Genetic Response to Acute Hypoxia in Channel Catfish (Ictaluruspunctatus), Blue Catfish (Ictalurusfurcatus) and Hybrid Catfish by Michael William Gyengo A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Masters of Science Auburn, Alabama August 4, 2012 Keywords: Hypoxia, Catfish, Aquaculture, Gene Expression, Stress Copyright 2012 by Michael William Gyengo Approved by Eric Peatman, Chair, Assistant Professor of Fisheries and Allied Aquacultures Rex A. Dunham, Alumni Professor of Fisheries and Allied Aquacultures Ronald P. Phelps,Associate Professor of Fisheries and Allied Aquacultures ii Abstract The catfish industry is one of the largestdomestic aquaculture markets in the United States. In aggregate, the industry was valued at $423 million dollars in 2011 and is dominated by the production of channel catfish (Ictaluruspunctatus) and, to a lesser extant, hybrid catfish, a cross between channel female and blue male catfish (Ictalurusfurcatus). Dissolved oxygen (DO) levels are a critical component governing the success and profitability of catfish pond aquaculture. Low DO levels are known to negatively impact feed utilization/growth, health/stress levels, and ultimately survival. However, major gaps remain in our understanding of differential susceptibilities of channel, blue, and hybrid catfish to low DO and the molecular consequences of these events on critical genes governing metabolism/growth, stress/immunity, and overall physiological functions. Here, therefore, we examined both phenotypic and genotypic responses to acute hypoxia in the three catfish groups. It was determined that genotypic reaction to hypoxia is highly variable between the different catfish families, the various tissues and at between time intervals. Six different known catfish genes were investigated at time points of 2, 4, and 8 hr at 2mg/l dissolved oxygen and at 2 and 4 hr at 1.5mg/L in liver and gill tissue. The observed genes HIF-1, HIF-2, BPI, Ferritin, Myostatin and NKEF showed highly variable regulation changes at different time points and oxygen levels. Channel and hybrid catfish showed almost identical phenotypic stress response times while blue catfish were significantly quicker to show observable stress. This pattern of hybrid, channel similarity held true across the majority of treatments tested with this pairing showing iii much greater sensitivity to the HIF family of genes than their blue counterparts. Hybrid and channel catfish also showed similar genotypic response for BPI genes with multiple significant down regulated time points for BPI genes in gill and liver tissue. Both hybrid and channel catfish recorded their largest fold change of any gene at the 8 hr at 2mg/L time point in the ferritin liver trial reporting an up regulation of 26.9 fold and 75 fold respectively. The only tested gene that showed any similarity between blue and hybrid catfish was myostatin. Blue catfish showed a 24.1 fold up regulation in liver at 4 hr and 1.5mg/L oxygen level while hybrid catfish showed a 21 fold increase in liver tissue at 8 hr at 2mg/L oxygen. Outside of the myostatin gene blue catfish showed muted sensitivity to the treatments compared to channel and hybrid catfish. This study is one of the few investigating acute hypoxia as it relates to genetic change and so there are few other results to compare to. Our findings provide an early foundation of understanding of the consequences of low oxygen events and should provide a scientific basis upon which to set minimum DO thresholds for catfish aquaculture. iv Acknowledgments I want to thank my family, friends, and committee members for their assistance in completing this project. I want to thank Dr. Peatman for his guidance and patience. Thanks is also given to Chao Li for his assistance in the molecular aspects of the project. v Table of Contents Abstract ......................................................................................................................................... ii Acknowledgments........................................................................................................................ iv List of Tables ............................................................................................................................... vi List of Figures ............................................................................................................................. vii List of Abbreviations ................................................................................................................... ix Chapter 1 ..................................................................................................................................... 1 Introduction ..................................................................................................................... 1 Literature Cited ............................................................................................................... 10 Materials and Methods .................................................................................................... 16 Results ............................................................................................................................. 27 Discussion ....................................................................................................................... 53 Literature Cited ............................................................................................................... 65 Appendix 1 .................................................................................................................................. 69 Appendix 2 .................................................................................................................................. 71 Appendix 3 .................................................................................................................................. 73 vi List of Tables Table 1. Raw data fish weights .............................................................................................. 16 Table 2. Summary statistics fish weights ............................................................................... 18 Table 3. Gene names and primer sequences .......................................................................... 21 Table 4. Observed phenotypic stress ..................................................................................... 24 Table 5. Summary statistics phenotypic stress ..................................................................... 26 vii List of Figures Figure 1. Diagram of water flows for experimental trials ........................................................ 19 Figure 2. HIF-1 gene in gill tissue relative expression over time and intensity ....................... 29 Figure 3. HIF-1 gene in liver tissue relative expression over time and intensity ..................... 30 Figure 4. HIF-2 gene in gill tissue relative expression over time and intensity ....................... 32 Figure 5. HIF-2 gene in liver tissue relative expression over time and intensity ..................... 35 Figure 6. BPIgene in gill tissue relative expression over time and intensity ........................... 36 Figure 7. BPIgene in liver tissue relative expression over time and intensity ......................... 38 Figure8. Ferritingene in gill tissue relative expression over time and intensity ..................... 39 Figure9. Ferritingene in liver tissue relative expression over time and intensity ................... 41 Figure10. Myostatingene in gill tissue relative expression over time and intensity ................. 42 Figure11. Myostatingene in liver tissue relative expression over time and intensity ............... 44 Figure12. NKEFgene in gill tissue relative expression over time and intensity ....................... 45 Figure13. NKEFgene in liver tissue relative expression over time and intensity ..................... 46 Figure14. Side by side comparison fold change 1.5mg/L vs. 2mg/L all genes blue gill ......... 47 Figure15. Side by side comparison fold change 1.5mg/L vs. 2mg/L all genes channel gill .... 48 Figure16. Side by side comparison fold change 1.5mg/L vs. 2mg/L all genes hybrid gill ...... 49 Figure17. Side by side comparison fold change 1.5mg/L vs. 2mg/L all genes blue liver ........ 50 Figure18. Side by side comparison fold change 1.5mg/L vs. 2mg/L all genes blue liver ........ 51 Figure19. Side by side comparison fold change 1.5mg/L vs. 2mg/L all genes blue liver ........ 52 viii List of Abbreviations MS222- TricaineMethanesulfonate HIF-1- Hypoxia Inducible Factor ? subunit HIF-2- Hypoxia Inducible Factor 2 ? subunit BPI- Bactericidal Permeability-Increasing NKEF- Natural Killer Enhancing Factor ESC- Enteric Septicemia of Catfish LPS- Lipopolysaccharide m-RNA- messenger Ribonucleic acid q-RT PCR- Quantitative Real Time Polymerase Chain Reaction TGF?- Transforming Growth Factor Beta ROS- Reactive Oxygen Species MG/L-Milligrams per Liter 1 Introduction Catfish aquaculture is one of the oldest and nationally important forms of aquaculture in North America. Commercial culture of the channel catfish, Ictaluruspunctatus, began in the 1960?s in Arkansas and Alabama and quickly spread throughout the region with Mississippi becoming the largest producer. This trend continues through today with Mississippi still accounting for the highest percentage of farmed catfish, followed by Alabama, Arkansas, and South Carolina respectively (Hanson and Sites 2011). Domestic production steadily increased to a peak in 2003 of 662 million pounds processed and has since dropped precipitously in 2010 by 327 million pounds, a decrease of 49% and a level of domestic production not seen since the late 1980?s (Hanson and Sites 2011). A combination of events have contributed to the decline of the domestic catfish industry include rise in feed costs, increasing labor and land costs, and perhaps most importantly a massive increase in the amount of imported catfish and tilapia from Asian producers. Imported frozen fillets of Asian catfish were of minimal impact to the industry until 2005 when import quantity reached 30 million pounds of frozen fillet. Since 2005 imported catfish has increased every year to its current total of 204 million pounds (Hanson and Sites 2011). This has represented an almost complete reversal of frozen fillet percentages which in 2005 stood at 80 percent domestic and 20 percent imported for total frozen fillets to the current 76 percent imported and 26 percent domestically produced frozen catfish fillets. Even with this decline, the sales value of domestic catfish products (food fish, brood fish, stockers, fry and fingerlings) was $423 million dollars and 2011 showed profitability in the industry for the first time in 2 years(Hanson and Sites 2011). Pressure from overseas competitors combined with volatile feed and fuel prices have required domestic producers toimprove their farming practices and efficiency or leave the industry altogether. The only viable ways to accomplish this is through better and different farming practices, such as in-pond raceways, or through genetic improvements that lead to increased yields with minimal additional input costs. Genetic enhancement has been occurring in aquaculture for as long as farmers have been able to control the conditions under which fish spawn. From the beginning of the industry farmers have chosen the largest and hardiest fish as the parents for future generations. The production of channel catfish initially dominated the industry due to its superior growth to market size of all ictalurid species studied(Dunham et al. 1993). However, it does not necessarily contain a superior genotype for all aspects of aquaculture. Other species of ictalurids have traits that, taken independent of other aspects, would make them appropriate for culture systems andhave been considered as alternatives for culture or viacross breedingintroduced into existing channel catfish genomes. These species include the bullhead catfishes (genus Ameiurus), which tolerate low dissolved oxygen levels but have extremely slow growth and poor resistance to diseases, the white catfish (Ameiuruscatus), which has accelerated initial growth, relatively good growth at cold temperatures, and have shown resistance against low dissolved oxygen concentrations, but have slow growth during the adult grow out phase, low dress-out percentage, and poor survival. The flathead catfish (Pylodictisolivaris), which exhibitfast growth to market, but are cannibalistic and difficult to harvest. The blue catfish (Ictalurusfurcatus) exhibit relatively fast growth, high dress-out percentage, good resistance to enteric septicemia or (ESC), and are easy to harvest via seining, but are considered to have relatively poor resistance to pathogens (Dunham et al. 1993). Taken 3 in aggregate no other species of ictalurid shows as many characteristics amenable to commercial production as the mainstay of the industry, the channel catfish. After the channelcatfish is the blue catfish in terms of desirable traits for the industry and thus research to increase the production of these two species continues (Dunham et al. 1993). Both of these species show behavior and physical characteristics that, if specifically chosen independent of other deleterious traits and combined, would make the optimal aquaculture catfish. Traits inwhich channel catfish are superior to the blue catfish are growth, tolerance to handling stress, tolerance of high ammonia, ability to withstand high nitrite, resistance to pathogens, particularlyFlavobacterium columnarisand the parasite Ichthyophthiriusmultfiliis, and earlier sexual maturity (Dunham et al. 1993; Dunham and Argue 2000). Traits for which the blue catfish displays superiority are uniformity of growth, reduced susceptibility to channel catfish virus and Edwardsiellaictaluri (ESC), increased seinabilityover channel catfish, and increased dress-out percentage (Dunham et al. 1993; Dunham and Argue 2000). With each species showing different strengths and weaknesses neither can be considered optimal for every culture situation (Dunham et al. 1993).To further cloud the choice of species to culture, both exhibit a high degree of variability in culture traits that arises due to strain variation within each species (Dunham et al. 1993). With both species showing superiority for various traits culturists have turned to genetic enhancement as a means of improvement. Channel catfish, being the dominant culture species, have received the majority of attention for enhancement research. Genetic research to improve the culture traits of catfish officially began in the 1960?s although less rigorous selection for growth and size has likely occurred from the moment captive spawning was achieved. Multiple techniques have been employed in order to increase desirable production characteristics, most 4 notably mass selection of channel catfish for faster growth to market size (Dunham et al. 1987;Dunham and Brummett 1999; Dunham et al. 1999, Rezk et al. 2003), intraspecific breeding programs to isolate and combine preferred characteristics of different strains of channel catfish (Dunham et al. 1983; Dunham et al. 1987), creation of sterile triploid channel catfish (Lilyestrom et al. 1999), and interspecific hybridization (Dunham et al. 1987; Dunham and Brummett 1999; Dunham et al. 1999; Argue et al. 2003). These methods, with the exception of the development of triploid channel catfish (Lilyestrom et al. 1999), have resulted in significantly improved culture traits for the species examined. Mass selection has shown to be one of the most powerful methods for improvements in the growth of channel catfish (Bondari 1983; Dunham and Smitherman 1983b; Dunham et al. 1987; Dunham and Smitherman 1987; Dunham and Brummett 1999; Dunham et al. 1999).Studies have reported up to 50% increase in body weight after four generations of mass selection (Padi 1995). This impressive increase in growth as a response to selection for body weight also resulted in increased survival, feed conversion ratios, and disease resistance (Dunham and Smitherman 1983). While these improvements are substantial, a comparison of two channel catfish lines selected for faster growth for two generations compared to the interspecific cross of channel catfish female X blue catfish male hybrid (CB hybrid) indicated that the hybrid exhibited faster growth than either of the two select lines (Dunham and Brummet 1999). Intraspecific breeding of various strains of channel catfish have also shown significant improvements for the species. Studies have reported that the intraspecific crossbreed from the pairing of a Marion strain female channel catfish with a Kansas strain male channel catfish (MK) exhibited faster growth to 100g than the CB hybrid. However these gains were mitigated once both fish reached 500g in the same time frame(Dunham et al. 1987). Other studies have 5 shown67% of intraspecific crossbreeds examined exhibited improved growth compared to parental controls, but reciprocal intraspecific hybrids did not grow at the same rates (Dunham and Smitherman 1983). From the beginning of controlled selection in the 1960?s, a total of fifty different types of ictalurid hybrids havebeencreated (Dupree and Green 1969; Dupree et al. 1969; Dunham et al. 1987; Goudieet al. 1993; Dunham et al. 2000). These hybrids were created as a result of various crosses of channel catfish with other members of the ictalurid family, including the following species: white catfish,brown bullhead (Ameiurusnebulosus), yellow bullhead (A.natalis),black bullhead (Ameiurusmelas), flathead catfish, and blue catfish (Goudie et al. 1993). Offspring from these combinations produced organisms with characteristics of each of the parents but not always of the desirable trait (Goudie et al. 1993). The majority of these crosses resulted in inferior offspring in opposition to the desired result of an improved organism for culture in a commercial food production setting. The notable exception to these breeding outcomes being the cross between a female channel catfish and a male blue catfish which exhibits over dominance for traits desirable for intensive aquaculture (Dunham et al. 1982; Dunham and Smitherman 1983;Giudice 1966; Dunham et al. 2000). This hybrid cross of the blue catfish male and channel catfish female has shown significant improvements in a variety of traits.Improvements include growth uniformity (Dunham et al. 1982; Smitherman et al.1983; Argue et al. 2003), accelerated grow out (Giudice 1966; Dunham and Smitherman1981; Dunham et al. 1987; Dunham et al. 1990; Dunham and Brummett1999), enhanced tolerance to lower dissolved oxygen concentrations (Dunham et al. 1983), greater resistance to some diseases (Dunham et al.1990),in particular the major bacterial disease of catfish ESC(Wolters et al. 1996), higher dress-out percentage (Smitherman et al. 1983; 6 Argue et al.2003), higher catchability or seinability (Tave et al. 1981; Dunham et al. 1982;Smitherman et al. 1983; Dunham et al. 1986), greater feed efficiency (Li et al. 2004), and lower mortality rates (Dunham et al. 1987). Studies have also shown the hybrid to exhibit increased body weight yields of 18-100% over channel catfish (Smitherman et al. 1983; Dunham et al.1987; Dunham et al. 1990; Dunham and Brummett 1999). One of, if not, the most important aspects in the rearing of aquatic organisms is dissolved oxygen. Low dissolved oxygen levels in water and the resultant physiological stress on almost all fish is well documented. In catfish, hypoxia has been linked to increased susceptibility to Edwardsiellaictaluri, Aeromonus hydrophila andEdwardsiellatarda(Welker et al. 2007),in addition to being implicated as the stress stimulus resulting inhistopathological lesions in the gills, liver, spleen, trunk and head kidneys (Walters and Plumb,1980). In addition to being a causative agent for a wide variety of diseases, hypoxic conditions have been linked to reduced feed consumption and metabolic rate in a range of fish. For any aquacultured species the ultimate goal is growth of that organism. Hypoxia has been linked to suppressed growth in largemouth bass (Stewart et al. 1967), common carp (Chiba 1966), Coho salmon (Hermann et al. 1962; Fisher 1963), northern pike (Adelman and Smith 1970) brook trout (Whitworth 1968), yellow perch (Carlson et al. 1980), and most importantly for this study catfish (Andrews et al. 1973; Buentello et al. 1999 ; Carlson et al. 1980; Green et al. 2012). The list of species impacted by reduced feed intake, feed efficiency, overall metabolic rate and therefore growth as a result of these factors pursuant to hypoxic conditions could extend to nearly every aquatic species ever investigated. However, there are some species that exhibit a high degree of tolerance to hypoxic conditions. 7 Fish have evolved the ability to cope with a wide variety of physiological stressors present in aquatic environments and, based on the organism?s native habitat, have adapted varying degrees of sensitivity to hypoxic stress. Bottom dwelling fish such as flounder often show good hypoxia tolerance (Weber and Dewilde 1975), compared to fish that live in moving, more oxygenated, water such as Chinese sucker (Myxocyprinusasiaticus) which exhibits poor hypoxia tolerance (Pan et al. 2007). Hypoxia sensitivity can also be variable within related fish, for example, Grayling is a salmonid with high-oxygen requirements, whereas the related pike (both species belong to Protacanthopterygii) is hypoxia tolerant (Cameron 1973). Some species have evolved highly specialized mechanisms for dealing with hypoxic stress. The crucian carp (Carassiuscarassius) possesses blood with extreme affinity for oxygen allowing it to maintain aroutine rate of oxygen consumption down toa water oxygen level of 5?10% of air saturation (Sollid et al.2003). Other species such as the epaulette shark (Hemiscylliumocellatum) employ a strategy of extreme metabolic depression (Renshaw et al. 2002). Whatever strategy an organism employs for hypoxia tolerance there is likely an accompanying array of internal genetic events that coincide with the response. There are a variety of techniques for creating hypoxic conditions in a controlled manner for experimentation. These include the bubbling of nitrogen into water to strip it of oxygen (Nilson 1990; Gracey et al. 2001; Burleson et al. 2002), placing fish in ponds with known hypoxic conditions (Green et al. 2012), removing artificially supplied oxygen from a closed system (Rahman and Thomas 2007; Chen et al. 2012) andthe addition of anhydrous sodium sulfite to water to reduce oxygen (Kramer and McClure 1982; Melnychuck and Chapman 2002). Each technique carries with it pros and cons. Long running experiments with durations of weeks or months generally opt to use ponds due to the cost and complication of continuously adding 8 supplemental chemicals to the water. However, this technique does not allow tight control of the environment and oxygen levels may fluctuate greatly, both spatially and temporally. Medium duration experiments with high specificity of conditions are most easily achieved via nitrogen bubbling and, with a lesser degree of control, removal of supplemental oxygen. For an acute hypoxic experiment with a high level of control of oxygen levels the simplest method is the addition of anhydrous sodium sulfite to water. The drawback of this technique is that the desired dissolved oxygen level cannot be maintained indefinitely without the continued addition of more sulfite. Our study of genetic response to relatively brief, acute, hypoxic conditions used anhydrous sodium sulfite due to the short duration of the stress event and the necessity of highly controlled oxygen levels. Genetic research in aquatic species is relatively new compared to mammalian studies and thus there is a less comprehensive understanding of genetic phenomenon. Terrestrial and aquatic environments are extremely different with highly variable selective pressures between the two suggesting that an aquatic organism?s genetic response may not be identical to similar stress events encountered by terrestrial animals. This experiment sought to investigate the relative change in mRNA transcripts in six known genes in the three most highly represented species in catfish aquaculture. Channel, blue and hybrid catfish were subjected to acute hypoxic conditions at the end of which liver and gill tissue were harvested for molecular genetic testing. RNA was extracted and subjected to analysis by qRT-PCR to determine relative changes in gene expression between different tissues, time points and oxygen levels. The genes selected for this experiment, HIF-1, HIF-2, myostatin, ferritin, NKEF and BPI, have all been sequenced in the catfish genome, however there is little to no research on the effects of acute hypoxic stress and if regulation and function of these genes in catfish is similar to orthologs in other species. This 9 study seeks to investigate the relative change in these genes across multiple time points at two different levels of hypoxic stress in order to improve our understanding of the catfish genome and potentially lead to improvements in future selective breeding in aquacultured catfish. 10 LITERATURE CITED: Adelman, I.R., and L.L. Smith. 1970. Effect of oxygen on growth and food conversion efficiency ofnorthern pike. Progressive Fish-Culturalist 32, 93?96. Andrews, J., W.Murai, T., G. Gibbons. 1973. The influence of dissolved oxygen on the growth of channel catfish. Transactions of the American Fisheries Society 102,835?838. Argue B.J., Z. Liu, and R.A. Dunham. 2003. 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Smitherman. 1983. Crossbreeding channel catfish for improvement of body weight in earthen ponds. Growth 47: 97-103. Dunham R.A. and R.O. Smitherman. 1983. Response to selection and realized heritability for body weight in three strains of channel catfish, Ictalurus punctatus, grown in earthen ponds. Aquaculture 33: 89-96. Dunham R.A., R.O. Smitherman and C. Webber. 1983. Relative tolerance of channel X blue hybrid and channel catfish to low oxygen concentrations. Progressive Fish-Culturist 45: 55-56. Dunham R.A., R.O. Smitherman and R.K. Goodman. 1987. Comparison of mass selection, crossbreeding, and hybridization for improving growth of channel catfish. Progressive Fish-Culturist 49: 293-296. Dunham R.A., C. Hyde, M. Masser, J.A. Plumb, R.O. Smitherman, R. Perez and A.C. Ramboux. 1993. Comparison of culture traits of channel catfish, 12 Ictalurus punctatus, and blue catfish, I. furcatus. Journal of Applied Aquaculture 3: 257-267. Dunham R.A. and R.E. Brummett. 1999. Response of two generations of selection to increased body weight in channel catfish, Ictalurus punctatus, compared to hybridization with blue catfish, I. furcatus, males. Journal of Applied Aquaculture 9: 37-45. Dunham R.A., A.N. Bart, H. Kucuktas. 1999. Effects of fertilization method and of selection for body weight and species on fertilization efficiency of channel catfish eggs with blue or channel catfish sperm. North American Journal of Aquaculture 61: 156-161. Dunham R.A. and B.J. Argue. 2000. Reproduction among channel catfish, blue catfish, and their F1 and F2 hybrids. Transactions of the American Fisheries Society 129: 222-231. Dupree H. K. and O. L. Green. 1969. Comparison of feed conversion and growth rate of six catfish species and their hybrids. Southeastern Fish Cult. Lab., Marion, Ala. 13 pp. Fisher R.J. 1963. Influence of oxygen concentration and of its diurnal fluctuations on the growth of juvenile Coho salmon. MS. Thesis. Oregon State University, Corvallis, OR, USA, 48 pp. Giudice J. 1966. Growing of a blue X channel catfish hybrid as compared to its parent species. Progress Fish-Culturist 28: 142-154. Goudie C.A., T.R. Tiersch, B.A. Simco, K.B. Davis and Q. Liu. 1993. Early growth and morphology among hybrids of ictalurid catfishes. Journal of Applied 13 Aquaculture 3: 235-255. Gracey, A.Y., Troll, J.V., andSomero, G., 2001. Hypoxia-induced gene expression profiling in theeuryoxic fish Gillichthys mirabilis. Proc. Natl. Acad. Sci. U. S. A. 98, 1993?1998 Green B.W., S.D. Rawles., and B.H Beck. 2012. Response of channel X blue hybrid catfish to chronic diurnal hypoxia. Aquaculture 350-353: 183-191. Hanson, T., andD. Sites.2011 U.S Catfish Database. Mississippi State University Department ofAgricultural Economics Information Report 2012. Hermann, R.B., C.E. Warren, and P.Doudoroff. 1962. Influence of oxygen concentration on the growthof juvenileCoho salmon. Trans. Am. Fish. Soc. 91, 155?167. Lilyestrom C.G., W.R. Wolters, D. Bury, M. Rezk and R.A. Dunham. 1999. Growth, carcass traits, and oxygen tolerance of diploid and triploid catfish hybrids. North American Journal of Aquaculture 61: 293-303. Melnychuck M.C. and L.J. Chapman. 2002. Hypoxia Tolerance of Two Haplochloramine Cichlids: Swamp Leakage and Potential Interlacustrine Dispersal. Environmental Biology of Fishes65: 99-110 Nilsson G. E. (1990). Long-term anoxia in crucian carp: changes in the levels of amino acid and monoamine neurotransmitters in the brain, catecholamine?s in chromaffin tissue, and liver glycogen. J. Exp. Biol. 150, 295-320. Padi J. N. 1995. Response and correlated responses to four generations of selection for increased body weight in the Kansas strain channel catfish, Ictalurus punctatus, grown in earthen ponds. M.S. Thesis. Auburn University, AL. Pan Y., W.Q. Tang, andY.J.Zhang. 2007. Study on the oxygen consumption rate and suffocation point of Myxocyprinusasiaticus. Freshwater.Fish. (China) 37, 48?51. 14 RahmanM.S., and P. Thomas. 2007. Molecular cloning, characterization and expression of two hypoxia inducible factor alpha subunits, HIF-1? and HIF-2?, in a hypoxia tolerant marine teleost, Atlantic croaker (Micropogoniasundulatus). Gene 396,273?282. Rezk M.A., R.O. Smitherman, J.C. Williams, A. Nichols, H. Kucuktas and R.A. Dunham. 2003. Response to three generations of selection for increased bodyweight in channel catfish, Ictalurus punctatus, grown in earthen ponds.Aquaculture 228: 69-79. Renshaw G. M. C., C.B.Kerrisk, and G.E. Nilsson.2002. The role of adenosine in the anoxic survival of the epaulette shark, Hemiscylliumocellatum. Comp. Biochem. Physiol. B 131, 133-41. Sollid J., P. De Angelis, K.Gundersen, and G.E. Nilsson. 2003. Hypoxiainduces adaptive and reversible gross morphological changes in cruciancarpgills. J. Exp. Biol. 206, 3667-3673. Smitherman R.O., R.A. Dunham, and D. Tave. 1983. Review of catfish breeding research 1961-1981 at Auburn University. Aquaculture 33: 197-205. Stewart N.E., D.L. Shumway, and P.Douddoroff. 1967. Influence of oxygen concentration on thegrowth oflargemouth bass. J. Fish. Res. Board Can. 24, 475?494. Welker T.L, S.T Mcnulty, and P.H. Klesius. 2007. Effect of Sub lethal Hypoxia on the Immune Response and Susceptibility of Channel Catfish, Ictalurus punctatus, to Enteric Septicemia. Journal of the World Aquaculture Society 38: 12?23, March 2007 Walters G.R. and J.A. Plumb.1980.Environmental stress and bacterial infection in channel catfish, Ictalurus punctatus Rafinesque. Journal of Fish Biology 17: 177-185 August 1980 Weber, R.E., and J.A.M. Dewilde. 1975. Oxygenation properties of hemoglobins from flatfish Plaice(Pleuronectesplatessa) and flounder (Platichthysflesus). J. Comp. Physiol. 101, 15 99?110 Whitworth W.R. 1968. Effects of diurnal fluctuations of dissolved oxygen on the growth of brook trout. J.Fish. Res. Board Can. 25, 579?584. Wolters W.R., D.J. Wise and P.H. Klesius. 1996. Survival and antibody response of channel catfish, blue catfish, and channel catfish female X blue catfish malehybrids after exposure to Edwardsiella ictaluri. Journal of Aquatic Animal Health 8: 24 16 MATERIALS AND METHODS: Experimental fish and design Fingerling size fish for this experiment were of the obtained from existing brood stock at North Auburn Fisheries Research Unit, Alabama Agricultural Experiment Station, Auburn University. Fish had initially been raised in PAS systems at the Clemson University Fisheries unit and subsequently moved to and maintained in the S-6 indoor recirculating system and had been held at uniform temperature and water quality for the entirety of the fish?s lives. All fish were raised in extremely similar environments for the entirety of their lives. Channel catfish used in this experiment were of mixed sex and ranged in size from 12 to 22g with a mean size of 14.4g . All were from a single family of the Marion strain catfish and any fish showing signs of deformities and or erratic behavior were discarded before trials began. Blue catfish were of mixed sex and ranged in size between 13g and 24g with a mean size of 16.2g. All blue catfish were from a single family of the Rio Grande strain and were checked for physical and behavioral abnormalities pre-trial. The hybrid catfish used in this trial were of mixed sex and ranged in size from 10.5g to 21g with and average size of 13.6g.The hybrids were from a single family and a cross of Marion strain channel catfish female with Rio Grande strain blue male. Hybrid catfishwere also checked for abnormalities before trials began. Table 1.Raw fish weight data. Family, Duration, Oxygen level. Trial# Fish1 weight (g) Fish2 weight (g) Fish3 weight (g) Fish4 weight (g) Fish5 weight (g) Blue 2hr 2mg/L 1 12.4 16 14.3 20.1 17 Blue 2hr 2mg/L 2 13.2 19 17.7 16.2 14.4 Blue 2hr 2mg/L 3 16 17.2 13 14 14.3 Blue 4 hr 2mg/L 1 24.2 14 16 17.7 17 Blue 4 hr 2mg/L 2 16 15.1 19 17 14.6 17 Blue 4 hr 2mg/L 3 16 20.2 16 13.2 15 Blue 8 hr 2mg/L 1 14 24 14.4 16.2 17 Blue 8 hr 2mg/L 2 17.3 14 15 13 14.3 Blue 8 hr 2mg/L 3 15 16.4 19.2 16 15 Blue 2hr 1.5mg/L 1 17.2 14.3 13 21.2 14 Blue 2hr 1.5mg/L 2 16 20.3 16 14 17.3 Blue 2hr 1.5mg/L 3 16.7 14 20 20.4 14 Blue 4hr 1.5mg/L 1 17 15 14.3 17 15 Blue 4hr 1.5mg/L 2 15.6 14 21.3 14 20.9 Blue 4hr 1.5mg/L 3 16 14.1 17.4 15.2 14 Channel 2hr 2mg/L 1 16.3 12.3 12.3 13.2 13.1 Channel 2hr 2mg/L 2 15 14.5 14.6 15.6 14 Channel 2hr 2mg/L 3 14.5 13 13.1 16.4 14 Channel 4hr 2mg/L 1 16.4 14.7 12 14.2 14.4 Channel 4hr 2mg/L 2 14.5 13.2 16.3 12.1 14.7 Channel 4hr 2mg/L 3 15.2 13 12.1 13.3 15.7 Channel 8hr 2mg/L 1 13.3 14.6 13.4 16.2 13 Channel 8hr 2mg/L 2 14.3 22.1 16.3 13.3 14 Channel 8hr 2mg/L 3 13.8 13.3 15.1 16.2 15.8 Channel 2hr 1.5mg/L 1 14 15 14.2 13.8 14 Channel 2hr 1.5mg/L 2 14.4 14.3 15 13.6 17 Channel 2hr 1.5mg/L 3 12.4 14.1 14.3 16.7 14.2 Channel 4hr 1.5mg/L 1 17.2 13.6 14.1 14.3 12.2 Channel 4hr 1.5mg/L 2 15.5 13.2 15.1 12 15.2 Channel 4hr 1.5mg/L 3 13 17.1 16.1 14.3 14.5 Hybrid 2hr 2mg/L 1 11.2 15.3 12 13.6 15.2 Hybrid 2hr 2mg/L 2 15 14.8 13.2 11 14.3 Hybrid 2hr 2mg/L 3 14.1 12.9 15.3 13.4 13 Hybrid 4hr 2mg/L 1 13.2 14 12.2 14.3 16.2 Hybrid 4hr 2mg/L 2 14.4 15.1 11.5 12.5 14.3 Hybrid 4hr 2mg/L 3 15.2 12.4 12.2 13 15 Hybrid 8 hr 2mg/L 1 15.6 14.2 12.1 12.3 13.6 Hybrid 8 hr 2mg/L 2 11.8 13.2 13.1 13.5 14 Hybrid 8 hr 2mg/L 3 18.5 13.1 13.9 12.2 12.1 Hybrid 2hr 1.5mg/L 1 14.6 15.3 14 10.5 14.2 Hybrid 2hr 1.5mg/L 2 14.2 12.3 13.1 15.2 11.4 Hybrid 2hr 1.5mg/L 3 12.3 12.1 15.2 14 14.2 Hybrid 4hr 1.5mg/L 1 15.1 11.1 14.2 14 12.3 Hybrid 4hr 1.5mg/L 2 13.2 13.1 14 11.3 21.2 Hybrid 4hr 1.5mg/L 3 13.4 12.1 12.6 11.9 14 18 Table 2. Summary statistics for fish weight data. Blue (g) Channel (g) Hybrid (g) All Fish (g) Mean 16.21 14.44 13.58 14.74 SD 2.54? 1.59? 1.67? 2.25? Variance 6.44 2.55 2.8 5.096 Min 12.4 12 10.5 10.5 Max 24.2 22.1 21.2 24.2 A 2-way ANOVA analysis of fish weights determined that there was a significant difference between aggregate fish weights for each family tested but size disparities were spread evenly enough between trials so there was no statistical difference between treatments among fish family weights. ( F, Fcrit, and tables in appendix 1) Two 1,135 L tanks were placed on constructed support tables of 1.2meters in height in order to give each tank enough head pressure to gravity flow water through smaller holding units for the duration of each trial. Fivecm diameter PVC pipe was plumbed into each tank and equipped with a ball valve for flow regulation. Both tanks piping led to a centralized 10cm Y joint that allowed for both to flow to the same end point yetbe operated independently of one another. From this the Y joint extended to a line of 3 1.9cm ball valves with brass nipples fitted to each. Attached to each brass nipple was ? inch flexible plastic tubing which led into isolated 19 liter plastic buckets. Water could then be fed into each individual tank at the set rate of 1.25 liters per minute per tank to maintain uniform dissolved oxygen levels. All dissolved oxygen levels were checked every 10 minutes using a YSI Pro20 dissolved oxygen sensor with galvanic probe and 4m cable to determine oxygen levels were held at desired levels.(Figure 1). 19 Figure 1. Diagram of tank set up and water flows. Two independent tanks were necessary due to the fact that one reservoir tank did not have enough capacity for the 8 hr trials. Each 1,135 L tank was filled to capacity with well water and heated to 24? C to match the temperature at which fish were being held. Anhydrous sodium sulfite was added to the filled reserve tanks until desired dissolved oxygen levels of 2 mg/L or 1.5 mg/L were achieved, dependant on the treatment desired. These relative oxygen levels were determined by observing multiple test runs comparing time to show visible stress against a range of oxygen Reservoir Tanks Y-Joint Hypoxia Tanks 20 levels.Phenotypic stress was defined as fish leaving a resting state on the tank bottom and actively swimming on the surface. Oxygen levels above 2 mg/L did not achieve visible stress in channel and hybrid catfish while values below 1.5 mg/L produced mortality too quickly in blue catfish. At the selected time intervals and dissolved oxygen levels trials could achieve phenotypic stress in the majority of fish but not induce unwanted mortality. Fish were selected at random in groups of 5 from each of the aforementioned families and placed into the treatment tanks at normal oxygen levels. The valves were fully opened for 10 minutes allowing oxygen levels to quickly drop from 5.5-6.0 mg/L holding levels to the desired 1.5 or 2.0 mg/L level desired. After individual tank oxygen levels had stabilized at desired levels, flow was returned to the set rate of 1.25 liters per minute for the duration of the trial.2,4, and 8 hr trials were done at 2mg/L, at 1.5 mg/L only two and four hr trials were done as eight hr trials induced mortality in an unacceptable amount of blue catfish. Trials occurred over a three month period from October to December. All trials were done with 5 fish per family per tank and oxygen levels were monitored at 10 minute intervals. Control values were determine using the same protocols but under normal oxygen levels of 5.5 to 6.0 mg/L for a period of four hr. All treatments were repeated 3 times. Tissue removal and RNA extraction After the predetermined time point had been reached all valves were closed and MS-222 was added to each individual tank to anesthetize fish. Fish were then weighed and had their liver and gills removed to be placed into separateten ml test tubes containing 2ml of RNAlater (RNA stabilizing buffer) for storage. Each group of 5 fish per treatment had their organs pooled in to one communal vial per organ per treatment. These tubes were placed in a refrigerator at 4?C for 24 hr. After temporary storage at 4?C samples they were moved to a -80 C? freezer for long term 21 storage. Samples were prepared for RNA extraction by submersion in liquid nitrogen and then ground to a fine powder using a mortar and pestle. Once samples were ground, RNA was extracted using the RNAeasy mini kit (Qiagen, Valencia, CA). RNA concentrations were checked using an Amersham Bio Sciences ultrospec 1100 pro spectrophotometer. Any RNA with wavelength ratios outisde the accepted OD260/OD280 of 1.8-2.0 or with concentrations lower than 200?g/?l were discarded and the extraction process was run again. After RNA was obtained in sufficient quantity and quality samples were converted to cDNA using the BioRadiscriptcDNA synthesis kit via reverse transcription. Samples were then amplified using PCR in a BioRad thermal cycler and checked for cDNA quantity. Next, samples were diluted with RNAase free water to a uniform 250 ?g/?l ?30?g/?l. Primers for six different genes HIF-1, HIF-2, Ferritin, Myostatin, NKEF, and BPI genes were designed and obtained from Invitrogen Custom Primers and were tested for specific amplification prior to use in qRT-PCR (Table 3). Table 3. Primers used in this study. Gene Name Primer Sequence (5? to 3?) HIF-1 Upper ACCACCTCAGCAAGACACAT Lower TCCTCCTCCACAATACCACTG HIF-2 Upper TCACCAGAAGCCACCAGAAT Lower CACTCAGGACATAGTTGACACA 22 Myostatin Upper AGTATTGTGAGGAGTGTGAGAC Lower GACTCGCCTTCCTTATTCTTCT Ferritin Upper AAAGTCCAGAACCAGAGAGGA Lower ACCCAGTCAGAAAGCTCCTTA NKEF Upper ACAGATTTTGTAACGCACGTT Lower TGTTTCTCTGGATGAAATGCAG BPI Upper AGAAGCAGAGACAGAGACCAA Lower GCCAATCTGACGACCATACTC qRT-PCR was performed on a BioRad CFX 96 Touch? real time PCR detection machine using a qRT-PCR so fast EvaGreenSuppermix kit. CFX Manager Software version 1.6 was used for data collection and results were then exported unto Microsoft Excel spreadsheets for graphing and analysis. The Relative Expression Software Tool or R.E.S.T was used for statistical analysisand significance testing of the genetic data. Control samples were obtained after 3 replicates of 5 fish per family were placed in the test tanks for a duration of 4 hr at normal dissolved oxygen levels of 5.5-6.0 mg/L and had their organs extracted and pooled according to the protocol. These samples provided the base like C/T value to determine future fold regulation changes post hypoxic treatment. The housekeeping gene 18s was used for standardization of transcript expression during the statistical analysis. Hypoxia trials and phenotypic measurements 23 Prior to any trials documented in this experiment the groups of catfish were subjected to a range of dissolved oxygen levels and time periods to determine differences among the groups in terms of phenotypic stress. Normal behavior for all groups in this experiment, post placement in tanks, was to maintain an upright orientation, displaying little or no activity on the tank bottom. Phenotypic stress was defined as leaving this position on the tank bottom and swimmingerratically at the water?s surface. Fish that left the tank bottom never returned to this resting state in any trial. It was determined that dissolved oxygen levels greater than 2 mg/L was insufficiently stressful to induce behavioral changes consistently in all groups of fish tested. At the 2mg/L threshold a large degree of phenotypic variability was observed in the three families tested but there was observable responses in all families. In Table 2 we can see a marked difference in behavior between our groups at 2 mg/Lwith blue catfish showing phenotypic stress much sooner than their channel and hybrid counter parts. Oxygen levels of 2 mg/L elicited a range of responses for channel and hybrid catfish that were between no visible stress for 8 hr to showing clear agitation after only 105 minutes. An oxygen level of 1.5 mg/L was determined to be sufficiently stressful to induce phenotypic change in all fish within two hr of introduction. Oxygen levels below 1.5 mg/L induced mortality quickly, often within two hr, across all tested fish especially blue catfish. Table 5 shows mean time to stress at 2.0 mg/L for channel and hybrid catfish to be more than double that of the blue. Mean time to stress of channel and hybrid catfish was nearly quadrupled compared to blue?s at 1.5 mg/L. Based on these observations it was determinedthat time points of two, four and eight hrwere selected as bench marks for differences in phenotypic stress and used these set periods as intervals for tissue collection to determine molecular 24 changes. No 8 hr 1.5 mg/L time point was used as it caused a high degree of mortality in blue catfish. Genes examined in the study consisted of two known oxygen sensitive factors (HIF-1, HIF-2), two primarily metabolic effectors (ferritin, myostatin) and two immune related factors (NKEF, BPI) gill and liver tissue were harvested for molecular testing in this experiment due to their central role in all functions the selected genes are known to effect. Gill was selected due to its role as the major respiratory organ in fish. Oxygen is integrated into the fish body through the gills and therefore it is reasonable to believe that drastic changes in ambient oxygen would alter the molecular patterns in this organ. Gills can also be an induction point for diseases and catfish which may enhance the organs molecular sensitivity among immune related genes. Liver tissue was selected due to its major role in metabolism for all vertebrates, the process of which is highly oxygen dependent. Table 4. First signs of phenotypic stress in minutes, determined by time for fish behavior to change from lying on the bottom of the tank respiring to swimming at the surface apparently agitated. Species, Duration, Oxygen Level, Trial # Time to surface-- 1st fish Time to surface-- 2nd fish Time to surface-- 3rd fish Time to surface-- 4th fish Time to surface--last fish Blue 2hr 2mg/L 1 74 78 81 82 none Blue 2hr 2mg/L 2 62 74 76 none none Blue 2hr 2mg/L 3 65 69 73 81 98 Blue 4 hr 2mg/L 1 73 75 79 82 126 Blue 4 hr 2mg/L 2 52 58 76 96 131 Blue 4 hr 2mg/L 3 61 74 83 102 128 Blue 8 hr 2mg/L 1 62 69 74 98 105 Blue 8 hr 2mg/L 2 71 81 98 122 125 Blue 8 hr 2mg/L 3 74 87 92 101 142 25 Blue 2hr 1.5mg/L 1 12 15 17 18 20 Blue 2hr 1.5mg/L 2 9 11 11 14 16 Blue 2hr 1.5mg/L 3 15 17 17 18 21 Blue 4hr 1.5mg/L 1 6 10 10 12 14 Blue 4hr 1.5mg/L 2 12 13 13 13 13 Blue 4hr 1.5mg/L 3 9 12 15 16 19 Channel 2hr 2mg/L 1 107 none none none none Channel 2hr 2mg/L 2 111 118 none none none Channel 2hr 2mg/L 3 none none none none none Channel 4hr 2mg/L 1 121 145 none none none Channel 4hr 2mg/L 2 155 178 193 none none Channel 4hr 2mg/L 3 137 none none none none Channel 8hr 2mg/L 1 143 237 329 none none Channel 8hr 2mg/L 2 225 343 422 none none Channel 8hr 2mg/L 3 157 417 none none none Channel 2hr 1.5mg/L 1 45 49 57 68 76 Channel 2hr 1.5mg/L 2 51 53 55 71 73 Channel 2hr 1.5mg/L 3 49 62 74 77 77 Channel 4hr 1.5mg/L 1 52 62 65 66 74 Channel 4hr 1.5mg/L 2 31 45 61 62 68 Channel 4hr 1.5mg/L 3 42 52 56 62 77 Hybrid 2hr 2mg/L 1 111 none none none none Hybrid 2hr 2mg/L 2 none none none none none Hybrid 2hr 2mg/L 3 105 116 none none none Hybrid 4hr 2mg/L 1 123 168 none none none Hybrid 4hr 2mg/L 2 113 212 none none none Hybrid 4hr 2mg/L 3 137 178 none none none Hybrid 8 hr 2mg/L 1 111 214 400 none none Hybrid 8 hr 2mg/L 2 200 315 none none none Hybrid 8 hr 2mg/L 3 157 235 410 none none Hybrid 2hr 1.5mg/L 1 47 52 56 62 68 Hybrid 2hr 1.5mg/L 2 52 56 62 67 73 Hybrid 2hr 1.5mg/L 3 23 45 53 58 64 Hybrid 4hr 1.5mg/L 1 37 50 54 61 69 Hybrid 4hr 1.5mg/L 2 55 57 57 67 73 26 Table 5.Summary statistics of time to distress for each group of catfish. Fish showing no signs of stress were not included in these measures. Blue SD Channel SD Hybrid SD Mean? 2 mg/L 89.7 min ?21.7min 188.9 min ?105.6min 206.5 min ?97.2min Mean? 1.5 mg/L 13.95 min ?3.54min 60.4 min ?12 min 57.5 min ?11.2min Min time? 2 mg/L 52min 107 min 105 min Max time? 2 mg/L 142 min 422 min 410 min Min time--1.5 mg/L 6 min 31 min 23 min Max time--1.5 mg/L 15 min 77 min 78 min One way ANOVA analysis determined time to stress was significantly different between blue catfish and both channel and hybrid catfish at 2mg/L and 1.5mg/L dissolved oxygen content. ANOVA analysis also determined no significant difference between channel and hybrid catfish time to stress at both oxygen concentrations. (F and Fcritical values in appendix 2) 27 RESULTS: Molecular Expression Measurements The following graphs the represent the relative fold changes of the genes HIF-1, HIF-2, BPI, Ferritin, Myostatin and NKEF in gill and liver tissue at pre-determined oxygen levels and time intervals. Significance was determined using the REST qRT-PCR statistical analysis tool. Significant fold changes are denoted by stars. Hypoxia-inducible factor-1 (HIF-1) HIF-1 gene gill expression showed significant fold changes only in hybrid catfish at treatments of 2 hr at 2 mg/L oxygen, 4 hr at 2 mg/L oxygen, and 4 hr at 1.5 mg/L oxygen levels. The greatest fold change of an approximately 5-fold increase in expression came at 4 hr 1.5 mg/L oxygen in hybrid catfish. None of the results for channel or blue catfish were deemed to be significant for HIF-1 gene regulation in gill tissue. Notably, while not significant due to individual variation, blue catfish manifested an overall pattern of down-regulation of HIF-1 at all examined time points. HIF-1 regulation for channel catfish at these time points and oxygen levels did not show significant change. However, in general they showed a pattern of up- regulation, the generally expected response to hypoxic conditions (Figure 2) HIF-1 in liver tissue showed significant up and down- regulation in blue catfish.We see very erratic significant movement as well as the majority of blue time points showing down- regulation. Channel catfish showed the largest change with a significant up-regulation of 13.669 fold at 4 hr and 1.5 mg/L as well as a general overall trend of up-regulation although to a lesser 28 and not always significant degree. Hybrid catfish showed one significant result for HIF-1of down- regulation at 2 hr2 mg/Lin liver tissue however the overall trend was that of up-regulation. 29 Figure 2 ? Fold change in HIF-1 gene in gill tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -4 -2 0 2 4 6 8 10 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e HIF1 Gill Blue HIF1 Gill Channel HIF1 Gill Hybrid * * * * * * 30 Figure 3 ? Fold change in HIF-1 gene in Liver tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -10 -5 0 5 10 15 20 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e HIF 1 LIVER Blue HIF 1 LIVER Channel HIF 1 LIVER Hybrid * * * * * * * * * 31 Hypoxia inducible factor-2 (HIF-2) HIF-2 is believed to have similar affects to HIF-1 in regulation of internal cellular signaling cascades in response to hypoxia. HIF-2 has been shown to have differential sensitivity when compared across species, even those of similar evolutionary origin. Blue catfish showed significant up and down regulation while hybrid catfish?s only significant change was down regulation at 2 hr 2 mg/L . All other time points did not show significant change for HIF-2 in gill when analyzed with the REST program. Blue catfish show up and down-regulation with the majority being down. Channel and hybrid catfish showed an overall trend of up-regulation but not to any significant degree. Channel catfish showed significant up-regulation for HIF-2 in liver at 4 hr 1.5 mg/L and 8 hr2 mg/L when analyzed with the REST program. Blue catfish show up and down-regulation with the majority being down. Hybrid catfish showed an overall trend of up-regulation but not to any significant degree. Channel catfish showed initial down-regulation but to a small degree and then change to up-regulation that was significant. Only channel catfish showed significant molecular change for HIF-2 in either tissue examined. 32 Figure 4 ? Fold change in HIF-2 gene in gill tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -4 -3 -2 -1 0 1 2 3 4 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e HIF2 Gill Blue HIF2 Gill Channel HIF2 Gill Hybrid * 33 Figure 5 ? Fold change in HIF-2 gene in liver tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -3 -2 -1 0 1 2 3 4 5 6 7 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e HIF 2 LIVER Blue HIF 2 LIVER Channel HIF 2 LIVER Hybrid * * 34 Bactericidal permeability-increasing protein (BPI) BPI genes are believed to enhance an organisms response to Gram negative bacteria such as the common catfish pathogens Flavobacterium columnaris and Edwardsiella ictaluri. This response is mediated by the presence of LPS in the Gram negative bacterial cell wall. Hypoxia has been shown to increase catfish susceptibility to these pathogens after exposure. Blue catfish did not show any significant change in regulation and showed no real trend with 2 time points showing up-regulation with a large degree of error. Channel and hybrid catfish each showed multiple significant points of down- regulation. Both channel and hybrid catfish also showed a very large degree of down-regulation at the 8 hr2 mg/L time point with Channel showing a 9.9 negative fold change from the control and hybrid catfish showing an negative 8 fold change. These extremely large changes at the final time point suggest that there is a cumulative effect occurring that induces this large change overperiods prolonged stress. Blue catfish it should be noted while not having any significant regulation changes also had much lower levels of this gene present in the tissue at all time points. Blue catfish again showed no significant differences across any of the time points but did show an overall downward trend that had a closer resemblance to the other fish?s genetic profiles. Channel catfish with exclusion of the first time point showed an overall trend of down- regulation with one time point at 4 hr and 2 mg/L oxygen showing significance. Hybrid catfish showed significant down-regulation at 4 hr2 mg/L and 1.5 mg/L as well as the largest down- regulation of negative 8.4 fold at 8 hr and 2 mg/L. Just as with gill tissue blue catfish had a much lower initial expression quantity of the BPI genes. 35 Figure 6 ? Fold change in BPI gene in gill tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e BPI Gill Blue BPI Gill Channel BPI Gill Hybrid * * * * * * * * * * * 36 Figure 7 ? Fold change in BPI gene in liver tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -10 -8 -6 -4 -2 0 2 4 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e BPI Liver Blue BPI Liver Channel BPI Liver Hybrid * * * * * * * * 37 Ferritin Ferritin is a key factor in iron regulation in organisms. Ferritins iron binding activity prevents excessive free iron accumulation in cells and aids in preventing oxidative damage to cells from ROS. No significant changes were seen in gill trials until the final time point at 8 hr and 2 mg/L oxygen. At the 8 hr time point we see a large and significant up-regulation in channel and hybrid catfish of 8.2 times and 10.9 times respectively. The small and insignificant changes up until the 8 hr point suggest that up-regulation of the ferritin gene is the result of a cumulative effect of genetic change in the fish that does not manifest until the duration of the stress event crosses a certain threshold, in this instance at some time greater than 4 hr of hypoxia. Blue catfish only showed minor and insignificant regulation changes throughout the trial. Hybrid catfish showed significant down-regulation of about 4 fold at 2 and 4 hr at 2 mg/L oxygen and then significant and large up-regulation of ferritin at 8 hr and 2 mg/L oxygen. The large up-regulation of 26.9 fold shows a similar pattern of delayed large up-regulation in the gill tissue for hybrids. Blue catfish showed significant down-regulation of 2.7 fold at 4 hr and 2 mg/L oxygen and also significant up-regulation of 9.1 times at 4 hr and 1.5 mg/L oxygen levels suggesting that not only the duration of the stress even but the intensity plays a large role for blue catfish ferritin regulation. Channel catfish did not show significant gene regulation changes until a 4.4 fold up-regulation at the 4 hr 1.5 mg/L time point and then a massive 75 fold increase at the 8 hr2 mg/L time point. This is the largest fold change for any gene in this experiment and follows the pattern seen in channel gill tissue of initial small insignificant change followed by large up-regulation after a certain threshold has been reached. 38 Figure 8 ? Fold change in ferritin gene in gill tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -5 0 5 10 15 20 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e FERRITIN Gill Blue FERRITIN Gill Channel FERRITIN Gill Hybrid * * * 39 Figure 9 ? Fold change in ferritin gene in liver tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -20 0 20 40 60 80 100 120 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e FERRITIN Liver Blue FERRITIN Liver Channel FERRITIN Liver Hybrid * * * * * * * * * * * 40 Myostatin Myostatin is a major regulator of skeletal muscle growth directly effecting metabolism and recent evidence suggests it may possess other unknown regulatory functions. In gill tissue myostatin shows a general trend of down-regulation across all fish and time points with the exception of channels at 2 hr 1.5 mg/L and hybrids at 4 hr2 mg/L but both of these time points are not statistically significant. Channel catfish did show a significant down-regulation of 2.55 fold at 2hr and 2 mg/L oxygen. Hybrid catfish had the only other significant difference with down-regulation of 1.68 fold at 8 hr and 2 mg/L. Blue catfish while showing overall down- regulation of myostatin did not have any significant results in these trials. Myostatin showed significant up-regulation in liver tissue at the 4 hr 1.5 mg/L time point for both channel and blue catfish. It was generally up regulated in all time points and all fish except for the 8 hr2 mg/L time point in channel catfish. Blue catfish showed in general large up- regulation at both 2 hr, and the 4 hr 1.5 mg/L time points however, there is a very large amount of error at all 3 time points and only one was deemed to be significant. Hybrid catfish also showed a large degree of up-regulation at the 8 hr time point but it also has a large amount of error so as not to be considered significant.Myostatin in gill tissue moved opposite among all fish to liver samples, where we saw significant down-regulation in gill tissue at some time points, conversely significant up-regulation was observed in liver tissue. 41 Figure 10 ? Fold change in myostatin gene in gill tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -5 -4 -3 -2 -1 0 1 2 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R E l a t i v e F o l d C h a n g e Myostatin Gill Blue Myostatin Gill Channel Myostatin Gill Hybrid * * * * 42 Figure 11 ? Fold change in myostatin gene in liver tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -5 0 5 10 15 20 25 30 35 40 45 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e Myostatin Liver Blue Myostatin Liver Channel Myostatin Liver Hybrid * * * * * 43 Natural Killer enhancing factor (NKEF) Natural Killer enhancing factor (NKEF) is believed to be involved in the innate immune response of organisms and has been shown to up-regulate in response to challenge by pathogens. NKEF is also believed assist in the clearance of ROS which result as a byproduct of phagocytosis of Natural Killer cells. The only significant change in the NKEF gene occurred at the 8 hr2 mg/L time point in hybrid catfish. Overall movement for NKEF in gill tissue was very limited never up regulating over 1.5 fold and only down regulating slightly over 2 fold for any fish. It is interesting to note that channel and blue catfish moved together and opposite of hybrids in all but 1 time point. While these moves were not significant it is unusual the hybrid which is a product of channel and blue moved in a opposite direction at nearly all time points. NKEF showed significant down-regulation in blue catfish at 4 hr and 2 mg/L oxygen level. Channel catfish showed significant up-regulation at 4 hr1.5 mg/L time point. No other time points showed significant changes in regulation. It is interesting to note that channel catfish showed an overall trend of down-regulation in gill tissue and small and insignificant regulation changes at all time points for NKEF regulation in liver tissue with the one exception of up- regulation at the 4 hr1.5 mg/L time point in both trials. 44 Figure 12 ? Fold change in myostatin gene in liver tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e NKEF Gill Blue NKEF Gill Channel NKEF Gill Hybrid * * 45 Figure 13 ? Fold change in myostatin gene in liver tissue (y axis) at different time points and oxygen levels (x axis). Asterisks denote significant difference (p value<0.10) double asterisk denotes (p value<0.05) in fold change using REST software for gene quantification analysis relative to control, normalized to changes in 18S rRNA values among the same samples. -10 -5 0 5 10 15 20 2hours 2mg/L 2 Hours 1.5mg/L 4 hours 2mg/L 4 hours 1.5mg/L 8 hours 2mg/L R e l a t i v e F o l d C h a n g e NKEF LIVER Blue NKEF LIVER Channel NKEF LIVER Hybrid * * * 46 General Pattern of Hypoxia-Induced Regulation Research has shown that the level of intensity of a stress event can have profound effects on the relative gene expression pursuant to that event. Catfish are a relatively hypoxia tolerant family of fishes andan event that may induce large degrees of molecular change in other species of fish may not meet the necessary threshold to cause significant molecular change in catfish. The oxygen parameters in this experiment were specifically chosen because they caused varying degrees of observable phenotypic stress and as result produced measurable fluctuations in gene expression patterns. The following graphs show a side by side comparison of each group and tissues relative fold changes in all genes at the two different dissolved oxygen levels tested in this experiment. 47 Figure 14Side by side comparison relative fold changes1.5 mg/L time points vs.2 mg/L time points in all genes over all time points blue catfish gill tissue. -4 -3 -2 -1 0 1 2 3 4 Control 2 Hours 1.5mg/L 4 hours 1.5mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF -4 -3 -2 -1 0 1 2 3 Control 2hours 2mg/L 4 hours 2mg/L 8 hours 2mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF 48 Figure 15.Side by side comparison relative fold changes1.5 mg/L time points vs.2 mg/L time points in all genes over all time points channel catfish gill tissue. -4 -3 -2 -1 0 1 2 3 Control 2 Hours 1.5mg/L 4 hours 1.5mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 Control 2hours 2mg/L 4 hours 2mg/L 8 hours 2mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF 49 Figure 16.Side by side comparison relative fold changes1.5 mg/L time points vs.2 mg/L time points in all genes over all time points hybrid catfish gill tissue -4 -3 -2 -1 0 1 2 3 4 5 6 control 2 Hours 1.5mg/L 4 hours 1.5mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF -10 -5 0 5 10 15 control 2hours 2mg/L 4 hours 2mg/L 8 hours 2mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF 50 Figure 17.Side by side comparison relative fold changes1.5 mg/L time points vs.2 mg/L time points in all genes over all time points blue catfish liver tissue -10 -5 0 5 10 15 20 25 30 Control 2 Hours 1.5mg/L 4 hours 1.5mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF -8 -6 -4 -2 0 2 4 6 Control 2hours 2mg/L 4 hours 2mg/L 8 hours 2mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF 51 Figure 18.Side by side comparison relative fold changes1.5 mg/L time points vs.2 mg/L time points in all genes over all time points channel catfish liver tissue -4 -2 0 2 4 6 8 10 12 Control 2 Hours 1.5mg/L 4 hours 1.5mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF -10 0 10 20 30 40 50 60 70 80 Control 2hours 2mg/L 4 hours 2mg/L 8 hours 2mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF 52 Figure 19.Side by side comparison relative fold changes1.5 mg/L time points vs.2 mg/L time points in all genes over all time points hybrid catfish liver tissue -8 -6 -4 -2 0 2 4 6 Control 2 Hours 1.5mg/L 4 hours 1.5mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF -15 -10 -5 0 5 10 15 20 25 30 Control 2hours 2mg/L 4 hours 2mg/L 8 hours 2mg/L HIF-1 HIF-2 BPI Ferritin Myostatin NKEF 53 DISCUSSION Oxygen levels are a critical control point in aquaculture production upon which feed conversion, fish health, and production capacity often hinge. In spite of the importance of this area, relatively little research has been devoted to identification of phenotypic differences among strains/species of aquacultured organisms, to selection for superior oxygen tolerance, or to a greater understanding of the molecular regulation of these phenomena. In this study, comparisons of the tolerance capacities of channel catfish, blue catfish, and their strain-matched hybrid to acute hypoxic conditions. These conditions were designed to model low DO episodes these fish may experience in industry settings during a pond turnover event, phytoplankton bloom die-off, or failure of aerators to turn on. After initial characterization of the phenotypic differences between the catfish groups, we chose key DO levels and durations to examine expression levels of six genes chosen a prioridue to known roles in metabolism, immunity, and growth. This study represents, to our knowledge, the first molecular characterization of physiological events associated with acute hypoxia in catfish. The three groups of organisms (two ictalurid catfish species and their hybrid) in this trial showed a high phenotypic variability among individuals upon induction of acute hypoxic conditions with blue catfish across all time points and oxygen levels being the first to show signs of physical stress. Previous studies comparing differing oxygen tolerances between catfish species and strains (Dunham et al. 1983; Green and Rawles 2011; Green et al. 2012), have been pond-based and have examined the impacts of chronic hypoxic conditions. These studies, while important, introduce a myriad of opportunities for environmental variability to mask genetic differences. Additionally, they do not model responses to dramatic swings in DO levels that can occur over the course of a production cycle. Channel and hybrid catfish both showed a very low 54 degree of visible stress during 2 mg/L oxygen trials only showing phenotypic stress 35.5 and 37.7% respectively compared to blue catfish which showed stress in 93.3% of 2mg/L trials. Channel and hybrid catfish showed a similar pattern of significantly delayed stressresponse compared to blue catfish during 1.5 mg/L trials taking on average 4 times longer to manifest phenotypic stress. While channel and hybrid catfish commonly could tolerate 8 hrs at 2 mg/L with no visible signs of stress, no blue catfish tolerated greater than 2.5 hrs at this dissolved oxygen level. Some variability, however, was observed between blue catfish, raising the possibility that selection could improve this trait in blues. It should be noted that only a single strain of channel catfish (Marion) and blue catfish (Rio Grande) and their matched hybrid were used in this study. Further work would be needed to determine if these strains are good representatives of the phenotypic performance of their species. This study may serve as starting point for development of methods and baseline measurements with which to assess the range of cultured catfish oxygen tolerances. The six genes profiled in this study showed a large degree of variability in response to the stimulus encountered across the different species, time points, and oxygen levels. Some of the most studied genes in response to hypoxia are the appropriately named hypoxia inducible factor genes or HIF family of genes. These genes have been studied extensively in mammalian models and tissues (Wieneret al. 1996; Rossignol et al. 2002; Zhao et al. 2004; Wang et al. 2006) but have only been studied to a limited extent in teleost fishes (Soitamo et al. 2001; Lawet al. 2006; Rahmanand Thomas 2007) and to a less extensive degree in siluriforms.There is also limited study on rapid and acute changes in oxygen concentrations more akin to what is encountered in a commercial aquaculture setting in which a system contains an unnaturally high amount of respiring organism opposed to that of a natural system. 55 HIF-1 is an oxygen sensitive gene believed to initiate a cascade of cellular events upon exposure to hypoxia in all obligate aerobes. HIF-1 genes for hybrid catfish showed significant up- regulation in gill at 2 hr2 mg/L trial 4 hr2 mg/L and 4 hr1.5 mg/L. Channel and blue catfish did not show significant up-regulationat any points in gill tissue. It is interesting to note that that while none of the time points were deemed to be significant, HIF-1 appears to show an overall trend of down- regulation to hypoxic stress in blue catfish which is the opposite of the expected response observed in HIF-1 studies across most animals. The three groups also showed significant response differences in liver compared to gill with channel catfish showing the only significant movement in liver tissue. Channel catfish showed the only significant increase of HIF-1 in liver tissue at the 4 hr 1.5 mg/L time point with hybrids showing an overall trend of up- regulation but not to a significant degree. Blue catfish again showed an overall trend of down- regulation with significant down-regulation at 2 and 8 hr at 2 mg/L. Duration and intensity of stimulus have shown in other studies to be extremely important factors in determining genetic response.The Terova et al. (2008) study of sea bass showed significant up-regulation of HIF-1 at 2 days, 5 days and 15 days but not after 24 hr at4.3 ? .8 mg/L oxygen levels in liver tissue they also reported significant up-regulation at 4 hr under 1.9 ?.2 mg/L oxygen levels. A similar response was found in this study where the largest fold change at both oxygen concentrations occurred at the 4 hr 1.5 mg/L trial which was the most visibly stressful for all fish. HIF- 2 is even less studied than HIF-1 in fish, and so there is even less data to compare our results with(Raman and Thomas 2007; Shen et al 2010). In this study, HIF-2 only showed significant up regulation differences in channel catfish and in no other group. However the general trends in regulation of HIF-2 compared to HIF-1 were similar. Hybrid catfish showed up-regulation across all time points except for 2 hr 1.5 mg/L in gill tissue. Channel catfish also 56 showed an overall trend of up-regulation particularly in the gills as opposed to up and down- regulation in liver tissue leading to significant up-regulation after the two most stressful trials. Blue catfish again showed an overall trend of down-regulation across both tissues oxygen levels and time points although not to a significant degree. Other studies agree that HIF-1 and HIF-2 can move differentially across tissues and species. Soitamo et al. (2001) showed no significant changes in rainbow trout HIF-1 mRNA transcripts as a response to hypoxia. Shen et al. (2010) reported no significant up-regulation and even some insignificant down-regulation of HIF-1 across liver brain and kidney of the Wuchang bream under hypoxic conditions. They also reported significant up-regulation of HIF-2 in liver and kidney but not in brain. Remoldiet al. (2011) reported no change in HIF-1 in liver and kidney of Eurasian perch under hypoxic conditions but significant up-regulation for HIF-2 in these tissues. HIF gene family regulation has been observed to behave uniquely between different species and under different levels of dissolved oxygen, our results suggest that HIF-1 may be more important to hybrid catfish oxygen tolerance while HIF-2 may be more impactful to channel catfish. Hybrid catfish HIF-1 genes show the greatest degree of sensitivity to ambient conditions followed by channel catfish while channel catfish were the only group to show HIF-2 sensitivity.Based on the relative observable stress performance of the three catfish groups tested and their corresponding HIF gene reaction it is a reasonable assumption that HIF gene family sensitivity is beneficial to survival of catfish. Blue catfish showed the least degree of significant movement in HIF genesnever reporting larger than a 5 fold up or down change and consistently were the first to show observable phenotypic stress during trials. It is possible that the low degree of HIF gene family sensitivity in blue catfish contributes to the fish?s inability to withstand acute hypoxic 57 conditions.When discussing the lack of significant HIF movement across all time points and species it should also note the large amount of variability between manifestations of observable stress. Channel and hybrid catfish showed a wide range in time to surface between individual fish in the same trial. This high degree of variability in time to stress between trials and individual fish could explain the lack of significant movement at similar stress intensity but different time points. BPI genesorbactericidal permeability increasing protein (BPI) is an antimicrobial peptide belonging to the lipid transfer/LPS-binding protein family. It serves important roles in early protection against Gram-negative bacteria in the innate immune system. BPI genes showed highly differential expression levels between the three groups of fish tested. Channel and hybrid catfish showed similar levels of expression in both tested tissue whereas blue catfish had significantly less expression levels inall control samples of all tissues. BPI genes in catfish have been shown to up-regulate in response to challenge from the Gram-negative bacteria, Edwardsiellaictaluri,the causative agent of ESC in catfish (Xu et al. 2005) and have been linked to innate immune responses of other organisms. BPI genes were significantly down-regulated in both channel and hybrid catfish at multiple time points and in both tissues observed. In gill tissue, both channel and hybrid catfish showed by far the largest down-regulationwhich occurred at the 8 hr2 mg/L time point suggesting that duration of the stress event plays a large role in determining the magnitude of movement for BPI genes in gill tissue. In liver tissue channel catfish showed the largest down- regulation at 4 hrand1.5 mg/L and no significant movement for all other observed trials. Hybrid catfish showed steadily decreasing expression profiles with duration and intensity of the trials. The first significant down-regulation for BPI genes in hybrid liver occurred at 4 hr for both 2 58 mg/L and 1.5 mg/L and fell even lower at the 8 hr2 mg/L time point. These significant drops in BPI gene expression under hypoxic conditions could help explain the increased susceptibility to disease encountered by all fish that experience similar stress events. Blue catfish also showed an overall pattern of down-regulation however none of the time points across both tissues were deemed significant when contrasted against the low initial expression levels of the BPI gene. It cannot determine from this experiment if the movement in BPI genes exhibited by each class of fish is the optimal genotype for culture. Previous studies have shown blue catfish with superior resistance to ESC (Bosworth et al. 2003) and channel catfish with increased resistance to columnaris (Dunham et al. 1993; Dunham and Argue 2000),both Gram negative bacteria,and therefore should be affected by BPI production, because of this apparent difference between the species in both susceptibility and response to these bacterial infections there are likely other factors at work in determining optimal genetic response and genotype for disease resistance pursuant to hypoxia in all species of catfish. Iron regulation is critical in many physiological and biochemical processes such as oxygen transportation, electron transfer, DNA replication and photosynthesis (Theil1987). The concentration level of iron within an organism is vitally important for both cell growth and metabolism. High levels of iron in cells will lead to oxidative damage of proteins, lipids and DNA (Reif 1992; Linn 1998). Ferritin plays a key role in maintaining normal iron levels (Theil 1987). Its main functions are iron storage and detoxification (Harrison and Arosio 1996; Connolly and Guerinot 2002). Ferritin has also been suggested as an acute phase protein responding to a nonlethal injury to the organism (Beck et al. 2002). Ferritin regulation in the fish tested was variable and dramatic with channel catfish showing an over 70-fold up-regulation in liver tissue at the 8 hr2 mg/L time point. This was by 59 far the largest fold change for any gene, tissue or time point in the entire study. We also observed a slightly smaller but significant fold change in liver tissue for hybrid catfish with a 27- fold up-regulation at the 8 hr at 2 mg/L time point. Blue catfish also showed a significant 9-fold up-regulation of ferritin at the 4 hr at 1.5 mg/L time point. Interestingly both blue and hybrid catfish showed small but significant down-regulation of expression at the 2 hr and 4 hr2 mg/L trials. Similar results were observed for the ferritin gene in gill tissue as well. In channel and hybrid catfish no significant changes occurred in the early time points of the trials, and even showed small down-regulation, then at the 8 hr time point both species showed large and significant increases in ferritin regulation. No such increases were observed in blue catfish across all time points. These delayed increases in the ferritin gene suggest that the fish are over time accumulating iron, andas a result, reactive oxygen species (ROS)in their organs. Our results suggest that it takes a minimum of fourhr of hypoxic stress to induce significant changes in transcript levels and as the stress event continues ferritin up-regulation increases with it. It would have been interesting to see, if returned to normoxic conditions, how long ferritin levels would remain elevated in the affected fish. It should also be noted that the blue catfish showed a much lower degree of ferritin regulation than the other fish and while blue catfish showed outward stress signals long before any changes to ferritin regulation were observed, the inability to clear iron and ROS buildup in this species may be a major component of its poor hypoxia tolerance in general. Had recovery trials been observed it is likely that blue catfish would have shown much poorer ability to return to homeostasis than its channel and hybrid counterparts. Myostatin, a member of the TGF?super family of ligands,has been shown to be a negative regulator of skeletal musclemass during embryogenesis and early postnatal muscle 60 growth(Kambadur et al. 1997; McPherron et al. 1997).Myostatin has only recently been studied in aquatic species with much of our knowledge coming from mammalianmodels. However, its physical structure is highly conserved across all animals suggesting high levels of evolutionary constraint and the importance of its function (Kocabas et al. 2002). It was initially believed that myostatin was only expressed in skeletal muscle but other studies (Kocabas et al. 2002) and our study have shown myostatin to be expressed in other tissues as well. Myostatin in gill tissue showed significant down-regulation in channel catfish at the 2 hr at2 mg/L time point and in hybrid catfish at 8 hr and 2 mg/L. While there were no other significant time points, the overall trend in gill tissue was one of down-regulation across all time points and species. While none of the trials showed extreme changes in down-regulation,any decrease is the opposite effect of the typical results (Hayot et al. 2010) found for myostatin in other organisms subjected to hypoxia. Gills are the major respiratory organ in fish and lacking other acute hypoxic studies with which to make comparisons, we expected similar results in other animals. Contrarily, mammalian studies of mice and humans show up-regulation in lung tissue under conditions of hypoxic stress (Bartman and Speer 2004;Hayot et al. 2010). Myostatin showed significant increases in both channel and blue catfishliverat the 4 hr1.5 mg/L time point. While no other time points were deemed significant, it should be noted that blue catfish also showed a high degree of up-regulation at the 2 hr 1.5 mg/L time point with an 11-fold increase and a nearly linear progression to its peak expression at 4 hr 1.5 mg/Lwith a 24- fold increase. Hybrid catfish did not show significant change in myostatin,due to high variability,but showed a large 21 fold increase at the 8 hr at2 mg/L time point. Both hybrid and blue catfish showed very large degrees of myostatin change under different conditions with blue catfish changes being stronger at lower dissolved oxygen levels and hybrid changes occurring 61 after longer duration. All results for myostatin show a very high degree of error preventing more time points from achieving significance. Channel catfish myostatin also appears to be much less sensitive than that of the other species, never showing a greater than 4.5 times fold change in either direction across all treatmentsand tissues. Blue catfish showed the highest degree of change in myostatin as a response to hypoxia followed by the hybrid. Of all the genes examined, myostatin was the only gene in which hybrid expression patterns showed greater similarity to blue catfish than channel catfish. It is interesting to note that regulation patterns were reversed for liver and gill tissue suggesting that myostatin may have tissue specific function as well as regulation. Recent studies on myostatin in barramundi also showeddown-regulation in gill tissue with corresponding up- regulation in liver tissue as well as greater magnitude changes occurring in liver in response to fasting (De Santis and Jerry 2010). Studies such De Santis and Jerry (2010) and this study that show opposing regulation direction between tissues bolster the hypothesis that myostatin may be responsible for other factors of fish homeostasis, such as osmoregulation and not just an arbiter of muscle growth. De Santis and Jerry (2010)also determined that myostatin in barramundi contained two paralogs with 90% similarity which could show differential and often opposite expression between tissues. It is unknown if catfish contain these two paralogs or if the primers used in this experiment were specific enough to differentiate one from the other. NKEF or natural killer enhancing factor effects NK cells in mammals and natural killer- likecells in fish (also called non-specific cytotoxic cells). NK cells are important early effectors of the innate immune response. They may be essential for priming the adaptive immune response that plays an important role in defense against pathogens (Kuznetsov 1996). Catfish NKEF shares high levels of sequence identity with other teleost NKEFs and are expressed in all major 62 tissues of catfish (Li and Waldbieser 2004).NKEF has also been confirmed and shows a highly conserved sequence in rainbow trout, common carp, and puffer fish(Tetraodonnigroviridis)(Zhang et al. 2001; Shin et al. 2001;Dong et al. 2006). NKEF?s response to hypoxia has not been studied with the majority of research determining its regulation after challenge with disease of LPS (Shin et al.2001; Kim et al. 2011). In gill tissue, only hybrid catfish showed any significant change in NKEF which occurred at the 8 hr at2 mg/L time point. No other fish showed significant change in gill across all time points. It is interesting to note that hybrid catfish were the only fish to show an overall pattern of up-regulation in gill tissue. Channel and blue catfish showed general trends of down-regulation with channels only showing insignificant up-regulation at the 4 hrat 1.5 mg/L time point. This finding is unusual due to the fact that hybrids, being a combination of channel and blue catfish, showed a completely different expression pattern compared to their genetic source material. In this study hybrid expression patterns in general were relatively close to one of the other species challenged, NKEF is the only gene in this study in which hybrid catfish showed a total divergence in the general regulation trend of the other two species. In liver tissue only, blue catfish showed significant NKEF down-regulation at the 4 hr 1.5 mg/L time point. Channel catfish while not showing significant change did show an overall trend of up-regulation for NKEF in liver tissue including a large upward fold change of 10.6 times at the 4 hr 1.5 mg/L time point. In liver tissue again divergent trends were observed in overall regulation between the species with hybrid and channel catfish showing general trends of up-regulation and blue catfish showing the opposite. Kim et al. (2011), reported up-regulation in response to challenge from various pathogens and also suggested that NKEF regulation responds to ROS accumulation as a result of phagocytic activity of the immune system. If NKEF indeed 63 responds to ROS build up we would expect similar expression patterns to that of ferritin in catfish, but this was not the case. NKEF may indeed be involved in ROS mitigation, only more specifically targeted to byproducts of phagocytosis and not hypoxic stress-related buildup of metabolites that caused the aforementioned ferritin spike. The results of this study show the three genotypes of catfish tested exhibited a large degree of genetic variation in response to acute hypoxic stress, dependent on duration of the event, as well as the intensity. Directly opposite molecular responses were observed to the same stimulus which was interesting considering the high degree of genetic similarity between the fish challenged. The lack of consistently significant changes in regulation for genes, particularly in the HIF family, over similar stress levels also requires further investigation. When time to show visible stress, classified in this study as agitated swimming at the surface, a very large degree of variability was observed for individual fish at different time points and oxygen levels. This extreme amount of difference between individual fish could represent significantdifferences of genetic sensitivitythat become masked when tissues are taken in aggregate for a trial, introducing the large degree of error observed for some of the genes in this study. For future studies it may be beneficial to challenge fish individually noting the time to stress and investigating the resultant fold change. The catfish is also unique when compared to other species of teleosts in the fact that it has been an aquaculture species for decades and, therefore, faced an extremely different set of artificial selection pressures than other less domesticated species. Farmers selecting for growth, disease resistance, or dissolved oxygen tolerance based on the ambient culture conditions could have inadvertently changed the sensitivity of any of the investigated genes to hypoxic conditions. Future studies might benefit from obtaining wild caught fish for a control against unknown 64 selection bias introduced by aquaculturalists in the pre-genetics era. Future studies would also benefit from a subsequent disease challenge post hypoxia to determine if the fold changes of the immune specific genes tested were beneficial or harmful from a disease resistance standpoint. A genetic optimum is difficult to ascertain for aquaculture species due to the fact that no two sites have identical conditions. Therefore, the optimum genetic signature can change from farm to farm or even pond to pond. In this study, channel and hybrid catfish showed significantly less phenotypic stress than their blue counterparts measured in time to surface. Furthermore the genetic expression profiles of channel and hybrid catfish showed much higher similarity when compared to the blue catfish. This study gives us a basis for future marker assisted selection in catfish. As technology continues to improve making it easier and cheaper to examine the genetic makeup of an organism we will hone the ability to view large arrays of genes and their changes in sensitivity over generations helping to determine our optimal fish. Currently, it would appear that any immediate improvements to hybrid catfish hypoxia tolerance would come as a result of improvements in the channel mother in a hybrid cross. 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Anova: Two-Factor With Replication SUMMARY Blue Hybrid Total 2hr 2mg/L Count 15 15 30 Sum 234.8 204.3 439.1 Average 15.65333 13.62 14.63667 Variance 5.108381 2.043143 4.521713 4hr 2mg/L Count 15 15 30 Sum 251 205.5 456.5 Average 16.73333 13.7 15.21667 Variance 7.59381 1.904286 6.964885 8hr 2mg/L Count 15 15 30 Sum 240.8 203.2 444 Average 16.05333 13.54667 14.8 Variance 7.324095 2.902667 6.562069 2hr 1.5mg/L Count 15 15 30 Sum 248.4 202.6 451 Average 16.56 13.50667 15.03333 Variance 7.692571 2.186381 7.18023 4hr 1.5 mg/L Count 15 15 30 Sum 240.8 203.5 444.3 Average 16.05333 13.56667 14.81 Variance 5.522667 5.760952 7.046448 Total Count 75 75 Sum 1215.8 1019.1 Average 16.21067 13.588 Variance 6.442047 2.804043 70 ANOVA Source of Variation SS df MS F P-value F crit Sample 6.164933 4 1.541233 0.32083 0.863652 2.436317 Columns 257.9393 1 257.9393 53.69377 1.7E-11 3.908741 Interaction 5.5004 4 1.3751 0.286247 0.886512 2.436317 Within 672.5453 140 4.803895 Total 942.1499 149 71 Appendix 2. One and Two way ANOVA tables for time to phenotypic stress. Anova: Single Factor 2mg/L Channel vs.Hybrid catfish that surfaced SUMMARY Groups Count Sum Average Variance Channel 17 3538 208.1176 11157.61 Hybrid 17 3305 194.4118 9444.132 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 1596.735 1 1596.735 0.15501 0.696404 4.149097 Within Groups 329627.9 32 10300.87 Total 331224.6 33 Anova: Single Factor 1.5mg/L Channel vs Hybrid SUMMARY Groups Count Sum Average Variance Channel 30 1812 60.4 143.8345 Hybrid 30 1724 57.46667 125.5678 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 129.0667 1 129.0667 0.95817 0.331714 4.006873 Within Groups 7812.667 58 134.7011 Total 7941.733 59 72 Anova: Two-Factor With Replication 1.5mg/L All fish SUMMARY Blue Channel Hybrid Total 2hr 1.5mg/L Count 15 15 15 45 Sum 231 937 838 2006 Average 15.4 62.46667 55.86667 44.57778 Variance 11.97143 137.6952 144.6952 536.4313 4hr 1.5 mg/L Count 15 15 15 45 Sum 187 875 886 1948 Average 12.46667 58.33333 59.06667 43.28889 Variance 9.409524 151.0952 109.9238 571.9374 Total Count 30 30 30 Sum 418 1812 1724 Average 13.93333 60.4 57.46667 Variance 12.54713 143.8345 125.5678 ANOVA Source of Variation SS df MS F P-value F crit Sample 37.37778 1 37.37778 0.397079 0.530312 3.954568 Columns 40629.07 2 20314.53 215.8096 7.98E-34 3.105157 Interaction 232.0889 2 116.0444 1.232788 0.296698 3.105157 Within 7907.067 84 94.13175 Total 48805.6 89 73 Appendix 3.Raw Data with additional REST statistical data Well Fluor Threshold Cycle ( C(t) C(t) Mean Gene Fish, Duration, Intensity Tissue Direction of regulation, P value, Standard Error A01 FAM 24.42 HIF-1 blue 4 hr control gill A02 FAM 24.82 24.42 HIF-1 blue 4 hr control gill A03 FAM 24.01 HIF-1 blue 4 hr control gill B07 FAM 25.43 HIF-1 blue 2hr 2 mg/L gill down 2.399 B08 FAM 24.07 24.40 HIF-1 blue 2hr 2 mg/L gill p .512 B09 FAM 23.71 HIF-1 blue 2hr 2 mg/L gill 0.197 D01 FAM 23.24 HIF-1 blue 2hr 1.5 mg/L gill down 1.541 D02 FAM 24.15 23.76 HIF-1 blue 2hr 1.5 mg/L gill p.892 D03 FAM 23.90 HIF-1 blue 2hr 1.5 mg/L gill 0.22967 E07 FAM 23.23 HIF-1 blue 4hr 2 mg/L gill up 1.105 E08 FAM 22.83 22.99 HIF-1 blue 4hr 2 mg/L gill p.946 E09 FAM 22.92 HIF-1 blue 4hr 2 mg/L gill 0.37756 G01 FAM 23.68 HIF-1 blue 4hr 1.5 mg/L gill down 1.012 G02 FAM 22.52 23.16 HIF-1 blue 4hr 1.5 mg/L gill p. 946 G03 FAM 23.27 HIF-1 blue 4hr 1.5 mg/L gill 3.776 H07 FAM 24.53 HIF-1 blue 8hr 2 mg/L gill down 2.721 H08 FAM 24.95 24.58 HIF-1 blue 8hr 2 mg/L gill p.31 H09 FAM 24.28 HIF-1 blue 8hr 2 mg/L gill 0.1211 A04 FAM 26.03 HIF-1 blue 4 hr control liver A05 FAM 26.39 26.20 HIF-1 blue 4 hr control liver A06 FAM 26.19 HIF-1 blue 4 hr control liver B10 FAM 26.76 HIF-1 blue 2hr 2 mg/L liver down 2.37 B11 FAM 26.48 26.76 HIF-1 blue 2hr 2 mg/L liver p .001 B12 FAM 27.03 HIF-1 blue 2hr 2 mg/L liver 1.88 D04 FAM 26.14 HIF-1 blue2hr 1.5 mg/L liver down 1.916 D05 FAM 26.17 26.48 HIF-1 blue2hr 1.5 mg/L liver p .279 D06 FAM 27.13 HIF-1 blue2hr 1.5 mg/L liver 0.245 74 E10 FAM 26.06 HIF-1 blue4hr 2 mg/L liver down 2.171 E11 FAM 26.94 26.66 HIF-1 blue4hr 2 mg/L liver p .093 E12 FAM 26.99 HIF-1 blue4hr 2 mg/L liver 0.2166 G01 FAM 23.68 HIF-1 blue4hr 1.5 mg/L liver up 5.229 G02 FAM 22.52 23.16 HIF-1 blue4hr 1.5 mg/L liver p.062 G03 FAM 23.27 HIF-1 blue4hr 1.5 mg/L liver 2.53 H10 FAM 27.40 HIF-1 blue8hr 2 mg/L liver down 5.663 H11 FAM 27.67 27.88 HIF-1 blue8hr 2 mg/L liver p .001 H12 FAM 28.58 HIF-1 blue8hr 2 mg/L liver 0.09645 A07 FAM 25.35 HIF-1 channel 4hr control gill A08 FAM 24.24 24.83 HIF-1 channel 4hr control gill A09 FAM 24.89 HIF-1 channel 4hr control gill C01 FAM 24.42 HIF-1 channel 2hr 2 mg/L gill down 1.001 C02 FAM 24.19 24.25 HIF-1 channel 2hr 2 mg/L gill p .944 C03 FAM 24.15 HIF-1 channel 2hr 2 mg/L gill 0.351 D07 FAM 23.68 HIF-1 channel 2hr 1.5 mg/L gill up 1.364 D08 FAM 23.76 23.80 HIF-1 channel 2hr 1.5 mg/L gill p .653 D09 FAM 23.98 HIF-1 channel 2hr 1.5 mg/L gill 0.48 F01 FAM 23.99 HIF-1 channel 4hr 2 mg/L gill up 1.540 F02 FAM 23.52 23.63 HIF-1 channel 4hr 2 mg/L gill p .451 F03 FAM 23.38 HIF-1 channel 4hr 2 mg/L gill 0.568 G07 FAM 24.20 HIF-1 channel 4hr 1.5 mg/L gill up 1.408 G08 FAM 23.08 23.76 HIF-1 channel 4hr 1.5 mg/L gill p .527 G09 FAM 23.99 HIF-1 channel 4hr 1.5 mg/L gill 0.593 C01 FAM 24.22 HIF-1 channel 8hr 2 mg/L gill up 1.230 C02 FAM 24.50 23.95 HIF-1 channel 8hr 2 mg/L gill p .65 C03 FAM 23.15 HIF-1 channel 8hr 2 mg/L gill 0.551 A10 FAM 27.11 HIF-1 channel 4hr control liver A11 FAM 27.23 27.19 HIF-1 channel 4hr control liver A12 FAM 27.23 HIF-1 channel 4hr control liver C04 FAM 27.19 HIF-1 channel 2hr 2 mg/L liver up 1.158 C05 FAM 26.80 26.88 HIF-1 channel 2hr 2 mg/L liver p .243 75 C06 FAM 26.65 HIF-1 channel 2hr 2 mg/L liver 0.342 D10 FAM 26.19 HIF-1 channel 2hr 1.5 mg/L liver up 1.427 D11 FAM 26.45 26.58 HIF-1 channel 2hr 1.5 mg/L liver p .19 D12 FAM 27.08 HIF-1 channel 2hr 1.5 mg/L liver 0.47 F04 FAM 26.13 HIF-1 channel 4hr 2 mg/L liver up 1.67 F05 FAM 26.90 26.35 HIF-1 channel 4hr 2 mg/L liver p .104 F06 FAM 26.03 HIF-1 channel 4hr 2 mg/L liver 0.557 G10 FAM 23.34 HIF-1 channel 4hr 1.5 mg/L liver up 13.669 G11 FAM 23.86 23.32 HIF-1 channel 4hr 1.5 mg/L liver p.048 G12 FAM 22.75 HIF-1 channel 4hr 1.5 mg/L liver 4.814 C04 FAM 26.45 HIF-1 channel 8 hr2 mg/L liver up 1.772 C05 FAM 25.97 26.31 HIF-1 channel 8 hr2 mg/L liver p .056 C06 FAM 26.51 HIF-1 channel 8 hr2 mg/L liver 0.513 B01 FAM 24.14 HIF-1 hybrid 4hr control gill B02 FAM 26.20 25.32 HIF-1 hybrid 4hr control gill B03 FAM 25.61 HIF-1 hybrid 4hr control gill C07 FAM 23.97 HIF-1 hybrid 2hr 2 mg/L gill up 2.59 C08 FAM 23.84 23.95 HIF-1 hybrid 2hr 2 mg/L gill p .034 C09 FAM 24.04 HIF-1 hybrid 2hr 2 mg/L gill 1.502 E01 FAM 24.83 HIF-1 hybrid 2hr 1.5 mg/L gill up 1.739 E02 FAM 23.45 24.52 HIF-1 hybrid 2hr 1.5 mg/L gill p .349 E03 FAM 25.29 HIF-1 hybrid 2hr 1.5 mg/L gill 1.206 F07 FAM 23.76 HIF-1 hybrid 4hr 2 mg/L gill up 3.459 F08 FAM 23.14 23.53 HIF-1 hybrid 4hr 2 mg/L gill p .034 F09 FAM 23.70 HIF-1 hybrid 4hr 2 mg/L gill 2.057 H01 FAM 22.36 HIF-1 hybrid 4hr 1.5 mg/L gill up 5.161 H02 FAM 22.52 22.95 HIF-1 hybrid 4hr 1.5 mg/L gill p .034 H03 FAM 23.98 HIF-1 hybrid 4hr 1.5 mg/L gill 3.505 C07 FAM 24.34 HIF-1 hybrid 8hr 2 mg/L gill up 1.596 C08 FAM 23.90 24.65 HIF-1 hybrid 8hr 2 mg/L gill p .424 C09 FAM 25.70 HIF-1 hybrid 8hr 2 mg/L gill 1.101 B04 FAM 26.29 HIF-1 hybrid 4hr control liver 76 B05 FAM 26.26 26.77 HIF-1 hybrid 4hr control liver B06 FAM 27.74 HIF-1 hybrid 4hr control liver C10 FAM 26.69 HIF-1 hybrid 2hr 2 mg/L liver down 2.891 C11 FAM 28.84 27.71 HIF-1 hybrid 2hr 2 mg/L liver p.089 C12 FAM 27.60 HIF-1 hybrid 2hr 2 mg/L liver 0.236 E04 FAM 26.87 HIF-1 hybrid 2hr 1.5 mg/L liver down 1.229 E05 FAM 26.05 26.48 HIF-1 hybrid 2hr 1.5 mg/L liver p .664 E06 FAM 26.51 HIF-1 hybrid 2hr 1.5 mg/L liver 0.451 F10 FAM 26.41 HIF-1 hybrid 4hr 2 mg/L liver up 1.054 F11 FAM 26.33 26.10 HIF-1 hybrid 4hr 2 mg/L liver p.988 F12 FAM 25.57 HIF-1 hybrid 4hr 2 mg/L liver 0.5903 H04 FAM 26.51 HIF-1 hybrid 4hr 1.5 mg/L liver up 1.346 H05 FAM 26.46 25.75 HIF-1 hybrid 4hr 1.5 mg/L liver p .772 H06 FAM 24.27 HIF-1 hybrid 4hr 1.5 mg/L liver 0.99 C10 FAM 26.12 HIF-1 hybrid 8hr 2 mg/L liver up 2.672 C11 FAM 23.97 24.76 HIF-1 hybrid 8hr 2 mg/L liver p .215 C12 FAM 24.19 HIF-1 hybrid 8hr 2 mg/L liver 1.897 A01 FAM 23.99 HIF-2 blue 4 hr control gill A02 FAM 24.82 24.28 HIF-2 blue 4 hr control gill A03 FAM 24.01 HIF-2 blue 4 hr control gill B07 FAM 25.43 HIF-2 blue 2hr 2 mg/L gill up 1.402 B08 FAM 24.07 24.40 HIF-2 blue 2hr 2 mg/L gill p .622 B09 FAM 23.71 HIF-2 blue 2hr 2 mg/L gill 1.136 D01 FAM 23.24 HIF-2 blue 2hr 1.5 mg/L gill down 1.420 D02 FAM 24.15 23.76 HIF-2 blue 2hr 1.5 mg/L gill p .674 D03 FAM 23.90 HIF-2 blue 2hr 1.5 mg/L gill 0.571 E07 FAM 23.23 HIF-2 blue 4hr 2 mg/L gill up 1.269 E08 FAM 22.83 22.99 HIF-2 blue 4hr 2 mg/L gill p .479 E09 FAM 22.92 HIF-2 blue 4hr 2 mg/L gill 1.037 G01 FAM 23.68 HIF-2 blue 4hr 1.5 mg/L gill down 2.745 G02 FAM 22.52 23.16 HIF-2 blue 4hr 1.5 mg/L gill p .815 G03 FAM 23.27 HIF-2 blue 4hr 1.5 mg/L gill 0.313 77 H07 FAM 24.53 HIF-2 blue 8hr 2 mg/L gill down 2.842 H08 FAM 28.95 27.58 HIF-2 blue 8hr 2 mg/L gill p .418 H09 FAM 29.28 HIF-2 blue 8hr 2 mg/L gill 0.3251 A04 FAM 26.03 HIF-2 blue 4 hr control liver A05 FAM 26.39 26.20 HIF-2 blue 4 hr control liver A06 FAM 26.19 HIF-2 blue 4 hr control liver B10 FAM 26.76 HIF-2 blue 2hr 2 mg/L liver down 1.10 B11 FAM 26.48 26.76 HIF-2 blue 2hr 2 mg/L liver p .845 B12 FAM 27.03 HIF-2 blue 2hr 2 mg/L liver 0.436 D04 FAM 27.14 HIF-2 blue2hr 1.5 mg/L liver 1.075 D05 FAM 26.17 26.81 HIF-2 blue2hr 1.5 mg/L liver p.905 D06 FAM 27.13 HIF-2 blue2hr 1.5 mg/L liver 0.42641 E10 FAM 27.06 HIF-2 blue4hr 2 mg/L liver down 1.435 E11 FAM 26.94 26.99 HIF-2 blue4hr 2 mg/L liver p.554 E12 FAM 26.99 HIF-2 blue4hr 2 mg/L liver 0.37785 G01 FAM 23.68 HIF-2 blue4hr 1.5 mg/L liver down 1.966 G02 FAM 22.52 23.16 HIF-2 blue4hr 1.5 mg/L liver p.316 G03 FAM 23.27 HIF-2 blue4hr 1.5 mg/L liver 0.27226 H10 FAM 28.40 HIF-2 blue8hr 2 mg/L liver down 1.252 H11 FAM 27.67 28.55 HIF-2 blue8hr 2 mg/L liver p.759 H12 FAM 29.58 HIF-2 blue8hr 2 mg/L liver 0.60337 A07 FAM 25.35 HIF-2 channel 4hr control gill A08 FAM 24.24 24.49 HIF-2 channel 4hr control gill A09 FAM 23.89 HIF-2 channel 4hr control gill C01 FAM 24.42 HIF-2 channel 2hr 2 mg/L gill up 1.917 C02 FAM 24.19 24.25 HIF-2 channel 2hr 2 mg/L gill p.388 C03 FAM 24.15 HIF-2 channel 2hr 2 mg/L gill 1.051 D07 FAM 23.68 HIF-2 channel 2hr 1.5 mg/L gill up 1.740 D08 FAM 23.76 23.80 HIF-2 channel 2hr 1.5 mg/L gill p.312 D09 FAM 23.98 HIF-2 channel 2hr 1.5 mg/L gill 0.928 F01 FAM 23.99 HIF-2 channel 4hr 2 mg/L gill up 2.236 F02 FAM 23.52 23.63 HIF-2 channel 4hr 2 mg/L gill p.185 F03 FAM 23.38 HIF-2 channel 4hr 2 mg/L gill 1.175 78 G07 FAM 24.20 HIF-2 channel 4hr 1.5 mg/L gill up 1.282 G08 FAM 23.08 25.76 HIF-2 channel 4hr 1.5 mg/L gill p.80 G09 FAM 29.99 HIF-2 channel 4hr 1.5 mg/L gill 0.96343 D01 FAM 24.34 HIF-2 channel 8hr 2 mg/L gill down 3.332 D02 FAM 24.53 24.07 HIF-2 channel 8hr 2 mg/L gill p.054 D03 FAM 23.35 HIF-2 channel 8hr 2 mg/L gill 0.177 A10 FAM 27.11 HIF-2 channel 4hr control liver A11 FAM 27.23 27.19 HIF-2 channel 4hr control liver A12 FAM 27.23 HIF-2 channel 4hr control liver C04 FAM 27.19 HIF-2 channel 2hr 2 mg/L liver down 1.294 C05 FAM 26.80 26.88 HIF-2 channel 2hr 2 mg/L liver p.236 C06 FAM 26.65 HIF-2 channel 2hr 2 mg/L liver 0.436 D10 FAM 26.19 HIF-2 channel 2hr 1.5 mg/L liver down 1.337 D11 FAM 26.45 26.91 HIF-2 channel 2hr 1.5 mg/L liver p.236 D12 FAM 28.08 HIF-2 channel 2hr 1.5 mg/L liver 0.25917 F04 FAM 26.13 HIF-2 channel 4hr 2 mg/L liver down 1.145 F05 FAM 26.90 26.35 HIF-2 channel 4hr 2 mg/L liver p.74 F06 FAM 26.03 HIF-2 channel 4hr 2 mg/L liver 0.43194 G10 FAM 23.34 HIF-2 channel 4hr 1.5 mg/L liver up 2.308 G11 FAM 23.86 23.32 HIF-2 channel 4hr 1.5 mg/L liver p.055 G12 FAM 22.75 HIF-2 channel 4hr 1.5 mg/L liver 0.69906 D04 FAM 26.27 HIF-2 channel 8 hr2 mg/L liver up 3.951 D05 FAM 26.10 26.16 HIF-2 channel 8 hr2 mg/L liver p.068 D06 FAM 26.11 HIF-2 channel 8 hr2 mg/L liver 0.603 B01 FAM 24.14 HIF-2 hybrid 4hr control gill B02 FAM 26.20 HIF-2 hybrid 4hr control gill B03 FAM 25.61 25.32 HIF-2 hybrid 4hr control gill C07 FAM 23.97 HIF-2 hybrid 2hr 2 mg/L gill up 1.334 C08 FAM 23.84 HIF-2 hybrid 2hr 2 mg/L gill p.591 C09 FAM 24.04 23.95 HIF-2 hybrid 2hr 2 mg/L gill 1.051 E01 FAM 24.83 HIF-2 hybrid 2hr 1.5 mg/L gill down 1.053 E02 FAM 23.45 HIF-2 hybrid 2hr 1.5 mg/L gill p.952 79 E03 FAM 25.29 24.52 HIF-2 hybrid 2hr 1.5 mg/L gill 0.635 F07 FAM 23.76 HIF-2 hybrid 4hr 2 mg/L gill up 1.543 F08 FAM 23.14 HIF-2 hybrid 4hr 2 mg/L gill p.499 F09 FAM 23.70 23.53 HIF-2 hybrid 4hr 2 mg/L gill 1.058 H01 FAM 22.36 HIF-2 hybrid 4hr 1.5 mg/L gill up 1.534 H02 FAM 22.52 HIF-2 hybrid 4hr 1.5 mg/L gill p.478 H03 FAM 23.98 22.95 HIF-2 hybrid 4hr 1.5 mg/L gill 1.055 D07 FAM 23.58 HIF-2 hybrid 8hr 2 mg/L gill up 1.090 D08 FAM 24.15 HIF-2 hybrid 8hr 2 mg/L gill p.794 D09 FAM 23.71 23.82 HIF-2 hybrid 8hr 2 mg/L gill 0.732 B04 FAM 26.29 HIF-2 hybrid 4hr control liver B05 FAM 26.26 HIF-2 hybrid 4hr control liver B06 FAM 27.74 26.77 HIF-2 hybrid 4hr control liver C10 FAM 26.69 HIF-2 hybrid 2hr 2 mg/L liver up 1.890 C11 FAM 28.84 HIF-2 hybrid 2hr 2 mg/L liver p.609 C12 FAM 27.60 27.71 HIF-2 hybrid 2hr 2 mg/L liver 1.844 E04 FAM 26.87 HIF-2 hybrid 2hr 1.5 mg/L liver up 2.093 E05 FAM 26.05 HIF-2 hybrid 2hr 1.5 mg/L liver p.411 E06 FAM 26.51 26.48 HIF-2 hybrid 2hr 1.5 mg/L liver 1.927 F10 FAM 26.41 HIF-2 hybrid 4hr 2 mg/L liver up 1.262 F11 FAM 26.33 HIF-2 hybrid 4hr 2 mg/L liver p.751 F12 FAM 25.57 26.10 HIF-2 hybrid 4hr 2 mg/L liver 1.134 H04 FAM 26.51 HIF-2 hybrid 4hr 1.5 mg/L liver up 2,982 H05 FAM 26.46 HIF-2 hybrid 4hr 1.5 mg/L liver p.257 H06 FAM 24.27 25.75 HIF-2 hybrid 4hr 1.5 mg/L liver 2.713 D10 FAM 26.99 HIF-2 hybrid 8hr 2 mg/L liver up 2.695 D11 FAM 23.97 HIF-2 hybrid 8hr 2 mg/L liver p.308 D12 FAM 24.25 25.07 HIF-2 hybrid 8hr 2 mg/L liver 2.461 A01 FAM 35.52 BPI blue 4 hr control gill A02 FAM 38.58 36.03 BPI blue 4 hr control gill A03 FAM 34.00 BPI blue 4 hr control gill B07 FAM 35.81 BPI blue 2hr 2 mg/L gill down 1.838 80 B08 FAM 37.16 35.63 BPI blue 2hr 2 mg/L gill p .66 B09 FAM 33.91 BPI blue 2hr 2 mg/L gill 0.645 D01 FAM 31.63 BPI blue 2hr 1.5 mg/L gill up 3.346 D02 FAM 31.95 33.01 BPI blue 2hr 1.5 mg/L gill p .521 D03 FAM 35.45 BPI blue 2hr 1.5 mg/L gill 4.3 E07 FAM 35.58 BPI blue 4hr 2 mg/L gill down 3.046 E08 FAM 36.49 36.36 BPI blue 4hr 2 mg/L gill p .356 E09 FAM 37.01 BPI blue 4hr 2 mg/L gill 0.331 G01 FAM 37.17 BPI blue 4hr 1.5 mg/L gill down 3.214 G02 FAM 35.77 36.44 BPI blue 4hr 1.5 mg/L gill p.363 G03 FAM 36.37 BPI blue 4hr 1.5 mg/L gill 0.313 H07 FAM 34.06 BPI blue 8hr 2 mg/L gill up 1.997 H08 FAM 33.30 33.75 BPI blue 8hr 2 mg/L gill p.553 H09 FAM 33.90 BPI blue 8hr 2 mg/L gill 1.96 A04 FAM 35.23 BPI blue 4 hr control liver A05 FAM 33.31 35.23 BPI blue 4 hr control liver A06 FAM 37.15 BPI blue 4 hr control liver B10 FAM 34.52 BPI blue 2hr 2 mg/L liver down 1.227 B11 FAM 34.91 34.86 BPI blue 2hr 2 mg/L liver p .944 B12 FAM 35.15 BPI blue 2hr 2 mg/L liver 0.72 D04 FAM 37.66 BPI blue2hr 1.5 mg/L liver down 7.66 D05 FAM 36.98 37.50 BPI blue2hr 1.5 mg/L liver p.056 D06 FAM 37.87 BPI blue2hr 1.5 mg/L liver 0.117 E10 FAM 39.48 BPI blue4hr 2 mg/L liver down 5.33 E11 FAM 35.42 36.98 BPI blue4hr 2 mg/L liver p.144 E12 FAM 36.04 BPI blue4hr 2 mg/L liver 0.232 G01 FAM 37.17 BPI blue4hr 1.5 mg/L liver down 3.564 G02 FAM 35.77 36.44 BPI blue4hr 1.5 mg/L liver p.144 G03 FAM 36.37 BPI blue4hr 1.5 mg/L liver 0.251 H10 FAM 34.61 BPI blue8hr 2 mg/L liver down 1.448 H11 FAM 34.39 35.10 BPI blue8hr 2 mg/L liver p .642 H12 FAM 36.31 BPI blue8hr 2 mg/L liver 0.67 81 A07 FAM 24.92 BPI channel 4hr control gill A08 FAM 24.39 24.39 BPI channel 4hr control gill A09 FAM 23.87 BPI channel 4hr control gill C01 FAM 25.39 BPI channel 2hr 2 mg/L gill down 2.866 C02 FAM 25.27 25.34 BPI channel 2hr 2 mg/L gill p .001 C03 FAM 25.34 BPI channel 2hr 2 mg/L gill 0.118 D07 FAM 25.62 BPI channel 2hr 1.5 mg/L gill down 2.85 D08 FAM 26.00 25.33 BPI channel 2hr 1.5 mg/L gill p.095 D09 FAM 24.37 BPI channel 2hr 1.5 mg/L gill 0.168 F01 FAM 24.55 BPI channel 4hr 2 mg/L gill down 1.887 F02 FAM 25.12 24.73 BPI channel 4hr 2 mg/L gill p 0.144 F03 FAM 24.53 BPI channel 4hr 2 mg/L gill 0.192 G07 FAM 24.84 BPI channel 4hr 1.5 mg/L gill down 1.822 G08 FAM 24.35 24.68 BPI channel 4hr 1.5 mg/L gill p .307 G09 FAM 24.86 BPI channel 4hr 1.5 mg/L gill 0.195 G01 FAM 27.23 BPI channel 8hr 2 mg/L gill down 9.964 G02 FAM 27.67 27.13 BPI channel 8hr 2 mg/L gill p.001 G03 FAM 26.50 BPI channel 8hr 2 mg/L gill 0.041 A10 FAM 24.33 BPI channel 4hr control liver A11 FAM 25.93 25.61 BPI channel 4hr control liver A12 FAM 26.58 BPI channel 4hr control liver C04 FAM 25.15 BPI channel 2hr 2 mg/L liver up 1.863 C05 FAM 24.39 24.61 BPI channel 2hr 2 mg/L liver p .313 C06 FAM 24.30 BPI channel 2hr 2 mg/L liver 1.06 D10 FAM 25.15 BPI channel 2hr 1.5 mg/L liver down 1.672 D11 FAM 27.27 26.25 BPI channel 2hr 1.5 mg/L liver p .424 D12 FAM 26.33 BPI channel 2hr 1.5 mg/L liver 0.409 F04 FAM 27.18 BPI channel 4hr 2 mg/L liver down 3.16 F05 FAM 27.12 27.17 BPI channel 4hr 2 mg/L liver p .015 F06 FAM 27.21 BPI channel 4hr 2 mg/L liver 0.169 G10 FAM 26.24 BPI channel 4hr 1.5 mg/L liver down 2.041 G11 FAM 26.67 26.54 BPI channel 4hr 1.5 mg/L liver p.116 G12 FAM 26.71 BPI channel 4hr 1.5 mg/L liver 0.268 82 G04 FAM 26.37 BPI channel 8 hr2 mg/L liver down 1.711 G05 FAM 26.21 26.28 BPI channel 8 hr2 mg/L liver p.297 G06 FAM 26.28 BPI channel 8 hr2 mg/L liver 0.314 B01 FAM 25.67 BPI hybrid 4hr control gill B02 FAM 24.20 25.03 BPI hybrid 4hr control gill B03 FAM 25.21 BPI hybrid 4hr control gill BP C07 FAM 24.39 BPI hybrid 2hr 2 mg/L gill down 3.214 C08 FAM 27.67 26.72 BPI hybrid 2hr 2 mg/L gill p .279 C09 FAM 28.09 BPI hybrid 2hr 2 mg/L gill 0.296 E01 FAM 26.00 BPI hybrid 2hr 1.5 mg/L gill down 3.433 E02 FAM 28.30 26.81 BPI hybrid 2hr 1.5 mg/L gill p .05 E03 FAM 26.13 BPI hybrid 2hr 1.5 mg/L gill 0.269 F07 FAM 26.24 BPI hybrid 4hr 2 mg/L gill down 2.44 F08 FAM 26.57 26.32 BPI hybrid 4hr 2 mg/L gill p.05 F09 FAM 26.15 BPI hybrid 4hr 2 mg/L gill 0.206 H01 FAM 25.60 BPI hybrid 4hr 1.5 mg/L gill down 1.817 H02 FAM 25.18 25.89 BPI hybrid 4hr 1.5 mg/L gill p.464 H03 FAM 26.90 BPI hybrid 4hr 1.5 mg/L gill 0.3367 G07 FAM 28.09 BPI hybrid 8hr 2 mg/L gill down 8.006 G08 FAM 28.16 28.03 BPI hybrid 8hr 2 mg/L gill p.05 G09 FAM 27.85 BPI hybrid 8hr 2 mg/L gill 0.062 B04 FAM 24.65 BPI hybrid 4hr control liver B05 FAM 25.15 25.81 BPI hybrid 4hr control liver B06 FAM 27.62 BPI hybrid 4hr control liver C10 FAM 25.37 BPI hybrid 2hr 2 mg/L liver down 1.714 C11 FAM 26.89 26.00 BPI hybrid 2hr 2 mg/L liver p .504 C12 FAM 25.72 BPI hybrid 2hr 2 mg/L liver 0.479 E04 FAM 26.11 BPI hybrid 2hr 1.5 mg/L liver down 3.53 E05 FAM 26.82 27.04 BPI hybrid 2hr 1.5 mg/L liver p.095 E06 FAM 28.19 BPI hybrid 2hr 1.5 mg/L liver 0.245 F10 FAM 28.04 BPI hybrid 4hr 2 mg/L liver down 5.95 F11 FAM 28.90 27.79 BPI hybrid 4hr 2 mg/L liver p.051 83 F12 FAM 26.44 BPI hybrid 4hr 2 mg/L liver 0.152 H04 FAM 27.53 BPI hybrid 4hr 1.5 mg/L liver down 6.456 H05 FAM 27.92 27.91 BPI hybrid 4hr 1.5 mg/L liver p.051 H06 FAM 28.27 BPI hybrid 4hr 1.5 mg/L liver 0.119 G10 FAM 28.05 BPI hybrid 8hr 2 mg/L liver down 8.385 G11 FAM 28.37 BPI hybrid 8hr 2 mg/L liver p.001 G12 FAM 28.44 BPI hybrid 8hr 2 mg/L liver 0.091 A01 FAM 26.62 Ferritin blue 4 hr control gill A02 FAM 25.78 26.46 Ferritin blue 4 hr control gill A03 FAM 26.96 Ferritin blue 4 hr control gill B07 FAM 25.43 Ferritin blue 2hr 2 mg/L gill down 1.394 B08 FAM 25.41 25.65 Ferritin blue 2hr 2 mg/L gill p.921 B09 FAM 26.12 Ferritin blue 2hr 2 mg/L gill 0.277 D01 FAM 25.79 Ferritin blue 2hr 1.5 mg/L gill down 1.875 D02 FAM 26.13 26.08 Ferritin blue 2hr 1.5 mg/L gill p.76 D03 FAM 26.33 Ferritin blue 2hr 1.5 mg/L gill 0.196 E07 FAM 26.39 Ferritin blue 4hr 2 mg/L gill down 1.454 E08 FAM 25.15 25.71 Ferritin blue 4hr 2 mg/L gill p .92 E09 FAM 25.60 Ferritin blue 4hr 2 mg/L gill 0.296 G01 FAM 24.78 Ferritin blue 4hr 1.5 mg/L gill up 1.287 G02 FAM 25.03 24.81 Ferritin blue 4hr 1.5 mg/L gill p.846 G03 FAM 24.63 Ferritin blue 4hr 1.5 mg/L gill 0.462 H07 FAM 24.47 Ferritin blue 8hr 2 mg/L gill up 1.415 H08 FAM 24.97 24.67 Ferritin blue 8hr 2 mg/L gill p.788 H09 FAM 24.58 Ferritin blue 8hr 2 mg/L gill 0.517 A04 FAM 29.04 Ferritin blue 4 hr control liver A05 FAM 28.44 28.66 Ferritin blue 4 hr control liver A06 FAM 28.50 Ferritin blue 4 hr control liver B10 FAM 28.94 Ferritin blue 2hr 2 mg/L liver down 1.511 B11 FAM 27.66 28.59 Ferritin blue 2hr 2 mg/L liver p.538 B12 FAM 29.18 Ferritin blue 2hr 2 mg/L liver 0.361 D04 FAM 27.26 Ferritin blue2hr 1.5 mg/L liver down 1.401 84 D05 FAM 29.16 28.48 Ferritin blue2hr 1.5 mg/L liver p 0.526 D06 FAM 29.04 Ferritin blue2hr 1.5 mg/L liver 0.435 E10 FAM 29.46 Ferritin blue4hr 2 mg/L liver down 2.708 E11 FAM 29.69 29.44 Ferritin blue4hr 2 mg/L liver p.001 E12 FAM 29.16 Ferritin blue4hr 2 mg/L liver 0.166 G01 FAM 24.78 Ferritin blue4hr 1.5 mg/L liver up 9.106 G02 FAM 25.03 24.81 Ferritin blue4hr 1.5 mg/L liver p.053 G03 FAM 24.63 Ferritin blue4hr 1.5 mg/L liver 4.04 H10 FAM 27.85 Ferritin blue8hr 2 mg/L liver up 1.182 H11 FAM 27.70 27.76 Ferritin blue8hr 2 mg/L liver p.799 H12 FAM 27.72 Ferritin blue8hr 2 mg/L liver 0.517 A07 FAM 26.03 Ferritin channel 4hr control gill A08 FAM 25.75 25.95 Ferritin channel 4hr control gill A09 FAM 26.07 Ferritin channel 4hr control gill C01 FAM 26.37 Ferritin channel 2hr 2 mg/L gill down 1.692 C02 FAM 26.36 26.13 Ferritin channel 2hr 2 mg/L gill p.255 C03 FAM 25.67 Ferritin channel 2hr 2 mg/L gill 0.187 D07 FAM 26.44 Ferritin channel 2hr 1.5 mg/L gill down 1.457 D08 FAM 26.04 25.92 Ferritin channel 2hr 1.5 mg/L gill p.577 D09 FAM 25.27 Ferritin channel 2hr 1.5 mg/L gill 0.249 F01 FAM 25.40 Ferritin channel 4hr 2 mg/L gill down 1.219 F02 FAM 25.87 25.66 Ferritin channel 4hr 2 mg/L gill p.89 F03 FAM 25.71 Ferritin channel 4hr 2 mg/L gill 0.237 G07 FAM 24.13 Ferritin channel 4hr 1.5 mg/L gill up 1.859 G08 FAM 24.25 24.48 Ferritin channel 4hr 1.5 mg/L gill p.293 G09 FAM 25.06 Ferritin channel 4hr 1.5 mg/L gill 0.634 E01 FAM 22.99 Ferritin channel 8hr 2 mg/L gill up 8.21 E02 FAM 23.14 22.37 Ferritin channel 8hr 2 mg/L gill p.052 E03 FAM 20.98 Ferritin channel 8hr 2 mg/L gill 4.437 A10 FAM 29.18 Ferritin channel 4hr control liver A11 FAM 27.95 28.67 Ferritin channel 4hr control liver A12 FAM 28.89 Ferritin channel 4hr control liver 85 C04 FAM 29.08 Ferritin channel 2hr 2 mg/L liver down 1.243 C05 FAM 29.13 28.88 Ferritin channel 2hr 2 mg/L liver p.497 C06 FAM 28.43 Ferritin channel 2hr 2 mg/L liver 0.327 D10 FAM 29.74 Ferritin channel 2hr 1.5 mg/L liver down 1.423 D11 FAM 28.43 29.08 Ferritin channel 2hr 1.5 mg/L liver p.35 D12 FAM 29.05 Ferritin channel 2hr 1.5 mg/L liver 0.321 F04 FAM 28.56 Ferritin channel 4hr 2 mg/L liver down 1.74 F05 FAM 29.30 28.80 Ferritin channel 4hr 2 mg/L liver p.674 F06 FAM 28.53 Ferritin channel 4hr 2 mg/L liver 0.354 G10 FAM 26.30 Ferritin channel 4hr 1.5 mg/L liver up 4.387 G11 FAM 26.56 26.43 Ferritin channel 4hr 1.5 mg/L liver p.054 G12 FAM 26.45 Ferritin channel 4hr 1.5 mg/L liver 1.66 E04 FAM 21.90 Ferritin channel 8 hr2 mg/L liver up 74.965 E05 FAM 22.33 22.34 Ferritin channel 8 hr2 mg/L liver p.054 E06 FAM 22.78 Ferritin channel 8 hr2 mg/L liver 21.071 B01 FAM 24.51 Ferritin hybrid 4hr control gill B02 FAM 25.55 25.33 Ferritin hybrid 4hr control gill B03 FAM 25.93 Ferritin hybrid 4hr control gill C07 FAM 27.91 Ferritin hybrid 2hr 2 mg/L gill down 2.314 C08 FAM 25.52 26.55 Ferritin hybrid 2hr 2 mg/L gill p.251 C09 FAM 26.20 Ferritin hybrid 2hr 2 mg/L gill 0.301 E01 FAM 26.56 Ferritin hybrid 2hr 1.5 mg/L gill down 1.455 E02 FAM 26.11 25.88 Ferritin hybrid 2hr 1.5 mg/L gill p 0.35 E03 FAM 24.96 Ferritin hybrid 2hr 1.5 mg/L gill 0.407 F07 FAM 25.79 Ferritin hybrid 4hr 2 mg/L gill up 1.1 F08 FAM 24.48 25.20 Ferritin hybrid 4hr 2 mg/L gill p .585 F09 FAM 25.33 Ferritin hybrid 4hr 2 mg/L gill 0.614 H01 FAM 24.76 Ferritin hybrid 4hr 1.5 mg/L gill down 1.243 H02 FAM 25.59 25.65 Ferritin hybrid 4hr 1.5 mg/L gill p.678 H03 FAM 26.60 Ferritin hybrid 4hr 1.5 mg/L gill 0.494 E07 FAM 21.51 Ferritin hybrid 8hr 2 mg/L gill up 10.875 E08 FAM 22.56 21.89 Ferritin hybrid 8hr 2 mg/L gill p.001 E09 FAM 21.61 Ferritin hybrid 8hr 2 mg/L gill 5.896 86 B04 FAM 27.56 Ferritin hybrid 4hr control liver B05 FAM 27.15 27.50 Ferritin hybrid 4hr control liver B06 FAM 27.79 Ferritin hybrid 4hr control liver C10 FAM 27.60 Ferritin hybrid 2hr 2 mg/L liver down 4.034 C11 FAM 29.75 28.93 Ferritin hybrid 2hr 2 mg/L liver p.04 C12 FAM 29.43 Ferritin hybrid 2hr 2 mg/L liver 0.156 E04 FAM 28.31 Ferritin hybrid 2hr 1.5 mg/L liver down 1.8 E05 FAM 26.75 27.76 Ferritin hybrid 2hr 1.5 mg/L liver p.247 E06 FAM 28.22 Ferritin hybrid 2hr 1.5 mg/L liver 0.307 F10 FAM 29.26 Ferritin hybrid 4hr 2 mg/L liver down 4.377 F11 FAM 28.85 29.04 Ferritin hybrid 4hr 2 mg/L liver p.001 F12 FAM 29.03 Ferritin hybrid 4hr 2 mg/L liver 0.099 H04 FAM 28.54 Ferritin hybrid 4hr 1.5 mg/L liver down 1.046 H05 FAM 28.32 26.98 Ferritin hybrid 4hr 1.5 mg/L liver p 0.968 H06 FAM 24.07 Ferritin hybrid 4hr 1.5 mg/L liver 1.046 E10 FAM 22.82 Ferritin hybrid 8hr 2 mg/L liver up 26.902 E11 FAM 21.83 22.17 Ferritin hybrid 8hr 2 mg/L liver p.05 E12 FAM 21.85 Ferritin hybrid 8hr 2 mg/L liver 13.017 A01 FAM 31.67 Myostatin blue 4 hr control gill A02 FAM 32.77 32.76 Myostatin blue 4 hr control gill A03 FAM 33.85 Myostatin blue 4 hr control gill B07 FAM 33.28 Myostatin blue 2hr 2 mg/L gill down 1.957 B08 FAM 32.05 32.45 Myostatin blue 2hr 2 mg/L gill p .698 B09 FAM 32.02 Myostatin blue 2hr 2 mg/L gill 0.296 D01 FAM 30.94 Myostatin blue 2hr 1.5 mg/L gill down 1.441 D02 FAM 33.10 32.01 Myostatin blue 2hr 1.5 mg/L gill p .79 D03 FAM 31.99 Myostatin blue 2hr 1.5 mg/L gill 0.461 E07 FAM 33.22 Myostatin blue 4hr 2 mg/L gill down 1.964 E08 FAM 31.98 32.46 Myostatin blue 4hr 2 mg/L gill p .687 E09 FAM 32.16 Myostatin blue 4hr 2 mg/L gill 0.29 G01 FAM 31.96 Myostatin blue 4hr 1.5 mg/L gill down 2.125 G02 FAM 32.89 32.57 Myostatin blue 4hr 1.5 mg/L gill p .565 87 G03 FAM 32.87 Myostatin blue 4hr 1.5 mg/L gill 0.257 H07 FAM 31.35 Myostatin blue 8hr 2 mg/L gill down3.164 H08 FAM 34.83 33.14 Myostatin blue 8hr 2 mg/L gill p.395 H09 FAM 33.25 Myostatin blue 8hr 2 mg/L gill 0.272 A04 FAM 36.43 Myostatin blue 4 hr control liver A05 FAM 37.68 37.82 Myostatin blue 4 hr control liver A06 FAM 39.37 Myostatin blue 4 hr control liver B10 FAM 33.52 Myostatin blue 2hr 2 mg/L liver up 4.626 B11 FAM 37.24 34.95 Myostatin blue 2hr 2 mg/L liver p.209 B12 FAM 34.10 Myostatin blue 2hr 2 mg/L liver 4.99 D04 FAM 35.98 Myostatin blue2hr 1.5 mg/L liver up 11.246 D05 FAM 32.20 33.67 Myostatin blue2hr 1.5 mg/L liver p.194 D06 FAM 32.83 Myostatin blue2hr 1.5 mg/L liver 12.214 E10 FAM 39.54 Myostatin blue4hr 2 mg/L liver up 1.849 E11 FAM 34.73 36.28 Myostatin blue4hr 2 mg/L liver p 0.704 E12 FAM 34.56 Myostatin blue4hr 2 mg/L liver 2.481 G01 FAM 31.96 Myostatin blue4hr 1.5 mg/L liver up 24.133 G02 FAM 32.89 32.57 Myostatin blue4hr 1.5 mg/L liver p.048 G03 FAM 32.87 Myostatin blue4hr 1.5 mg/L liver 18.163 H10 FAM 36.83 Myostatin blue8hr 2 mg/L liver up 1.579 H11 FAM 37.03 36.50 Myostatin blue8hr 2 mg/L liver p.064 H12 FAM 35.65 Myostatin blue8hr 2 mg/L liver 1.234 A07 FAM 26.22 Myostatin channel 4hr control gill A08 FAM 26.37 26.31 Myostatin channel 4hr control gill A09 FAM 26.35 Myostatin channel 4hr control gill C01 FAM 27.89 Myostatin channel 2hr 2 mg/L gill down 2.55 C02 FAM 26.66 27.09 Myostatin channel 2hr 2 mg/L gill p.001 C03 FAM 26.72 Myostatin channel 2hr 2 mg/L gill 1.51 D07 FAM 25.17 Myostatin channel 2hr 1.5 mg/L gill up 1.104 D08 FAM 25.62 25.60 Myostatin channel 2hr 1.5 mg/L gill p.791 D09 FAM 26.00 Myostatin channel 2hr 1.5 mg/L gill 0.3466 F01 FAM 26.49 Myostatin channel 4hr 2 mg/L gill down 1.406 88 F02 FAM 26.10 26.23 Myostatin channel 4hr 2 mg/L gill p.71 F03 FAM 26.10 Myostatin channel 4hr 2 mg/L gill 0.2 G07 FAM 26.03 Myostatin channel 4hr 1.5 mg/L gill down 1.406 G08 FAM 27.38 26.40 Myostatin channel 4hr 1.5 mg/L gill p.71 G09 FAM 25.79 Myostatin channel 4hr 1.5 mg/L gill 0.275 B01 FAM 26.64 Myostatin channel 8hr 2 mg/L gill down 1.974 B02 FAM 26.05 26.72 Myostatin channel 8hr 2 mg/L gill p.22 B03 FAM 27.47 Myostatin channel 8hr 2 mg/L gill 0.197 A10 FAM 29.33 Myostatin channel 4hr control liver A11 FAM 29.87 30.10 Myostatin channel 4hr control liver A12 FAM 31.11 Myostatin channel 4hr control liver C04 FAM 30.11 Myostatin channel 2hr 2 mg/L liver up 1.22 C05 FAM 30.40 29.83 Myostatin channel 2hr 2 mg/L liver p .81 C06 FAM 28.99 Myostatin channel 2hr 2 mg/L liver 0.612 D10 FAM 28.18 Myostatin channel 2hr 1.5 mg/L liver up 2.079 D11 FAM 29.38 28.94 Myostatin channel 2hr 1.5 mg/L liver p 0.185 D12 FAM 29.27 Myostatin channel 2hr 1.5 mg/L liver 0.098 F04 FAM 30.57 Myostatin channel 4hr 2 mg/L liver up 1.314 F05 FAM 29.06 29.60 Myostatin channel 4hr 2 mg/L liver p.539 F06 FAM 29.19 Myostatin channel 4hr 2 mg/L liver 0.743 G10 FAM 27.67 Myostatin channel 4hr 1.5 mg/L liver up 4.484 G11 FAM 27.75 27.83 Myostatin channel 4hr 1.5 mg/L liver p.044 G12 FAM 28.08 Myostatin channel 4hr 1.5 mg/L liver 2.083 B04 FAM 30.52 Myostatin channel 8 hr2 mg/L liver down 1.204 B05 FAM 30.45 30.60 Myostatin channel 8 hr2 mg/L liver p.712 B06 FAM 30.83 Myostatin channel 8 hr2 mg/L liver 0.305 B01 FAM 27.03 Myostatin hybrid 4hr control gill B02 FAM 26.75 26.72 Myostatin hybrid 4hr control gill B03 FAM 26.37 Myostatin hybrid 4hr control gill C07 FAM 26.20 Myostatin hybrid 2hr 2 mg/L gill down 2.087 C08 FAM 28.62 27.78 Myostatin hybrid 2hr 2 mg/L gill p.345 C09 FAM 28.53 Myostatin hybrid 2hr 2 mg/L gill 0.33 89 E01 FAM 28.31 Myostatin hybrid 2hr 1.5 mg/L gill down 2.653 E02 FAM 29.06 28.13 Myostatin hybrid 2hr 1.5 mg/L gill p.167 E03 FAM 27.02 Myostatin hybrid 2hr 1.5 mg/L gill 0.221 F07 FAM 26.31 Myostatin hybrid 4hr 2 mg/L gill up 1.088 F08 FAM 26.65 26.60 Myostatin hybrid 4hr 2 mg/L gill p.622 F09 FAM 26.84 Myostatin hybrid 4hr 2 mg/L gill 0.467 H01 FAM 27.07 Myostatin hybrid 4hr 1.5 mg/L gill down 1.043 H02 FAM 27.26 26.78 Myostatin hybrid 4hr 1.5 mg/L gill p.846 H03 FAM 26.01 Myostatin hybrid 4hr 1.5 mg/L gill 0.475 B07 FAM 27.16 Myostatin hybrid 8hr 2 mg/L gill down 1.68 B08 FAM 27.15 27.47 Myostatin hybrid 8hr 2 mg/L gill p.043 B09 FAM 28.10 Myostatin hybrid 8hr 2 mg/L gill 0.279 B04 FAM 31.02 Myostatin hybrid 4hr control liver B05 FAM 32.67 32.31 Myostatin hybrid 4hr control liver B06 FAM 33.25 Myostatin hybrid 4hr control liver C10 FAM 30.30 Myostatin hybrid 2hr 2 mg/L liver up 3.878 C11 FAM 29.00 29.77 Myostatin hybrid 2hr 2 mg/L liver p.175 C12 FAM 30.00 Myostatin hybrid 2hr 2 mg/L liver 2.62 E04 FAM 30.34 Myostatin hybrid 2hr 1.5 mg/L liver up 5.397 E05 FAM 28.56 29.29 Myostatin hybrid 2hr 1.5 mg/L liver p .122 E06 FAM 28.97 Myostatin hybrid 2hr 1.5 mg/L liver 3.889 F10 FAM 32.22 Myostatin hybrid 4hr 2 mg/L liver up 1.059 F11 FAM 31.98 31.64 Myostatin hybrid 4hr 2 mg/L liver p.906 F12 FAM 30.73 Myostatin hybrid 4hr 2 mg/L liver 0.736 H04 FAM 31.23 Myostatin hybrid 4hr 1.5 mg/L liver up 1.101 H05 FAM 31.07 31.59 Myostatin hybrid 4hr 1.5 mg/L liver p.817 H06 FAM 32.46 Myostatin hybrid 4hr 1.5 mg/L liver 0.758 B10 FAM 28.32 Myostatin hybrid 8hr 2 mg/L liver up 21.025 B11 FAM 26.67 27.33 Myostatin hybrid 8hr 2 mg/L liver p.082 B12 FAM 27.00 Myostatin hybrid 8hr 2 mg/L liver 14.894 A01 FAM 29.41 NKEF blue 4 hr control gill A02 FAM 28.84 28.97 NKEF blue 4 hr control gill A03 FAM 28.66 NKEF blue 4 hr control gill 90 B07 FAM 27.67 NKEF blue 2hr 2 mg/L gill down 1.128 B08 FAM 28.53 27.86 NKEF blue 2hr 2 mg/L gill p.968 B09 FAM 27.39 NKEF blue 2hr 2 mg/L gill 0.338 D01 FAM 28.35 NKEF blue 2hr 1.5 mg/L gill down 1.46 D02 FAM 28.90 28.24 NKEF blue 2hr 1.5 mg/L gill p0.873 D03 FAM 27.46 NKEF blue 2hr 1.5 mg/L gill 0.285 E07 FAM 27.92 NKEF blue 4hr 2 mg/L gill down 1.147 E08 FAM 27.84 27.89 NKEF blue 4hr 2 mg/L gill p.968 E09 FAM 27.91 NKEF blue 4hr 2 mg/L gill 0.26 G01 FAM 29.77 NKEF blue 4hr 1.5 mg/L gill down 1.936 G02 FAM 28.09 28.64 NKEF blue 4hr 1.5 mg/L gill p.725 G03 FAM 28.07 NKEF blue 4hr 1.5 mg/L gill 0.254 H07 FAM 27.97 NKEF blue 8hr 2 mg/L gill down 2.155 H08 FAM 29.07 28.80 NKEF blue 8hr 2 mg/L gill p0.609 H09 FAM 29.35 NKEF blue 8hr 2 mg/L gill 0.194 A04 FAM 30.53 NKEF blue 4 hr control liver A05 FAM 30.34 30.34 NKEF blue 4 hr control liver A06 FAM 30.14 NKEF blue 4 hr control liver B10 FAM 31.78 NKEF blue 2hr 2 mg/L liver down 2.362 B11 FAM 30.04 30.92 NKEF blue 2hr 2 mg/L liver p.16 B12 FAM 30.93 NKEF blue 2hr 2 mg/L liver 0.232 D04 FAM 30.77 NKEF blue2hr 1.5 mg/L liver down 1.409 D05 FAM 29.74 30.17 NKEF blue2hr 1.5 mg/L liver p.659 D06 FAM 30.00 NKEF blue2hr 1.5 mg/L liver 0.336 E10 FAM 32.13 NKEF blue4hr 2 mg/L liver down 6.971 E11 FAM 31.79 32.48 NKEF blue4hr 2 mg/L liver p.001 E12 FAM 33.51 NKEF blue4hr 2 mg/L liver 0.08 G01 FAM 29.77 NKEF blue4hr 1.5 mg/L liver up 2.047 G02 FAM 28.09 28.64 NKEF blue4hr 1.5 mg/L liver p.233 G03 FAM 28.07 NKEF blue4hr 1.5 mg/L liver 1.179 H10 FAM 28.28 NKEF blue8hr 2 mg/L liver up 1.237 H11 FAM 29.44 29.37 NKEF blue8hr 2 mg/L liver p.724 91 H12 FAM 30.40 NKEF blue8hr 2 mg/L liver 0.741 A07 FAM 28.75 NKEF channel 4hr control gill A08 FAM 28.38 28.57 NKEF channel 4hr control gill A09 FAM 28.59 NKEF channel 4hr control gill C01 FAM 28.53 NKEF channel 2hr 2 mg/L gill down 1.265 C02 FAM 28.41 28.34 NKEF channel 2hr 2 mg/L gill p.842 C03 FAM 28.07 NKEF channel 2hr 2 mg/L gill 0.229 D07 FAM 28.45 NKEF channel 2hr 1.5 mg/L gill down 1.077 D08 FAM 28.40 28.10 NKEF channel 2hr 1.5 mg/L gill p0.842 D09 FAM 27.46 NKEF channel 2hr 1.5 mg/L gill 0.328 F01 FAM 28.97 NKEF channel 4hr 2 mg/L gill down 1.431 F02 FAM 28.44 28.52 NKEF channel 4hr 2 mg/L gill p .55 F03 FAM 28.14 NKEF channel 4hr 2 mg/L gill 0.224 G07 FAM 28.07 NKEF channel 4hr 1.5 mg/L gill up 1.076 G08 FAM 26.41 27.89 NKEF channel 4hr 1.5 mg/L gill p0.927 G09 FAM 29.20 NKEF channel 4hr 1.5 mg/L gill 0.673 F01 FAM 29.36 NKEF channel 8hr 2 mg/L gill down 1.674 F02 FAM 29.62 28.74 NKEF channel 8hr 2 mg/L gill p.446 F03 FAM 27.24 NKEF channel 8hr 2 mg/L gill 0.353 A10 FAM 31.13 NKEF channel 4hr control liver A11 FAM 30.75 31.59 NKEF channel 4hr control liver A12 FAM 32.89 NKEF channel 4hr control liver C04 FAM 30.92 NKEF channel 2hr 2 mg/L liver up 1.606 C05 FAM 30.45 30.81 NKEF channel 2hr 2 mg/L liver p.53 C06 FAM 31.04 NKEF channel 2hr 2 mg/L liver 0.878 D10 FAM 32.32 NKEF channel 2hr 1.5 mg/L liver down 1.005 D11 FAM 31.38 31.50 NKEF channel 2hr 1.5 mg/L liver p.949 D12 FAM 30.79 NKEF channel 2hr 1.5 mg/L liver 0.612 F04 FAM 30.61 NKEF channel 4hr 2 mg/L liver up 1.882 F05 FAM 30.96 30.58 NKEF channel 4hr 2 mg/L liver p.31 F06 FAM 30.15 NKEF channel 4hr 2 mg/L liver 1.048 G10 FAM 27.92 NKEF channel 4hr 1.5 mg/L liver up 10.618 92 G11 FAM 28.09 28.08 NKEF channel 4hr 1.5 mg/L liver p.061 G12 FAM 28.23 NKEF channel 4hr 1.5 mg/L liver 5.69 F04 FAM 31.91 NKEF channel 8 hr2 mg/L liver up 1.407 F05 FAM 30.48 31.00 NKEF channel 8 hr2 mg/L liver p.586 F06 FAM 30.59 NKEF channel 8 hr2 mg/L liver 0.873 B01 FAM 28.51 NKEF hybrid 4hr control gill B02 FAM 28.91 28.65 NKEF hybrid 4hr control gill B03 FAM 28.53 NKEF hybrid 4hr control gill C07 FAM 29.58 NKEF hybrid 2hr 2 mg/L gill up 1.104 C08 FAM 27.78 28.51 NKEF hybrid 2hr 2 mg/L gill p.716 C09 FAM 28.17 NKEF hybrid 2hr 2 mg/L gill 0.611 E01 FAM 28.39 NKEF hybrid 2hr 1.5 mg/L gill up 1.269 E02 FAM 29.18 28.31 NKEF hybrid 2hr 1.5 mg/L gill p.604 E03 FAM 27.35 NKEF hybrid 2hr 1.5 mg/L gill 0.691 F07 FAM 28.34 NKEF hybrid 4hr 2 mg/L gill up 1.031 F08 FAM 28.70 28.61 NKEF hybrid 4hr 2 mg/L gill p.866 F09 FAM 28.78 NKEF hybrid 4hr 2 mg/L gill 0.427 H01 FAM 30.58 NKEF hybrid 4hr 1.5 mg/L gill down 1.186 H02 FAM 27.09 28.90 NKEF hybrid 4hr 1.5 mg/L gill p.655 H03 FAM 29.03 NKEF hybrid 4hr 1.5 mg/L gill 0.681 F07 FAM 28.29 NKEF hybrid 8hr 2 mg/L gill up 1.441 F08 FAM 28.31 28.13 NKEF hybrid 8hr 2 mg/L gill p.001 F09 FAM 27.78 NKEF hybrid 8hr 2 mg/L gill 0.607 B04 FAM 31.57 NKEF hybrid 4hr control liver B05 FAM 31.31 31.31 NKEF hybrid 4hr control liver B06 FAM 31.07 NKEF hybrid 4hr control liver C10 FAM 31.62 NKEF hybrid 2hr 2 mg/L liver up 1.032 C11 FAM 30.25 30.68 NKEF hybrid 2hr 2 mg/L liver p.94 C12 FAM 30.17 NKEF hybrid 2hr 2 mg/L liver 0.549 E04 FAM 31.38 NKEF hybrid 2hr 1.5 mg/L liver up 1.247 E05 FAM 29.45 30.41 NKEF hybrid 2hr 1.5 mg/L liver p.695 E06 FAM 30.39 NKEF hybrid 2hr 1.5 mg/L liver 0.711 93 F10 FAM 31.52 NKEF hybrid 4hr 2 mg/L liver down 1.697 F11 FAM 31.53 31.49 NKEF hybrid 4hr 2 mg/L liver p.151 F12 FAM 31.42 NKEF hybrid 4hr 2 mg/L liver 0.248 H04 FAM 30.65 NKEF hybrid 4hr 1.5 mg/L liver up 2.068 H05 FAM 31.22 29.68 NKEF hybrid 4hr 1.5 mg/L liver p.562 H06 FAM 27.16 NKEF hybrid 4hr 1.5 mg/L liver 2.015 F10 FAM 32.51 NKEF hybrid 8hr 2 mg/L liver up 1.393 F11 FAM 29.19 30.25 NKEF hybrid 8hr 2 mg/L liver p.657 F12 FAM 29.04 NKEF hybrid 8hr 2 mg/L liver 1.24 A01 FAM 10.99 S18 blue 4 hr control gill A02 FAM 11.51 10.96 S18 blue 4 hr control gill A03 FAM 10.37 S18 blue 4 hr control gill B07 FAM 9.59 S18 blue 2hr 2 mg/L gill B08 FAM 9.85 9.86 S18 blue 2hr 2 mg/L gill B09 FAM 10.14 S18 blue 2hr 2 mg/L gill D01 FAM 9.90 S18 blue 2hr 1.5 mg/L gill D02 FAM 9.69 9.60 S18 blue 2hr 1.5 mg/L gill D03 FAM 9.21 S18 blue 2hr 1.5 mg/L gill E07 FAM 9.03 S18 blue 4hr 2 mg/L gill E08 FAM 9.96 9.70 S18 blue 4hr 2 mg/L gill E09 FAM 10.11 S18 blue 4hr 2 mg/L gill G01 FAM 9.92 S18 blue 4hr 1.5 mg/L gill G02 FAM 9.52 9.72 S18 blue 4hr 1.5 mg/L gill G03 FAM 9.73 S18 blue 4hr 1.5 mg/L gill H07 FAM 9.30 S18 blue 8hr 2 mg/L gill H08 FAM 9.51 9.50 S18 blue 8hr 2 mg/L gill H09 FAM 9.68 S18 blue 8hr 2 mg/L gill A04 FAM 10.53 S18 blue 4 hr control liver A05 FAM 11.62 10.62 S18 blue 4 hr control liver A06 FAM 9.71 S18 blue 4 hr control liver B10 FAM 10.07 S18 blue 2hr 2 mg/L liver B11 FAM 9.89 10.21 S18 blue 2hr 2 mg/L liver B12 FAM 10.65 S18 blue 2hr 2 mg/L liver 94 D04 FAM 10.62 S18 blue2hr 1.5 mg/L liver D05 FAM 8.52 9.49 S18 blue2hr 1.5 mg/L liver D06 FAM 9.33 S18 blue2hr 1.5 mg/L liver E10 FAM 10.09 S18 blue4hr 2 mg/L liver E11 FAM 10.27 10.34 S18 blue4hr 2 mg/L liver E12 FAM 10.66 S18 blue4hr 2 mg/L liver G04 FAM 10.64 S18 blue4hr 1.5 mg/L liver G05 FAM 10.14 10.06 S18 blue4hr 1.5 mg/L liver G06 FAM 9.40 S18 blue4hr 1.5 mg/L liver H10 FAM 9.03 S18 blue8hr 2 mg/L liver H11 FAM 9.96 9.70 S18 blue8hr 2 mg/L liver H12 FAM 10.11 S18 blue8hr 2 mg/L liver A07 FAM 10.38 S18 channel 4hr control gill A08 FAM 10.59 10.45 S18 channel 4hr control gill A09 FAM 10.38 S18 channel 4hr control gill C01 FAM 9.23 S18 channel 2hr 2 mg/L gill C02 FAM 10.52 9.92 S18 channel 2hr 2 mg/L gill C03 FAM 10.01 S18 channel 2hr 2 mg/L gill D07 FAM 9.42 S18 channel 2hr 1.5 mg/L gill D08 FAM 9.76 9.41 S18 channel 2hr 1.5 mg/L gill D09 FAM 9.06 S18 channel 2hr 1.5 mg/L gill F01 FAM 10.06 S18 channel 4hr 2 mg/L gill F02 FAM 10.13 10.23 S18 channel 4hr 2 mg/L gill F03 FAM 10.49 S18 channel 4hr 2 mg/L gill G07 FAM 9.75 S18 channel 4hr 1.5 mg/L gill G08 FAM 9.83 9.89 S18 channel 4hr 1.5 mg/L gill G09 FAM 10.09 S18 channel 4hr 1.5 mg/L gill A01 FAM 10.70 S18 channel 8hr 2 mg/L gill A02 FAM 9.39 9.91 S18 channel 8hr 2 mg/L gill A03 FAM 9.64 S18 channel 8hr 2 mg/L gill A10 FAM 10.40 S18 channel 4hr control liver A11 FAM 10.04 10.08 S18 channel 4hr control liver 95 A12 FAM 9.81 S18 channel 4hr control liver C04 FAM 8.91 S18 channel 2hr 2 mg/L liver C05 FAM 9.10 9.35 S18 channel 2hr 2 mg/L liver C06 FAM 10.05 S18 channel 2hr 2 mg/L liver D10 FAM 10.81 S18 channel 2hr 1.5 mg/L liver D11 FAM 9.23 9.84 S18 channel 2hr 1.5 mg/L liver D12 FAM 9.48 S18 channel 2hr 1.5 mg/L liver F04 FAM 9.68 S18 channel 4hr 2 mg/L liver F05 FAM 9.51 9.62 S18 channel 4hr 2 mg/L liver F06 FAM 9.68 S18 channel 4hr 2 mg/L liver G10 FAM 10.11 S18 channel 4hr 1.5 mg/L liver G11 FAM 10.28 10.19 S18 channel 4hr 1.5 mg/L liver G12 FAM 10.18 S18 channel 4hr 1.5 mg/L liver A04 FAM 11.37 S18 channel 8 hr2 mg/L liver A05 FAM 10.51 10.89 S18 channel 8 hr2 mg/L liver A06 FAM 10.78 S18 channel 8 hr2 mg/L liver B01 FAM 9.92 S18 hybrid 4hr control gill B02 FAM 10.07 9.98 S18 hybrid 4hr control gill B03 FAM 9.94 S18 hybrid 4hr control gill C07 FAM 11.00 S18 hybrid 2hr 2 mg/L gill C08 FAM 9.07 9.93 S18 hybrid 2hr 2 mg/L gill C09 FAM 9.72 S18 hybrid 2hr 2 mg/L gill E01 FAM 9.75 S18 hybrid 2hr 1.5 mg/L gill E02 FAM 9.38 9.53 S18 hybrid 2hr 1.5 mg/L gill E03 FAM 9.47 S18 hybrid 2hr 1.5 mg/L gill F07 FAM 9.40 S18 hybrid 4hr 2 mg/L gill F08 FAM 10.11 9.58 S18 hybrid 4hr 2 mg/L gill F09 FAM 9.24 S18 hybrid 4hr 2 mg/L gill H01 FAM 10.72 S18 hybrid 4hr 1.5 mg/L gill H02 FAM 9.82 10.09 S18 hybrid 4hr 1.5 mg/L gill H03 FAM 9.74 S18 hybrid 4hr 1.5 mg/L gill A07 FAM 11.03 S18 hybrid 8hr 2 mg/L gill 96 A08 FAM 10.80 10.77 S18 hybrid 8hr 2 mg/L gill A09 FAM 10.49 S18 hybrid 8hr 2 mg/L gill B04 FAM 10.39 S18 hybrid 4hr control liver B05 FAM 10.09 10.52 S18 hybrid 4hr control liver B06 FAM 11.07 S18 hybrid 4hr control liver C10 FAM 8.95 S18 hybrid 2hr 2 mg/L liver C11 FAM 10.62 9.95 S18 hybrid 2hr 2 mg/L liver C12 FAM 10.29 S18 hybrid 2hr 2 mg/L liver E04 FAM 10.79 S18 hybrid 2hr 1.5 mg/L liver E05 FAM 8.87 10.08 S18 hybrid 2hr 1.5 mg/L liver E06 FAM 10.57 S18 hybrid 2hr 1.5 mg/L liver F10 FAM 10.62 S18 hybrid 4hr 2 mg/L liver F11 FAM 10.33 10.12 S18 hybrid 4hr 2 mg/L liver F12 FAM 9.41 S18 hybrid 4hr 2 mg/L liver H04 FAM 9.63 S18 hybrid 4hr 1.5 mg/L liver H05 FAM 9.79 9.49 S18 hybrid 4hr 1.5 mg/L liver H06 FAM 9.06 S18 hybrid 4hr 1.5 mg/L liver A10 FAM 9.37 S18 hybrid 8hr 2 mg/L liver A11 FAM 10.25 10.01 S18 hybrid 8hr 2 mg/L liver A12 FAM 10.42 S18 hybrid 8hr 2 mg/L liver