanin synthesis cascade have been documented in a number of Echinoderms (Canicatti and D’Ancona, 1998). Previous experiments using sheep erythrocytes as an immune challenge in the sea cucumber Holothuria polii resulted in both clearance of the antigen and the production of ‘brown masses’ which were positive for Schmorl’s, Lillie’s, and Hueck’s reactions, indicating the presence of melanin (Canicatti and D’Ancona, 1998). Furthermore, various urchins, sea stars, and sea cucumbers are characterized by low levels of phenoloxidase, an important part of the melanin synthesis cascade (Smith and Soderhall, 1991). Our results serve as the first genetic evidence of melanin synthesis in an echnioderm and suggest that melanin production may play an important role in the healing of lesions on infected sea stars. Clotting response and ECM remodeling [x][y] Focal adhesion complexes are essential for binding cell surfaces to the extracellular matrix (ECM) and have roles in ECM remodeling and signal transduction. Integrins are a large family of transmembrane receptors in the focal adhesion complex that interact with a variety of ligand binding factors (Plow et al, 2000).They provide structure by linking the ECM to the actin cytoskeleton, contribute to growth factor receptor signaling transduction, and regulate the extracellular affinity of receptors to ECM proteins, leading to the focal adhesion “inside-out” signaling theory (Widmaier et al, 2012). The focal adhesion complex also has a role in initiation of the clotting response through the large EGF- and pentraxin-domain containing protein vWF, and can be affected by ECM connective tissue remodeling through metalloproteinase and disintegrin proteolysis (Page-McCaw et al, 2007). In the treated P. helianthoides, we report differential expression in 3 integrin contigs, annotating to integrin beta-PS, CD antigen CD29 and CD antigen CD49d. Additionally, there are changes in 12 integrin-binding contigs, including collagenase 3, stromylesin-1, the disintegrins ADAM-10, ADAM-TS3, ADAM12, ADAM-TS6, ADAM-TS13, the metalloproteinases MMP-16, MMP-17, MMP19, MMP24, and the clotting factors vWF, FVII, and FXI. Log2 fold change for the clotting factors, MMPs, and ADAM disintegrins are all increased in the treated samples, while three out of the four contigs for protein vWF (cleaved by the anticlotting ADAM superfamily) also increase expression. Three out of the four annotated integrins also have increased differential expression. Matrix metalloproteinases degrade components of the ECM such as fibronectinin and laminin and are shown to be essential in connective tissue remodeling in the sea cucumber Holothuria glaberrima (Quinones 2004). These metalloproteinases may have a role in connective tissue mutability and lead to some of the signs of SSWD. G protein coupled receptors Guanosine nucleotide-binding proteins (G proteins) are signal transducers attached to the plasma membrane of the cell, which act as molecular switches, communicating signals from a variety of stimuli within and outside the cell (Neves et al., 2002). G protein coupled receptors (GPCRs) are the largest family of cell-surface molecules involved with the transmission of signals (Dorsam & Gutkin, 2007). While the family is large, enriched GPCRs were exclusively down in the treatment samples and show suppression of particular signalling cascades in contrast to healthy samples. GPCRs involved with adenylate cyclase activity such as Luteinizing hormone receptor (contig_8658), Metabotropic glutamate receptor 7 (contig_2786), Muscarinic acetylcholine receptor M (contig_7895), and Diuretic hormone receptor (contig_11919) were down in the treatment condition. This enrichment is important as adenylate cyclase has been shown to cause muscle relaxation in sea stars (Elphick & Melarange, 2001), which parallels one physiological clinical sign of SSWD - loss of structural turgor (Eckert et al., 2000). GPCRs involved with neural processes such as GABA-B receptor 2 (contig_29418), calcium-independent alpha-latrotoxin receptor 3 (contig_8096), and GRL101 (contig_15446) were also down in treatment samples. These among others were found in the sea urchin, Strongylocentrotus purpuratus, as well as Saccoglossus kowalevskii, the acorn worm, genomes and were linked to neural processes (Burke et al., 2006; Krishna et al., 2013). Additional proteins, such as Ras-related and estrogen-regulated growth inhibitor (contig_8892) and Ras-related protein Rab-15 (contig_1757) both bind GTP for activation of G protein were also down in treatment samples. GTP activates G proteins of subsequent signaling pathways within the cell (Dorsam & Gutkin, 2007) and therefor without these processes being activated the subsequent signaling cascades cannot occur. Insights into Sea Star Wasting Disease Pathology Observable signs of SSWD also include a deflated appearance, twisting arms, lesions, and in advanced cases arm autotomy and death (Eckert et al. 2000). These suggest a role of nervous system and connective tissues in the disease pathology. The nervous system is the primary control of adhesion and connective tissues, including the mutable collagenous tissues (MCT), which are key in maintaining sea star structure (Sugni et al 2014). Our results yielded several differentially expressed genes that suggest a disruption of neural function, possibly having downstream effects to connective tissue function. Norepinephrine transporter, responsible for the uptake of the stress hormone norepinephrine into synaptic terminals, had strongly opposite expression between two contigs. BCL-2-like protein 1 (BCLx) was over-expressed. While it has a role in apoptosis (Adams & Cory 1998, Boise et al 1993), BCLx also regulates synaptic activity (Li et al 2013). Also, gamma-aminobutyric acid B receptor 2 (GABABR2), a neuroinhibitor that helps fine-tune neural function (Bettler et al 2004) was under-expressed. There is also evidence for neurogenesis: nerve guide and growth protein transcripts such as Blocks Notch protein (Botch, Chi et al 2012), neurensin (Araki & Taketani 2009), and netrin receptor UNC5D (Katow 2008) were highly expressed in treatment stars. Neural changes in tandem with or mediating focal adhesion complexes are likely affecting MCT through manipulation of matrix metalloproteinases. The neurotransmitter acetylcholine is known to be a stiffening modulator of MCT in urchins (Ribeiro et al 2012, Hidaka & Takahashi 2009), and its degrading enzyme, acetylcholinesterase, is under-expressed in the treatment stars. While this suggests a greater stiffening response, which is opposite to what is observed, MCT response to neurotransmitters is often varied between echinoderm classes (Sugni et al 2014) or could be an attempt by the star to maintain structure. Matrix metalloproteinases such as collagenase and stromelysin degrade components MCT and soften it (Ribeiro et al 2012, Quinones et al 2002), while their antagonists Tissue Inhibitors of Metalloproteinases (TIMPs) stiffen it (Sugni et al 2014, Tipper et al 2003). Both collagenase-3 and stromelysin were over-expressed, which is consistent with the “melted” appearance of diseased stars. TIMPs had mixed expressions. Interestingly, collagen alpha-1(XIV) chain, a component of ECM/MCT, was over-expressed. However, we expected to observe some opposing expression in our transcriptome as some DEGs may represent disease signs while others could be the sea stars’ attempt to resist further development of the disease. Insights into Sea Star Wasting Disease Pathology[z] Observable signs of SSWD also include a deflated appearance, twisting arms, lesions and, in advanced cases, arm autotomy and death (Eckert et al. 2000). The nervous system is the primary control of adhesion and connective tissues, including the mutable collagenous tissues (MCT), which are key in maintaining help maintain sea star structure (Sugni et al 2014). Therefore the signs of SSWD suggest a role of for the nervous system and connective tissues in the disease pathology. Our[aa] results yielded differentially expressed genes that suggest a disruption of neural function, possibly having downstream effects to connective tissue function. Norepinephrine transporter, which is responsible for the uptake of the stress hormone norepinephrine into synaptic terminals, had opposite expression between two contigs[ab] (contig_1368, contig_6750). BCL-2-like protein 1 (BCLx, contig_3774) was over-expressed in treatment stars and has a role in apoptosis (Adams & Cory 1998, Boise et al 1993), but BCLx also regulates synaptic activity (Li et al 2013). There is also evidence for neurogenesis, as nerve guide and growth protein transcripts such as Blocks Notch protein (Botch, Chi et al 2012, contig_7409), neurensin (Araki & Taketani 2009, contig_18669), and netrin receptor UNC5D (Katow 2008, contig_4949) were all highly expressed in the treatment stars. Enriched G-protein coupled receptors (GPCRs) were exclusively down in the treatment samples and show suppression of particular signaling cascades in contrast to healthy samples. [Sentence on what G-proteins CAN do - list a few things. Otherwise it jumps in too quickly]. Gamma-aminobutyric acid B receptor 2 (GABABR2, contig_29418), a neuroinhibitor that helps fine-tune neural function (Bettler et al 2004), was under-expressed. Other GPCRs involved with neural processes such as calcium-independent alpha-latrotoxin receptor 3 (contig_8096), and GRL101 (contig_15446) were also down in the treatment samples. These among others were found in the S. purpuratus (sea urchin) and Saccoglossus kowalevskii (acorn worm) genomes and were linked to neural processes (Burke et al., 2006; Krishna et al., 2013). Additionally, Ras-related and estrogen-regulated growth inhibitor (contig_8892) and Ras-related protein Rab-15 (contig_1757), both of which bind GTP for activation of G protein, were also down in treatment samples. GTP activates G proteins of subsequent signaling pathways within the cell (Dorsam & Gutkin, 2007) and therefore Without these processes being activated the activation of these processes, the subsequent signaling cascades cannot occur. The neurotransmitter acetylcholine is known to be a stiffening modulator of MCT in urchins (Ribeiro et al 2012, Hidaka & Takahashi 2009). Its degrading enzyme, acetylcholinesterase (contig_16508, contig_17140, contig_24134), is down in the treatment stars while the GPCR Muscarinic acetylcholine receptor M (contig_7895) is down in the treatment condition[ac], indicating a reduction of acetylcholine-mediated MCT stiffening. While this suggests greater stiffening response, which is opposite to what is observed[ad], the treatment stars may be over-expressing this as an attempt to maintain turgor. GPCRs involved with adenylate cyclase activity such as Luteinizing hormone receptor (contig_8658), Metabotropic glutamate receptor 7 (contig_2786), and Diuretic hormone receptor (contig_11919) were down in the treatment condition. This enrichment is important, as adenylate cyclase has been shown to cause muscle relaxation in sea stars by possibly mediating the nitric oxide signaling pathway (Elphick & Melarange, 2001). Matrix metalloproteinases (MMPs) and the clotting factor vWF have a physiological role in extracellular matrix (ECM) remodeling and the clotting response involved in wound healing, respectively cleaving [ae]or binding the focal adhesion complex. They are essential for connecting cell surfaces to the ECM, for providing tissue structure by linking the ECM to the actin cytoskeleton, and for initiation of the clotting response (Widmaier et al, 2012). In the treated P. helianthoides, we report changes in the clotting factors vWF (contig_1077, contig_12224, contig_163, contig_5796), FVII (contig_3408), and FXI[af] (contig_1871, contig_20104). Three of the four contigs for protein vWF (cleaved by the anticlotting ADAM superfamily) and both clotting factors FVII and FXI show increased expression in the treated conditions stars. Additionally, there is increased expression in the protein integrins, annotating to integrin beta-PS (contig_1522), CD antigen CD29 and CD antigen CD49d (contig_13337, contig_2142). ECM connective tissue remodeling occurs through matrix metalloproteinases (MMPs) and disintegrin proteolysis (Page-McCaw et al, 2007). MMPs, such as collagenase and stromelysin, degrade components of the ECM and are shown to be essential in tissue remodeling in the sea cucumber Holothuria glaberrima, indicating a key role in mutable connective tissue (MCT) changes that may lead to some of the signs exhibited in SSWD[CC3][ag] [k4][ah] (Ribeiro et al 2012, Quinones et al 2002, Quinones et al 2004). In the treated stars, both collagenase-3 (contig_235, contig_296, contig_475) and stromelysin (contig_360) were over-expressed. Additionally, 9 other proteases change expression with treatment, disintegrins ADAM-10 (contig_1865) and ADAM12 (contig_111[ai]2), the ADAMTS proteins ADAM-TS3, (contig_1128), ADAM-TS6 (contig_6569), and ADAM-TS13 (contig_7590), and the membrane-associated metalloproteinases MMP-3 (contig_360), MMP-13 (contig_235, contig_296, contig_475), MMP-16 (contig_202), MMP-17 (contig_1508), MMP19 (contig_6080), and MMP24 (contig_61). Differential expression for the disintegrins and MMPs are all increased in the treated samples[aj] except for ADAM-TS6, indicating a massive MCT response. This physiological response is likely linked to the physical “melted” appearance of diseased stars. The antagonists Tissue Inhibitors of Metalloproteinases (TIMPs) have been shown to stiffen MCT (Sugni et al 2014, Tipper et al 2003) and had mixed expression changes between treated and control (contig_1066, contig_2994, contig_4458, contig_27280). Interestingly, collagen alpha-1(XIV) chain (contig_2464), a component of the ECM, was over-expressed, possibly due to the sea stars’ attempt to reconstruct tissues affected by immense changes in tissue organization[CC5][ak][al] . This analysis of differentially expressed genes and enriched pathways in the sea star transcriptome shows that, similar to urchins, sea stars have a complex immune response involving thousands of genes. Pycnopodia helianthoides exhibiting clinical signs of SSWD had differential expression in the toll pathway, complement cascades, melanization, clotting response, ECM remodeling, G protein coupled receptors, and neural involvement, highlighting that the experimental treatment caused a significant host response. While further studies are needed to differentiate the gene expression changes following infection with SSWD from other pathogens, this insight into the P. helianthoides transcriptome lays the foundations for better characterization of sea star immune responses. Comparisons with the few other echinoderm transcriptomes can provide insight into the evolution of immunity-related genes in deuterostomes[am]. Citations Adams, J. M., & Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science, 281(5381), 1322-1326. Aderem, A., & Ulevitch, R. J. (2000). Toll-like receptors in the induction of the innate immune response. Nature, 406(6797), 782-787. Akira, S., Uematsu, S., & Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell, 124(4), 783-801. Anders S and Huber W (2010). “Differential expression analysis for sequence count data.” Genome Biology, 11, pp. R106. http://dx.doi.org/10.1186/gb-2010-11-10-r106,http://genomebiology.com/2010/11/10/R106/. Andrade, W. A., Silva, A. M., Alves, V. S., Salgado, A. P. C., Melo, M. B., Andrade, H. M., ... & Gazzinelli, R. T. (2010). Early endosome localization and activity of RasGEF1b, a toll-like receptor-inducible Ras guanine-nucleotide exchange factor. Genes and immunity, 11(6), 447-457. Araki, M., & Taketani, S. Neurensin: A novel neuron-specific gene and its role in membrane trafficking and neurite outgrowth. Bates, A. E., B. J. Hilton, and C. D. G. Harley. 2009. Effects of temperature, season and locality on wasting disease in the keystone predatory sea star Pisaster ochraceus. Diseases of aquatic organisms 86:245–51. Beck, G., & Habicht, G. S. (1996). CHARACTERIZATION OF AN IL-6-LIKE MOLECULE FROM AN ECHINODERM (< i> ASTERIAS FORBESI).Cytokine, 8(7), 507-512. Bettler, B., Kaupmann, K., Mosbacher, J., & Gassmann, M. (2004). Molecular structure and physiological functions of GABAB receptors. Physiological reviews, 84(3), 835-867. Bitto, A., C. A. Lerner, et al. (2014). "P62/SQSTM1 at the interface of aging, autophagy, and disease." Age (Dordr) 36(3): 9626. Blair, J. E., and S. B. Hedges. 2005. Molecular phylogeny and divergence times of deuterostome animals. Molecular biology and evolution 22:2275–84. Bloom, A. M., Kask, L., and Dahlback, B. (2003). CCP1–4 of the C4b-binding protein -chain are required for factor I mediated cleavage of complement factor C3b, Molecular Immunology, 39, 547–556. Boise, L. H., González-Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., ... & Thompson, C. B. (1993). Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. cell, 74(4), 597-608. Buckley KM, Terwilliger DP, Smith LC (2008) Sequence variations in 185/333 messages from the purple sea urchin suggest posttranscriptional modifications to increase immune diversity. J Immunol 181: 8585–8594. Burge, C. a, C. Mark Eakin, C. S. Friedman, B. Froelich, P. K. Hershberger, E. E. Hofmann, L. E. Petes, K. C. Prager, E. Weil, B. L. Willis, S. E. Ford, and C. D. Harvell. 2014. Climate change influences on marine infectious diseases: implications for management and society. Annual review of marine science 6:249–77. Burke, R. D., Angerer, L. M., Elphick, M. R., Humphrey, G. W., Yaguchi, S., Kiyama, T., ... & Thorndyke, M. C. (2006). A genomic view of the sea urchin nervous system. Developmental biology, 300(1), 434-460. Cameron, R. Andrew, et al. "SpBase: the sea urchin genome database and web site." Nucleic acids research 37.suppl 1 (2009): D750-D754. Canicatti C and D’Ancona G (1988). “Cellular Aspects of Holothuria polii Immune Response.” Journal of Invertebrate Pathology, 53, pp. 152-158. Cerenius L and Söderhäll K (2004). “The prophenoloxidase-activating system in invertebrates.” Immunological Reviews, 198, pp. 116–26. Chi, Z., Zhang, J., Tokunaga, A., Harraz, M. M., Byrne, S. T., Dolinko, A., ... & Dawson, V. L. (2012). Botch promotes neurogenesis by antagonizing Notch. Developmental cell, 22(4), 707-720. Chun, S. J., Rasband, M. N., Sidman, R. L., Habib, A. A., & Vartanian, T. (2003). Integrin-linked kinase is required for laminin-2–induced oligodendrocyte cell spreading and CNS myelination. The Journal of cell biology, 163(2), 397-408. Clow L, Raftos D, Gross P, Smith C (2004). “The sea urchin complement homologue, SpC3, functions as an opsonin.” Journal of Experimental Biology, 207, pp. 2147-2155. Cohen-Sfady, M., G. Nussbaum, et al. (2005). "Heat Shock Protein 60 Activates B Cells via the TLR4-MyD88 Pathway." The Journal of Immunology 175(6): 3594-3602. Coscia, M. R., Giacomelli, S., & Oreste, U. (2011). Toll-like receptors: an overview from invertebrates to vertebrates. Inv Surv J, 8, 210-226. Cuadrado, A., Martín-Moldes, Z., Ye, J., & Lastres-Becker, I. (2014). Transcription Factors NRF2 and NF-κB Are Coordinated Effectors of the Rho Family, GTP-binding Protein RAC1 during Inflammation. Journal of Biological Chemistry, 289(22), 15244-15258. Dheilly, N. M., D. a Raftos, P. a Haynes, L. C. Smith, and S. V Nair. 2013. Shotgun proteomics of coelomic fluid from the purple sea urchin, Strongylocentrotus purpuratus. Developmental and comparative immunology 40:35–50. Dorsam, R. T., & Gutkind, J. S. (2007). G-protein-coupled receptors and cancer. Nature Reviews Cancer, 7(2), 79-94. Du, H., Z. Bao, R. Hou, S. Wang, H. Su, J. Yan, M. Tian, Y. Li, W. Wei, W. Lu, X. Hu, S. Wang, and J. Hu. 2012. Transcriptome sequencing and characterization for the sea cucumber Apostichopus japonicus (Selenka, 1867). PloS one 7:e33311. Duggins, D. 1983. Starfish predation and the creation of mosaic patterns in a kelp-dominated community. Ecology 64:1610–1619. Eckert, G., J. Engle, and D. Kushner. 2000. Sea star disease and population declines at the Channel Islands. Pages 390–393 Proceedings of the Fifth California Islands Symposium. Minerals Management Service 99-0038. Elenkov, I. J., Webster, E., Papanicolaou, D. A., Fleisher, T. A., Chrousos, G. P., & Wilder, R. L. (1998). Histamine potently suppresses human IL-12 and stimulates IL-10 production via H2 receptors. The Journal of Immunology,161(5), 2586-2593. Elphick, M. R., & Melarange, R. I. C. H. A. R. D. (2001). Neural control of muscle relaxation in echinoderms. Journal of Experimental Biology, 204(5), 875-885. Finberg, R. W., J. P. Wang, et al. (2007). "Toll like receptors and viruses." Rev Med Virol 17(1): 35-43. Franco, C. F., R. Santos, and A. V Coelho. 2011. Proteome characterization of sea star coelomocytes--the innate immune effector cells of echinoderms. Proteomics 11:3587–92. Grohmann, U., & Bronte, V. (2010). Control of immune response by amino acid metabolism. Immunological reviews, 236(1), 243-264. Harvell, C. D., K. Kim, J. M. Burkholder, R. P. Colwell, D. J. Grimes, E. E. Hofmann, E. K. Lipp, A. D. M. E. Osterhaus, R. M. Overstreet, J. W. Porter, G. W. Smith, and G. R. Vasta. 1999. Emerging Marine Diseases--Climate Links and Anthropogenic Factors. Science 285:1505–1510. Hibino, T., M. Loza-Coll, C. Messier, A. J. Majeske, A. H. Cohen, D. P. Terwilliger, K. M. Buckley, V. Brockton, S. V Nair, K. Berney, S. D. Fugmann, M. K. Anderson, Z. Pancer, R. A. Cameron, L. C. Smith, and J. P. Rast. 2006. The immune gene repertoire encoded in the purple sea urchin genome. Developmental biology 300:349–65. Hidaka, M., and Takahashi, K. (1983). Fine structure and mechanical properties of the catch apparatus of the sea-urchin spine, a collagenous connective tissue with muscle-like holding capacity. Journal of Experimental Biology, 103(1), 1-14. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009;4(1):44-57. (got this from the “how to cite section on DAVID website) Into, T., Inomata, M., Niida, S., Murakami, Y., & Shibata, K. I. (2010). Regulation of MyD88 aggregation and the MyD88-dependent signaling pathway by sequestosome 1 and histone deacetylase 6. Journal of Biological Chemistry, 285(46), 35759-35769. Janeway, C. A., Travers, P., Walport, M. J., & Shlomchik, M. J. (2001).Immunobiology: the immune system in health and disease (Vol. 2). Churchill Livingstone. Katow, H. (2008). Spatio-temporal expression of a Netrin homolog in the sea urchin Hemicentrotus pulcherrimus (HpNetrin) during serotonergic axon extension. International Journal of Developmental Biology, 52(8), 1077. Kemper C, Mitchell L, Zhang L, Hourcade D (2008). “The complement protein properdin binds apoptotic T cells and promotes complement activation and phagocytosis.” PNAS, 105, pp. 9023-9028. Krishnan, A., Almén, M. S., Fredriksson, R., & Schiöth, H. B. (2013). Remarkable similarities between the hemichordate (< i> Saccoglossus kowalevskii) and vertebrate GPCR repertoire. Gene, 526(2), 122-133. lectin pathways.” Immunopharmacology, 42, pp. 107-120 Li, H., Alavian, K. N., Lazrove, E., Mehta, N., Jones, A., Zhang, P., ... & Jonas, E. A. (2013). A Bcl-xL–Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis. Nature cell biology, 15(7), 773-785. MARINe. 2013. Unprecedented Sea Star Mass Mortality along the West Coast of North America due to Wasting Syndrome. Press Release. Pages 1–3. Santa Cruz, CA. Matsushita, M., Endo, Y., Fujita, T. (2000). Cutting Edge: Complement-Activating Complex of Ficolin and Mannose-Binding Lectin-Associated Serine Protease. The Journal of Immunology, 164(5), 2281-2284. Moreira, R., Milan, M., Balseiro, P., Romero, A., Babbucci, M., Figueras, A., ... & Novoa, B. (2014). Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray. BMC genomics, 15(1), 267. Morozova, Olena, Martin Hirst, and Marco A. Marra. "Applications of new sequencing technologies for transcriptome analysis." Annual review of genomics and human genetics 10 (2009): 135-151. Muroi, M., & Tanamoto, K. I. (2008). TRAF6 distinctively mediates MyD88-and IRAK-1-induced activation of NF-κB. Journal of leukocyte biology, 83(3), 702-707. Muzio, M., Ni, J., Feng, P., & Dixit, V. M. (1997). IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science,278(5343), 1612-1615. Neves, S. R., Ram, P. T., & Iyengar, R. (2002). G protein pathways. Science,296(5573), 1636-1639. Page-McCaw, A., Ewald, A. J., & Werb, Z. (2007). Matrix metalloproteinases and the regulation of tissue remodelling. Nature reviews Molecular cell biology,8(3), 221-233. Paine, R. T. 1974. Intertidal Community Structure. Experimental Studies on the Relationship between a Dominant Competitor and Its Principal Predator. Oecologia 15:93–120. Plow, E. F., Haas, T. A., Zhang, L., Loftus, J., & Smith, J. W. (2000). Ligand binding to integrins. Journal of Biological Chemistry, 275(29), 21785-21788. Quiñones, J. L., Rosa, R., Ruiz, D. L., & Garcı́a-Arrarás, J. E. (2002). Extracellular Matrix Remodeling and Metalloproteinase Involvement during Intestine Regeneration in the Sea Cucumber Holothuria glaberrima. Developmental biology, 250(1), 181-197. Rast, J. P., Smith, L. C., Loza-Coll, M., Hibino, T., & Litman, G. W. (2006). Genomic insights into the immune system of the sea urchin. Science,314(5801), 952-956. Rauta, P. R., Samanta, M., Dash, H. R., Nayak, B., & Das, S. (2014). Toll-like receptors (TLRs) in aquatic animals: Signaling pathways, expressions and immune responses. Immunology letters, 158(1), 14-24. Reid, K. B. and Porter, R. R. (1981). The Proteolytic activation system of complement. Annual Reviews of Biochemistry, 50, 433-64. Rojo, I., de Ilárduya, Ó. M., Estonba, A., & Pardo, M. Á. (2007). Innate immune gene expression in individual zebrafish after Listonella anguillarum inoculation. Fish & shellfish immunology, 23(6), 1285-1293. Runza V, Schwaeble W, and Mannel D (2008). Ficolins: Novel pattern recognition molecules of the innate immune response. Immunobiology, 213, pp. 297-306. Sarkar, D., R. Desalle, et al. (2008). Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting. Proc Natl Acad Sci U S A 105(44): 17040-17045. Shin, H. J., & Youn, H. S. (2013). TBK1-targeted suppression of TRIF-dependent signaling pathway of Toll-like receptors by helenalin. Life sciences,93(22), 847-854. Smith C, Azmui K, Nonaka M (1999). “Complement systems in invertebrates. The ancient alternative and Smith, L. C. 2010. Diversification of innate immune genes: lessons from the purple sea urchin. Disease models & mechanisms 3:274–9. Smith, V. J., & Söderhäll, K. (1991). A comparison of phenoloxidase activity in the blood of marine invertebrates. Developmental & Comparative Immunology,15(4), 251-261. Sugni, M., Fassini, D., Barbaglio, A., Biressi, A., Di Benedetto, C., Tricarico, S., ... & Candia Carnevali, M. D. (2014). Comparing dynamic connective tissue in echinoderms and sponges: Morphological and mechanical aspects and environmental sensitivity. Marine environmental research, 93, 123-132. Sun, L., Deng, L., Ea, C. K., Xia, Z. P., & Chen, Z. J. (2004). The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Molecular cell, 14(3), 289-301. Supek F, Bošnjak M, Škunca N, Šmuc T (2011) REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms. PLoS ONE 6(7): e21800. Takeda, K., & Akira, S. (2004, February). TLR signaling pathways. In Seminars in immunology (Vol. 16, No. 1, pp. 3-9). Academic Press. Terwilliger D, Clow L, Gross P, and Smith C (2004). “Constitutive expression and alternative splicing of the exons encoding SCRs in Sp152, the sea urchin homologue of complement factor B. Implications on the evolution of the Bf/C2 gene family.” Immunogenetics, 56, pp. 531-543. Tipper, J. P., Lyons-Levy, G., Atkinson, M. A., & Trotter, J. A. (2002). Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis. Matrix biology, 21(8), 625-635. Valanne, S., Wang, J. H., & Rämet, M. (2011). The Drosophila toll signaling pathway. The Journal of Immunology, 186(2), 649-656. Vouret-Craviari, V., Boulter, E., Grall, D., Matthews, C., & Van Obberghen-Schilling, E. (2004). ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells. Journal of cell science, 117(19), 4559-4569. Wang, J., Hu, Y., Deng, W. W., & Sun, B. (2009). Negative regulation of Toll-like receptor signaling pathway. Microbes and Infection, 11(3), 321-327. Wang, P. H., Gu, Z. H., Wan, D. H., Zhang, M. Y., Weng, S. P., Yu, X. Q., & He, J. G. (2011). The shrimp NF-κB pathway is activated by white spot syndrome virus (WSSV) 449 to facilitate the expression of WSSV069 (ie1), WSSV303 and WSSV371. PloS one, 6(9), e24773. Widmaier, M., Rognoni, E., Radovanac, K., Azimifar, S. B., & Fässler, R. (2012). Integrin-linked kinase at a glance. Journal of cell science, 125(8), 1839-1843. Zambon, R. A., Nandakumar, M., Vakharia, V. N., & Wu, L. P. (2005). The Toll pathway is important for an antiviral response in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 102(20), 7257-7262. Zervas, C. G., Psarra, E., Williams, V., Solomon, E., Vakaloglou, K. M., & Brown, N. H. (2011). A central multifunctional role of integrin-linked kinase at muscle attachment sites. Journal of cell science, 124(8), 1316-1327. [a]Add in the more of the collection/site methods - so we can talk about the differences in exposure. [b]add version numbers? [c]Add to the table and change the columns so all are clearly labeled. Change the numbers to site codes. [d]+fuess@uta.edu +gw.sarah@gmail.com - I think you might be using wrong starting file - do you have url to notebook that has details on count file? [e]make plot bigger [f]We have duplicate contigs in here, do we want them? Is there a real reason they're here or is it an oversight? I forget how we made these lists exactly [g]end of day this info is going into supp File- should think of it in that context [h]what is this about? [i]I think this is in reference to talking about the transcriptome in general. I tried to work it into the first paragraph but it needs some revision. [j]Do we want this part here?? Definitely need a short paragraph in the discussion in regards to general transcriptome description. May flow better as the second paragraph though. [k]I kind of added this information in to the first paragraph. Might need some flow work though [l]Also check out Buckley 2008 - relevant [m]Are we only saying when things are differentially expressed or what? [n]simplified for now but HSP60 also has an IL-6 connection [o]See LeClerc 2000 - "antibody-like" (ABL) kappa in Asterias [p]do we want to say enriched or DE? [q]I don't know... [r]me neither...haha [s]what if we say differentially expressed for now and then note at the beginning that all the differentially expressed genes we're talking about are part of enriched pathways? We'll all have to figure out a coordinated system soon for this [t]that sounds good [u]KEGG - http://www.genome.jp/kegg-bin/show_pathway?hsa04620 [v]Need to investigate this further to make a solid comment. [w]Including in sea stars - Mogilenko et al. 2010 with LPS (sea star immunity papers folder) [x]Ruth, is this yours? (or Morgan?) I'm going to try and combine our sections since we have some considerable overlap with ECM/MCTs. [y]okey dokes. let me know if you have any questions. [z]Ruth's, Collin's, & Reyn's new section [aa]Overall I'm following - the reorganization of this seems to be successful to me! [ab]Rephrase - like: "was differentially expressed in treatment stars for two contigs (contig_, contig_). Interestingly, this disruption went in different directions for each contig. [ac]the way this is written makes it seem like the "treatment stars" are not the same thing as the "treatment condition"… chose one? [ad]rephrase this sounds forced [ae]so MMPs cleave and vWF cleave? Lacking clarity… I think you just need two sentences [af]Perhaps you should use full protein names first, then use abbreviations?? (This is assuming the name of the protein isn't just vWF clotting factor). [ag]Collin: Spell this out more. The link is missing between sea cucumber tissue remodeling and signs of SSWD. [ah]How about “…in both tissue remodeling and the modulation of mutable connective tissue in echinoderms, processes that are likely implicated in the signs of SSWD” ? [ai]?? [aj]Going back and forth between treated stars/conditions/samples is confusing - just stars I think [ak]Collin: Looks good! I like the paragraphs joined. We may want to finesse the order of some of these statements, so that they build upon one another more, but that can be done once we know where the placement of these paragraphs will be. [al]Agreed, especially in leading up to the big point that this is all behind the "melting" [am]This ending is pretty good, I like the wrapping up by bringing back themes. Follows up on introduction well.