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• 1561. [Article] Statistical Methods for Tissue Culture Medium Optimization and A Multiplexed Fingerprinting Set for Hazelnuts

  Hazelnut is one of the most important nuts in worldwide production and the European hazelnut (Corylus avellana L.) is the most economically important of the 11 recognized hazelnut species. Development ...

  Citation × Citation Citation Abstract Copy Title: Statistical Methods for Tissue Culture Medium Optimization and A Multiplexed Fingerprinting Set for Hazelnuts Author: Akin, Melekşen Hazelnut is one of the most important nuts in worldwide production and the European hazelnut (Corylus avellana L.) is the most economically important of the 11 recognized hazelnut species. Development of new cultivars is continuous, with emphasis on better nut quality, high yield and disease resistance. Hazelnuts are highly heterozygous, and clonally propagated. Traditional propagation methods in hazelnut are not rapid enough to provide the required nursery stock for newly released hazelnut cultivars, but micropropagation can provide rapid production of hazelnut planting stock. Several growth media are available for specific cultivars, but many are not suitable for the wide range of germplasm used in new cultivars. Micropropagation of hazelnuts remains challenging due to the various responses of diverse genotypes to in vitro growth. Several studies incrimentally improved the growth medium, but determining exact nutrient requirements was difficult. The aim of this study was to determine which statistical methods would make the growth medium optimization process more practical and to develop an optimal micropropagation medium for diverse hazelnuts by testing salts and ions as factors within the experimental design. In addition an SSR fingerprinting set suitable for a diverse group of hazelnuts was developed. The first study was designed to test the effect of salts on three hazelnut genotypes and compare two methods of data analysis. Driver and Kuniyuki Walnut medium (DKW) macro-salts (NH₄NO₃, Ca(NO₃)₂∙4H₂O, CaCl₂∙2H₂O, MgSO₄∙7H₂O, KH₂PO₄ and K₂SO₄) were varied from 0.5× to 3× DKW concentrations with 42 combinations in a IV-optimal design. Shoot quality, shoot length, multiplication and callus formation were rated and analyzed using Response Surface Methodology (RSM) and the Chi-Squared Automatic Interaction Detection (CHAID) data mining algorithm. Both analyses indicated that NH₄NO₃ was a predominant nutrient factor. RSM results were genotype dependent while CHAID included genotype as a factor in the analysis, allowing development of a common medium rather than several genotype-specific media. Overall, CHAID results were more specific and easier to interpret than RSM graphs. The optimal growth medium for diverse hazelnut genotypes was formulated as: 0.5× NH₄NO₃, 3× KH₂PO₄, 1.5× Ca(NO₃)₂ and and the rest of the macro salts set at 1× DKW with modified minor nutrients [4× H₃BO₃, 4× Na₂MoO₄∙2H₂O, 4× Zn(NO₃)₂∙6H₂O, 0.5× MnSO₄∙H₂O, 0.5× CuSO₄∙5H₂O]. The second study was to determine the effects of ions on tissue culture medium optimization. NH₄⁺, Ca²⁺, Mg²⁺, SO₄²⁻ and PO₄³⁻ ions were used as factors in a D-optimal design. K⁺ and NO₃⁻ ions were used to bring the pH level to neutral, and as factors in the statistical analysis. The CHAID data mining algorithm was used to analyze shoot growth responses of three hazelnut genotypes. The algorithm trees revealed significant variables and their interactions, and provided exact cut-off amounts for each of the ions for the related growth response by incorporating genotype as an independent factor. The critical cut-off values for good shoot quality, elongation, multiplication and medium callus formation were suggested to be: NO₃⁻ <88 mM, NH₄⁺ <20 mM, Ca²⁺ <5 mM, Mg²⁺ >5 mM and K⁺ <46 mM. Another step of the research was to develop a reliable and economical fingerprinting set consisting of high core-repeat SSRs (≥3) for genotype identification of 102 hazelnut accessions. Identification of trueness-to-type by phenotypic observation is very difficult and labeling mistakes during the several steps of micropropagation can result in costly errors. The use of SSRs for plant identification is preferred over other molecular markers because they are reproducible across laboratories, exhibit co-dominant inheritance, have a large number of alleles per locus and are randomly distributed throughout the genome. Twenty SSRs containing repeat motifs of three or more nucleotides distributed throughout the hazelnut genome were screened on eight genetically diverse cultivars to assess polymorphism, allele size range, and ease of scoring. Six SSRs were discarded after genotyping 96 hazelnut samples, either due to large allele bin widths and/or alleles that do not match the motifs, complicating allele scoring. Fourteen polymorphic, easy-to-score SSRs with non-overlapping alleles were selected and amplified in a single multiplex. The multiplexed set generated the same alleles that were obtained when amplifying each SSR individually in the eight test accessions. SSR primer concentrations were then optimized to generate a clear signal for each locus. This 14-SSR fingerprinting set was used to genotype 102 hazelnut accessions, and distinguished unique accessions mainly according to parentage and in some cases based on geographic origin. As a result of these studies, salt- and ion-based optimized tissue culture medium formulations were developed for diverse hazelnuts. The importance of salts and ions as factors within the experimental design and analysis was examined, and using salts as factors results in complexity within the design as the effects of ions can not be determined. Although salt optimization studies are still a powerful tool, and are experimentally easier, optimization at the ionic level provided a clearer evaluation of the ion-based growth responses, because the plants take up minerals as ions of the corresponding salts. Data mining (CHAID) was used to make the tissue culture optimization process more practical compared to analysis with the standard ANOVA, regression and RSM. CHAID delineated specific concentrations that were effective and allowed easier analysis of nutrient content for an improved medium. A reliable and cost-effective multiplexed fingerprinting set of 14 SSR markers was developed for confirming identity and paternity in diverse hazelnut cultivars and species and 102 accessions were fingerprinted. Title: Statistical Methods for Tissue Culture Medium Optimization and A Multiplexed Fingerprinting Set for Hazelnuts Author: Akin, Melekşen Hazelnut is one of the most important nuts in worldwide production and the European hazelnut (Corylus avellana L.) is the most economically important of the 11 recognized hazelnut species. Development of new cultivars is continuous, with emphasis on better nut quality, high yield and disease resistance. Hazelnuts are highly heterozygous, and clonally propagated. Traditional propagation methods in hazelnut are not rapid enough to provide the required nursery stock for newly released hazelnut cultivars, but micropropagation can provide rapid production of hazelnut planting stock. Several growth media are available for specific cultivars, but many are not suitable for the wide range of germplasm used in new cultivars. Micropropagation of hazelnuts remains challenging due to the various responses of diverse genotypes to in vitro growth. Several studies incrimentally improved the growth medium, but determining exact nutrient requirements was difficult. The aim of this study was to determine which statistical methods would make the growth medium optimization process more practical and to develop an optimal micropropagation medium for diverse hazelnuts by testing salts and ions as factors within the experimental design. In addition an SSR fingerprinting set suitable for a diverse group of hazelnuts was developed. The first study was designed to test the effect of salts on three hazelnut genotypes and compare two methods of data analysis. Driver and Kuniyuki Walnut medium (DKW) macro-salts (NH₄NO₃, Ca(NO₃)₂∙4H₂O, CaCl₂∙2H₂O, MgSO₄∙7H₂O, KH₂PO₄ and K₂SO₄) were varied from 0.5× to 3× DKW concentrations with 42 combinations in a IV-optimal design. Shoot quality, shoot length, multiplication and callus formation were rated and analyzed using Response Surface Methodology (RSM) and the Chi-Squared Automatic Interaction Detection (CHAID) data mining algorithm. Both analyses indicated that NH₄NO₃ was a predominant nutrient factor. RSM results were genotype dependent while CHAID included genotype as a factor in the analysis, allowing development of a common medium rather than several genotype-specific media. Overall, CHAID results were more specific and easier to interpret than RSM graphs. The optimal growth medium for diverse hazelnut genotypes was formulated as: 0.5× NH₄NO₃, 3× KH₂PO₄, 1.5× Ca(NO₃)₂ and and the rest of the macro salts set at 1× DKW with modified minor nutrients [4× H₃BO₃, 4× Na₂MoO₄∙2H₂O, 4× Zn(NO₃)₂∙6H₂O, 0.5× MnSO₄∙H₂O, 0.5× CuSO₄∙5H₂O]. The second study was to determine the effects of ions on tissue culture medium optimization. NH₄⁺, Ca²⁺, Mg²⁺, SO₄²⁻ and PO₄³⁻ ions were used as factors in a D-optimal design. K⁺ and NO₃⁻ ions were used to bring the pH level to neutral, and as factors in the statistical analysis. The CHAID data mining algorithm was used to analyze shoot growth responses of three hazelnut genotypes. The algorithm trees revealed significant variables and their interactions, and provided exact cut-off amounts for each of the ions for the related growth response by incorporating genotype as an independent factor. The critical cut-off values for good shoot quality, elongation, multiplication and medium callus formation were suggested to be: NO₃⁻ <88 mM, NH₄⁺ <20 mM, Ca²⁺ <5 mM, Mg²⁺ >5 mM and K⁺ <46 mM. Another step of the research was to develop a reliable and economical fingerprinting set consisting of high core-repeat SSRs (≥3) for genotype identification of 102 hazelnut accessions. Identification of trueness-to-type by phenotypic observation is very difficult and labeling mistakes during the several steps of micropropagation can result in costly errors. The use of SSRs for plant identification is preferred over other molecular markers because they are reproducible across laboratories, exhibit co-dominant inheritance, have a large number of alleles per locus and are randomly distributed throughout the genome. Twenty SSRs containing repeat motifs of three or more nucleotides distributed throughout the hazelnut genome were screened on eight genetically diverse cultivars to assess polymorphism, allele size range, and ease of scoring. Six SSRs were discarded after genotyping 96 hazelnut samples, either due to large allele bin widths and/or alleles that do not match the motifs, complicating allele scoring. Fourteen polymorphic, easy-to-score SSRs with non-overlapping alleles were selected and amplified in a single multiplex. The multiplexed set generated the same alleles that were obtained when amplifying each SSR individually in the eight test accessions. SSR primer concentrations were then optimized to generate a clear signal for each locus. This 14-SSR fingerprinting set was used to genotype 102 hazelnut accessions, and distinguished unique accessions mainly according to parentage and in some cases based on geographic origin. As a result of these studies, salt- and ion-based optimized tissue culture medium formulations were developed for diverse hazelnuts. The importance of salts and ions as factors within the experimental design and analysis was examined, and using salts as factors results in complexity within the design as the effects of ions can not be determined. Although salt optimization studies are still a powerful tool, and are experimentally easier, optimization at the ionic level provided a clearer evaluation of the ion-based growth responses, because the plants take up minerals as ions of the corresponding salts. Data mining (CHAID) was used to make the tissue culture optimization process more practical compared to analysis with the standard ANOVA, regression and RSM. CHAID delineated specific concentrations that were effective and allowed easier analysis of nutrient content for an improved medium. A reliable and cost-effective multiplexed fingerprinting set of 14 SSR markers was developed for confirming identity and paternity in diverse hazelnut cultivars and species and 102 accessions were fingerprinted. Close

• 1562. [Article] Evaluating the Efficacy of Predicting Bycatch Mortality Using Reflex Impairment through an Assessment of Crab Discards

  All animals that interact with fishing gear are not necessarily captured, and all animals that are captured are not necessarily retained. Fishing practices and gear configuration, management regulations, ...

  Citation × Citation Citation Abstract Copy Title: Evaluating the Efficacy of Predicting Bycatch Mortality Using Reflex Impairment through an Assessment of Crab Discards Author: Yochum, Noëlle All animals that interact with fishing gear are not necessarily captured, and all animals that are captured are not necessarily retained. Fishing practices and gear configuration, management regulations, and markets dictate which animals ultimately are retained or discarded. The impact of a fishery and the efficacy of management regulations can depend on the mortality rate of the animals that interact with the gear or are discarded. The Reflex Action Mortality Predictor (RAMP) is a simple, non-invasive, and inexpensive approach that has been used to evaluate this component of fishing mortality. The RAMP approach relates the degree of reflex impairment in an animal to the probability the animal will die. Since its introduction in 2006, the RAMP approach has been utilized in the U.S. and abroad to evaluate mortality for a variety of species, fishing gear types, and stressors. Although there have been numerous applications of the RAMP approach in mortality estimation studies, there has been limited research to directly evaluate RAMP estimates and some skepticism remains in the fisheries science and management communities about the reliability and accuracy of the approach. The goal of this dissertation was to conduct research to assess RAMP and to synthesize findings from previously completed RAMP studies. The three research studies described in this dissertation consider: (1) the accuracy of applying an established relationship between reflex impairment and mortality probability to predict overall mortality attributed to novel stressors; (2) the development and utilization of a RAMP relationship to evaluate discard mortality in a fishery with management regulations that mandate discarding of certain categories of animals; and (3) whether the RAMP approach produces accurate estimates of mortality if survival is determined through laboratory captive holding. The first study estimated a relationship between reflex impairment and mortality probability for Tanner crab (Chionoecetes bairdi) discarded from the groundfish bottom trawl fishery in the Gulf of Alaska. This relationship was then compared to one previously established for Tanner crab in the Bering Sea bottom trawl fishery that encountered the fishing gear, but remained on the seafloor ('unobserved bycatch'). While mortality probabilities were similar between the two studies for crab with no or full reflex impairment, discarded crab with intermediate levels of reflex impairment had lower mortality probabilities than those from the unobserved bycatch study. Results from this study indicate the importance of describing all stressors to which animals are exposed and detailing the study methodology when initially creating a RAMP relationship. Failure to do so may result in inaccurate mortality estimates when the RAMP is applied to animals exposed to stressors not included in the original calibration. The second study developed a RAMP relationship using laboratory captive holding for Dungeness crab (Cancer magister) discarded in the Oregon commercial and recreational Dungeness fisheries and estimated that the discard mortality rate is lower than previously determined. This supports the goal of the '3-S' management strategy currently employed for these fisheries to protect sub-legal males (Size), females (Sex), and soft-shell (Season) crab by discarding them from the catch. For the commercial ocean Dungeness fishery, the estimated overall discard mortality rates (five days after release) varied by sex and shell-hardness, and reflex impairment was a significant predictor of mortality for both the commercial and recreational fisheries. In addition, results indicated that, when evaluating the role of discard mortality in '3-S' management with respect to fishery impact and sustainability, it is important to look not only at mortality rates, but also at the mortality- and bycatch- per retained ratios, and temporal trends relative to changes in effort, animal condition, and catch composition. This study also highlighted the (i) importance of evaluating the influence of biological, environmental, and fishing variables on mortality, (ii) complications that arise when establishing a RAMP relationship for a low impact fishery, and (iii) limitations of determining mortality through laboratory captive holding. The third study used mark-recapture methods to evaluate the reliability of results generated using the RAMP relationship established in the second study, which was based on the survival of crab held in captivity in the laboratory. Given the unnatural conditions for determining survival in captivity and the short-term duration of the experiment, mortality probability estimates may be biased. Similarities in patterns of relative survival rates between the studies lend support to the ability of the RAMP relationship to estimate discard mortality rates using captive holding. The laboratory-based RAMP approach was superior in its ability to provide direct estimates of mortality rates, whereas the mark-recapture study was limited to providing relative survival rates between reflex impairment levels that were imprecise due to low numbers of recaptured crab. This study highlighted the complications associated with tagging discarded animals and conducting a RAMP study with a fishery that has highly variable seasonal fishing effort. A synthesis of the research described in this dissertation and published work by other researchers highlights the limitations of the RAMP approach so that future researchers can avoid pitfalls in its application, and leads to suggestions on how to standardize some of the methodological steps. This analysis aims to increase the reliability of future RAMP studies and their production of high quality estimates of discard mortality rates that promote sustainable fisheries. Title: Evaluating the Efficacy of Predicting Bycatch Mortality Using Reflex Impairment through an Assessment of Crab Discards Author: Yochum, Noëlle All animals that interact with fishing gear are not necessarily captured, and all animals that are captured are not necessarily retained. Fishing practices and gear configuration, management regulations, and markets dictate which animals ultimately are retained or discarded. The impact of a fishery and the efficacy of management regulations can depend on the mortality rate of the animals that interact with the gear or are discarded. The Reflex Action Mortality Predictor (RAMP) is a simple, non-invasive, and inexpensive approach that has been used to evaluate this component of fishing mortality. The RAMP approach relates the degree of reflex impairment in an animal to the probability the animal will die. Since its introduction in 2006, the RAMP approach has been utilized in the U.S. and abroad to evaluate mortality for a variety of species, fishing gear types, and stressors. Although there have been numerous applications of the RAMP approach in mortality estimation studies, there has been limited research to directly evaluate RAMP estimates and some skepticism remains in the fisheries science and management communities about the reliability and accuracy of the approach. The goal of this dissertation was to conduct research to assess RAMP and to synthesize findings from previously completed RAMP studies. The three research studies described in this dissertation consider: (1) the accuracy of applying an established relationship between reflex impairment and mortality probability to predict overall mortality attributed to novel stressors; (2) the development and utilization of a RAMP relationship to evaluate discard mortality in a fishery with management regulations that mandate discarding of certain categories of animals; and (3) whether the RAMP approach produces accurate estimates of mortality if survival is determined through laboratory captive holding. The first study estimated a relationship between reflex impairment and mortality probability for Tanner crab (Chionoecetes bairdi) discarded from the groundfish bottom trawl fishery in the Gulf of Alaska. This relationship was then compared to one previously established for Tanner crab in the Bering Sea bottom trawl fishery that encountered the fishing gear, but remained on the seafloor ('unobserved bycatch'). While mortality probabilities were similar between the two studies for crab with no or full reflex impairment, discarded crab with intermediate levels of reflex impairment had lower mortality probabilities than those from the unobserved bycatch study. Results from this study indicate the importance of describing all stressors to which animals are exposed and detailing the study methodology when initially creating a RAMP relationship. Failure to do so may result in inaccurate mortality estimates when the RAMP is applied to animals exposed to stressors not included in the original calibration. The second study developed a RAMP relationship using laboratory captive holding for Dungeness crab (Cancer magister) discarded in the Oregon commercial and recreational Dungeness fisheries and estimated that the discard mortality rate is lower than previously determined. This supports the goal of the '3-S' management strategy currently employed for these fisheries to protect sub-legal males (Size), females (Sex), and soft-shell (Season) crab by discarding them from the catch. For the commercial ocean Dungeness fishery, the estimated overall discard mortality rates (five days after release) varied by sex and shell-hardness, and reflex impairment was a significant predictor of mortality for both the commercial and recreational fisheries. In addition, results indicated that, when evaluating the role of discard mortality in '3-S' management with respect to fishery impact and sustainability, it is important to look not only at mortality rates, but also at the mortality- and bycatch- per retained ratios, and temporal trends relative to changes in effort, animal condition, and catch composition. This study also highlighted the (i) importance of evaluating the influence of biological, environmental, and fishing variables on mortality, (ii) complications that arise when establishing a RAMP relationship for a low impact fishery, and (iii) limitations of determining mortality through laboratory captive holding. The third study used mark-recapture methods to evaluate the reliability of results generated using the RAMP relationship established in the second study, which was based on the survival of crab held in captivity in the laboratory. Given the unnatural conditions for determining survival in captivity and the short-term duration of the experiment, mortality probability estimates may be biased. Similarities in patterns of relative survival rates between the studies lend support to the ability of the RAMP relationship to estimate discard mortality rates using captive holding. The laboratory-based RAMP approach was superior in its ability to provide direct estimates of mortality rates, whereas the mark-recapture study was limited to providing relative survival rates between reflex impairment levels that were imprecise due to low numbers of recaptured crab. This study highlighted the complications associated with tagging discarded animals and conducting a RAMP study with a fishery that has highly variable seasonal fishing effort. A synthesis of the research described in this dissertation and published work by other researchers highlights the limitations of the RAMP approach so that future researchers can avoid pitfalls in its application, and leads to suggestions on how to standardize some of the methodological steps. This analysis aims to increase the reliability of future RAMP studies and their production of high quality estimates of discard mortality rates that promote sustainable fisheries. Close

• 1563. [Article] Mitochondrial decay in the aging rat heart : changes in fatty acid-supported bioenergetics and macromolecular organization of the electron transport system

  Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved ...

  Citation × Citation Citation Abstract Copy Title: Mitochondrial decay in the aging rat heart : changes in fatty acid-supported bioenergetics and macromolecular organization of the electron transport system Author: Gomez Ramirez, Luis A. (Luis Alejandro) Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved in the chemiosmotic process of energy transduction and lower capacity to maintain fatty acid-driven respiration are identified as intrinsic factors of mitochondrial decay in the aged myocardium. Age-associated destabilization of electron transport supercomplexes as a potential factor of mitochondrial decay in the rat heart. Defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Nevertheless, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not fully known. As recent biophysical evidence shows that the ETC may form large macromolecular assemblies (i.e. supercomplexes) that disintegrate in certain pathologies (e.g. heart failure or Barth syndrome) reminiscent of aging, we investigated the hypothesis that alterations in supercomplexes are partly responsible for the age-related loss of cardiac ETC function. In this dissertation, age-associated changes in supercomplex organization and stability were investigated in subsarcolemmal (SSM) and interfibrillary (IFM) mitochondria isolated from cardiac tissue from young (3-5 months) and old (24-28 months) male Fischer 344 rats. Blue native-PAGE (BN-PAGE) analysis of digitonin-solubilized mitochondrial membranes coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate supercomplex organization. Results show that both SSM and IFM display supercomplexes comprised of various stoichiometries of complexes I, III and IV (never complex II), which typically organize as high mass (1500-2300 kDa) assemblies containing up to four copies of complex IV (i.e. I₁III₂IV[subscript N]-type supercomplexes). Interestingly, analysis of IFM proteins showed that, in general, supercomplex levels declined by up to 15 % (p < 0.05) with age; however, different degrees of supercomplex deterioration were observed, depending on the particular supercomplex investigated. Supercomplexes of the highest molecular weights (i.e. 1900-2300 kDa), which were also composed of the most complex stoichiometries (i.e. I1III2IVN, N ≥ 2), were primarily lost with age. In particular, I₁III₂IV₂, I₁III₂IV₃ and I₁III₂IV₄ supercomplexes were found to decline by 13% (p < 0.05), 30% (p < 0.05) and 45% (p < 0.05), respectively, on an age basis. Therefore, the age-associated loss of supercomplexes in IFM stems from destabilization of the assemblies that comprise several copies of complex IV, which could partially limit proper electron transfer to O₂ for its reduction, affecting mitochondrial respiratory capacity. In contrast to IFM, the aging defects of SSM supercomplexes appeared to be confined to the assembly comprised of only one copy of complex IV (I₁III₂IV₁, 1700 kDa) (37% loss; p = 0.06), while the higher molecular weight supercomplex sub-types that were most affected in IFM (i.e. I₁III₂IV[subscript N], N ≥ 2) were not significantly altered with age. Thus, the results from this dissertation indicate that mitochondria from different subcellular locations in the myocyte show different degrees of supercomplex destabilization in the aging rat heart. The more robust supercomplex deficits noted for IFM fit well with previous observations that electron transport characteristics of this subpopulation are more adversely affected with age than SSM. Although the underlying factor(s) of supercomplex deterioration are not fully known, the hypothesis that age-related alterations of certain constituents of the IMM (e.g. cardiolipin) may be important factors of supercomplex destabilization in cardiac mitochondria was investigated in this dissertation. To this end, LC-MS/MS characterization of supercomplex proteins and HPLC analysis of cardiolipin were used as approaches to elucidate potential factor(s) of supercomplex destabilization in the aging rat heart. Age-related alterations of cardiolipin levels and its acyl-chain content showed a strong parallel to the age-associated destabilization of supercomplexes. Specifically, cardiolipin levels declined by 10% (p < 0.05) in IFM, the mitochondrial subpopulation displaying the highest degree of supercomplex deterioration. In addition, the content of (18:2)₄-cardiolipin, the predominant species in the heart, was found to decline by 50% (p < 0.05) on average in both populations of cardiac mitochondria. Therefore, the data presented in this dissertation indicate that changes in cardiolipin may be at least one of the factors involved in supercomplex destabilization in the aging heart. Age-related decline in carnitine palmitoyltransferase I (CPT1) activity as a mitochondrial lesion that limits fatty acid catabolism in the rat heart. Loss of fatty acid utilization, another intrinsic factor of mitochondrial decay in the aged myocardium, has been associated with age-related alterations in the activity of carnitine palmitoyltransferase 1 (CPT1), the rate-controlling enzyme for overall fatty acid β-oxidation. Nevertheless, the exact molecular mechanism involved in the age-related loss of fatty acid-driven bioenergetics is not fully understood. In this dissertation, it was also investigated whether the aging lesion for fatty oxidation lies in a particular mitochondrial subpopulation or more generally results from cardiac decrements in L-carnitine levels. In order to clarify the role of each one of these factors, the effect of long-term dietary supplementation with the L-carnitine analogue, acetyl-L-carnitine (ALCAR), was also investigated. Results show that aging selectively decreases CPT1 activity in IFM by reducing enzyme catalytic efficiency for palmitoyl-CoA. IFM displayed a 28% (p < 0.05) loss of CPT1 activity, which correlated with a decline (41%, p < 0.05) in palmitoyl-CoA-driven state 3 respiration. Interestingly, SSM had preserved enzyme function and efficiently utilized palmitate. Analysis of IFM CPT1 kinetics showed both diminished V[subscript max] and K[subscript m] (60% and 49% respectively, p < 0.05) when palmitoyl-CoA was the substrate. However, no age-related changes in enzyme kinetics were evident with respect to L-carnitine. ALCAR supplementation restored CPT1 activity in heart IFM, but not apparently through remediation of L-carnitine levels. Rather, ALCAR influenced enzyme activity over time, potentially by modulating conditions in the aging heart that ultimately affect palmitoyl-CoA binding and CPT1 kinetics. In conclusion, this dissertation presents a characterization of age-associated alterations in the macromolecular organization of the IMM components that could partly explain the loss of mitochondrial oxidative capacity that affects the aging heart. In addition, the characterization of an age-related lesion of the controlling enzyme for β-oxidation is presented as another important factor that limits mitochondrial function and energy metabolism in cardiac mitochondria. Title: Mitochondrial decay in the aging rat heart : changes in fatty acid-supported bioenergetics and macromolecular organization of the electron transport system Author: Gomez Ramirez, Luis A. (Luis Alejandro) Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved in the chemiosmotic process of energy transduction and lower capacity to maintain fatty acid-driven respiration are identified as intrinsic factors of mitochondrial decay in the aged myocardium. Age-associated destabilization of electron transport supercomplexes as a potential factor of mitochondrial decay in the rat heart. Defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Nevertheless, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not fully known. As recent biophysical evidence shows that the ETC may form large macromolecular assemblies (i.e. supercomplexes) that disintegrate in certain pathologies (e.g. heart failure or Barth syndrome) reminiscent of aging, we investigated the hypothesis that alterations in supercomplexes are partly responsible for the age-related loss of cardiac ETC function. In this dissertation, age-associated changes in supercomplex organization and stability were investigated in subsarcolemmal (SSM) and interfibrillary (IFM) mitochondria isolated from cardiac tissue from young (3-5 months) and old (24-28 months) male Fischer 344 rats. Blue native-PAGE (BN-PAGE) analysis of digitonin-solubilized mitochondrial membranes coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate supercomplex organization. Results show that both SSM and IFM display supercomplexes comprised of various stoichiometries of complexes I, III and IV (never complex II), which typically organize as high mass (1500-2300 kDa) assemblies containing up to four copies of complex IV (i.e. I₁III₂IV[subscript N]-type supercomplexes). Interestingly, analysis of IFM proteins showed that, in general, supercomplex levels declined by up to 15 % (p < 0.05) with age; however, different degrees of supercomplex deterioration were observed, depending on the particular supercomplex investigated. Supercomplexes of the highest molecular weights (i.e. 1900-2300 kDa), which were also composed of the most complex stoichiometries (i.e. I1III2IVN, N ≥ 2), were primarily lost with age. In particular, I₁III₂IV₂, I₁III₂IV₃ and I₁III₂IV₄ supercomplexes were found to decline by 13% (p < 0.05), 30% (p < 0.05) and 45% (p < 0.05), respectively, on an age basis. Therefore, the age-associated loss of supercomplexes in IFM stems from destabilization of the assemblies that comprise several copies of complex IV, which could partially limit proper electron transfer to O₂ for its reduction, affecting mitochondrial respiratory capacity. In contrast to IFM, the aging defects of SSM supercomplexes appeared to be confined to the assembly comprised of only one copy of complex IV (I₁III₂IV₁, 1700 kDa) (37% loss; p = 0.06), while the higher molecular weight supercomplex sub-types that were most affected in IFM (i.e. I₁III₂IV[subscript N], N ≥ 2) were not significantly altered with age. Thus, the results from this dissertation indicate that mitochondria from different subcellular locations in the myocyte show different degrees of supercomplex destabilization in the aging rat heart. The more robust supercomplex deficits noted for IFM fit well with previous observations that electron transport characteristics of this subpopulation are more adversely affected with age than SSM. Although the underlying factor(s) of supercomplex deterioration are not fully known, the hypothesis that age-related alterations of certain constituents of the IMM (e.g. cardiolipin) may be important factors of supercomplex destabilization in cardiac mitochondria was investigated in this dissertation. To this end, LC-MS/MS characterization of supercomplex proteins and HPLC analysis of cardiolipin were used as approaches to elucidate potential factor(s) of supercomplex destabilization in the aging rat heart. Age-related alterations of cardiolipin levels and its acyl-chain content showed a strong parallel to the age-associated destabilization of supercomplexes. Specifically, cardiolipin levels declined by 10% (p < 0.05) in IFM, the mitochondrial subpopulation displaying the highest degree of supercomplex deterioration. In addition, the content of (18:2)₄-cardiolipin, the predominant species in the heart, was found to decline by 50% (p < 0.05) on average in both populations of cardiac mitochondria. Therefore, the data presented in this dissertation indicate that changes in cardiolipin may be at least one of the factors involved in supercomplex destabilization in the aging heart. Age-related decline in carnitine palmitoyltransferase I (CPT1) activity as a mitochondrial lesion that limits fatty acid catabolism in the rat heart. Loss of fatty acid utilization, another intrinsic factor of mitochondrial decay in the aged myocardium, has been associated with age-related alterations in the activity of carnitine palmitoyltransferase 1 (CPT1), the rate-controlling enzyme for overall fatty acid β-oxidation. Nevertheless, the exact molecular mechanism involved in the age-related loss of fatty acid-driven bioenergetics is not fully understood. In this dissertation, it was also investigated whether the aging lesion for fatty oxidation lies in a particular mitochondrial subpopulation or more generally results from cardiac decrements in L-carnitine levels. In order to clarify the role of each one of these factors, the effect of long-term dietary supplementation with the L-carnitine analogue, acetyl-L-carnitine (ALCAR), was also investigated. Results show that aging selectively decreases CPT1 activity in IFM by reducing enzyme catalytic efficiency for palmitoyl-CoA. IFM displayed a 28% (p < 0.05) loss of CPT1 activity, which correlated with a decline (41%, p < 0.05) in palmitoyl-CoA-driven state 3 respiration. Interestingly, SSM had preserved enzyme function and efficiently utilized palmitate. Analysis of IFM CPT1 kinetics showed both diminished V[subscript max] and K[subscript m] (60% and 49% respectively, p < 0.05) when palmitoyl-CoA was the substrate. However, no age-related changes in enzyme kinetics were evident with respect to L-carnitine. ALCAR supplementation restored CPT1 activity in heart IFM, but not apparently through remediation of L-carnitine levels. Rather, ALCAR influenced enzyme activity over time, potentially by modulating conditions in the aging heart that ultimately affect palmitoyl-CoA binding and CPT1 kinetics. In conclusion, this dissertation presents a characterization of age-associated alterations in the macromolecular organization of the IMM components that could partly explain the loss of mitochondrial oxidative capacity that affects the aging heart. In addition, the characterization of an age-related lesion of the controlling enzyme for β-oxidation is presented as another important factor that limits mitochondrial function and energy metabolism in cardiac mitochondria. Close

• 1564. [Article] Brown Marmorated Stink Bug (BMSB), Halyomorpha halys Taint in Wine : Impact on Wine Sensory, Effect of Wine-processing and Management Techniques

  Brown marmorated stink bug (BMSB), (Halyomorpha halys, Hemiptera: Pentatomidae) is an invasive species that damages numerous agricultural crops including grapes. Related damage include lower ...

  Citation × Citation Citation Abstract Copy Title: Brown Marmorated Stink Bug (BMSB), Halyomorpha halys Taint in Wine : Impact on Wine Sensory, Effect of Wine-processing and Management Techniques Author: Mohekar, Pallavi, 1985- Brown marmorated stink bug (BMSB), (Halyomorpha halys, Hemiptera: Pentatomidae) is an invasive species that damages numerous agricultural crops including grapes. Related damage include lower berry weight with increased exposure and cracked berries as a result of BMSB feeding activity. The insect is currently detected in 43 US states including Oregon, Washington, California, and New York. The grape and wine industries in these areas hold significant economic value. BMSB damage has already resulted in major economic loss in the agricultural industry. Current data indicates that the infestation is spreading to new regions and its population density is increasing in regions where it has been detected. Surveys show BMSB in vineyards of Oregon, Virginia and New York where BMSB can damage grapes, lowering their yield and quality. When harvested with grape clusters, BMSB can introduce volatile compounds, trans-2-decenal and tridecane, into wine. Prior work has shown that the presence of these compounds alters wine sensory. In this thesis, the focus is on analyzing BMSB’s impact on wine quality and consumer preference. Additionally, this work determines the action threshold (AT) for BMSB in the vineyard, which is likely to prove important to the grape and wine industry in designing control limits and maintaining wine quality. The first contribution of this work establishes the sensory detection threshold (DT) and consumer rejection thresholds (CRT) for trans-2-decenal in red wine. Trans-2-decenal is one of the main aroma compounds in BMSB taint, having green, cilantro-like aroma characteristics that is undesirable in wine. Results conclusively show that trans-2-decenal in wine has a negative effect on its quality. In Pinot noir, consumers were able to perceive trans-2-decenal at 1.92 µg/L (DT). Consumer preference for Pinot noir and Merlot containing trans-2-decenal decreased significantly above the concentration of 4.8 µg/L (CRT). Pinot noir containing trans-2-decenal above CRT was described as green, herbal, musty and less fruity by wine professionals. Based on such findings, the use of CRT is recommended when establishing consumer tolerance levels of trans-2-decenal in wine. The second contribution relates BMSB presence in vineyard with sensory threshold of trans-2-decenal in the finished wine. Pinot noir, Merlot and Pinot gris were produced using different densities of BMSB in grape clusters. The results of this study indicate BMSB density of three per cluster can result in Pinot noir containing trans-2-decenal at its DT and below its CRT. This density can be used as AT for BMSB in the vineyard since wines made from grapes contaminated at or greater than 3 BMSB per cluster are likely to experience low preference by wine consumers. The same BMSB density can be used to devise control measures for Merlot since trans-2-decenal CRT was found to be similar for both Pinot noir and Merlot. Pinot gris was found to be free of trans-2-decenal even at BMSB density of 1 per cluster. Therefore, we believe that BMSB may not be a concern for white wines. The third contribution provides methods to reduce BMSB taint in finished wine. This can be done by modifying the winemaking process or by applying corrective measures in the wine. During winemaking, destemming and pressing were identified to be the steps responsible for increasing BMSB taint levels in wine whereas alcoholic fermentation decreases taint levels. Consequently, finished white wine was found to be free of trans-2-decenal since fermentation occurs after pressing. However, trans-2-decenal was present in finished red wine since pressing occurs after fermentation. Taint levels in finished red wine are also affected by different pressing variants (free run versus press fraction, bladder press versus basket press), with press fraction and bladder press introducing more BMSB taint compared to free run and basket press. This information will allow winemakers to adjust processing steps in order to minimize BMSB taint levels in red wines. Further reduction in taint levels was shown to be possible through the use of reverse osmosis filtration. Alternatively, oak addition can be used to mask the sensory attributes of trans-2-decenal at the risk of introducing spicy notes into wine. None of the other common fining agents tested were found to be effective against trans-2-decenal. Taken together, this thesis contributes a number of novel insights into the impact of BMSB on red and white wine. By relating consumer thresholds and descriptive analysis with chemical and wine processing, we are able to establish control densities for the pest in the vineyard, identify key processing steps and post-fermentation treatments to reduce BMSB taint. The information contained in this thesis is likely to prove valuable to the wine industry in its struggle against BMSB. Title: Brown Marmorated Stink Bug (BMSB), Halyomorpha halys Taint in Wine : Impact on Wine Sensory, Effect of Wine-processing and Management Techniques Author: Mohekar, Pallavi, 1985- Brown marmorated stink bug (BMSB), (Halyomorpha halys, Hemiptera: Pentatomidae) is an invasive species that damages numerous agricultural crops including grapes. Related damage include lower berry weight with increased exposure and cracked berries as a result of BMSB feeding activity. The insect is currently detected in 43 US states including Oregon, Washington, California, and New York. The grape and wine industries in these areas hold significant economic value. BMSB damage has already resulted in major economic loss in the agricultural industry. Current data indicates that the infestation is spreading to new regions and its population density is increasing in regions where it has been detected. Surveys show BMSB in vineyards of Oregon, Virginia and New York where BMSB can damage grapes, lowering their yield and quality. When harvested with grape clusters, BMSB can introduce volatile compounds, trans-2-decenal and tridecane, into wine. Prior work has shown that the presence of these compounds alters wine sensory. In this thesis, the focus is on analyzing BMSB’s impact on wine quality and consumer preference. Additionally, this work determines the action threshold (AT) for BMSB in the vineyard, which is likely to prove important to the grape and wine industry in designing control limits and maintaining wine quality. The first contribution of this work establishes the sensory detection threshold (DT) and consumer rejection thresholds (CRT) for trans-2-decenal in red wine. Trans-2-decenal is one of the main aroma compounds in BMSB taint, having green, cilantro-like aroma characteristics that is undesirable in wine. Results conclusively show that trans-2-decenal in wine has a negative effect on its quality. In Pinot noir, consumers were able to perceive trans-2-decenal at 1.92 µg/L (DT). Consumer preference for Pinot noir and Merlot containing trans-2-decenal decreased significantly above the concentration of 4.8 µg/L (CRT). Pinot noir containing trans-2-decenal above CRT was described as green, herbal, musty and less fruity by wine professionals. Based on such findings, the use of CRT is recommended when establishing consumer tolerance levels of trans-2-decenal in wine. The second contribution relates BMSB presence in vineyard with sensory threshold of trans-2-decenal in the finished wine. Pinot noir, Merlot and Pinot gris were produced using different densities of BMSB in grape clusters. The results of this study indicate BMSB density of three per cluster can result in Pinot noir containing trans-2-decenal at its DT and below its CRT. This density can be used as AT for BMSB in the vineyard since wines made from grapes contaminated at or greater than 3 BMSB per cluster are likely to experience low preference by wine consumers. The same BMSB density can be used to devise control measures for Merlot since trans-2-decenal CRT was found to be similar for both Pinot noir and Merlot. Pinot gris was found to be free of trans-2-decenal even at BMSB density of 1 per cluster. Therefore, we believe that BMSB may not be a concern for white wines. The third contribution provides methods to reduce BMSB taint in finished wine. This can be done by modifying the winemaking process or by applying corrective measures in the wine. During winemaking, destemming and pressing were identified to be the steps responsible for increasing BMSB taint levels in wine whereas alcoholic fermentation decreases taint levels. Consequently, finished white wine was found to be free of trans-2-decenal since fermentation occurs after pressing. However, trans-2-decenal was present in finished red wine since pressing occurs after fermentation. Taint levels in finished red wine are also affected by different pressing variants (free run versus press fraction, bladder press versus basket press), with press fraction and bladder press introducing more BMSB taint compared to free run and basket press. This information will allow winemakers to adjust processing steps in order to minimize BMSB taint levels in red wines. Further reduction in taint levels was shown to be possible through the use of reverse osmosis filtration. Alternatively, oak addition can be used to mask the sensory attributes of trans-2-decenal at the risk of introducing spicy notes into wine. None of the other common fining agents tested were found to be effective against trans-2-decenal. Taken together, this thesis contributes a number of novel insights into the impact of BMSB on red and white wine. By relating consumer thresholds and descriptive analysis with chemical and wine processing, we are able to establish control densities for the pest in the vineyard, identify key processing steps and post-fermentation treatments to reduce BMSB taint. The information contained in this thesis is likely to prove valuable to the wine industry in its struggle against BMSB. Close

• 1565. [Article] Conservation genetics of Kincaid's lupine : a threatened plant of western Oregon and southwest Washington grasslands

  Kincaid's lupine (Lupinus oreganus Heller) is a federally listed threatened species native to remnant grassland of western Oregon and southwestern Washington, and is the primary larval host plant of a ...

  Citation × Citation Citation Abstract Copy Title: Conservation genetics of Kincaid's lupine : a threatened plant of western Oregon and southwest Washington grasslands Author: Severns, Paul M. Kincaid's lupine (Lupinus oreganus Heller) is a federally listed threatened species native to remnant grassland of western Oregon and southwestern Washington, and is the primary larval host plant of a once thought extinct butterfly, Plebejus icarioides fenderi Macy. Past studies concerning Kincaid's lupine reproduction suggested that populations may suffer reductions in fitness and progeny vigor due to inbreeding depression, but no direct investigation into range-wide patterns of genetic variation has been undertaken. I used nuclear DNA and chloroplast DNA simple sequence repeat (SSR) markers to determine genet size and patterns of non-adventitious rhizomatous lupine spread, to estimate the number of genets within Kincaid's lupine populations, and to assess whether seed transfer for the purpose of genetic rescue is an appropriate genetics management strategy for Kincaid's lupine. Patterns of allelic diversity at nDNA SSR loci within study patches revealed that non-adventitious spread of rhizomes can extend to at least 27 m and may dominate a portion of a lupine patch or small population. However, genet spread and arrangement in study patches were sufficiently integrated such that interplantlet Bombus foraging flights exceeding 2 m had > 90% probability of occurring between different genets. Within-lupine patch genetic diversity was well-undersampled, refuting the supposition that Kincaid's lupine populations suffer from inbreeding depression due to small effective population sizes. Estimation of Kincaid's lupine abundance through leaf cover and inflorescence number was tightly correlated with plantlet number, a unit of vegetative and sexual growth, within lupine patches but the relationship was not consistent between patches within populations or between populations. We used genet to plantlet ratios (determined through genotyping) and plantlet density to estimate genet population size in Kincaid's lupine patches. Because of the strong correlation between cover and plantlet density, historically collected lupine abundance data could be used to estimate genet population size provided that plantlet density is calibrated to patch-specific cover measurements. Within patches and populations across the range of Kincaid's lupine there was little DNA evidence suggesting severe inbreeding. Only one of 24 populations and five study patches had strong statistical evidence of a recent genetic bottleneck despite the range-wide fragmentation of lupine populations and habitat. Mean population fixation index values for nearly half of the populations were near Hardy-Weinberg equilibrium expectations and only one small lupine population had a F-value > 0.20, suggestive of high inbreeding levels. Half of the populations actually had an excess of heterozygotes, suggesting that genetic diversity is not being lost. Chloroplast DNA coincides well with the observation that genetic diversity is not being lost through inbreeding or genetic bottlenecking in Kincaid's lupine. The mean number of cpDNA haplotypes per population was approximately 4 maternal lineages, which is very high for an animal pollinated plant with heavy seeds that have limited dispersal. Even relatively small populations of Kincaid's lupine had 2 or more cpDNA haplotypes, indicating that populations are not severely inbred. Both nuclear and chloroplast DNA SSR genetic marker diversity suggests that Kincaid's lupine does not require genetic rescue for effective conservation. Due to the longevity of Kincaid's lupine and the apparently large amount of within population genetic diversity, the encouragement of natural recruitment from vegetation management that improves habitat conditions is likely to maintain the relatively large amount of genetic diversity within Kincaid's lupine populations. Title: Conservation genetics of Kincaid's lupine : a threatened plant of western Oregon and southwest Washington grasslands Author: Severns, Paul M. Kincaid's lupine (Lupinus oreganus Heller) is a federally listed threatened species native to remnant grassland of western Oregon and southwestern Washington, and is the primary larval host plant of a once thought extinct butterfly, Plebejus icarioides fenderi Macy. Past studies concerning Kincaid's lupine reproduction suggested that populations may suffer reductions in fitness and progeny vigor due to inbreeding depression, but no direct investigation into range-wide patterns of genetic variation has been undertaken. I used nuclear DNA and chloroplast DNA simple sequence repeat (SSR) markers to determine genet size and patterns of non-adventitious rhizomatous lupine spread, to estimate the number of genets within Kincaid's lupine populations, and to assess whether seed transfer for the purpose of genetic rescue is an appropriate genetics management strategy for Kincaid's lupine. Patterns of allelic diversity at nDNA SSR loci within study patches revealed that non-adventitious spread of rhizomes can extend to at least 27 m and may dominate a portion of a lupine patch or small population. However, genet spread and arrangement in study patches were sufficiently integrated such that interplantlet Bombus foraging flights exceeding 2 m had > 90% probability of occurring between different genets. Within-lupine patch genetic diversity was well-undersampled, refuting the supposition that Kincaid's lupine populations suffer from inbreeding depression due to small effective population sizes. Estimation of Kincaid's lupine abundance through leaf cover and inflorescence number was tightly correlated with plantlet number, a unit of vegetative and sexual growth, within lupine patches but the relationship was not consistent between patches within populations or between populations. We used genet to plantlet ratios (determined through genotyping) and plantlet density to estimate genet population size in Kincaid's lupine patches. Because of the strong correlation between cover and plantlet density, historically collected lupine abundance data could be used to estimate genet population size provided that plantlet density is calibrated to patch-specific cover measurements. Within patches and populations across the range of Kincaid's lupine there was little DNA evidence suggesting severe inbreeding. Only one of 24 populations and five study patches had strong statistical evidence of a recent genetic bottleneck despite the range-wide fragmentation of lupine populations and habitat. Mean population fixation index values for nearly half of the populations were near Hardy-Weinberg equilibrium expectations and only one small lupine population had a F-value > 0.20, suggestive of high inbreeding levels. Half of the populations actually had an excess of heterozygotes, suggesting that genetic diversity is not being lost. Chloroplast DNA coincides well with the observation that genetic diversity is not being lost through inbreeding or genetic bottlenecking in Kincaid's lupine. The mean number of cpDNA haplotypes per population was approximately 4 maternal lineages, which is very high for an animal pollinated plant with heavy seeds that have limited dispersal. Even relatively small populations of Kincaid's lupine had 2 or more cpDNA haplotypes, indicating that populations are not severely inbred. Both nuclear and chloroplast DNA SSR genetic marker diversity suggests that Kincaid's lupine does not require genetic rescue for effective conservation. Due to the longevity of Kincaid's lupine and the apparently large amount of within population genetic diversity, the encouragement of natural recruitment from vegetation management that improves habitat conditions is likely to maintain the relatively large amount of genetic diversity within Kincaid's lupine populations. Close

• 1566. [Article] The determination of 4-Pyridoxic acid in urine ; an investigation into the recovery of 4-pyridoxic acid and the lactone of 4-pyridoxic acid from two Dowex ion exchange resins

  4-Pyridoxic acid is the major metabolite of all forms of vitamin B₆ in mammalian species. The determination of 4-pyridoxic acid, therefore, is important in the investigation of vitamin B₆ metabolism and ...

  Citation × Citation Citation Abstract Copy Title: The determination of 4-Pyridoxic acid in urine ; an investigation into the recovery of 4-pyridoxic acid and the lactone of 4-pyridoxic acid from two Dowex ion exchange resins Author: Rohrer, Martha Jane 4-Pyridoxic acid is the major metabolite of all forms of vitamin B₆ in mammalian species. The determination of 4-pyridoxic acid, therefore, is important in the investigation of vitamin B₆ metabolism and for the establishment of human requirements for the vitamin. Although 4-pyridoxic acid is itself fluorescent, Huff and Perlzweig (1944) developed a classic method for its determination based on the conversion of the metabolite to the even more highly fluorescent lactone. Sarett in 1951 recognized that there are many substances in urine other than 4-pyridoxic acid which are fluorescent and which may interfere with its determination. His attempts to remove the extraneous fluorescent compounds with Decalso or with charcoal were not entirely satisfactory. He proposed, therefore, the administration of a test dose of 4-pyridoxic acid to subjects whose urine was to be analyzed for the metabolite of vitamin B₆, thus rendering the foreign fluorescence less significant by dilution. In 1955, Fujita and associates employed ion exchange chromatography as a means of separating 4-pyridoxic acid from other fluorescent compounds in urine and in 1958 Reddy, Reynolds and Price developed a chromatographic method using Dowex 1 (Cl⁻), a strongly basic anion exchange resin and Dowex 5OW (H⁺), a strongly acidic cation exchange resin for the separation of 4-pyridoxic acid from extraneous fluorescent materials in urine. Eluates from the latter of the two columns, used in sequence, were subjected to a chemical procedure to oxidize the 4-pyridoxic acid to the more highly fluorescent lactone form. The Reddy, Reynolds and Price procedure has provided the means for achieving more valid results than had been possible previously. Because of the importance of this procedure, the dynamics of the ion exchange method of Reddy and associates was investigated. Standards of 4-pyridoxic acid, or 4-pyridoxic acid lactone, and of urine were subjected to ion exchange chromatography using their procedure. Conversion of the 4-pyridoxic acid to the lactone was accomplished by means of the microprocedure of Woodring, Fisher and Storvick (1964). Effluent, wash, and eluate fractions were collected to determine the pattern of elution of the 4-pyridoxic acid, as well as to determine the effect of interfering fluorescence from the resins and from reagents. Eluates of urine from the second in the series of two Dowex ion exchange resins were subjected to paper and thin layer chromatography along with standards of 4-pyridoxic acid and the lactone of 4-pyridoxic acid. Eluates from influents of hydrolyzed urine produced highly fluorescent zones which did not correspond to those of standards of 4-pyridoxic acid or its lactone. A tryptophan load test is often used to diagnose vitamin B₆ deficiency but some of the metabolites of tryptophan are highly fluorescent and may interfere with the fluorometric measurement of 4-pyridoxic acid even after its conversion to the more fluorescent lactone. To study the effect of the presence of tryptophan metabolites on the determination of 4-pyridoxic acid in urine, a test dose of L-tryptophan was administered to one subject and the subsequent 24-hour urine collection was treated according to the chromatographic procedure of Reddy et al. The final eluate was analyzed for 4- pyridoxic acid using the raicroprocedure of Woodring and associates. Readings of fluorescence indicated that much foreign fluorescence remained even after the lactonization procedure. Recovery of 4- pyridoxic acid was 65.5 percent. Therefore, for the determination of 4-pyridoxic acid following a test dose of L-tryptophan, a recovery curve would have to be used for calculation of 4-pyridoxic acid excretion. It was made emphatically clear that there are highly fluorescent compounds present in the resin itself, even after extensive treatment, which cannot be removed. The results of this study emphasize the great importance of maintaining a constant rate of flow, and thus a constant rate of leaching, during chromatography to minimize the effect of the interfering fluorescence. Dowex 5OW (H⁺) will not retain 4-pyridoxic acid well unless the resin has been rendered strongly acidic. It was ascertained that this procedure should be accomplished just before use. 4-Pyridoxic acid and the lactone of 4-pyridoxic acid are eluted almost at once from the Dowex 1 resin but are released in the middle fractions during elution from freshly activated Dowex 50W, with 50 ml. of elutriant. It was determined that chromatography could be undertaken at a relatively rapid flow rate, thus allowing the entire chromatogtaphic procedure and analysis of eluates to be completed in one day. Title: The determination of 4-Pyridoxic acid in urine ; an investigation into the recovery of 4-pyridoxic acid and the lactone of 4-pyridoxic acid from two Dowex ion exchange resins Author: Rohrer, Martha Jane 4-Pyridoxic acid is the major metabolite of all forms of vitamin B₆ in mammalian species. The determination of 4-pyridoxic acid, therefore, is important in the investigation of vitamin B₆ metabolism and for the establishment of human requirements for the vitamin. Although 4-pyridoxic acid is itself fluorescent, Huff and Perlzweig (1944) developed a classic method for its determination based on the conversion of the metabolite to the even more highly fluorescent lactone. Sarett in 1951 recognized that there are many substances in urine other than 4-pyridoxic acid which are fluorescent and which may interfere with its determination. His attempts to remove the extraneous fluorescent compounds with Decalso or with charcoal were not entirely satisfactory. He proposed, therefore, the administration of a test dose of 4-pyridoxic acid to subjects whose urine was to be analyzed for the metabolite of vitamin B₆, thus rendering the foreign fluorescence less significant by dilution. In 1955, Fujita and associates employed ion exchange chromatography as a means of separating 4-pyridoxic acid from other fluorescent compounds in urine and in 1958 Reddy, Reynolds and Price developed a chromatographic method using Dowex 1 (Cl⁻), a strongly basic anion exchange resin and Dowex 5OW (H⁺), a strongly acidic cation exchange resin for the separation of 4-pyridoxic acid from extraneous fluorescent materials in urine. Eluates from the latter of the two columns, used in sequence, were subjected to a chemical procedure to oxidize the 4-pyridoxic acid to the more highly fluorescent lactone form. The Reddy, Reynolds and Price procedure has provided the means for achieving more valid results than had been possible previously. Because of the importance of this procedure, the dynamics of the ion exchange method of Reddy and associates was investigated. Standards of 4-pyridoxic acid, or 4-pyridoxic acid lactone, and of urine were subjected to ion exchange chromatography using their procedure. Conversion of the 4-pyridoxic acid to the lactone was accomplished by means of the microprocedure of Woodring, Fisher and Storvick (1964). Effluent, wash, and eluate fractions were collected to determine the pattern of elution of the 4-pyridoxic acid, as well as to determine the effect of interfering fluorescence from the resins and from reagents. Eluates of urine from the second in the series of two Dowex ion exchange resins were subjected to paper and thin layer chromatography along with standards of 4-pyridoxic acid and the lactone of 4-pyridoxic acid. Eluates from influents of hydrolyzed urine produced highly fluorescent zones which did not correspond to those of standards of 4-pyridoxic acid or its lactone. A tryptophan load test is often used to diagnose vitamin B₆ deficiency but some of the metabolites of tryptophan are highly fluorescent and may interfere with the fluorometric measurement of 4-pyridoxic acid even after its conversion to the more fluorescent lactone. To study the effect of the presence of tryptophan metabolites on the determination of 4-pyridoxic acid in urine, a test dose of L-tryptophan was administered to one subject and the subsequent 24-hour urine collection was treated according to the chromatographic procedure of Reddy et al. The final eluate was analyzed for 4- pyridoxic acid using the raicroprocedure of Woodring and associates. Readings of fluorescence indicated that much foreign fluorescence remained even after the lactonization procedure. Recovery of 4- pyridoxic acid was 65.5 percent. Therefore, for the determination of 4-pyridoxic acid following a test dose of L-tryptophan, a recovery curve would have to be used for calculation of 4-pyridoxic acid excretion. It was made emphatically clear that there are highly fluorescent compounds present in the resin itself, even after extensive treatment, which cannot be removed. The results of this study emphasize the great importance of maintaining a constant rate of flow, and thus a constant rate of leaching, during chromatography to minimize the effect of the interfering fluorescence. Dowex 5OW (H⁺) will not retain 4-pyridoxic acid well unless the resin has been rendered strongly acidic. It was ascertained that this procedure should be accomplished just before use. 4-Pyridoxic acid and the lactone of 4-pyridoxic acid are eluted almost at once from the Dowex 1 resin but are released in the middle fractions during elution from freshly activated Dowex 50W, with 50 ml. of elutriant. It was determined that chromatography could be undertaken at a relatively rapid flow rate, thus allowing the entire chromatogtaphic procedure and analysis of eluates to be completed in one day. Close

• 1567. [Article] Epichloë typhina (fungus) - Botanophila lobata (fly) interaction : an invasive "pollinator" system in its introduced range in western Oregon

  Epichloë typhina (Ascomycetes: Clavicipitaceae) is an endophytic fungus that infects perennial Pooid grasses and is the causal agent of choke disease. It is endemic to Europe and was inadvertently introduced ...

  Citation × Citation Citation Abstract Copy Title: Epichloë typhina (fungus) - Botanophila lobata (fly) interaction : an invasive "pollinator" system in its introduced range in western Oregon Author: Kaser, Joseph M. Epichloë typhina (Ascomycetes: Clavicipitaceae) is an endophytic fungus that infects perennial Pooid grasses and is the causal agent of choke disease. It is endemic to Europe and was inadvertently introduced into orchardgrass seed production fields in western Oregon. Choke disease, which was first recorded in Oregon in 1996, currently infects ~90% of cultivated orchardgrass seed fields in the region, resulting in yield losses >65%. Infective propagules (i.e. ascospores) are produced sexually by the bipolar heterothallic fungus and gamete outcross has been shown to be facilitated in the wild by Botanophila spp. (Diptera: Anthomyiidae), including Botanophila lobata. The fly - fungus interaction is generally considered to be one of obligatory mutualism based on studies conducted in areas endemism. However, recent evidence suggests that the fungus is able to sexually outcross in cultivated Oregon orchardgrass fields without the aid of fly "pollinators." Additionally, ascosporic fertilization has recently been implicated as an alternative mechanism for gamete transfer and might have important impacts on fungal reproduction in Oregon. The objectives of this study were to: 1) explore how two tightly linked species, which appear to have an obligate mutualistic relationship in areas of endemism, interact in a non-native context; 2) quantitatively examine the seasonal and diurnal presence of E. typhina ascospores in a cultivated Oregon orchardgrass field; and 3) test alternative transfer mechanisms of fungal spermatia for E. typhina. To address objective 1), the spatial variability and reproductive success of E. typhina and B. lobata were estimated during surveys of ten cultivated orchardgrass fields in 2008 and four fields in 2009. Fungal distributions were spatially aggregated at five of the study sites in 2008 and three in 2009. Fly distributions were spatially aggregated at three sites in 2008 and one in 2009. Botanophila lobata density exhibited a positive linear relationship with E. typhina density, suggestive of positive density dependence of fly oviposition with fungal density. However, fungal reproductive success was not affected by fly density or fungal density within the range of distributions observed in this study. To address objective 2), airborne ascospores were monitored in a single cultivated orchardgrass field during 2008 and 2009 using a Burkard volumetric spore trap. Ascospore production began in early to middle May and continued into late July during both years of the study. Daily ascospore production exhibited a circadian rhythm, with production peaking on average at 1:08 am and 12:36 am, in 2008 and 2009, respectively. The prolonged duration and high intensity of ascospore production during the growing season suggest a large window within which new plants are at risk to infection, and within which preventative management strategies must be adopted. To address objective 3), splash fertilization, contact fertilization and B. lobata fertilization were tested in the greenhouse. Although only two replicates were completed, both contact and splash fertilization appeared to be viable mechanisms of sexual outcross for E. typhina. The results of this study strongly indicate that E. typhina can successfully reproduce without the presence of B. lobata. Splash and contact fertilization, as well as ascosporic fertilization, provide opportunities for reproduction of the fungus in absence of fly pollinators. It appears that the fly - fungus interaction has shifted from an obligatory mutualism to facultative mutualism or simple fungivory within the introduced range in western Oregon. Title: Epichloë typhina (fungus) - Botanophila lobata (fly) interaction : an invasive "pollinator" system in its introduced range in western Oregon Author: Kaser, Joseph M. Epichloë typhina (Ascomycetes: Clavicipitaceae) is an endophytic fungus that infects perennial Pooid grasses and is the causal agent of choke disease. It is endemic to Europe and was inadvertently introduced into orchardgrass seed production fields in western Oregon. Choke disease, which was first recorded in Oregon in 1996, currently infects ~90% of cultivated orchardgrass seed fields in the region, resulting in yield losses >65%. Infective propagules (i.e. ascospores) are produced sexually by the bipolar heterothallic fungus and gamete outcross has been shown to be facilitated in the wild by Botanophila spp. (Diptera: Anthomyiidae), including Botanophila lobata. The fly - fungus interaction is generally considered to be one of obligatory mutualism based on studies conducted in areas endemism. However, recent evidence suggests that the fungus is able to sexually outcross in cultivated Oregon orchardgrass fields without the aid of fly "pollinators." Additionally, ascosporic fertilization has recently been implicated as an alternative mechanism for gamete transfer and might have important impacts on fungal reproduction in Oregon. The objectives of this study were to: 1) explore how two tightly linked species, which appear to have an obligate mutualistic relationship in areas of endemism, interact in a non-native context; 2) quantitatively examine the seasonal and diurnal presence of E. typhina ascospores in a cultivated Oregon orchardgrass field; and 3) test alternative transfer mechanisms of fungal spermatia for E. typhina. To address objective 1), the spatial variability and reproductive success of E. typhina and B. lobata were estimated during surveys of ten cultivated orchardgrass fields in 2008 and four fields in 2009. Fungal distributions were spatially aggregated at five of the study sites in 2008 and three in 2009. Fly distributions were spatially aggregated at three sites in 2008 and one in 2009. Botanophila lobata density exhibited a positive linear relationship with E. typhina density, suggestive of positive density dependence of fly oviposition with fungal density. However, fungal reproductive success was not affected by fly density or fungal density within the range of distributions observed in this study. To address objective 2), airborne ascospores were monitored in a single cultivated orchardgrass field during 2008 and 2009 using a Burkard volumetric spore trap. Ascospore production began in early to middle May and continued into late July during both years of the study. Daily ascospore production exhibited a circadian rhythm, with production peaking on average at 1:08 am and 12:36 am, in 2008 and 2009, respectively. The prolonged duration and high intensity of ascospore production during the growing season suggest a large window within which new plants are at risk to infection, and within which preventative management strategies must be adopted. To address objective 3), splash fertilization, contact fertilization and B. lobata fertilization were tested in the greenhouse. Although only two replicates were completed, both contact and splash fertilization appeared to be viable mechanisms of sexual outcross for E. typhina. The results of this study strongly indicate that E. typhina can successfully reproduce without the presence of B. lobata. Splash and contact fertilization, as well as ascosporic fertilization, provide opportunities for reproduction of the fungus in absence of fly pollinators. It appears that the fly - fungus interaction has shifted from an obligatory mutualism to facultative mutualism or simple fungivory within the introduced range in western Oregon. Close

• 1568. [Article] Integrating traceability with onboard handling to enhance product quality and marketability of Eastern Pacific troll-caught albacore tuna (Thunnus alalunga)

  The fishing industry in the Pacific Northwest is a dynamic and highly competitive industry that must constantly respond to rapidly changing resource, regulatory and market forces. These forces have already ...

  Citation × Citation Citation Abstract Copy Title: Integrating traceability with onboard handling to enhance product quality and marketability of Eastern Pacific troll-caught albacore tuna (Thunnus alalunga) Author: Thompson, Michael J. The fishing industry in the Pacific Northwest is a dynamic and highly competitive industry that must constantly respond to rapidly changing resource, regulatory and market forces. These forces have already had a significant impact on our fisheries and will continue to do so as we learn more about the biology and ecology of harvested species, and how this information can be used to effectively manage our fisheries. In addition, market forces, both here and abroad, including the push by some governments to improve food safety and consumer confidence, are also having an impact on the U.S. seafood industry. In an attempt to address these issues, the European Union (E.U.) recently enacted traceability legislation, now mandatory for seafood, which will require all U.S. businesses exporting to the E.U. to provide adequate traceability documentation on their products. This legislation will have an impact on many U.S. fisheries, including the troll-caught albacore fishery in the Eastern Pacific Ocean, which, after losing its traditional market, has recently relied heavily on exporting. Although the term traceability has been around for some time it is relatively new to the U.S. food industry and many in the seafood sector, who now face the possibility of mandatory traceability, have not yet considered all its implications. Another issue facing the Eastern Pacific albacore fishery is that of product quality. Traditionally, this fishery has concentrated on producing albacore for the high-volume cannery market, which does not require the same stringent quality control standards as other market sectors. Albacore tuna (Thunnus alalunga), members of the Scrombroid family, are capable of thermoregulation and can exhibit internal temperatures of more than 15º C above ambient seawater. High internal temperatures, if sustained, and residual blood content can stimulate bacterial growth, which can eventually lead to scrombroid poisoning in humans. This makes the time immediately after landing critical in attaining and preserving important quality traits. In order to develop new markets for Eastern Pacific troll-caught albacore, quality needs to be improved and/or maintained within the industry at a sufficient level to supply market demands. This would require at least a portion of the industry to implement a set of onboard handling procedures designed to preserve and maintain quality. Although guidelines for handling albacore are available there is no industry “standard” for producing high quality products and only limited research presently exists into preserving quality traits of albacore onboard a vessel. The first paper in this thesis explores the concepts of traceability, current traceability legislation, and the potential applications of traceability systems to improve quality and marketing of U.S. seafood products. The second paper evaluates different onboard bleeding and handling techniques, which can affect product quality, currently used in the albacore industry and their ability to remove blood from the muscle tissues. Thirty-two different combinations of handling techniques and their affect on residual blood content were investigated by examining blood concentrations within the muscle tissue, using spectrophotometry methods, and cutaneous blood vessel coverage, by computer-aided digital color analysis. The results of the statistical analysis indicate that three of the five factors investigated had some influence on residual blood content. The third paper in this thesis evaluates the design and development of a computer-based onboard traceability system with integrated barcode technology, capable of efficiently recording capture and handling data. Two system trials were conducted, during normal fishing operations, where a total of 450 albacore tuna were successfully entered into the traceability database through the use of the Fishery Data Interface application. Documentation supplied by this system, which includes both spatial and temporal data, as well as information on product activities, will enable businesses to meet existing traceability requirements while also providing a new tool for the marketing of seafood products produced in the United States. Title: Integrating traceability with onboard handling to enhance product quality and marketability of Eastern Pacific troll-caught albacore tuna (Thunnus alalunga) Author: Thompson, Michael J. The fishing industry in the Pacific Northwest is a dynamic and highly competitive industry that must constantly respond to rapidly changing resource, regulatory and market forces. These forces have already had a significant impact on our fisheries and will continue to do so as we learn more about the biology and ecology of harvested species, and how this information can be used to effectively manage our fisheries. In addition, market forces, both here and abroad, including the push by some governments to improve food safety and consumer confidence, are also having an impact on the U.S. seafood industry. In an attempt to address these issues, the European Union (E.U.) recently enacted traceability legislation, now mandatory for seafood, which will require all U.S. businesses exporting to the E.U. to provide adequate traceability documentation on their products. This legislation will have an impact on many U.S. fisheries, including the troll-caught albacore fishery in the Eastern Pacific Ocean, which, after losing its traditional market, has recently relied heavily on exporting. Although the term traceability has been around for some time it is relatively new to the U.S. food industry and many in the seafood sector, who now face the possibility of mandatory traceability, have not yet considered all its implications. Another issue facing the Eastern Pacific albacore fishery is that of product quality. Traditionally, this fishery has concentrated on producing albacore for the high-volume cannery market, which does not require the same stringent quality control standards as other market sectors. Albacore tuna (Thunnus alalunga), members of the Scrombroid family, are capable of thermoregulation and can exhibit internal temperatures of more than 15º C above ambient seawater. High internal temperatures, if sustained, and residual blood content can stimulate bacterial growth, which can eventually lead to scrombroid poisoning in humans. This makes the time immediately after landing critical in attaining and preserving important quality traits. In order to develop new markets for Eastern Pacific troll-caught albacore, quality needs to be improved and/or maintained within the industry at a sufficient level to supply market demands. This would require at least a portion of the industry to implement a set of onboard handling procedures designed to preserve and maintain quality. Although guidelines for handling albacore are available there is no industry “standard” for producing high quality products and only limited research presently exists into preserving quality traits of albacore onboard a vessel. The first paper in this thesis explores the concepts of traceability, current traceability legislation, and the potential applications of traceability systems to improve quality and marketing of U.S. seafood products. The second paper evaluates different onboard bleeding and handling techniques, which can affect product quality, currently used in the albacore industry and their ability to remove blood from the muscle tissues. Thirty-two different combinations of handling techniques and their affect on residual blood content were investigated by examining blood concentrations within the muscle tissue, using spectrophotometry methods, and cutaneous blood vessel coverage, by computer-aided digital color analysis. The results of the statistical analysis indicate that three of the five factors investigated had some influence on residual blood content. The third paper in this thesis evaluates the design and development of a computer-based onboard traceability system with integrated barcode technology, capable of efficiently recording capture and handling data. Two system trials were conducted, during normal fishing operations, where a total of 450 albacore tuna were successfully entered into the traceability database through the use of the Fishery Data Interface application. Documentation supplied by this system, which includes both spatial and temporal data, as well as information on product activities, will enable businesses to meet existing traceability requirements while also providing a new tool for the marketing of seafood products produced in the United States. Close

• 1569. [Article] Caspian tern (Hydroprogne caspia) foraging ecology and predation on juvenile salmonids in San Francisco Bay, California

  Brooks Island, located in central San Francisco Bay, California, currently supports the largest breeding colony of Caspian terns (Hydroprogne caspia) in the Bay Area, and is one of several proposed relocation ...

  Citation × Citation Citation Abstract Copy Title: Caspian tern (Hydroprogne caspia) foraging ecology and predation on juvenile salmonids in San Francisco Bay, California Author: Adrean, Lindsay J. Brooks Island, located in central San Francisco Bay, California, currently supports the largest breeding colony of Caspian terns (Hydroprogne caspia) in the Bay Area, and is one of several proposed relocation sites for some Caspian terns from the world's largest colony in the Columbia River estuary of Oregon. Juvenile salmonids have been identified in the diet of Caspian terns nesting at Brooks Island, so I investigated whether the colony, at its current or an enhanced size, poses a threat to the recovery of several runs of salmonids (Oncorhynchus spp.) in San Francisco Bay that are listed under the U.S. Endangered Species Act (ESA). I also examined the foraging ecology of Brooks Island Caspian terns to 1) determine whether the colony is suitable for expansion based on availability of forage fish resources, a factor potentially limiting the size and productivity of the colony, and 2) investigate how Caspian terns nesting at this colony exploit forage fish resources. I used a bioenergetics modeling approach, employing estimates of tern energy requirements and proportions of energy supplied by various prey types, to estimate consumption of juvenile salmonids by Caspian terns nesting on Brooks Island during 2008 and 2009. Estimated salmonid consumption was 205,000 smolts (95% CI: 175,000 – 245,000 smolts) in 2008 and 167,000 smolts (95% CI: 144,000 – 191,000 smolts) in 2009. Predation rates on ESA-listed Central Valley spring-run Chinook salmon (O. tshawytscha; 0.08%) were lower than those on unlisted fall-run Chinook salmon (1.0%). Average per capita predation rates on juvenile salmonids by Brooks Island Caspian terns (2008: 126 fish; 2009: 123 fish) were less than half those of Caspian terns nesting in the Columbia River estuary. If the current downward trend in the number of Caspian terns nesting on Brooks Island continues until the colony is no longer extant, the resulting declines in predation on salmonids would lead to increases in annual population growth rates (Δλ) of salmonid runs of just small fractions of one percentage point. The proposed enhancement of the Brooks Island Caspian tern colony to 3,000 individuals would at most cause declines in annual population growth rates of 0.3% for fall-run Chinook salmon and 0.02% for threatened spring-run Chinook salmon, assuming that smolt mortality from tern predation is 100% additive. This level of impact to the ESA-listed spring-run Chinook salmon stock is less than the level considered acceptable by the National Marine Fisheries Service (Δλ = 0.05%). Radio-tracking of Caspian terns nesting on Brooks Island revealed that the maximum foraging distance from the colony was 80 km. The median foraging distance from the colony was greater in 2009 compared to 2008 (20.6 km vs. 14.0 km), average number of foraging trips per day was higher (4.4 vs. 3.4 foraging trips), and average adult colony attendance was lower (43% vs. 52% of daylight hours). These results indicate that the colony was more food-limited during the 2009 breeding season, concurrent with anomalous downwelling along the coast of northern California and reduced availability and size of marine forage fishes, such as herring, sardines, and anchovies. Caspian terns used a number of core foraging areas within 30 km of the Brooks Island colony, both inside and just outside the Bay. Two large core foraging areas were close to or overlapped with the release site for juvenile salmonids in eastern San Pablo Bay, where > 10 million hatchery-raised smolts were released from net pens during both the 2008 and 2009 tern nesting seasons. This finding supports the hypothesis that most juvenile salmonids consumed by terns nesting at the Brooks Island colony were captured at or near the release site. Individual Caspian terns displayed foraging site fidelity, suggesting that foraging at the release site for hatchery-raised salmonids was a learned behavior by some terns. The Brooks Island colony site is within foraging distance of adequate marine forage fish resources in most years, and Caspian terns nesting there are not dependent on juvenile salmonids as a food resource. Consumption of juvenile salmonids by Brooks Island terns would be largely curtailed by modification of hatchery release practices. Title: Caspian tern (Hydroprogne caspia) foraging ecology and predation on juvenile salmonids in San Francisco Bay, California Author: Adrean, Lindsay J. Brooks Island, located in central San Francisco Bay, California, currently supports the largest breeding colony of Caspian terns (Hydroprogne caspia) in the Bay Area, and is one of several proposed relocation sites for some Caspian terns from the world's largest colony in the Columbia River estuary of Oregon. Juvenile salmonids have been identified in the diet of Caspian terns nesting at Brooks Island, so I investigated whether the colony, at its current or an enhanced size, poses a threat to the recovery of several runs of salmonids (Oncorhynchus spp.) in San Francisco Bay that are listed under the U.S. Endangered Species Act (ESA). I also examined the foraging ecology of Brooks Island Caspian terns to 1) determine whether the colony is suitable for expansion based on availability of forage fish resources, a factor potentially limiting the size and productivity of the colony, and 2) investigate how Caspian terns nesting at this colony exploit forage fish resources. I used a bioenergetics modeling approach, employing estimates of tern energy requirements and proportions of energy supplied by various prey types, to estimate consumption of juvenile salmonids by Caspian terns nesting on Brooks Island during 2008 and 2009. Estimated salmonid consumption was 205,000 smolts (95% CI: 175,000 – 245,000 smolts) in 2008 and 167,000 smolts (95% CI: 144,000 – 191,000 smolts) in 2009. Predation rates on ESA-listed Central Valley spring-run Chinook salmon (O. tshawytscha; 0.08%) were lower than those on unlisted fall-run Chinook salmon (1.0%). Average per capita predation rates on juvenile salmonids by Brooks Island Caspian terns (2008: 126 fish; 2009: 123 fish) were less than half those of Caspian terns nesting in the Columbia River estuary. If the current downward trend in the number of Caspian terns nesting on Brooks Island continues until the colony is no longer extant, the resulting declines in predation on salmonids would lead to increases in annual population growth rates (Δλ) of salmonid runs of just small fractions of one percentage point. The proposed enhancement of the Brooks Island Caspian tern colony to 3,000 individuals would at most cause declines in annual population growth rates of 0.3% for fall-run Chinook salmon and 0.02% for threatened spring-run Chinook salmon, assuming that smolt mortality from tern predation is 100% additive. This level of impact to the ESA-listed spring-run Chinook salmon stock is less than the level considered acceptable by the National Marine Fisheries Service (Δλ = 0.05%). Radio-tracking of Caspian terns nesting on Brooks Island revealed that the maximum foraging distance from the colony was 80 km. The median foraging distance from the colony was greater in 2009 compared to 2008 (20.6 km vs. 14.0 km), average number of foraging trips per day was higher (4.4 vs. 3.4 foraging trips), and average adult colony attendance was lower (43% vs. 52% of daylight hours). These results indicate that the colony was more food-limited during the 2009 breeding season, concurrent with anomalous downwelling along the coast of northern California and reduced availability and size of marine forage fishes, such as herring, sardines, and anchovies. Caspian terns used a number of core foraging areas within 30 km of the Brooks Island colony, both inside and just outside the Bay. Two large core foraging areas were close to or overlapped with the release site for juvenile salmonids in eastern San Pablo Bay, where > 10 million hatchery-raised smolts were released from net pens during both the 2008 and 2009 tern nesting seasons. This finding supports the hypothesis that most juvenile salmonids consumed by terns nesting at the Brooks Island colony were captured at or near the release site. Individual Caspian terns displayed foraging site fidelity, suggesting that foraging at the release site for hatchery-raised salmonids was a learned behavior by some terns. The Brooks Island colony site is within foraging distance of adequate marine forage fish resources in most years, and Caspian terns nesting there are not dependent on juvenile salmonids as a food resource. Consumption of juvenile salmonids by Brooks Island terns would be largely curtailed by modification of hatchery release practices. Close

• 1570. [Article] Chemical labeling methods and mass spectrometry of mitochondrial thiol proteomes

  Dysfunction of mitochondria has been linked to aging and the pathogenesis of many degenerative diseases. Aside from their primary function in energy production, mitochondria are considered as a major source ...

  Citation × Citation Citation Abstract Copy Title: Chemical labeling methods and mass spectrometry of mitochondrial thiol proteomes Author: Wang, Jing Dysfunction of mitochondria has been linked to aging and the pathogenesis of many degenerative diseases. Aside from their primary function in energy production, mitochondria are considered as a major source and target of reactive oxygen and nitrogen species (ROS/RNS) in cells as well. The mitochondrial thiol proteome is a subset of proteins inside mitochondria particularly vulnerable to oxidative modifications often caused by ROS/RNS due to the high reactivity of thiol groups. In this dissertation, we used chemoselective labeling approaches to target mitochondrial thiol proteomes with two distinct purposes. In the first part of our study, a mitochondria-targeted cationic sulfhydryl reagent, (4-iodobutyl)triphenyl- phosphonium (IBTP), was used to label the protein thiols. This study was focused on the investigation of the tandem mass spectrometric behaviors of the thiol peptides modified by IBTP. In the latter part of the dissertation, we tested the hypothesis that the thiol proteomes of cardiac mitochondria undergo chemical changes as a consequence of aging. The thiol-reactive isotope-coded affinity tags (ICATs) were utilized to estimate the possible age-related impacts on the relative abundances of protein thiols in two mitochondrial subpopulations present in rat myocardium, respectively. Fixed charge chemical modifications on peptides and proteins can have significant effects on the ion fragmentation behaviors in tandem mass spectrometry (MS/MS). The MS/MS fragmentation behavior of butyltriphenylphosphonium (BTP)-modified peptides was evaluated via the comparison to their carbamidomethylated (CAM) analogues using a quadrupole ion trap mass spectrometer (LCQ) and/or a quadrupole time-of-flight (QTOF) instrument under conditions of low energy collision-induced dissociation (CID). Besides the expected higher charge states observed in peptides and fragment ions containing the BTP moiety, the charged BTP group also had a significant effect on the amide bond fragmentation products of modified cysteine-containing peptides. The presence of a phosphonium ion was speculated to reduce the tendency for the protonation of the proximal amide bonds in the peptide backbones, and consequently decrease the product ion abundances at the corresponding cleavage sites when compared to those from the CAM-modified derivatives. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. Calculations indicated that proton affinities were generally 20-50 kcal/mol lower for BTP-modified peptides in contrast to the respective unmodified or CAM-modified analogues. In the latter part of the dissertation, we focused on the evaluation of age-related effects on protein thiol abundances in cardiac subsarcolemmal and interfibrillar mitochondria (SSM/IFM). A total of 243 cysteine residues from 115 proteins in SSM and 149 cysteine sites from 65 proteins in IFM were identified from both young and old rats using ICAT labeling approach, respectively. Significant age-related differences (p-value < 0.05) in protein thiol abundances were only observed for five proteins from IFM. These proteins were associated with the functional pathways of the oxidative phosphorylation (NDUV1 and QCR1), the tricarboxylic acid cycle (MDHM and ODPB) and fatty acid beta-oxidation (ACSL1). Additionally, five other proteins in SSM and two proteins in IFM were detected with potentially significant age-related effects (0.05 < p-value < 0.1) with regard to their respective protein thiol abundances, however, no common protein was identified from both subpopulations. These results demonstrated the differences in age-related effects on protein thiol abundances between cardiac SSM and IFM. Further, the reactivities of cysteine residues, identified in the current ICAT reaction, were evaluated and sorted into high (78 in SSM vs. 47 in IFM), moderate (30 vs. 32) and low (135 vs. 70) labeling frequency groups. Thirty eight thiol groups were also identified from other cysteine-targeted studies conducted in our group, i.e., Michael addition by 2-alkenals and disulfide exchange via glutathionylation. The comparative results from these three studies revealed eight cysteine residues as the common modification sites targeted by three distinct reaction mechanisms. This resulted in the identification of candidates of vulnerable cysteine residues in the aging heart mitochondria. Title: Chemical labeling methods and mass spectrometry of mitochondrial thiol proteomes Author: Wang, Jing Dysfunction of mitochondria has been linked to aging and the pathogenesis of many degenerative diseases. Aside from their primary function in energy production, mitochondria are considered as a major source and target of reactive oxygen and nitrogen species (ROS/RNS) in cells as well. The mitochondrial thiol proteome is a subset of proteins inside mitochondria particularly vulnerable to oxidative modifications often caused by ROS/RNS due to the high reactivity of thiol groups. In this dissertation, we used chemoselective labeling approaches to target mitochondrial thiol proteomes with two distinct purposes. In the first part of our study, a mitochondria-targeted cationic sulfhydryl reagent, (4-iodobutyl)triphenyl- phosphonium (IBTP), was used to label the protein thiols. This study was focused on the investigation of the tandem mass spectrometric behaviors of the thiol peptides modified by IBTP. In the latter part of the dissertation, we tested the hypothesis that the thiol proteomes of cardiac mitochondria undergo chemical changes as a consequence of aging. The thiol-reactive isotope-coded affinity tags (ICATs) were utilized to estimate the possible age-related impacts on the relative abundances of protein thiols in two mitochondrial subpopulations present in rat myocardium, respectively. Fixed charge chemical modifications on peptides and proteins can have significant effects on the ion fragmentation behaviors in tandem mass spectrometry (MS/MS). The MS/MS fragmentation behavior of butyltriphenylphosphonium (BTP)-modified peptides was evaluated via the comparison to their carbamidomethylated (CAM) analogues using a quadrupole ion trap mass spectrometer (LCQ) and/or a quadrupole time-of-flight (QTOF) instrument under conditions of low energy collision-induced dissociation (CID). Besides the expected higher charge states observed in peptides and fragment ions containing the BTP moiety, the charged BTP group also had a significant effect on the amide bond fragmentation products of modified cysteine-containing peptides. The presence of a phosphonium ion was speculated to reduce the tendency for the protonation of the proximal amide bonds in the peptide backbones, and consequently decrease the product ion abundances at the corresponding cleavage sites when compared to those from the CAM-modified derivatives. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. Calculations indicated that proton affinities were generally 20-50 kcal/mol lower for BTP-modified peptides in contrast to the respective unmodified or CAM-modified analogues. In the latter part of the dissertation, we focused on the evaluation of age-related effects on protein thiol abundances in cardiac subsarcolemmal and interfibrillar mitochondria (SSM/IFM). A total of 243 cysteine residues from 115 proteins in SSM and 149 cysteine sites from 65 proteins in IFM were identified from both young and old rats using ICAT labeling approach, respectively. Significant age-related differences (p-value < 0.05) in protein thiol abundances were only observed for five proteins from IFM. These proteins were associated with the functional pathways of the oxidative phosphorylation (NDUV1 and QCR1), the tricarboxylic acid cycle (MDHM and ODPB) and fatty acid beta-oxidation (ACSL1). Additionally, five other proteins in SSM and two proteins in IFM were detected with potentially significant age-related effects (0.05 < p-value < 0.1) with regard to their respective protein thiol abundances, however, no common protein was identified from both subpopulations. These results demonstrated the differences in age-related effects on protein thiol abundances between cardiac SSM and IFM. Further, the reactivities of cysteine residues, identified in the current ICAT reaction, were evaluated and sorted into high (78 in SSM vs. 47 in IFM), moderate (30 vs. 32) and low (135 vs. 70) labeling frequency groups. Thirty eight thiol groups were also identified from other cysteine-targeted studies conducted in our group, i.e., Michael addition by 2-alkenals and disulfide exchange via glutathionylation. The comparative results from these three studies revealed eight cysteine residues as the common modification sites targeted by three distinct reaction mechanisms. This resulted in the identification of candidates of vulnerable cysteine residues in the aging heart mitochondria. Close