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• 1581. [Article] Host range and chemical composition of lactic streptococcal bacteriophages

  Comparisons were made of the abilities of Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetilactis bacteriophages to endure various laboratory isolation and storage treatments. Neutralization ...

  Citation × Citation Citation Abstract Copy Title: Host range and chemical composition of lactic streptococcal bacteriophages Author: Henning, David Ralph Comparisons were made of the abilities of Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetilactis bacteriophages to endure various laboratory isolation and storage treatments. Neutralization of Cottage cheese whey containing phages offered no survival advantage over unneutralized samples, refrigeration of neutral and acid whey samples increased the survival of only the S. diacetilactis phage. About 50 percent of the phages could be recovered from milk coagulated by addition of lactic acid to provide casein-free, clear, phage-containing whey. Sterilization of phage lysates by membrane filtration allowed collection of 92 to 99 percent of the phages; Seitz filtration allowed collection of a maximum of 24 percent. Whey samples containing phages could be concentrated by removing water using polyethylene glycol. Lyophilization decreased phage titers at least 50 percent. Cross reaction patterns of phages for strains of lactic streptococci revealed that similar strains were present in cultures supplied by several companies. For example, phages which were lytic for strains isolated from one brand of commercial mixed strain starter cultures would lyse strains isolated from as many as three different brands of starter cultures. The lytic patterns of 60 bacteriophage races toward 100 single lactic streptococcal strains allowed the establishment of eight phage groups, A through H. The phages within a particular group were generally species specific. However several exceptions were noted. Group A phages attacked primarily strains of S. diacetilactis. Groups B through H phages mainly attacked strains of S. lactis and S. cremoris. Groups G and H consisted of one phage that attacked only the homologous host. Lytic reactions determined by multiple printing of phages on lawns seeded with possible hosts were employed for selecting strains to be used in mixed-strain starter cultures. A computer was used in selecting strains for blending into mixed-strain cultures with resulting maximum protection against phage. The strains selected by the computer were arranged in a sequence or rotation system suitable for use in dairy plant fermentations. Protection from phage infection as determined by a resistance index remained high for six successive cultures, but the addition of the seventh culture to the rotation system increased the likelihood of phage attack significantly. Neutralization of representatives of each host range phage group by standardized rabbit antisera prepared against four different phages revealed similarity among several groups. However the neutralization of phages was not constant within each group. Electron photomicrographs of a phage for S. diacetilactis revealed a tadpole-shaped particle. The head width and length were each about 60 mμ , the tail width was about 10mμ and the tail length was about 170 mμ . There also were suggestions for a polyhedral head structure in enlarged (84,000X) pictures of the viruses. Chemical compositional studies of the phage studied by electron microscopy revealed that phosphorus represented 4.45 percent of the phage dry weight. Nitrogen was found to constitute 12.8 percent of the phage protein. A ratio of phage phosphorus to phage protein nitrogen of 2.7 was determined. Bacteriophages prepared in broth medium were recovered by differential centrifugation and purified in cesium chloride density gradients. An amino acid analysis revealed the following mole percentages for the amino acids quantitated: lysine, 8.1; histidine, 1.7; arginine, 3.8; aspartic acid and asparagine, 10.0; threonine, 7.5; serine, 6.8; glutamic acid and glutamine, 12.1; proline, 3.4; glycine, 9.0; alanine, 9.5; valine, 8.7; methionine, 1.1; isoleucine, 7.9; tyrosine, 2.2 and phenylalanine, 2.8. The nucleic acid of the phage was determined to be double-stranded DNA with a thermal melting point of 84.3°C, indicating an average base composition of 35.7 percent guanine plus cytosine. Calculated values for percentage DNA and protein composition of the phage were 44 and 56 percent respectively. The density of the phage was calculated to be 1.47g/cc. Title: Host range and chemical composition of lactic streptococcal bacteriophages Author: Henning, David Ralph Comparisons were made of the abilities of Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetilactis bacteriophages to endure various laboratory isolation and storage treatments. Neutralization of Cottage cheese whey containing phages offered no survival advantage over unneutralized samples, refrigeration of neutral and acid whey samples increased the survival of only the S. diacetilactis phage. About 50 percent of the phages could be recovered from milk coagulated by addition of lactic acid to provide casein-free, clear, phage-containing whey. Sterilization of phage lysates by membrane filtration allowed collection of 92 to 99 percent of the phages; Seitz filtration allowed collection of a maximum of 24 percent. Whey samples containing phages could be concentrated by removing water using polyethylene glycol. Lyophilization decreased phage titers at least 50 percent. Cross reaction patterns of phages for strains of lactic streptococci revealed that similar strains were present in cultures supplied by several companies. For example, phages which were lytic for strains isolated from one brand of commercial mixed strain starter cultures would lyse strains isolated from as many as three different brands of starter cultures. The lytic patterns of 60 bacteriophage races toward 100 single lactic streptococcal strains allowed the establishment of eight phage groups, A through H. The phages within a particular group were generally species specific. However several exceptions were noted. Group A phages attacked primarily strains of S. diacetilactis. Groups B through H phages mainly attacked strains of S. lactis and S. cremoris. Groups G and H consisted of one phage that attacked only the homologous host. Lytic reactions determined by multiple printing of phages on lawns seeded with possible hosts were employed for selecting strains to be used in mixed-strain starter cultures. A computer was used in selecting strains for blending into mixed-strain cultures with resulting maximum protection against phage. The strains selected by the computer were arranged in a sequence or rotation system suitable for use in dairy plant fermentations. Protection from phage infection as determined by a resistance index remained high for six successive cultures, but the addition of the seventh culture to the rotation system increased the likelihood of phage attack significantly. Neutralization of representatives of each host range phage group by standardized rabbit antisera prepared against four different phages revealed similarity among several groups. However the neutralization of phages was not constant within each group. Electron photomicrographs of a phage for S. diacetilactis revealed a tadpole-shaped particle. The head width and length were each about 60 mμ , the tail width was about 10mμ and the tail length was about 170 mμ . There also were suggestions for a polyhedral head structure in enlarged (84,000X) pictures of the viruses. Chemical compositional studies of the phage studied by electron microscopy revealed that phosphorus represented 4.45 percent of the phage dry weight. Nitrogen was found to constitute 12.8 percent of the phage protein. A ratio of phage phosphorus to phage protein nitrogen of 2.7 was determined. Bacteriophages prepared in broth medium were recovered by differential centrifugation and purified in cesium chloride density gradients. An amino acid analysis revealed the following mole percentages for the amino acids quantitated: lysine, 8.1; histidine, 1.7; arginine, 3.8; aspartic acid and asparagine, 10.0; threonine, 7.5; serine, 6.8; glutamic acid and glutamine, 12.1; proline, 3.4; glycine, 9.0; alanine, 9.5; valine, 8.7; methionine, 1.1; isoleucine, 7.9; tyrosine, 2.2 and phenylalanine, 2.8. The nucleic acid of the phage was determined to be double-stranded DNA with a thermal melting point of 84.3°C, indicating an average base composition of 35.7 percent guanine plus cytosine. Calculated values for percentage DNA and protein composition of the phage were 44 and 56 percent respectively. The density of the phage was calculated to be 1.47g/cc. Close

• 1582. [Article] Effects of pollen collected by honey bees from pollination dependent agricultural cropping systems on honey bee nutrition

  Managed honey bee (Apis mellifera L.) colonies are important pollinators of many cultivated crops. Honey bee colony declines averaging 30% annually in the United States for the past 7 years have caused ...

  Citation × Citation Citation Abstract Copy Title: Effects of pollen collected by honey bees from pollination dependent agricultural cropping systems on honey bee nutrition Author: Topitzhofer, Ellen Managed honey bee (Apis mellifera L.) colonies are important pollinators of many cultivated crops. Honey bee colony declines averaging 30% annually in the United States for the past 7 years have caused significant concern and hence have been a topic of intensive investigation. These declines are reportedly due to multiple factors. Poor nutrition, which may be a result of current migratory pollination practices, is one such factor. Migratory pollination is a common practice of beekeepers from the Pacific Northwest and involves the placement of managed honey bee colonies within a series of cropping systems. There is a gap in knowledge on how migratory pollination practices impact honey bee nutrition. To understand the potential impacts of migratory pollination on honey bee nutrition, it is critical to assess the diversity of pollen collected by bees when colonies are placed adjacent to these cropping systems. In this study, we describe the diversity of pollen collected by honey bee colonies managed by beekeepers in the Pacific Northwest region of the United States when placed in seven major cropping systems. We quantified the percent of target crop pollen and overall diversity of pollen collected by honey bees when colonies were placed in these cropping systems. We collected and identified pollen in almond (Prunus dulcis (Mill.)), cherry (Prunus avium L.), highbush blueberry (Vaccinium corymbosum L.), meadowfoam (Limnanthes alba Benth.), white clover (Trifolium repens L.), radish (Raphanus sativus L.), and carrot (Daucus carota (Hoffm.)) cropping systems. We found that pollen collected from colonies placed in almond cropping systems was predominately Prunus sp., and hence, low in pollen diversity. At the other end of the spectrum, pollen collected from colonies placed adjacent to blueberry cropping systems did not yield any target pollen types (Highbush blueberry, Vaccinium corymbosum L.), but was high in overall pollen diversity. The pollen collected from colonies placed in other cropping systems was largely intermediate in diversity between these two extremes. There were not many plant species in bloom when pollen was collected from colonies placed in almond cropping system, whereas more plants were in bloom when we collected pollen from colonies placed in blueberry cropping system. The results of this study demonstrate that honey bees collected pollen from plants in the surrounding environment and collected different degrees of pollen diversity across different cropping systems in which the colonies were placed. We further determined if pollen diversity influences colony-level protein utilization and biosynthesis of protein in nurse bees by conducting a pollen feeding experiment. Pollen collected from four different cropping systems in the first study was used to formulate four different diets, each varying in pollen diversity. We measured protein consumption in experimental colonies and by sampling nurse bees from each colony to estimate hypopharyngeal gland protein content and proteolytic enzyme activity after five weeks of feeding on the experimental pollen diets. Experimental colonies fed on pollen collected from almond cropping system exhibited a high protein consumption rate. However, low protein content as found in hypopharyngeal glands of nurse bees in these colonies. The nurse bees in these colonies also had low proteolytic enzyme activity, which indicates a lower rate of protein digestion. Overall, these results suggest that the diet representing pollen collected from almond cropping systems had low digestion rate and may have resulted in lower nurse bee hypopharyngeal gland protein. However, we cannot say this with certainty, as there were other confounding factors involved, such as presence of pesticides in the pollen collected from the cropping systems. Title: Effects of pollen collected by honey bees from pollination dependent agricultural cropping systems on honey bee nutrition Author: Topitzhofer, Ellen Managed honey bee (Apis mellifera L.) colonies are important pollinators of many cultivated crops. Honey bee colony declines averaging 30% annually in the United States for the past 7 years have caused significant concern and hence have been a topic of intensive investigation. These declines are reportedly due to multiple factors. Poor nutrition, which may be a result of current migratory pollination practices, is one such factor. Migratory pollination is a common practice of beekeepers from the Pacific Northwest and involves the placement of managed honey bee colonies within a series of cropping systems. There is a gap in knowledge on how migratory pollination practices impact honey bee nutrition. To understand the potential impacts of migratory pollination on honey bee nutrition, it is critical to assess the diversity of pollen collected by bees when colonies are placed adjacent to these cropping systems. In this study, we describe the diversity of pollen collected by honey bee colonies managed by beekeepers in the Pacific Northwest region of the United States when placed in seven major cropping systems. We quantified the percent of target crop pollen and overall diversity of pollen collected by honey bees when colonies were placed in these cropping systems. We collected and identified pollen in almond (Prunus dulcis (Mill.)), cherry (Prunus avium L.), highbush blueberry (Vaccinium corymbosum L.), meadowfoam (Limnanthes alba Benth.), white clover (Trifolium repens L.), radish (Raphanus sativus L.), and carrot (Daucus carota (Hoffm.)) cropping systems. We found that pollen collected from colonies placed in almond cropping systems was predominately Prunus sp., and hence, low in pollen diversity. At the other end of the spectrum, pollen collected from colonies placed adjacent to blueberry cropping systems did not yield any target pollen types (Highbush blueberry, Vaccinium corymbosum L.), but was high in overall pollen diversity. The pollen collected from colonies placed in other cropping systems was largely intermediate in diversity between these two extremes. There were not many plant species in bloom when pollen was collected from colonies placed in almond cropping system, whereas more plants were in bloom when we collected pollen from colonies placed in blueberry cropping system. The results of this study demonstrate that honey bees collected pollen from plants in the surrounding environment and collected different degrees of pollen diversity across different cropping systems in which the colonies were placed. We further determined if pollen diversity influences colony-level protein utilization and biosynthesis of protein in nurse bees by conducting a pollen feeding experiment. Pollen collected from four different cropping systems in the first study was used to formulate four different diets, each varying in pollen diversity. We measured protein consumption in experimental colonies and by sampling nurse bees from each colony to estimate hypopharyngeal gland protein content and proteolytic enzyme activity after five weeks of feeding on the experimental pollen diets. Experimental colonies fed on pollen collected from almond cropping system exhibited a high protein consumption rate. However, low protein content as found in hypopharyngeal glands of nurse bees in these colonies. The nurse bees in these colonies also had low proteolytic enzyme activity, which indicates a lower rate of protein digestion. Overall, these results suggest that the diet representing pollen collected from almond cropping systems had low digestion rate and may have resulted in lower nurse bee hypopharyngeal gland protein. However, we cannot say this with certainty, as there were other confounding factors involved, such as presence of pesticides in the pollen collected from the cropping systems. Close

• 1583. [Article] Rheological and spectroscopic characterization of surimi under various comminuting and heating conditions

  Optimization of comminuting and heating conditions for surimi gel preparation obtained from three fish species: Alaska pollock (AP) (Theragra chalcogramma), Pacific whiting (PW) (Merluccius productus), ...

  Citation × Citation Citation Abstract Copy Title: Rheological and spectroscopic characterization of surimi under various comminuting and heating conditions Author: Poowakanjana, Samanan Optimization of comminuting and heating conditions for surimi gel preparation obtained from three fish species: Alaska pollock (AP) (Theragra chalcogramma), Pacific whiting (PW) (Merluccius productus), and threadfin bream (TB) (Nemipterus spp.) was the focus of this study. Three parameters during comminution were separately evaluated: chopping time, chopping temperature, and salting time. Results from fracture gel analysis suggested a strong relation between the fish's environmental habitat and optimal final chopping temperature. Extending chopping time to 15 min under strictly controlled temperature at 0 °C was preferable for cold water fish AP surimi. Even though high chopping temperature (20 °C) for a shorter time (12 min) surprisingly resulted in strong gel texture similar to that of 0 °C for 15 min, high chopping temperature should not be employed for AP surimi. AP could set as a gel at this temperature within a shorter time in a holding tank which could subsequently cause a problem when extruded on the cooking belt. Temperate water fish Pacific whiting, demonstrated its maximum gel strength when chopped at 15-20 °C. The optimum comminution condition for warm water fish threadfin bream surimi was to chop the surimi until the paste temperature reached between 25-30 °C. Prolongation of chopping once the surimi hit its threshold (optimum) temperature diminished the quality of the resulting protein gel. Cooling system connected to the chopping bowl is strongly recommended as it will allow the comminution process to be extended as long as possible until the surimi paste reaches its target temperature. Raman spectroscopy disclosed the different level of protein unfolding based on secondary structure of α-helix and β-sheet during various comminuting conditions. Unfolding of protein was facilitated by increased chopping temperature to a greater degree than extended chopping time. Extending chopping could denature the light meromyosin structure as it could not form a semi gel-like structure at temperatures between 32-40 °C. Protein solubility of surimi paste in salt solution always decreased with prolonged chopping time. The decrease rate accelerated with increased chopping temperature. The formation of disulfide interchange gradually took place during chopping as observed from Raman spectroscopy. Also the surface hydrophobicity increased with extended chopping time. However, gel strength behaved differently according to the various chopping conditions indicating the lack of its relationship between salt soluble protein, disulfide formation, and surface hydrophobicity to gel strength. During extending chopping time, not only more mechanical force is applied to unfold protein structure, but proteins also have longer time to be extracted more by salt. Addition of salt at a different time during chopping process was therefore conducted using threadfin bream surimi due to its higher thermostability. Extending chopping time without salt followed by salt addition at the last step resulted in lower gel texture compared to the conventional chopping protocol where salt is always added at the early stage of comminution. Mechanical chopping could unfold protein structure; however, proteins, rather than staying solubilized, would precipitate and form a randomized structure under the chopping condition without salt. The heating condition greatly affected the gelation and rheological properties of AP surimi. The highest elastic modulus was obtained with the slowest heating rate at 1 °C/min. Increased heating rate did not only shorten the time for proteins to unfold and form a well-organized network, it also interfered with the protein network through the vibration of water molecules as phase angle increased. This suggested that AP surimi gained more viscous properties and failed to form an elastic gel. Adjusting moisture content along with applying various frequencies did not alter the pattern of G' formation when paste was heated at different heating rates. AP surimi favored the slow heating. Title: Rheological and spectroscopic characterization of surimi under various comminuting and heating conditions Author: Poowakanjana, Samanan Optimization of comminuting and heating conditions for surimi gel preparation obtained from three fish species: Alaska pollock (AP) (Theragra chalcogramma), Pacific whiting (PW) (Merluccius productus), and threadfin bream (TB) (Nemipterus spp.) was the focus of this study. Three parameters during comminution were separately evaluated: chopping time, chopping temperature, and salting time. Results from fracture gel analysis suggested a strong relation between the fish's environmental habitat and optimal final chopping temperature. Extending chopping time to 15 min under strictly controlled temperature at 0 °C was preferable for cold water fish AP surimi. Even though high chopping temperature (20 °C) for a shorter time (12 min) surprisingly resulted in strong gel texture similar to that of 0 °C for 15 min, high chopping temperature should not be employed for AP surimi. AP could set as a gel at this temperature within a shorter time in a holding tank which could subsequently cause a problem when extruded on the cooking belt. Temperate water fish Pacific whiting, demonstrated its maximum gel strength when chopped at 15-20 °C. The optimum comminution condition for warm water fish threadfin bream surimi was to chop the surimi until the paste temperature reached between 25-30 °C. Prolongation of chopping once the surimi hit its threshold (optimum) temperature diminished the quality of the resulting protein gel. Cooling system connected to the chopping bowl is strongly recommended as it will allow the comminution process to be extended as long as possible until the surimi paste reaches its target temperature. Raman spectroscopy disclosed the different level of protein unfolding based on secondary structure of α-helix and β-sheet during various comminuting conditions. Unfolding of protein was facilitated by increased chopping temperature to a greater degree than extended chopping time. Extending chopping could denature the light meromyosin structure as it could not form a semi gel-like structure at temperatures between 32-40 °C. Protein solubility of surimi paste in salt solution always decreased with prolonged chopping time. The decrease rate accelerated with increased chopping temperature. The formation of disulfide interchange gradually took place during chopping as observed from Raman spectroscopy. Also the surface hydrophobicity increased with extended chopping time. However, gel strength behaved differently according to the various chopping conditions indicating the lack of its relationship between salt soluble protein, disulfide formation, and surface hydrophobicity to gel strength. During extending chopping time, not only more mechanical force is applied to unfold protein structure, but proteins also have longer time to be extracted more by salt. Addition of salt at a different time during chopping process was therefore conducted using threadfin bream surimi due to its higher thermostability. Extending chopping time without salt followed by salt addition at the last step resulted in lower gel texture compared to the conventional chopping protocol where salt is always added at the early stage of comminution. Mechanical chopping could unfold protein structure; however, proteins, rather than staying solubilized, would precipitate and form a randomized structure under the chopping condition without salt. The heating condition greatly affected the gelation and rheological properties of AP surimi. The highest elastic modulus was obtained with the slowest heating rate at 1 °C/min. Increased heating rate did not only shorten the time for proteins to unfold and form a well-organized network, it also interfered with the protein network through the vibration of water molecules as phase angle increased. This suggested that AP surimi gained more viscous properties and failed to form an elastic gel. Adjusting moisture content along with applying various frequencies did not alter the pattern of G' formation when paste was heated at different heating rates. AP surimi favored the slow heating. Close

• 1584. [Article] Effect of feeding concentrates of Lactobacillus organisms on intestinal colonization by Escherichia coli in swine

  A Lactobacillus species of human intestinal origin (strain MLC) used in swine feeding experiments was characterized using biochemical, genetic and serological techniques and found to be Lactobacillus lactis. ...

  Citation × Citation Citation Abstract Copy Title: Effect of feeding concentrates of Lactobacillus organisms on intestinal colonization by Escherichia coli in swine Author: Muralidhara, K. S. A Lactobacillus species of human intestinal origin (strain MLC) used in swine feeding experiments was characterized using biochemical, genetic and serological techniques and found to be Lactobacillus lactis. Bottle feeding of the MLC strain in concentrate form (> 10⁹ cfu/ml) resulted in a reduction in both fecal coliforms and the incidence of scouring. In one group of pigs which received concentrate for 54 days, the Lactobacillus to coliform ratio was 1280:1; in the control group the ratio was 2.3:1. To increase the sample size, a herd of 125 swine was fed concentrates of Lactobacillus lactis MLC through the drinking water system using a water proportioner. After 90 days of such treatment, the coliform counts were reduced by 95%. The scouring incidence in the treated pigs was 13% as compared to 35% in the control group. However numbers of fecal lactobacilli were not increased. The influence of Lactobacillus MLC feeding on the bacterial flora of different parts of gastrointestinal tract was studied. In the case of scouring pigs, enteropathogenic Escherichia coli (EEC) were present in larger numbers in the tissue homogenate of different parts of the tract than in the lumen. The virulence of the EEC found present was confirmed by experimental infection in pigs. In control, non-scouring pigs only non-EEC were found in the tissue. In Lactobacillus MLC-fed pigs, E. coli both in the tissue and lumen was reduced to low numbers; also, the few E. coli observed were non-enteropathogenic. Thus by feeding Lactobacillus MLC concentrate, it was possible to reduce the E. coli to less than 10² /gm There were higher numbers of lactobacilli in the tissues of Lactobacillus-fed pigs than in control and scouring pigs. The lactobacilli isolated from tissue homogenate of the treated animals resembled biochemically and serologically (fluorescent antibody staining) the Lactobacillus MLC which was fed. Histological studies were done to show direct evidence of colonization in frozen sections of intestine obtained from Lactobacillus MLC-fed pigs. Gram and toluidine blue-staining revealed large numbers of Gram-positive bacilli colonizing the surface epithelium of the villi. On the other hand, control pigs which died of scouring revealed many EEC colonizing the small intestine. Pigs in groups receiving colostrum and lactobacilli performed very well. No symptoms of diarrhea was seen and many lactobacilli colonized throughout the small intestine. Even after the challenge with EEC serotype 09:K:NM, these two groups of pigs did not show any signs of disease and very few EEC colonized the intestines even after the challenge. Pigs not receiving colostrum but only lactobacilli did not scour before challenge with EEC 09:K:NM and many lactobacilli colonized the small intestine. However, 72 hours after challenge these latter animals revealed symptoms of diarrhea and EEC were seen colonizing the small intestine in addition to lactobacilli. The possible role of surface antigens in colonization by lactobacilli was studied. Data revealed that Lactobacillus lactis MLC and L. salivarius did not have any antigens in common. On the other hand, Lactobacillus FHS isolated from pig intestine had three antigens in common with the MLC strain. However, in vivo tests showed that all three strains colonized the small intestine to the same degree. This indicated that surface antigens were not involved in the colonization mechanism. The ability of Lactobacillus MLC to inhibit a variety of intestinal pathogens in broth cultures was demonstrated. Organisms inhibited included E. coli, Staphylococcus aureus, Clostridium perfringens and Bacterotdes sp. The mechanism of inhibition of S. aureus and E. coli in milk and broth was examined. These organisms did not grow in cell-free culture supernatants (whey at pH 4.0) after growth of the Lactobacillus MLC but they grew well in broth adjusted to pH 4.0 Supernatant from cultures of Lactobacillus MLC concentrate was found to contain 2-Deoxy-D-glucose in addition to glucose and galactose. Studies using 2-Deoxy-D-glucose alone and with glucose and galactose showed that the former was inhibitory to E. coli, S. aureus and Salmonella typhimurium. Possible applications of these findings in the animal industry as a substitute to antibiotics are discussed. A greater use of Lactobacillus organisms in preventive treatment of intestinal diseases is suggested. Title: Effect of feeding concentrates of Lactobacillus organisms on intestinal colonization by Escherichia coli in swine Author: Muralidhara, K. S. A Lactobacillus species of human intestinal origin (strain MLC) used in swine feeding experiments was characterized using biochemical, genetic and serological techniques and found to be Lactobacillus lactis. Bottle feeding of the MLC strain in concentrate form (> 10⁹ cfu/ml) resulted in a reduction in both fecal coliforms and the incidence of scouring. In one group of pigs which received concentrate for 54 days, the Lactobacillus to coliform ratio was 1280:1; in the control group the ratio was 2.3:1. To increase the sample size, a herd of 125 swine was fed concentrates of Lactobacillus lactis MLC through the drinking water system using a water proportioner. After 90 days of such treatment, the coliform counts were reduced by 95%. The scouring incidence in the treated pigs was 13% as compared to 35% in the control group. However numbers of fecal lactobacilli were not increased. The influence of Lactobacillus MLC feeding on the bacterial flora of different parts of gastrointestinal tract was studied. In the case of scouring pigs, enteropathogenic Escherichia coli (EEC) were present in larger numbers in the tissue homogenate of different parts of the tract than in the lumen. The virulence of the EEC found present was confirmed by experimental infection in pigs. In control, non-scouring pigs only non-EEC were found in the tissue. In Lactobacillus MLC-fed pigs, E. coli both in the tissue and lumen was reduced to low numbers; also, the few E. coli observed were non-enteropathogenic. Thus by feeding Lactobacillus MLC concentrate, it was possible to reduce the E. coli to less than 10² /gm There were higher numbers of lactobacilli in the tissues of Lactobacillus-fed pigs than in control and scouring pigs. The lactobacilli isolated from tissue homogenate of the treated animals resembled biochemically and serologically (fluorescent antibody staining) the Lactobacillus MLC which was fed. Histological studies were done to show direct evidence of colonization in frozen sections of intestine obtained from Lactobacillus MLC-fed pigs. Gram and toluidine blue-staining revealed large numbers of Gram-positive bacilli colonizing the surface epithelium of the villi. On the other hand, control pigs which died of scouring revealed many EEC colonizing the small intestine. Pigs in groups receiving colostrum and lactobacilli performed very well. No symptoms of diarrhea was seen and many lactobacilli colonized throughout the small intestine. Even after the challenge with EEC serotype 09:K:NM, these two groups of pigs did not show any signs of disease and very few EEC colonized the intestines even after the challenge. Pigs not receiving colostrum but only lactobacilli did not scour before challenge with EEC 09:K:NM and many lactobacilli colonized the small intestine. However, 72 hours after challenge these latter animals revealed symptoms of diarrhea and EEC were seen colonizing the small intestine in addition to lactobacilli. The possible role of surface antigens in colonization by lactobacilli was studied. Data revealed that Lactobacillus lactis MLC and L. salivarius did not have any antigens in common. On the other hand, Lactobacillus FHS isolated from pig intestine had three antigens in common with the MLC strain. However, in vivo tests showed that all three strains colonized the small intestine to the same degree. This indicated that surface antigens were not involved in the colonization mechanism. The ability of Lactobacillus MLC to inhibit a variety of intestinal pathogens in broth cultures was demonstrated. Organisms inhibited included E. coli, Staphylococcus aureus, Clostridium perfringens and Bacterotdes sp. The mechanism of inhibition of S. aureus and E. coli in milk and broth was examined. These organisms did not grow in cell-free culture supernatants (whey at pH 4.0) after growth of the Lactobacillus MLC but they grew well in broth adjusted to pH 4.0 Supernatant from cultures of Lactobacillus MLC concentrate was found to contain 2-Deoxy-D-glucose in addition to glucose and galactose. Studies using 2-Deoxy-D-glucose alone and with glucose and galactose showed that the former was inhibitory to E. coli, S. aureus and Salmonella typhimurium. Possible applications of these findings in the animal industry as a substitute to antibiotics are discussed. A greater use of Lactobacillus organisms in preventive treatment of intestinal diseases is suggested. Close

• 1585. [Article] Application of transport-reaction modeling to constrain biogeochemical processes in marine sediments

  Quantifying the mass transport through marine sediments, and the geochemical response to such flow with numerical models has become a common and powerful approach for geochemical data interpretation. In ...

  Citation × Citation Citation Abstract Copy Title: Application of transport-reaction modeling to constrain biogeochemical processes in marine sediments Author: Hong, Wei-Li Quantifying the mass transport through marine sediments, and the geochemical response to such flow with numerical models has become a common and powerful approach for geochemical data interpretation. In this dissertation, I developed and applied transport-reaction models to unravel complex and interdependent reactions involving carbon, sulfur and silica transformations in shallow marine sediments, and the impact of physical (mass transport deposits) and depositional events (volcanic ash input) on the overall geochemical state of the system. Carbon cycling in the gas hydrate bearing sediments of the Ulleung Basin was quantified using both box and kinetic modeling approaches. The box model balances mass, flux, and carbon isotopes of carbon (Chapter 2), and led to a better understanding of how methane is cycled in the marine sediments of this area. This effort demonstrates the significance of CO₂ reduction, a previously overlooked reaction. The picture of reaction network derived from this work serves as the foundation for a transport-reaction model (Chapter 3). The kinetic model results revealed a very different biogeochemistry between two distinct fluid-flow environments. At sites where transport is predominantly diffusive (non-chimney environments), organic matter decomposition is the dominant process driving production of methane, dissolved inorganic carbon (DIC) and consumption of sulfate. In contrast, anaerobic oxidation of methane (AOM) drives both carbon and sulfur cycles in the advective settings characterized by acoustic chimneys indicative of gas transport. I show that methane produced within the model domain, through CO₂ reduction and methanogenesis, fuels AOM in the non-chimney sites while AOM is primarily induced by methane from external sources at the chimney sites. A simulation of the system evolution from a non-chimney to a chimney condition was developed by increasing the bottom methane supply to an originally diffusion-controlled site. Results from this exercise show that the higher methane flux leads to a higher AOM activity, and enhanced organic matter decomposition through methanogenesis. Organic carbon cycling is also affected by changes in the depositional environment, as shown by application of the kinetic model to the sediments from the Krishna-Godavary (K-G) basin along the eastern Indian margin (Chapter 4). Proximity to large rivers results in the widespread occurrence of mass transport deposits (MTD) throughout the basin. In this work, MTD is defined as a fluidized sediment block whose pore water composition is identical to sea water value to reflect the homogenization process during sediment transport. The pore water sulfate and ammonium profiles measured at seven sites drilled in the K-G Basin during the NGHP-01 expedition were simulated to provide a quantitative description of how MTDs can affect geochemistry profiles, not only for sulfate and ammonium but potentially all pore water species. This model provides reliable estimates of the MTDs thickness, the time elapsed after the most recent event, and the organoclastic sulfate reduction rate at these seven sites. A transport-reaction modeling approach was also applied to investigate the silica diagenetic reactions fueled by volcanic ash decomposition in Shikuko Basin, Nankai Trough (Chapter 5). The model developed for this setting reproduces a silica diagenetic boundary (SDB) at each site, which is defined by marked decreases in reactive volcanic ash, pore water silica and potassium. Volcanic ash alteration was constrained by modeling pore water ⁸⁷Sr/⁸⁶Sr profiles. Below the SDB, formation of clinoptilolite consumes potassium and regulates the extension of amorphous silica by consuming SiO₂(aq). The observed low SiO₂(aq) and dissolved potassium in these deep sequences require continuous precipitation of clinoptilolite; however in order to maintain oversaturation of this mineral at the low SiO₂(aq) in sediments below the SDB, an increase in pH is required, consistent with pore water observations. Thermal history, rather than temperature alone, controls the inferred reaction network as shown by the convergence of the thermal maturity of sediments at the SDB from all studied sites and is consistent with other locations documented onshore Japan. These results are valuable as we move forward in understanding the mechanisms and consequences of ash alteration in convergent margins worldwide. Title: Application of transport-reaction modeling to constrain biogeochemical processes in marine sediments Author: Hong, Wei-Li Quantifying the mass transport through marine sediments, and the geochemical response to such flow with numerical models has become a common and powerful approach for geochemical data interpretation. In this dissertation, I developed and applied transport-reaction models to unravel complex and interdependent reactions involving carbon, sulfur and silica transformations in shallow marine sediments, and the impact of physical (mass transport deposits) and depositional events (volcanic ash input) on the overall geochemical state of the system. Carbon cycling in the gas hydrate bearing sediments of the Ulleung Basin was quantified using both box and kinetic modeling approaches. The box model balances mass, flux, and carbon isotopes of carbon (Chapter 2), and led to a better understanding of how methane is cycled in the marine sediments of this area. This effort demonstrates the significance of CO₂ reduction, a previously overlooked reaction. The picture of reaction network derived from this work serves as the foundation for a transport-reaction model (Chapter 3). The kinetic model results revealed a very different biogeochemistry between two distinct fluid-flow environments. At sites where transport is predominantly diffusive (non-chimney environments), organic matter decomposition is the dominant process driving production of methane, dissolved inorganic carbon (DIC) and consumption of sulfate. In contrast, anaerobic oxidation of methane (AOM) drives both carbon and sulfur cycles in the advective settings characterized by acoustic chimneys indicative of gas transport. I show that methane produced within the model domain, through CO₂ reduction and methanogenesis, fuels AOM in the non-chimney sites while AOM is primarily induced by methane from external sources at the chimney sites. A simulation of the system evolution from a non-chimney to a chimney condition was developed by increasing the bottom methane supply to an originally diffusion-controlled site. Results from this exercise show that the higher methane flux leads to a higher AOM activity, and enhanced organic matter decomposition through methanogenesis. Organic carbon cycling is also affected by changes in the depositional environment, as shown by application of the kinetic model to the sediments from the Krishna-Godavary (K-G) basin along the eastern Indian margin (Chapter 4). Proximity to large rivers results in the widespread occurrence of mass transport deposits (MTD) throughout the basin. In this work, MTD is defined as a fluidized sediment block whose pore water composition is identical to sea water value to reflect the homogenization process during sediment transport. The pore water sulfate and ammonium profiles measured at seven sites drilled in the K-G Basin during the NGHP-01 expedition were simulated to provide a quantitative description of how MTDs can affect geochemistry profiles, not only for sulfate and ammonium but potentially all pore water species. This model provides reliable estimates of the MTDs thickness, the time elapsed after the most recent event, and the organoclastic sulfate reduction rate at these seven sites. A transport-reaction modeling approach was also applied to investigate the silica diagenetic reactions fueled by volcanic ash decomposition in Shikuko Basin, Nankai Trough (Chapter 5). The model developed for this setting reproduces a silica diagenetic boundary (SDB) at each site, which is defined by marked decreases in reactive volcanic ash, pore water silica and potassium. Volcanic ash alteration was constrained by modeling pore water ⁸⁷Sr/⁸⁶Sr profiles. Below the SDB, formation of clinoptilolite consumes potassium and regulates the extension of amorphous silica by consuming SiO₂(aq). The observed low SiO₂(aq) and dissolved potassium in these deep sequences require continuous precipitation of clinoptilolite; however in order to maintain oversaturation of this mineral at the low SiO₂(aq) in sediments below the SDB, an increase in pH is required, consistent with pore water observations. Thermal history, rather than temperature alone, controls the inferred reaction network as shown by the convergence of the thermal maturity of sediments at the SDB from all studied sites and is consistent with other locations documented onshore Japan. These results are valuable as we move forward in understanding the mechanisms and consequences of ash alteration in convergent margins worldwide. Close

• 1586. [Article] The role of near-stream zones on flow, chemistry and isotopic composition at the headwater scale

  Stream discharge is a key water balance component and important factor in global change evaluations. Nevertheless, the mechanisms for streamflow generation are poorly understood. Near- stream surface saturation ...

  Citation × Citation Citation Abstract Copy Title: The role of near-stream zones on flow, chemistry and isotopic composition at the headwater scale Author: Frentress, Jay Stream discharge is a key water balance component and important factor in global change evaluations. Nevertheless, the mechanisms for streamflow generation are poorly understood. Near- stream surface saturation during precipitation events is one of the most iconic, visible indicators of rapid runoff production in upland humid catchments around the world. Despite years of study, we lack understanding of what occurs within the near-stream saturated area, its mixing dynamics and how this affects catchment geochemical- and flow-response dynamics during events. This thesis explores the mechanisms that control near-stream saturated area behavior in a headwater catchment. First, I explore the relation between catchment geochemical response and the flow duration curve (FDC) for the 46-ha Weierbach catchment in Luxembourg across 10 years of runoff monitoring. The shape of the Weierbach FDC suggested a two-phase system, a high-flow, precipitation-driven period and a dry, evapotranspiration-driven, low-flow period. I hypothesized that the two phases were correlated with activation of shallow hillslope and subsurface streamflow sources and that the activation of these sources would be reflected in stream chemistry and surface saturation dynamics. During high-flow periods of the FDC, stream geochemistry was largely unchanging, lacking a dilution effect and appeared a mix of the highly variable soil and groundwater. Thermal infrared (TIR) imagery suggested large surface saturation dynamics at high flows. The geochemical signature of streamflow and soil riparian water during low-flow periods most closely resembled groundwater chemistry and led to increasing base cation concentrations and electrical conductivity. Secondly, to better understand the effect of rain falling on saturated areas and the contribution of rainfall to saturation excess overland flow, I quantified surface saturation dynamics in a near- stream area during rainfall events using high-frequency TIR imagery. During 10 rainfall events across a 34-day period starting December 2013, a total of 161 mm of rainfall elicited 133 mm of runoff at the 6-ha outlet. Surface saturation within a 25-m² thermal infrared imaged area increased from 2 to 20% but was highly variable and weakly correlated to discharge and precipitation. Rainfall onto mapped, near-stream saturated areas accounted for little of the flow generated within a headwater reach. Streamflow isotopic composition at the 6-ha, headwater outlet deflected little throughout the 30-day rainfall period, 0.7 and 1.2 ‰ for δ¹⁸O and δ²H, respectively. Groundwater exfiltration within the saturated area generated nearly all of the streamflow as well the persistent saturation throughout the event. Thirdly, I examined the underlying controls on streamwater chemostasis in a forested, headwater catchment. Thermal infrared imagery was simultaneously used to quantify saturation expansion and groundwater exfiltration hotspots within the headwater reach. Streamflow during a series of rainfall events responded chemostatically, most measured geochemical species (Ca²⁺, Mg²⁺, Na⁺, SiO₂, Cl⁻, SO₄²⁻ and NO₃⁻) varied little (< 0.5 mg/L), despite discharge increases from 0.2 to 4 L/s. Groundwater levels within the saturated zone increased after an initial 24 mm of event rainfall and remained within 0.05 m of the soil surface throughout the runoff period. TIR imagery identified consistent groundwater exfiltration zones from temperature differences across the event-period in the saturated zone. This suggested that unlike many headwater systems, the alluvial aquifer was well connected to groundwater outside the riparian zone and the mapped seepage area was a focused discharge point for the catchment-scale groundwater flow system. Overall this work suggests that for this catchment, groundwater exfiltration in the near-stream zone strongly controls stream geochemical response as well as the timing, duration and quantity of streamflow generation. Title: The role of near-stream zones on flow, chemistry and isotopic composition at the headwater scale Author: Frentress, Jay Stream discharge is a key water balance component and important factor in global change evaluations. Nevertheless, the mechanisms for streamflow generation are poorly understood. Near- stream surface saturation during precipitation events is one of the most iconic, visible indicators of rapid runoff production in upland humid catchments around the world. Despite years of study, we lack understanding of what occurs within the near-stream saturated area, its mixing dynamics and how this affects catchment geochemical- and flow-response dynamics during events. This thesis explores the mechanisms that control near-stream saturated area behavior in a headwater catchment. First, I explore the relation between catchment geochemical response and the flow duration curve (FDC) for the 46-ha Weierbach catchment in Luxembourg across 10 years of runoff monitoring. The shape of the Weierbach FDC suggested a two-phase system, a high-flow, precipitation-driven period and a dry, evapotranspiration-driven, low-flow period. I hypothesized that the two phases were correlated with activation of shallow hillslope and subsurface streamflow sources and that the activation of these sources would be reflected in stream chemistry and surface saturation dynamics. During high-flow periods of the FDC, stream geochemistry was largely unchanging, lacking a dilution effect and appeared a mix of the highly variable soil and groundwater. Thermal infrared (TIR) imagery suggested large surface saturation dynamics at high flows. The geochemical signature of streamflow and soil riparian water during low-flow periods most closely resembled groundwater chemistry and led to increasing base cation concentrations and electrical conductivity. Secondly, to better understand the effect of rain falling on saturated areas and the contribution of rainfall to saturation excess overland flow, I quantified surface saturation dynamics in a near- stream area during rainfall events using high-frequency TIR imagery. During 10 rainfall events across a 34-day period starting December 2013, a total of 161 mm of rainfall elicited 133 mm of runoff at the 6-ha outlet. Surface saturation within a 25-m² thermal infrared imaged area increased from 2 to 20% but was highly variable and weakly correlated to discharge and precipitation. Rainfall onto mapped, near-stream saturated areas accounted for little of the flow generated within a headwater reach. Streamflow isotopic composition at the 6-ha, headwater outlet deflected little throughout the 30-day rainfall period, 0.7 and 1.2 ‰ for δ¹⁸O and δ²H, respectively. Groundwater exfiltration within the saturated area generated nearly all of the streamflow as well the persistent saturation throughout the event. Thirdly, I examined the underlying controls on streamwater chemostasis in a forested, headwater catchment. Thermal infrared imagery was simultaneously used to quantify saturation expansion and groundwater exfiltration hotspots within the headwater reach. Streamflow during a series of rainfall events responded chemostatically, most measured geochemical species (Ca²⁺, Mg²⁺, Na⁺, SiO₂, Cl⁻, SO₄²⁻ and NO₃⁻) varied little (< 0.5 mg/L), despite discharge increases from 0.2 to 4 L/s. Groundwater levels within the saturated zone increased after an initial 24 mm of event rainfall and remained within 0.05 m of the soil surface throughout the runoff period. TIR imagery identified consistent groundwater exfiltration zones from temperature differences across the event-period in the saturated zone. This suggested that unlike many headwater systems, the alluvial aquifer was well connected to groundwater outside the riparian zone and the mapped seepage area was a focused discharge point for the catchment-scale groundwater flow system. Overall this work suggests that for this catchment, groundwater exfiltration in the near-stream zone strongly controls stream geochemical response as well as the timing, duration and quantity of streamflow generation. Close

• 1587. [Article] The influence of local fidelity and recruitment on population dynamics and specialized foraging of humpback whales in Glacier Bay and Icy Strait, Alaska

  Humpback whales (Megaptera novaeangliae, Borowski 1781) in the North Pacific migrate from mid- to high- latitude summer feeding grounds along the Pacific Rim, including areas off the coasts of the U.S., ...

  Citation × Citation Citation Abstract Copy Title: The influence of local fidelity and recruitment on population dynamics and specialized foraging of humpback whales in Glacier Bay and Icy Strait, Alaska Author: Pierszalowski, Sophie Penny Humpback whales (Megaptera novaeangliae, Borowski 1781) in the North Pacific migrate from mid- to high- latitude summer feeding grounds along the Pacific Rim, including areas off the coasts of the U.S., Canada, Russia and eastern Asia, to tropical breeding grounds each winter along Pacific coasts of Mexico and Central America as well as the offshore islands of Mexico, Hawaii, and Japan. Humpback whales in the North Pacific and elsewhere were reduced to very low numbers during a period of intense commercial exploitation that ended in 1965. As the population recovers in abundance, the range of cultural and genetic diversity that survived the exploitation-driven bottleneck is able to adapt, endure and evolve. My work uses genetic tools and photo identification data to investigate the population dynamics, mitochondrial (mt) DNA control region evolution and potential drivers of a specialized feeding behavior in a recovering subpopulation of humpback whales in the Glacier Bay and Icy Strait (GBIS) sub-region of the southeastern Alaska (SEAK) feeding ground. I first collated and reconciled available DNA profiles (mtDNA control region, 10 microsatellite loci and sex) from 556 individuals using tissue samples collected from 1987 to 2012. Photo identification records associated with 692 of 1,026 total genetic samples collected in SEAK (now archived within the SEAK DNA Register and Tissue Database) corresponded to extensive life-history information, extending back to the early 1970s, as archived within the SEAK Regional Database, curated by the National Park Service (NPS) and University of Alaska, Southeast (UAS). Changes in population structure in GBIS over 32 years (1973-2005) were investigated in order to determine whether the increase in local abundance was attributable to local fidelity and recruitment or immigration from outside of SEAK. Two temporal strata were defined: 'Founder' individuals identified between 1973-1985 (n = 74, n = 46 with DNA profiles) and 'Contemporary' individuals identified between 2004-2005 (n = 171, n = 114 with DNA profiles). There was no significant genetic differentiation between the strata, indicating that it is unlikely that the population increase within GBIS was due largely to immigration of whales from elsewhere in the North Pacific. However, two additional haplotypes were documented in the Contemporary stratum at low frequency, one of which was previously unreported in the North Pacific (haplotype A8, see below). This relative stability in haplotype frequencies over time argues for strong regional fidelity of the maternal lineages represented in GBIS between 1973 and 1985. After excluding the 42 Contemporary whales with no photo ID record of a mother or genotype available for maternity inference, at least 73.6% (n = 95) of the Contemporary stratum was either a returning Founder or a recruited descendant of a Founder female. Of all genetically confirmed females with genotypes in the Founder stratum, 96% (n = 24) were either represented in the Contemporary stratum, had at least one confirmed descendant in the Contemporary stratum, or both. This high proportion, in addition to the large proportion of the verifiable Contemporary stratum that were either returning Founders or a descendant of a Founder female, provides clear evidence for local fidelity and recruitment to GBIS. The discovery of the A8 haplotype, which differs by one base pair from a common haplotype referred to as A-, represents an increase in mtDNA diversity for the North Pacific humpback whale from 28 to 29 haplotypes. To investigate the origin of this new haplotype, we re-evaluated n = 1089 electropherograms of n = 710 individuals with A- haplotypes from both the SEAK DNA Register and Tissue Database and the ocean-wide program, SPLASH (Baker et al. 2013). From this review, we identified two individuals with the A8 haplotype (a cow and calf, both sampled in GBIS) and n = 20 individuals with clear heteroplasmy for haplotypes A-/A8. The majority of A-/A8 individuals (n = 15) were sampled in SEAK. Genotype exclusion and likelihood were used to identify one of the heteroplasmic females, #196 (first sighted in SEAK in 1982), as the likely mother of the A8 cow and grandmother of the A8 calf, establishing the inheritance and germ-line fixation of the new haplotype from the parental heteroplasmy. Based on life history records and estimates of pairwise relatedness from microsatellite genotypes, it appears likely that the A-/A8 and the A8 individuals are descendants from a common maternal ancestor one or more generations prior to the three generations documented here. Humpback whale sociality takes a distinct form in Icy Strait, where whales form large, coordinated groups with repeated membership across several decades. Twenty-one years of group association records (1985-2005, n = 2,204 groups) were used to investigate the hypothesis that kin selection influences membership in large, stable groups. Of the 2204 groups recorded, 113 consisted of 6 or more individuals; a size considered unexpectedly large assuming a Poisson distribution of group size with a mean of 1.7. A total of n = 71 individuals (n = 48 with DNA profiles) were encountered in a large group in at least one year, n = 38 individuals (n = 34 with DNA profiles) were encountered in a large group in at least two years, n = 29 individuals (n = 27 with DNA profiles) were encountered in a large group in at least three years, decreasing to n = 2 individuals (n = 2 with DNA profiles) that were encountered in a large group in at least 20 years. There were no significant differences in mtDNA frequencies between large group feeders and the Founder and Contemporary strata or when compared to whales never encountered in large groups in Icy Strait, indicating that group membership is not predominately passed through one maternal lineage. Sex ratios did not deviate significantly from 1:1 for those feeding in large groups over an increasing number of years, as would be expected if females were actively recruiting offspring into large groups. The average pairwise relatedness for large group feeders was not significantly greater than expected by chance and did not increase for those feeding in large groups over an increasing number of years. Of the 179 known offspring of females encountered in a large group, only 6% were also encountered in a large group in Icy Strait as an adult and only 2.2% in the same large group as their mother. These results indicate that kin selection is not the primary driver of membership in large, stable groups and pose an interesting dynamic in local habitat use: individuals are recruited to GBIS through local maternal fidelity but do not usually associate closely with direct maternal kin. The extensive collection of DNA profiles now archived with the individual-based data within the SEAK Regional Database allowed us to integrate genetics and photo ID to answer ecologically relevant questions regarding the whales in GBIS. Together, these results demonstrate that GBIS provide habitat for a distinct collection of individuals that exhibit strong fidelity and local recruitment, some of which engage in a highly specialized feeding behavior. Further, GBIS is a local feeding habitat for two individuals with a newly arising North Pacific mtDNA haplotype. These findings reveal local genotypic and cultural variation and highlight the importance of habitat protection for species with fine-scale habitat use and strong fidelity to local migratory destinations. Title: The influence of local fidelity and recruitment on population dynamics and specialized foraging of humpback whales in Glacier Bay and Icy Strait, Alaska Author: Pierszalowski, Sophie Penny Humpback whales (Megaptera novaeangliae, Borowski 1781) in the North Pacific migrate from mid- to high- latitude summer feeding grounds along the Pacific Rim, including areas off the coasts of the U.S., Canada, Russia and eastern Asia, to tropical breeding grounds each winter along Pacific coasts of Mexico and Central America as well as the offshore islands of Mexico, Hawaii, and Japan. Humpback whales in the North Pacific and elsewhere were reduced to very low numbers during a period of intense commercial exploitation that ended in 1965. As the population recovers in abundance, the range of cultural and genetic diversity that survived the exploitation-driven bottleneck is able to adapt, endure and evolve. My work uses genetic tools and photo identification data to investigate the population dynamics, mitochondrial (mt) DNA control region evolution and potential drivers of a specialized feeding behavior in a recovering subpopulation of humpback whales in the Glacier Bay and Icy Strait (GBIS) sub-region of the southeastern Alaska (SEAK) feeding ground. I first collated and reconciled available DNA profiles (mtDNA control region, 10 microsatellite loci and sex) from 556 individuals using tissue samples collected from 1987 to 2012. Photo identification records associated with 692 of 1,026 total genetic samples collected in SEAK (now archived within the SEAK DNA Register and Tissue Database) corresponded to extensive life-history information, extending back to the early 1970s, as archived within the SEAK Regional Database, curated by the National Park Service (NPS) and University of Alaska, Southeast (UAS). Changes in population structure in GBIS over 32 years (1973-2005) were investigated in order to determine whether the increase in local abundance was attributable to local fidelity and recruitment or immigration from outside of SEAK. Two temporal strata were defined: 'Founder' individuals identified between 1973-1985 (n = 74, n = 46 with DNA profiles) and 'Contemporary' individuals identified between 2004-2005 (n = 171, n = 114 with DNA profiles). There was no significant genetic differentiation between the strata, indicating that it is unlikely that the population increase within GBIS was due largely to immigration of whales from elsewhere in the North Pacific. However, two additional haplotypes were documented in the Contemporary stratum at low frequency, one of which was previously unreported in the North Pacific (haplotype A8, see below). This relative stability in haplotype frequencies over time argues for strong regional fidelity of the maternal lineages represented in GBIS between 1973 and 1985. After excluding the 42 Contemporary whales with no photo ID record of a mother or genotype available for maternity inference, at least 73.6% (n = 95) of the Contemporary stratum was either a returning Founder or a recruited descendant of a Founder female. Of all genetically confirmed females with genotypes in the Founder stratum, 96% (n = 24) were either represented in the Contemporary stratum, had at least one confirmed descendant in the Contemporary stratum, or both. This high proportion, in addition to the large proportion of the verifiable Contemporary stratum that were either returning Founders or a descendant of a Founder female, provides clear evidence for local fidelity and recruitment to GBIS. The discovery of the A8 haplotype, which differs by one base pair from a common haplotype referred to as A-, represents an increase in mtDNA diversity for the North Pacific humpback whale from 28 to 29 haplotypes. To investigate the origin of this new haplotype, we re-evaluated n = 1089 electropherograms of n = 710 individuals with A- haplotypes from both the SEAK DNA Register and Tissue Database and the ocean-wide program, SPLASH (Baker et al. 2013). From this review, we identified two individuals with the A8 haplotype (a cow and calf, both sampled in GBIS) and n = 20 individuals with clear heteroplasmy for haplotypes A-/A8. The majority of A-/A8 individuals (n = 15) were sampled in SEAK. Genotype exclusion and likelihood were used to identify one of the heteroplasmic females, #196 (first sighted in SEAK in 1982), as the likely mother of the A8 cow and grandmother of the A8 calf, establishing the inheritance and germ-line fixation of the new haplotype from the parental heteroplasmy. Based on life history records and estimates of pairwise relatedness from microsatellite genotypes, it appears likely that the A-/A8 and the A8 individuals are descendants from a common maternal ancestor one or more generations prior to the three generations documented here. Humpback whale sociality takes a distinct form in Icy Strait, where whales form large, coordinated groups with repeated membership across several decades. Twenty-one years of group association records (1985-2005, n = 2,204 groups) were used to investigate the hypothesis that kin selection influences membership in large, stable groups. Of the 2204 groups recorded, 113 consisted of 6 or more individuals; a size considered unexpectedly large assuming a Poisson distribution of group size with a mean of 1.7. A total of n = 71 individuals (n = 48 with DNA profiles) were encountered in a large group in at least one year, n = 38 individuals (n = 34 with DNA profiles) were encountered in a large group in at least two years, n = 29 individuals (n = 27 with DNA profiles) were encountered in a large group in at least three years, decreasing to n = 2 individuals (n = 2 with DNA profiles) that were encountered in a large group in at least 20 years. There were no significant differences in mtDNA frequencies between large group feeders and the Founder and Contemporary strata or when compared to whales never encountered in large groups in Icy Strait, indicating that group membership is not predominately passed through one maternal lineage. Sex ratios did not deviate significantly from 1:1 for those feeding in large groups over an increasing number of years, as would be expected if females were actively recruiting offspring into large groups. The average pairwise relatedness for large group feeders was not significantly greater than expected by chance and did not increase for those feeding in large groups over an increasing number of years. Of the 179 known offspring of females encountered in a large group, only 6% were also encountered in a large group in Icy Strait as an adult and only 2.2% in the same large group as their mother. These results indicate that kin selection is not the primary driver of membership in large, stable groups and pose an interesting dynamic in local habitat use: individuals are recruited to GBIS through local maternal fidelity but do not usually associate closely with direct maternal kin. The extensive collection of DNA profiles now archived with the individual-based data within the SEAK Regional Database allowed us to integrate genetics and photo ID to answer ecologically relevant questions regarding the whales in GBIS. Together, these results demonstrate that GBIS provide habitat for a distinct collection of individuals that exhibit strong fidelity and local recruitment, some of which engage in a highly specialized feeding behavior. Further, GBIS is a local feeding habitat for two individuals with a newly arising North Pacific mtDNA haplotype. These findings reveal local genotypic and cultural variation and highlight the importance of habitat protection for species with fine-scale habitat use and strong fidelity to local migratory destinations. Close

• 1588. [Article] Defect Mechanisms in Bismuth-based Perovskites

  The aim of this research is to develop a fundamental understanding of the dominant defect species and the relevant defect equilibrium conditions for bismuth-containing perovskites to help guide the development ...

  Citation × Citation Citation Abstract Copy Title: Defect Mechanisms in Bismuth-based Perovskites Author: Kumar, Nitish The aim of this research is to develop a fundamental understanding of the dominant defect species and the relevant defect equilibrium conditions for bismuth-containing perovskites to help guide the development of these materials for emerging applications. This is of paramount importance for many demanding applications, because ultimately the defect equilibria have a profound influence on phenomena such as piezoelectric fatigue, reliability, and leakage current. At the same time, they can be used to tailor properties to make these materials better suited for specific applications. Perovskite materials with Bi³⁺ on the A-site have been the focus of great technical interest over the last decade. A number of compositions based on Bi-perovskites (e.g. (Bi,Ba)(B²⁺,Ti)O₃) are being studied for high energy density (or high temperature) capacitor applications. In addition, a number of Bi-based perovskite materials have shown great promise as a replacement for Pb(Zr,Ti)O₃ (PZT) for piezoelectric applications. Compounds such as (Bi₁/₂K₁/₂)TiO₃ (BKT) and (Bi₁/₂Na₁/₂)TiO₃ (BNT), and their solid solutions with BaTiO₃ and other tetragonal perovskites exhibit useful piezoelectric properties and are considered to be promising candidates to replace Pb-based materials if the underlying defect chemistry can be controlled. The technological impact of these materials is expected to grow because research in this area is being driven by increased environmental regulations and energy efficiency considerations. While much of the current research and progress on Bi-perovskites have been focused on primary materials properties like the piezoelectric coefficient, relative permittivity, etc., there have been few studies on the underlying fundamental defect chemistry and they are not fully understood. This research focuses mainly on two material systems to study their defect chemistry and transport properties. First is Bi(Zn₁/₂Ti₁/₂)O₃–BaTiO₃, for high performance capacitor applications. Conventional approaches to this technical challenge include utilizing ferroelectric or antiferroelectric materials with permittivities in excess of 1000. However, these non-linear materials derive their high permittivity from domain contributions that saturate at relatively low fields ultimately resulting in limited energy densities. However, solid solutions based on BiMO₃-BaTiO₃ that exhibit relaxor behavior can potentially demonstrate high energy densities. The second material system investigated was BNT-BKT- Bi(Mg₁/₂Ti₁/₂)O₃. This material features a field-induced relaxor-to-ferroelectric transition that is accompanied by a large piezoelectric strain values. The first part of the thesis focuses on polycrystalline BaTiO₃-Bi(Zn₁/₂Ti₁/₂)O₃ (BT-BZT) ceramics, which have been shown to exhibit superior dielectric properties for high temperature and high energy density applications as compared to the existing materials. As miniaturization without compromising cost and performance is vital for several applications, chapter 9.1 shows the results on multilayer ceramic capacitors based on relaxor BT-BZT ceramics. In bulk ceramic embodiments, BT-BZT has been shown to exhibit relative permittivities greater than 1000, high resistivities (ρ > 1 GΩ-cm at 300°C), and negligible saturation up to fields as high as 150 kV/cm. The multilayer capacitor embodiments exhibited similar dielectric and resistivity properties. The energy density for the multilayer ceramics reached values of ~2.8 J/cm³ at room temperature at an applied electric field of ~330 kV/cm. This represents a significant improvement compared to commercially available multilayer capacitors. The dielectric properties were also found to be stable over a wide range of temperatures with a temperature coefficient of approximately -2000 ppm/K measured from 50 to 350 °C, an important criteria for high temperature applications. Finally, the compatibility of inexpensive Ag-Pd electrodes with these ceramics was also demonstrated, which can have implications on minimizing the device cost. Having demonstrated that BT-BZT exhibits promising properties, the primary focus of this thesis research is developing a fundamental understanding of the transport properties and defect chemistry. A significant improvement in insulation properties was measured with the addition of BZT to BT. Both low-field AC impedance and high field direct DC measurements indicated an increase in resistivity of at least 2 orders of magnitude at 400 °C with the addition of just 3% BZT (~10⁷ Ω-cm) into the solid solution as compared to pure BT (~10⁵ Ω-cm). This effect was also evident in dielectric loss data, which remained low at higher temperatures as the BZT content increased. In conjunction with band gap measurements, it was also concluded that the conduction mechanism transitioned from extrinsic for pure BT to intrinsic-like for 7.5% BZT suggesting a change in the fundamental defect equilibrium conditions. It was also shown that this improvement in insulation properties was not limited to BT-BZT, but could also be observed in SrTiO₃-BZT system. While pure BT exhibits extrinsic p-type conduction, it is reported that BT-BZT ceramics exhibit intrinsic-like n-type conduction using atmosphere dependent conductivity measurements. Annealing studies and Seebeck measurements were performed and confirmed this result. For BT, resistivity values were higher for samples annealed in nitrogen as compared to oxygen, while the opposite responses were observed for BZT-containing solid solutions. This suggested a possibile unintentional donor doping upon addition of BZT to the solid solution, which may also be linked to the improvement in resistivity in BT-BZT ceramics as compared to pure BT. Impedance spectroscopy in conjunction with small DC-bias provided further proof of the p-type to n-type transition and also demonstrated the field-stable properties of BT-BZT ceramics. For p-type BaTiO₃, the ceramics deviated from Ohm’s law behavior at very low voltage levels along with a reversible drop in bulk resistivity by several orders of magnitude starting at bias fields as low as 0.1 kV/cm (~8 V). In contrast, n-type BT-BZT ceramics exhibited a small (i.e. less than one order of magnitude) increase in resistivity on application of small field levels. These data indicate a hole-generation mechanism which becomes active at a low voltage threshold. The bulk capacitance values calculated using AC impedance spectroscopy, however, were relatively unaffected (<15% change) by this application of a DC bias (up to ~0.25 kV/cm). These findings provide further insights into the electric transport mechanisms in BT-based ceramics. To investigate the possible presence of Bi⁵⁺ in BT-BZT ceramics, which was postulated to be one of the possible mechanisms for n-type behavior in BT-BZT ceramics, some BT-BaBiO₃ solid solutions were fabricated. The BaBiO₃ ceramics were sintered in oxygen to obtain a single phase with monoclinic I2/m symmetry as suggested by high-resolution x-ray diffraction. X-ray photoelectron spectroscopy confirmed the presence of bismuth in two valence states – 3+ and 5+. Optical spectroscopy showed presence of a direct band gap at ~2.2 eV and a possible indirect band gap at ~0.9 eV. This combined with determination of the activation energy for conduction of 0.25 eV, as obtained from ac impedance spectroscopy, suggested that a polaron-mediated conduction mechanism was prevalent in BaBiO₃. These BaBiO₃ ceramics were crushed, mixed with BaTiO₃, and sintered to obtain BaTiO₃-BaBiO₃ solid solutions. All the ceramics had tetragonal symmetry and exhibited a normal ferroelectric-like dielectric response. Using ac impedance and optical spectroscopy, it was shown that resistivity values of BaTiO₃-BaBiO₃ were orders of magnitude higher than BaTiO₃ or BaBiO₃ alone, indicating a change in the fundamental defect equilibrium conditions. A shift in the site occupancy of Bi to the A-site is proposed to be the mechanism for the increased electrical resistivity. To investigate the effect of A-site nonstoichiometry in BT-BiMO₃ ceramics, BaTiO₃-BiScO₃ (BT-BS) and SrTiO₃-Bi(Zn₁/₂Ti₁/₂)O₃ (ST-BZT) were fabricated. The effect of nonstoichiometry on the dielectric and transport properties was studied using temperature- and oxygen partial pressure-dependent AC impedance spectroscopy. For p-type BT-BS ceramics, the addition of excess Bi led to effective donor doping along with a significant improvement in insulation properties. A similar effect was observed on introducing Ba vacancies onto the A-sublattice. However, Bi deficiency had an opposite effect with effective acceptor doping and a deterioration in the bulk resistivity values. For n-type intrinsic ST-BZT ceramics, the addition of excess Sr onto the A-sublattice resulted in a decrease in resistivity values, as expected. Introduction of Sr vacancies or addition of excess Bi on A-site did not appear to affect the insulation properties in air. These results indicate that minor levels of non-stoichiometry can have an important impact on the material properties and furthermore it demonstrates the difficulties encountered in trying to establish a general model for the defect chemistry of Bi-containing perovskite systems. Finally, the other prospective candidates for n-type behavior in BT-BZT were studied—loss of volatile cations, oxygen vacancies, bismuth present in multiple valence states and precipitation of secondary phases. Combined x-ray and neutron diffraction, prompt gamma neutron activation analysis and electron energy loss spectroscopy suggested much higher oxygen vacancy concentration in BT-BZT ceramics as compared to BT alone. X-ray photoelectron spectroscopy and x-ray absorption spectroscopy did not suggest presence of bismuth in multiple valence states. At the same time, using transmission electron microscopy, some secondary phases were observed, whose compositions were such that they could result in effective donor doping in BT-BZT ceramics. Using experimentally determined thermodynamic parameters for BT and slopes of conductivity-oxygen partial pressure curves, it has been suggested that an ionic compensation mechanism is prevalent in these ceramics instead of electronic compensation. However, these defects in BT-BZT ceramics have an effect of shifting the conductivity minimum in conductivity-oxygen partial pressure curves to higher oxygen partial pressure values, resulting in significantly higher resistivity values in air atmosphere. This provides an important tool to tailor transport properties and defects in BT-BiMO₃ ceramics, to make them better suited for dielectric applications. The second Bi-based ceramic system which was looked at was lead-free Bi(Mg₁/₂Ti₁/₂)O₃-(Bi₁/₂K₁/₂)TiO₃-(Bi₁/₂Na₁/₂)TiO₃ for sensors and actuator applications. There has been a huge drive to replace Pb from existing ceramics (e.g. lead zirconate titanate) due to health and environmental concerns. The dielectric spectra showed a T[subscript max] of more than 320 °C for all compositions and the transitions became increasingly diffuse as the Bi(Mg₁/₂Ti₁/₂)O₃ content increased. A lower temperature transition, indicating a transformation from an ergodic to a non-ergodic relaxor state, was also seen for all compositions and this transition temperature decreased as the mole fraction of Bi(Mg₁/₂Ti₁/₂)O₃ increased. The composition with 1% Bi(Mg₁/₂Ti₁/₂)O₃ showed characteristic ferroelectric-like polarization and strain hysteresis. However, compositions with increased Bi(Mg₁/₂Ti₁/₂)O₃ content became increasingly ergodic at room temperature with pinched polarization loops and no negative strain. Among these compositions, the magnitude of d₃₃* increased with Bi(Mg₁/₂Ti₁/₂)O₃ content and the composition with 10% Bi(Mg₁/₂Ti₁/₂)O₃ exhibited a d₃₃* of 422 pm/V . Fatigue measurements were conducted on all compositions and while the 1% Bi(Mg₁/₂Ti₁/₂)O₃ composition exhibited a measurable, but small loss in maximum strain after a million cycles; all the other compositions from 2.5% to 10% Bi(Mg₁/₂Ti₁/₂)O₃ were essentially fatigue-free. Lastly, optical and AC impedance measurements were employed to identify intrinsic conduction as the dominant conduction mechanism. These compositions were also highly insulating with high resistivities (~10⁷ Ω-cm) at high temperatures (440 °C). To investigate the role of point defects on the fatigue characteristics, the composition 5%BMT-40%BKT-55%BNT was doped to incorporate acceptor and donor defects on the A and B sites by adjusting the Bi/Na and Ti/Mg stoichiometries. All samples had pseudo-cubic symmetries based on x-ray diffraction, typical of relaxors. Dielectric measurements showed that the high and low temperature phase transitions were largely unaffected by doping. Acceptor doping resulted in the observation of a typical ferroelectric-like polarization with a remnant polarization and strain hysteresis loops with significant negative strain. Donor-doped compositions exhibited characteristics that were indicative of an ergodic relaxor phase. Fatigue measurements were carried out on all of the compositions. While the A-site acceptor-doped composition showed a small degradation in maximum strain after 10⁶ cycles, the other compositions were essentially fatigue free. Impedance measurements were used to identify the important conduction mechanisms in these compositions. As expected, the presence of defects did not strongly influence the fatigue behavior in donor-doped compositions owing to the nature of their reversible field-induced phase transformation. Even for the acceptor-doped compositions, which had stable domains in the absence of an electric field at room temperature, there was negligible degradation in the maximum strain due to fatigue. This suggests that either the defects introduced through stoichiometric variations do not play a prominent role in fatigue in these systems or it is compensated by factors like decrease in coercive field, an increase in ergodicity, symmetry change, or other factors. The results obtained for these ceramic systems have provided significant insights in the defect chemistry and transport properties and are expected to help improve performance of these emerging materials for energy and MEMS technologies. Title: Defect Mechanisms in Bismuth-based Perovskites Author: Kumar, Nitish The aim of this research is to develop a fundamental understanding of the dominant defect species and the relevant defect equilibrium conditions for bismuth-containing perovskites to help guide the development of these materials for emerging applications. This is of paramount importance for many demanding applications, because ultimately the defect equilibria have a profound influence on phenomena such as piezoelectric fatigue, reliability, and leakage current. At the same time, they can be used to tailor properties to make these materials better suited for specific applications. Perovskite materials with Bi³⁺ on the A-site have been the focus of great technical interest over the last decade. A number of compositions based on Bi-perovskites (e.g. (Bi,Ba)(B²⁺,Ti)O₃) are being studied for high energy density (or high temperature) capacitor applications. In addition, a number of Bi-based perovskite materials have shown great promise as a replacement for Pb(Zr,Ti)O₃ (PZT) for piezoelectric applications. Compounds such as (Bi₁/₂K₁/₂)TiO₃ (BKT) and (Bi₁/₂Na₁/₂)TiO₃ (BNT), and their solid solutions with BaTiO₃ and other tetragonal perovskites exhibit useful piezoelectric properties and are considered to be promising candidates to replace Pb-based materials if the underlying defect chemistry can be controlled. The technological impact of these materials is expected to grow because research in this area is being driven by increased environmental regulations and energy efficiency considerations. While much of the current research and progress on Bi-perovskites have been focused on primary materials properties like the piezoelectric coefficient, relative permittivity, etc., there have been few studies on the underlying fundamental defect chemistry and they are not fully understood. This research focuses mainly on two material systems to study their defect chemistry and transport properties. First is Bi(Zn₁/₂Ti₁/₂)O₃–BaTiO₃, for high performance capacitor applications. Conventional approaches to this technical challenge include utilizing ferroelectric or antiferroelectric materials with permittivities in excess of 1000. However, these non-linear materials derive their high permittivity from domain contributions that saturate at relatively low fields ultimately resulting in limited energy densities. However, solid solutions based on BiMO₃-BaTiO₃ that exhibit relaxor behavior can potentially demonstrate high energy densities. The second material system investigated was BNT-BKT- Bi(Mg₁/₂Ti₁/₂)O₃. This material features a field-induced relaxor-to-ferroelectric transition that is accompanied by a large piezoelectric strain values. The first part of the thesis focuses on polycrystalline BaTiO₃-Bi(Zn₁/₂Ti₁/₂)O₃ (BT-BZT) ceramics, which have been shown to exhibit superior dielectric properties for high temperature and high energy density applications as compared to the existing materials. As miniaturization without compromising cost and performance is vital for several applications, chapter 9.1 shows the results on multilayer ceramic capacitors based on relaxor BT-BZT ceramics. In bulk ceramic embodiments, BT-BZT has been shown to exhibit relative permittivities greater than 1000, high resistivities (ρ > 1 GΩ-cm at 300°C), and negligible saturation up to fields as high as 150 kV/cm. The multilayer capacitor embodiments exhibited similar dielectric and resistivity properties. The energy density for the multilayer ceramics reached values of ~2.8 J/cm³ at room temperature at an applied electric field of ~330 kV/cm. This represents a significant improvement compared to commercially available multilayer capacitors. The dielectric properties were also found to be stable over a wide range of temperatures with a temperature coefficient of approximately -2000 ppm/K measured from 50 to 350 °C, an important criteria for high temperature applications. Finally, the compatibility of inexpensive Ag-Pd electrodes with these ceramics was also demonstrated, which can have implications on minimizing the device cost. Having demonstrated that BT-BZT exhibits promising properties, the primary focus of this thesis research is developing a fundamental understanding of the transport properties and defect chemistry. A significant improvement in insulation properties was measured with the addition of BZT to BT. Both low-field AC impedance and high field direct DC measurements indicated an increase in resistivity of at least 2 orders of magnitude at 400 °C with the addition of just 3% BZT (~10⁷ Ω-cm) into the solid solution as compared to pure BT (~10⁵ Ω-cm). This effect was also evident in dielectric loss data, which remained low at higher temperatures as the BZT content increased. In conjunction with band gap measurements, it was also concluded that the conduction mechanism transitioned from extrinsic for pure BT to intrinsic-like for 7.5% BZT suggesting a change in the fundamental defect equilibrium conditions. It was also shown that this improvement in insulation properties was not limited to BT-BZT, but could also be observed in SrTiO₃-BZT system. While pure BT exhibits extrinsic p-type conduction, it is reported that BT-BZT ceramics exhibit intrinsic-like n-type conduction using atmosphere dependent conductivity measurements. Annealing studies and Seebeck measurements were performed and confirmed this result. For BT, resistivity values were higher for samples annealed in nitrogen as compared to oxygen, while the opposite responses were observed for BZT-containing solid solutions. This suggested a possibile unintentional donor doping upon addition of BZT to the solid solution, which may also be linked to the improvement in resistivity in BT-BZT ceramics as compared to pure BT. Impedance spectroscopy in conjunction with small DC-bias provided further proof of the p-type to n-type transition and also demonstrated the field-stable properties of BT-BZT ceramics. For p-type BaTiO₃, the ceramics deviated from Ohm’s law behavior at very low voltage levels along with a reversible drop in bulk resistivity by several orders of magnitude starting at bias fields as low as 0.1 kV/cm (~8 V). In contrast, n-type BT-BZT ceramics exhibited a small (i.e. less than one order of magnitude) increase in resistivity on application of small field levels. These data indicate a hole-generation mechanism which becomes active at a low voltage threshold. The bulk capacitance values calculated using AC impedance spectroscopy, however, were relatively unaffected (<15% change) by this application of a DC bias (up to ~0.25 kV/cm). These findings provide further insights into the electric transport mechanisms in BT-based ceramics. To investigate the possible presence of Bi⁵⁺ in BT-BZT ceramics, which was postulated to be one of the possible mechanisms for n-type behavior in BT-BZT ceramics, some BT-BaBiO₃ solid solutions were fabricated. The BaBiO₃ ceramics were sintered in oxygen to obtain a single phase with monoclinic I2/m symmetry as suggested by high-resolution x-ray diffraction. X-ray photoelectron spectroscopy confirmed the presence of bismuth in two valence states – 3+ and 5+. Optical spectroscopy showed presence of a direct band gap at ~2.2 eV and a possible indirect band gap at ~0.9 eV. This combined with determination of the activation energy for conduction of 0.25 eV, as obtained from ac impedance spectroscopy, suggested that a polaron-mediated conduction mechanism was prevalent in BaBiO₃. These BaBiO₃ ceramics were crushed, mixed with BaTiO₃, and sintered to obtain BaTiO₃-BaBiO₃ solid solutions. All the ceramics had tetragonal symmetry and exhibited a normal ferroelectric-like dielectric response. Using ac impedance and optical spectroscopy, it was shown that resistivity values of BaTiO₃-BaBiO₃ were orders of magnitude higher than BaTiO₃ or BaBiO₃ alone, indicating a change in the fundamental defect equilibrium conditions. A shift in the site occupancy of Bi to the A-site is proposed to be the mechanism for the increased electrical resistivity. To investigate the effect of A-site nonstoichiometry in BT-BiMO₃ ceramics, BaTiO₃-BiScO₃ (BT-BS) and SrTiO₃-Bi(Zn₁/₂Ti₁/₂)O₃ (ST-BZT) were fabricated. The effect of nonstoichiometry on the dielectric and transport properties was studied using temperature- and oxygen partial pressure-dependent AC impedance spectroscopy. For p-type BT-BS ceramics, the addition of excess Bi led to effective donor doping along with a significant improvement in insulation properties. A similar effect was observed on introducing Ba vacancies onto the A-sublattice. However, Bi deficiency had an opposite effect with effective acceptor doping and a deterioration in the bulk resistivity values. For n-type intrinsic ST-BZT ceramics, the addition of excess Sr onto the A-sublattice resulted in a decrease in resistivity values, as expected. Introduction of Sr vacancies or addition of excess Bi on A-site did not appear to affect the insulation properties in air. These results indicate that minor levels of non-stoichiometry can have an important impact on the material properties and furthermore it demonstrates the difficulties encountered in trying to establish a general model for the defect chemistry of Bi-containing perovskite systems. Finally, the other prospective candidates for n-type behavior in BT-BZT were studied—loss of volatile cations, oxygen vacancies, bismuth present in multiple valence states and precipitation of secondary phases. Combined x-ray and neutron diffraction, prompt gamma neutron activation analysis and electron energy loss spectroscopy suggested much higher oxygen vacancy concentration in BT-BZT ceramics as compared to BT alone. X-ray photoelectron spectroscopy and x-ray absorption spectroscopy did not suggest presence of bismuth in multiple valence states. At the same time, using transmission electron microscopy, some secondary phases were observed, whose compositions were such that they could result in effective donor doping in BT-BZT ceramics. Using experimentally determined thermodynamic parameters for BT and slopes of conductivity-oxygen partial pressure curves, it has been suggested that an ionic compensation mechanism is prevalent in these ceramics instead of electronic compensation. However, these defects in BT-BZT ceramics have an effect of shifting the conductivity minimum in conductivity-oxygen partial pressure curves to higher oxygen partial pressure values, resulting in significantly higher resistivity values in air atmosphere. This provides an important tool to tailor transport properties and defects in BT-BiMO₃ ceramics, to make them better suited for dielectric applications. The second Bi-based ceramic system which was looked at was lead-free Bi(Mg₁/₂Ti₁/₂)O₃-(Bi₁/₂K₁/₂)TiO₃-(Bi₁/₂Na₁/₂)TiO₃ for sensors and actuator applications. There has been a huge drive to replace Pb from existing ceramics (e.g. lead zirconate titanate) due to health and environmental concerns. The dielectric spectra showed a T[subscript max] of more than 320 °C for all compositions and the transitions became increasingly diffuse as the Bi(Mg₁/₂Ti₁/₂)O₃ content increased. A lower temperature transition, indicating a transformation from an ergodic to a non-ergodic relaxor state, was also seen for all compositions and this transition temperature decreased as the mole fraction of Bi(Mg₁/₂Ti₁/₂)O₃ increased. The composition with 1% Bi(Mg₁/₂Ti₁/₂)O₃ showed characteristic ferroelectric-like polarization and strain hysteresis. However, compositions with increased Bi(Mg₁/₂Ti₁/₂)O₃ content became increasingly ergodic at room temperature with pinched polarization loops and no negative strain. Among these compositions, the magnitude of d₃₃* increased with Bi(Mg₁/₂Ti₁/₂)O₃ content and the composition with 10% Bi(Mg₁/₂Ti₁/₂)O₃ exhibited a d₃₃* of 422 pm/V . Fatigue measurements were conducted on all compositions and while the 1% Bi(Mg₁/₂Ti₁/₂)O₃ composition exhibited a measurable, but small loss in maximum strain after a million cycles; all the other compositions from 2.5% to 10% Bi(Mg₁/₂Ti₁/₂)O₃ were essentially fatigue-free. Lastly, optical and AC impedance measurements were employed to identify intrinsic conduction as the dominant conduction mechanism. These compositions were also highly insulating with high resistivities (~10⁷ Ω-cm) at high temperatures (440 °C). To investigate the role of point defects on the fatigue characteristics, the composition 5%BMT-40%BKT-55%BNT was doped to incorporate acceptor and donor defects on the A and B sites by adjusting the Bi/Na and Ti/Mg stoichiometries. All samples had pseudo-cubic symmetries based on x-ray diffraction, typical of relaxors. Dielectric measurements showed that the high and low temperature phase transitions were largely unaffected by doping. Acceptor doping resulted in the observation of a typical ferroelectric-like polarization with a remnant polarization and strain hysteresis loops with significant negative strain. Donor-doped compositions exhibited characteristics that were indicative of an ergodic relaxor phase. Fatigue measurements were carried out on all of the compositions. While the A-site acceptor-doped composition showed a small degradation in maximum strain after 10⁶ cycles, the other compositions were essentially fatigue free. Impedance measurements were used to identify the important conduction mechanisms in these compositions. As expected, the presence of defects did not strongly influence the fatigue behavior in donor-doped compositions owing to the nature of their reversible field-induced phase transformation. Even for the acceptor-doped compositions, which had stable domains in the absence of an electric field at room temperature, there was negligible degradation in the maximum strain due to fatigue. This suggests that either the defects introduced through stoichiometric variations do not play a prominent role in fatigue in these systems or it is compensated by factors like decrease in coercive field, an increase in ergodicity, symmetry change, or other factors. The results obtained for these ceramic systems have provided significant insights in the defect chemistry and transport properties and are expected to help improve performance of these emerging materials for energy and MEMS technologies. Close

• 1589. [Article] Reproductive implications of parasitic infections and immune challenges in garter snakes

  Parasitic infections and immune challenges can affect host reproductive fitness and, ultimately, the evolution of host populations in a myriad of ways. The fitness implications of parasitic infections ...

  Citation × Citation Citation Abstract Copy Title: Reproductive implications of parasitic infections and immune challenges in garter snakes Author: Uhrig, Emily J. Parasitic infections and immune challenges can affect host reproductive fitness and, ultimately, the evolution of host populations in a myriad of ways. The fitness implications of parasitic infections range from increased host mortality to subtle changes in reproductive investment. From alterations of behaviors, sexual signaling, and competitive ability to changes in gamete production and fertilization success, it is clear that parasites are capable of mediating sexual selection and influencing host reproductive fitness even without altering mortality. The mechanisms underlying fitness effects highlight the complexity of the host-parasite relationship which involves immune responses as well as a range of other, often interactive, physiological processes within the host. In some instances, it is not the direct effect of parasites per se, but rather the hosts' responses to infection that mediate fitness consequences. This dissertation presents studies designed to elucidate the implications of parasitism and immune responses for the reproductive fitness of garter snakes (genus Thamnophis). In chapter 2, "Alaria mesocercariae in the tails of red-sided garter snakes: evidence for parasite-mediated caudectomy", I focus on the histopathological changes associated with a trematode (Alaria sp.) infecting the tails of red-sided garter snakes (T. sirtalis parietalis). My results demonstrate that Alaria mesocercariae occur in high density within the tail tissue of both male and female snakes with as many as 2,000 mesocercariae in a single tail; infection prevalence was 100% in the snakes I examined. I found no evidence of intersexual variation in pathological changes or infection densities. For both sexes, external pathological manifestations include swelling of the tail while, internally, the aggregation of mesocercariae leads to the formation of mucus-filled pseudocysts and damage of muscle tissue. In severe cases, the extent of tissue destruction appeared to weaken the connection of the tail to the rest of the body, a condition that would facilitate tail breakage, which in turn negatively affects the snake's fitness by impairing mating success. From the parasite's perspective, tail breakage is likely beneficial by facilitating its transmission to subsequent hosts in its life cycle. Alaria sp. are not the only parasites commonly infecting garter snakes and in chapter 3, "Patterns in parasitism: interspecific and interpopulational variation in helminth assemblages and their reproductive fitness correlates in garter snakes", I broaden our investigation to include a suite of helminth parasites common in the garter snakes of Manitoba, Canada. My results demonstrate that helminth assemblages of two garter snake species (red-sided garter snakes, T. sirtalis parietalis, and plains garter snakes, T. radix) include Lechriorchis trematodes and Rhabdias nematodes in the lung, Alaria mesocercariae in the tail, and diplostomid trematode metacercariae in the visceral fat; red-sided garter snakes also had gastrointestinal cestodes. Helminth assemblages varied, mainly in terms of parasite density, among populations of red-sided garter snakes and between red-sided and plains garter snakes, but it is unclear whether this variation is due simply to diet-based differences in parasite exposure or whether variation in parasite resistance may have a role. Notably, for plains garter snakes and one red-sided garter snake population I found helminth densities to be predictive of male fitness correlates, namely body condition, testes mass, and sperm counts. Thus, parasitism in garter snakes clearly has important implications for reproductive fitness beyond just influencing tail loss. These results highlight the importance of considering more than a single parasite or single fitness correlate when exploring host-parasite relationships. The consequences of parasitic infections may arise simply through the activation of the host’s immune system rather than the presence of parasites. Thus, in chapter 4, "Changes in reproductive investment and hormone levels in response to an acute immune challenge", I use lipopolysaccharide (LPS) to assess immune-reproductive tradeoffs of male red-sided garter snakes during the breeding season. As LPS is non-pathogenic, I was able to assess the fitness implications of the immune activation itself. My results showed that males depress courtship behaviors and mating success when faced with a single acute immune challenge. For LPS-treated males that did mate, copulatory plug mass was significantly lower compared to controls, while sperm counts did not differ between treatments. This result likely reflects the dissociated breeding pattern of these snakes as spermatogenesis occurs outside the breeding season and, thus, sperm stores were already in place prior to the immune challenge whereas plug material is produced during the breeding season. Further, the LPS treatment was correlated with increased plasma levels of corticosterone, which were 1.8 times higher in LPS-treated males compared to controls, and decreased levels of androgens, which, in LPS-treated males, were only one third as high as androgen levels in control males. Thus, the observed immune-reproduction tradeoff appeared to be hormonally-mediated. Indeed, the low breeding season androgen levels characteristic of this dissociated breeder may have relaxed testosterone-mediated immunosuppression and so facilitate immune-induced suppression of reproductive behaviors. The results of this study highlight the influence of host life history on the consequences of immune activation and also emphasize the complex interactions between the immune, reproductive and endocrine systems. In chapter 5, "Implications of repeated immune challenges in a capital breeder with prolonged hibernation", I again utilized LPS as a means of investigating the implications of immune activation. In this study, I administered a series of LPS injections to male and mated female snakes throughout the summer feeding season, and, for males, into the autumn. Females give birth during the summer and males undergo testicular recrudescence and spermatogenesis during summer and into autumn so these seasons represent important reproductive periods for red-sided garter snakes. Also, as capital breeders, it is during the summer feeding season that snakes of both sexes accumulate the resources upon which they will rely throughout hibernation and the subsequent breeding season. For the most part, my results did not demonstrate clear immune-reproductive tradeoffs. It appears that the absence of tradeoffs may be due to immune-challenged males and gravid female compensating for the immune challenge and maintaining reproductive processes by increasing their food intake, which was not limited during the study. Indeed, LPS-treated gravid females actually had more offspring per litter compared to gravid control females, suggesting that the immune challenge led to greater investment in offspring. In contrast to gravid females, non-gravid females treated with LPS exhibited reduced food intake which may reflect a survival strategy as anorexia during infections tends to be beneficial for survival. Interestingly, the increased food consumption of males did not translate into greater fat stores, but rather higher liver masses which may be indicative of immunopathological changes which should be explored in future studies. Title: Reproductive implications of parasitic infections and immune challenges in garter snakes Author: Uhrig, Emily J. Parasitic infections and immune challenges can affect host reproductive fitness and, ultimately, the evolution of host populations in a myriad of ways. The fitness implications of parasitic infections range from increased host mortality to subtle changes in reproductive investment. From alterations of behaviors, sexual signaling, and competitive ability to changes in gamete production and fertilization success, it is clear that parasites are capable of mediating sexual selection and influencing host reproductive fitness even without altering mortality. The mechanisms underlying fitness effects highlight the complexity of the host-parasite relationship which involves immune responses as well as a range of other, often interactive, physiological processes within the host. In some instances, it is not the direct effect of parasites per se, but rather the hosts' responses to infection that mediate fitness consequences. This dissertation presents studies designed to elucidate the implications of parasitism and immune responses for the reproductive fitness of garter snakes (genus Thamnophis). In chapter 2, "Alaria mesocercariae in the tails of red-sided garter snakes: evidence for parasite-mediated caudectomy", I focus on the histopathological changes associated with a trematode (Alaria sp.) infecting the tails of red-sided garter snakes (T. sirtalis parietalis). My results demonstrate that Alaria mesocercariae occur in high density within the tail tissue of both male and female snakes with as many as 2,000 mesocercariae in a single tail; infection prevalence was 100% in the snakes I examined. I found no evidence of intersexual variation in pathological changes or infection densities. For both sexes, external pathological manifestations include swelling of the tail while, internally, the aggregation of mesocercariae leads to the formation of mucus-filled pseudocysts and damage of muscle tissue. In severe cases, the extent of tissue destruction appeared to weaken the connection of the tail to the rest of the body, a condition that would facilitate tail breakage, which in turn negatively affects the snake's fitness by impairing mating success. From the parasite's perspective, tail breakage is likely beneficial by facilitating its transmission to subsequent hosts in its life cycle. Alaria sp. are not the only parasites commonly infecting garter snakes and in chapter 3, "Patterns in parasitism: interspecific and interpopulational variation in helminth assemblages and their reproductive fitness correlates in garter snakes", I broaden our investigation to include a suite of helminth parasites common in the garter snakes of Manitoba, Canada. My results demonstrate that helminth assemblages of two garter snake species (red-sided garter snakes, T. sirtalis parietalis, and plains garter snakes, T. radix) include Lechriorchis trematodes and Rhabdias nematodes in the lung, Alaria mesocercariae in the tail, and diplostomid trematode metacercariae in the visceral fat; red-sided garter snakes also had gastrointestinal cestodes. Helminth assemblages varied, mainly in terms of parasite density, among populations of red-sided garter snakes and between red-sided and plains garter snakes, but it is unclear whether this variation is due simply to diet-based differences in parasite exposure or whether variation in parasite resistance may have a role. Notably, for plains garter snakes and one red-sided garter snake population I found helminth densities to be predictive of male fitness correlates, namely body condition, testes mass, and sperm counts. Thus, parasitism in garter snakes clearly has important implications for reproductive fitness beyond just influencing tail loss. These results highlight the importance of considering more than a single parasite or single fitness correlate when exploring host-parasite relationships. The consequences of parasitic infections may arise simply through the activation of the host’s immune system rather than the presence of parasites. Thus, in chapter 4, "Changes in reproductive investment and hormone levels in response to an acute immune challenge", I use lipopolysaccharide (LPS) to assess immune-reproductive tradeoffs of male red-sided garter snakes during the breeding season. As LPS is non-pathogenic, I was able to assess the fitness implications of the immune activation itself. My results showed that males depress courtship behaviors and mating success when faced with a single acute immune challenge. For LPS-treated males that did mate, copulatory plug mass was significantly lower compared to controls, while sperm counts did not differ between treatments. This result likely reflects the dissociated breeding pattern of these snakes as spermatogenesis occurs outside the breeding season and, thus, sperm stores were already in place prior to the immune challenge whereas plug material is produced during the breeding season. Further, the LPS treatment was correlated with increased plasma levels of corticosterone, which were 1.8 times higher in LPS-treated males compared to controls, and decreased levels of androgens, which, in LPS-treated males, were only one third as high as androgen levels in control males. Thus, the observed immune-reproduction tradeoff appeared to be hormonally-mediated. Indeed, the low breeding season androgen levels characteristic of this dissociated breeder may have relaxed testosterone-mediated immunosuppression and so facilitate immune-induced suppression of reproductive behaviors. The results of this study highlight the influence of host life history on the consequences of immune activation and also emphasize the complex interactions between the immune, reproductive and endocrine systems. In chapter 5, "Implications of repeated immune challenges in a capital breeder with prolonged hibernation", I again utilized LPS as a means of investigating the implications of immune activation. In this study, I administered a series of LPS injections to male and mated female snakes throughout the summer feeding season, and, for males, into the autumn. Females give birth during the summer and males undergo testicular recrudescence and spermatogenesis during summer and into autumn so these seasons represent important reproductive periods for red-sided garter snakes. Also, as capital breeders, it is during the summer feeding season that snakes of both sexes accumulate the resources upon which they will rely throughout hibernation and the subsequent breeding season. For the most part, my results did not demonstrate clear immune-reproductive tradeoffs. It appears that the absence of tradeoffs may be due to immune-challenged males and gravid female compensating for the immune challenge and maintaining reproductive processes by increasing their food intake, which was not limited during the study. Indeed, LPS-treated gravid females actually had more offspring per litter compared to gravid control females, suggesting that the immune challenge led to greater investment in offspring. In contrast to gravid females, non-gravid females treated with LPS exhibited reduced food intake which may reflect a survival strategy as anorexia during infections tends to be beneficial for survival. Interestingly, the increased food consumption of males did not translate into greater fat stores, but rather higher liver masses which may be indicative of immunopathological changes which should be explored in future studies. Close

• 1590. [Article] Vertebral elemental markers in elasmobranchs : potential for reconstructing environmental history and population structure

  Differences in the chemical composition of calcified structures can be used to reveal natal origins, connectivity, metapopulation structure, and reconstruct the environmental history or movement patterns ...

  Citation × Citation Citation Abstract Copy Title: Vertebral elemental markers in elasmobranchs : potential for reconstructing environmental history and population structure Author: Smith, Wade D. Differences in the chemical composition of calcified structures can be used to reveal natal origins, connectivity, metapopulation structure, and reconstruct the environmental history or movement patterns of many marine organisms. Sharks, skates, and rays (elasmobranchs) lack the calcified structures, known as otoliths, that are typically used for geochemical studies of dispersal and natal origin in fishes. If the incorporation of elements into shark and ray vertebrae is related to environmental conditions, the geochemical composition of cartilaginous vertebrae may also serve as natural tags and records of environmental history in elasmobranch populations. I used complementary laboratory and field studies to address several key assumptions regarding the incorporation of elements in elasmobranch vertebrae, providing the first detailed studies to assess relationships between water and vertebral chemical composition and the spatial and temporal variation of vertebral elemental signatures in this subclass of fishes. To validate the uptake and incorporation of elements from water to vertebrae, I conducted two laboratory studies using round stingrays, Urobatis halleri, as a model species. First, I examined the effects of temperature (16°, 18°, 24° C) on vertebral elemental incorporation (Li/Ca, Mg/Ca, Mn/Ca, Zn/Ca, Sr/Ca, Ba/Ca) and found that temperature had strong, negative effects on the uptake (measured as a partition coefficient, D[subscript Element]) of magnesium and Ba and positively influenced manganese incorporation. Second, I tested the relationship between water and vertebral elemental composition by manipulating dissolved barium (Ba) concentrations (1x, 3x, 6x ambient concentrations) and found significant differences among rays from each treament. I also evaluated the influence of natural variation in somatic growth and vertebral precipitation rates on elemental incorporation. Finally, I examined the accuracy of classifying individuals to known environmental histories (temperature and barium treatments) using vertebral elemental composition. There were no significant relationships between elemental incorporation and somatic growth or vertebral precipitation rates for any elements with the exception of Zn. Relationships between somatic growth rate and D[subscript Zn] were, however, inconsistent and inconclusive. Elemental variation of vertebrae reliably distinguished U. halleri based on temperature (85%) and [Ba] (96%) history. These results support the assumption that vertebral elemental composition reflects the environmental conditions during deposition and validates the use of vertebral elemental signatures as natural markers in an elasmobranch. To evaluate the utility of vertebral geochemistry as intrinsic markers of natal origin, I collected vertebrae of young-of-the-year scalloped hammerhead sharks (Sphyrna lewini) from artisanal fishery landings at six sites along the Pacific coast of Mexico and Costa Rica between 2007-2009. A total of 386 vertebrae were used to assess patterns of spatial and temporal variation in elemental composition using laser ablation-inductively coupled plasma mass spectrometry. A protracted pupping period was confirmed for S. lewini, with newborn pups being recorded from May through mid-October. Natal elemental signatures detected in the vertebrae of the sharks varied significantly among sites and could be used to identify source populations. All element-to-calcium ratios included in these analyses (Li/Ca, Mg/Ca, V/Ca, Cr/Ca, Mn/Ca, Rb/Ca, Sr/Ca, Ba/Ca, Pb/Ca) were useful for the discerning natal origins of sharks; however, Ba, Sr, Mn, and Mg ratios most consistently generated the greatest discriminatory power based on step-wise discriminant function analyses. Classification accuracy to putative nursery areas (natal signature) and location of capture (edge signature) based on step-wise discriminant function analysis ranged from low (30-60%) to high (80-100%) depending on the degree of spatial and temporal resolution by which the data were filtered for analysis (e.g. pooled across months, early season, late season). All classification accuracies exceeded chance expectations and assignment to putative nursery areas and sites of capture were accomplished with up to 100% accuracy in several models. I found significant intra-annual differences in natal elemental signatures within the three primary study sites, which likely contributed to the low assignment accuracies when data were grouped across months of collection. Significant differences in natal elemental signatures were also detected across years. However, pair-wise analyses revealed that site-specific inter-annual variation was driven by differences associated with samples collected in 2009. Natal elemental signatures were similar between 2007 and 2009, indicating some consistency in site-specific vertebral chemistry across years. These results confirmed that vertebral elemental signatures can be applied to distinguish individuals across small (5s km), moderate (100s km), and large spatial scales (>1000 km). The potential for intra-annual variation in natal signatures within a year-class highlights the importance of cohort-specific analyses and the development of a spatial atlas of natal vertebral elemental signatures for studies of natal origin and population connectivity. The findings of my laboratory validation experiments and field study establish that geochemical analyses of vertebrae can provide reliable information on the spatial ecology and environmental history of shark and ray populations. The use of elemental signatures offers a new approach for the study and conservation of this historically vulnerable group of fishes. Title: Vertebral elemental markers in elasmobranchs : potential for reconstructing environmental history and population structure Author: Smith, Wade D. Differences in the chemical composition of calcified structures can be used to reveal natal origins, connectivity, metapopulation structure, and reconstruct the environmental history or movement patterns of many marine organisms. Sharks, skates, and rays (elasmobranchs) lack the calcified structures, known as otoliths, that are typically used for geochemical studies of dispersal and natal origin in fishes. If the incorporation of elements into shark and ray vertebrae is related to environmental conditions, the geochemical composition of cartilaginous vertebrae may also serve as natural tags and records of environmental history in elasmobranch populations. I used complementary laboratory and field studies to address several key assumptions regarding the incorporation of elements in elasmobranch vertebrae, providing the first detailed studies to assess relationships between water and vertebral chemical composition and the spatial and temporal variation of vertebral elemental signatures in this subclass of fishes. To validate the uptake and incorporation of elements from water to vertebrae, I conducted two laboratory studies using round stingrays, Urobatis halleri, as a model species. First, I examined the effects of temperature (16°, 18°, 24° C) on vertebral elemental incorporation (Li/Ca, Mg/Ca, Mn/Ca, Zn/Ca, Sr/Ca, Ba/Ca) and found that temperature had strong, negative effects on the uptake (measured as a partition coefficient, D[subscript Element]) of magnesium and Ba and positively influenced manganese incorporation. Second, I tested the relationship between water and vertebral elemental composition by manipulating dissolved barium (Ba) concentrations (1x, 3x, 6x ambient concentrations) and found significant differences among rays from each treament. I also evaluated the influence of natural variation in somatic growth and vertebral precipitation rates on elemental incorporation. Finally, I examined the accuracy of classifying individuals to known environmental histories (temperature and barium treatments) using vertebral elemental composition. There were no significant relationships between elemental incorporation and somatic growth or vertebral precipitation rates for any elements with the exception of Zn. Relationships between somatic growth rate and D[subscript Zn] were, however, inconsistent and inconclusive. Elemental variation of vertebrae reliably distinguished U. halleri based on temperature (85%) and [Ba] (96%) history. These results support the assumption that vertebral elemental composition reflects the environmental conditions during deposition and validates the use of vertebral elemental signatures as natural markers in an elasmobranch. To evaluate the utility of vertebral geochemistry as intrinsic markers of natal origin, I collected vertebrae of young-of-the-year scalloped hammerhead sharks (Sphyrna lewini) from artisanal fishery landings at six sites along the Pacific coast of Mexico and Costa Rica between 2007-2009. A total of 386 vertebrae were used to assess patterns of spatial and temporal variation in elemental composition using laser ablation-inductively coupled plasma mass spectrometry. A protracted pupping period was confirmed for S. lewini, with newborn pups being recorded from May through mid-October. Natal elemental signatures detected in the vertebrae of the sharks varied significantly among sites and could be used to identify source populations. All element-to-calcium ratios included in these analyses (Li/Ca, Mg/Ca, V/Ca, Cr/Ca, Mn/Ca, Rb/Ca, Sr/Ca, Ba/Ca, Pb/Ca) were useful for the discerning natal origins of sharks; however, Ba, Sr, Mn, and Mg ratios most consistently generated the greatest discriminatory power based on step-wise discriminant function analyses. Classification accuracy to putative nursery areas (natal signature) and location of capture (edge signature) based on step-wise discriminant function analysis ranged from low (30-60%) to high (80-100%) depending on the degree of spatial and temporal resolution by which the data were filtered for analysis (e.g. pooled across months, early season, late season). All classification accuracies exceeded chance expectations and assignment to putative nursery areas and sites of capture were accomplished with up to 100% accuracy in several models. I found significant intra-annual differences in natal elemental signatures within the three primary study sites, which likely contributed to the low assignment accuracies when data were grouped across months of collection. Significant differences in natal elemental signatures were also detected across years. However, pair-wise analyses revealed that site-specific inter-annual variation was driven by differences associated with samples collected in 2009. Natal elemental signatures were similar between 2007 and 2009, indicating some consistency in site-specific vertebral chemistry across years. These results confirmed that vertebral elemental signatures can be applied to distinguish individuals across small (5s km), moderate (100s km), and large spatial scales (>1000 km). The potential for intra-annual variation in natal signatures within a year-class highlights the importance of cohort-specific analyses and the development of a spatial atlas of natal vertebral elemental signatures for studies of natal origin and population connectivity. The findings of my laboratory validation experiments and field study establish that geochemical analyses of vertebrae can provide reliable information on the spatial ecology and environmental history of shark and ray populations. The use of elemental signatures offers a new approach for the study and conservation of this historically vulnerable group of fishes. Close