Thesis (Ph.D.) - University of St Andrews, September 2008. / Electronic version restricted until 16th September 2009.

Thesis (Ph.D.)--University of Tasmania, 2000. / Library has additional copy on CD-ROM. Includes bibliographical references.

Thesis (Ph.D.) - University of St Andrews, May 2007.

Submitted by William Justo Figueiro (williamjf) on 2015-08-11T21:04:47Z No. of bitstreams: 1 13e.pdf: 3156102 bytes, checksum: da00a19d9782f6ac017289a5d272ac1f (MD5) / Made available in DSpace on 2015-08-11T21:04:47Z (GMT). No. of bitstreams: 1 13e.pdf: 3156102 bytes, checksum: da00a19d9782f6ac017289a5d272ac1f (MD5) Previous issue date: 2013 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPQ – Conselho Nacional de Desenvolvimento Científico e Tecnológico / INCT-APA - Instituto Nacional de Ciência e Tecnologia Antártico de Pesquisas Ambientais / PROANTAR - Programa Antártico Brasileiro / SECIRM - Secretaria Interministerial para os Recursos do Mar / MMA - Ministério do meio Ambiente / INCT-APA - Instituto Nacional de Ciência e Tecnologia Antártico de Pesquisas Ambientais / UNISINOS - Universidade do Vale do Rio dos Sinos / O nicho realizado de duas espécies precisa ser diferente, para que estas espécies possam coexistir de maneira estável. Pygoscelis papua e P. antarcticus reproduzem simpatricamente na Ilha Elefante, Antártica. Foram coletadas amostras de conteúdo estomacal e realizado biometria das duas espécies para analisar a dieta e a sobreposição de nicho. Um total de 56 amostras de P. papua e 71 amostras de P. antarcticus foram coletadas durante dois períodos de reprodução austral, 2010/11 e 2011/12, na região de Stinker Point. A proporção de itens variou entre as espécies de pinguins e entre os táxons representados. E. superba foi a presa de maior abundância comparado com outros itens alimentares, com aproximadamente 69% FO para P. papua e 98% FO para P. antarcticus. Foram encontradas nove espécies de peixe, seis espécies de crustáceos e uma espécie de cefalópode, identificados a nível específico. Existe uma diferença significativa entre os fatores, espécie de pinguim, tamanho da carapaça e sexo de krill antártico, quando comparados entre si. Além disso, a biometria indica que o comprimento do bico e peso de P. papua são maiores que em P. antarcticus, da mesma forma que o bico é mais alto e mais largo em P. antarcticus. Tais diferenças morfológicas podem explicar as diferenças no forrageio. Foi observada uma sobreposição de nicho trófico entre as espécies, pela utilização dos mesmos recursos, uma vez que estas espécies ocorrem simpatricamente na mesma região. Este estudo demonstra que a variação específica do nicho trófico ocupado pelas espécies pode ser definida pelo comportamento de forrageio e pela seleção dos recursos alimentares de cada espécie. É importante a recomendação destas espécies como indicadores de qualidade ambiental, adicionando questões como a variabilidade local, pois o nicho trófico pode alterar ao longo do tempo. / The two species realized niche needs to be different, since these species can coexist stably. Pygoscelis papua and P. antarcticus breed simpatricaly on Elephant Island, Antarctica. Stomach content samples were collected and biometrics of both species was measured in order to analyze the diet and the niche overlap. A total of 56 P. papua samples and 71 P. antarcticuss samples were collected, during the two austral breeding seasons, 2010/11 and 2011/12, on the Stinker Point region. The proportion of items ranged from the penguin species and among the taxa represented. E. superba was the most abundant prey compared with other food items, with approximately 69% FO for P. papua, and 98% FO for P. antarcticus. We found nine species of fish, six species of crustaceans and one species of cephalopod, identified by the specific level. There is a significant difference between the factors, species of penguin, carapace length and sex of antarctic krill, when compared with each other. In addition, the biometrics indicates that length of P. papua’s beak and weight are larger than P. antarcticus, but P. antarcticus has the highest and widest beak measures. Such morphological differences may explain differences in foraging. We observed a niche overlap of species, by the use of the same food resources, once these species occurs simpatricaly in the same region. This study also demonstrated that the specific variation of trophic niches occupied by the species may be defined by the foraging behavior and by the selection of the food resources. It is important the recommendation of these species as indicators of environmental quality, adding issues such as local variability since the trophic niche may change over the time.

1. Antarctic krill (Euphausia superba) form aggregations known as swarms that vary greatly in size and density. Six acoustic surveys were conducted as part of multidisciplinary studies at two study sites, the western and eastern core boxes (WCB and ECB), during the 1997, 1998 and 1999 austral summers, at South Georgia. A quantitative, automated, image processing algorithm was used to identify swarms, and calculate swarm descriptors, or metrics. In contrast to acoustic surveys of aggregations of other pelagic species, a strong correlation (r = 0.88, p = 0.02, 95% C.I.= 0.24 to 0.99) between the number of krill swarms and the mean areal krill density [rho.hat] was found. Multivariate analysis was used to partition swarms into three types, based on contrasting morphological and internal krill density parameters. Swarm types were distributed differently between inter-surveys and between on and off-shelf regions. This swarm type variation has implications for krill predators, by causing spatial heterogeneity in swarm detectability, suggesting that for optimal foraging to occur, predators must engage in some sort of adaptive foraging strategy. 2. Krill predator-prey interactions were found to occur at multiple spatial and temporal scales, in a nested, or hierarchical structure. At the largest inter-survey scale, an index of variability, I, was developed to compare variation in survey-scale predator sightings, sea temperature and [rho.hat]. Using I and a two-way ANOVA, core box, rather than year, was found to be a more important factor in determining species distribution. The absence of Blue-petrels (Halobaena caerulea) and the elevated number of Antarctic fur seals (Arctocephalus gazella) suggest that 1998 was a characterised by colder than average water surrounding South Georgia, and a high [rho.hat] in the ECB. At the smaller, intra-survey scales (<80 km, <5 day), the characteristic scale (distances in which predator group size, or krill density were similar, L_s) were determined. For krill and predators L_s varied by survey and the L_s of krill also varied by depth within a survey. Overlap in L_s were stronger between predator species than between a predator species and krill, indicating predators were taking foraging cues from the activity of predators, rather than from the underlying krill distribution. No relationship was found between swarm characteristics and predator activity, suggesting either there is no relationship between krill swarms and predators, or that the predator and acoustic observation techniques may not be appropriate to detect such a relationship. 3. To overcome the 2-D sampling limitations of conventional echosounders, a multibeam echosounder (MBE) observed entire swarms in three-dimensions. Swarms found in the nearshore environment of Livingston Island situated in the South Shetland Islands, exhibited only a narrow range of surface area to volume ratios or roughnesses (R = 3.3, CV = 0.23), suggesting that krill adopt a consistent group behaviour to maintain swarm shape. Generalized additive models (GAM) suggested that the presence of air-breathing predators influenced the shape of a krill swarm (R decreased in the presence of predators: the swarm became more spherical). A 2D distance sampling framework was used to estimate the abundance, N, and associated variance of krill swarms. This technique took into account angular and range detectability (half-normal, [sigma_r.hat] = 365.00 m, CV = 0.16) and determined the vertical distribution of krill swarms to be best approximated by a beta-distribution ([alpha.hat] = 2.62, [CV.hat] = 0.19; [beta.hat] = 2.41, [CV.hat] = 0.15), giving the abundance of swarms in survey region as [N.hat] = 5,062 ([CV.hat] = 0.35). This research represents a substantial contribution to developing estimation of pelagic biomass using MBEs. 4. When using a single- or split-beam missing pings occur when the transmit or receive cycles are interrupted, often by aeration of the water column, under the echosounder transducer during rough weather. A thin-plate regression spline based approach was used to model the missing krill data, with knots chosen using a branch and bound algorithm. This method performs well for acoustic observations of krill swarms where data are tightly clustered and change rapidly. For these data the technique outperformed the standard MGCV GAM, and the technique is applicable for estimating acoustically derived biomass from line transect surveys.

The zooplankton community of Marguerite Bay, western Antarctic Peninsula, was investigated in relation to variability in chlorophyll concentrations and sea ice dynamics, using a combination of satellite remote sensing techniques and plankton net data. SeaWiFS chlorophyll data were validated with concurrent in situ data measured by HPLC and fluoromentric methods, and results indicate that SeaWiFS chlorophyll is an accurate measure of in situ values when HPLC data are used as ground truth. Climatology data of SeaWiFS chlorophyll west of the Antarctic Peninsula showed that the Bellingshausen Sea and Marguerite Bay usually had higher and more persistent chlorophyll concentrations compared with northern regions. These predictable phytoplankton blooms could provide the Antarctic krill, Euphausia superba, with the food required for successful reproduction and larval survival. Unusually high krill reproduction in 2000/2001 was coincident with above-average chlorophyll concentrations throughout the study area and was followed by the largest juvenile recruitment since 1981. High larval densities at the shelf break along the Antarctic Peninsula may have resulted, in part, from krill spawning in the Bellingshausen Sea. Interannual differences in sea ice also probably contributed to the variability in larval krill abundances. Interannual differences were observed in the species composition of the zooplankton of Marguerite Bay during fall, and these were linked to variability in the environmental conditions. Thysanoessa macrura was the most abundant euphausiid in 2001, while Euphausia crystallorophias dominated in 2002, and E. superba had intermediate densities during both years. Copepods were more abundant in 2001 by a factor of 2.6. Copepods and T. macrura showed a rapid population response to unusually high chlorophyll concentrations in the Bellingshausen Sea and Marguerite Bay during spring-summer 2000/2001, whereas E. superba and E. crystallorophias had a longer term response and showed increased recruitment in fall 2002. There were no clear associations between the distribution of zooplankton and environmental conditions in fall; however there was a significant relationship between chlorophyll concentrations in the Bellingshausen Sea during the preceding spring and zooplankton patterns during fall.

Respiration, metabolic enzyme assays, and body composition parameters were measured in the Antarctic krill Euphausia superba during the summer, fall and winter on the Western Antarctic Peninsula (WAP). E. superba of all sizes decrease their metabolism from the summer to the winter. These same parameters were also measured along the WAP during the austral fall 2010. E. superba's enzyme activity indicated that there was a latitudinal gradient to the decline in metabolism along the WAP with the more northerly sites having significantly higher metabolic enzyme activities than the sites to the south. Carbon (δ13C) and nitrogen (δ15N) stable isotopes were measured in E. superba along the WAP to determine if there were any latitudinal trends. δ13C showed a significant trend with latitude with more depleted δ13C values in the southern portion of the WAP. Carbon (δ13C) and nitrogen (δ15N) stable isotopes were also measured in two important prey fishes along the WAP, the silverfish Pleuragramma antarcticum and the myctophid Electrona antarctica. P. antarcticum had a more variable and more enriched δ13C value than E. antarctica indicative of P. antarcticum's more neritic habitat. There were no significant differences between the δ15N values of the two fish, indicating that although they feed in different areas they were feeding at the same trophic level. Carbon (δ13C) and nitrogen (δ15N) stable isotopes were measured in twenty species in the marginal ice zone (MIZ) of the Weddell Sea at the beginning of the austral summer. Samples were taken from under the ice, at the ice edge and in the open ocean. A significant trend in the δ13C values of all species was found with the under-ice δ13C values being more depleted than those in the open ocean. This is most likely due to the reduced atmospheric exchange of CO2, upwelled water with depleted δ13C values, and continuous biological respiration under the ice, all of which contribute to very depleted δ13C values. δ15N values were significantly lower in the open ocean than the other ice conditions due to the increased reliance on primary production. The diapausing copepods Calanoides acutus and Rhincalanus gigas showed similar patterns in their isotopic signatures across the ice zones. Cluster analysis revealed trophic shifts between the different ice zones. The ice edge zone proved to contain the most species and was the best habitat for most species. The trophic shifts observed within species in the differing ice conditions mimicked the seasonal changes they undergo during the course of the productive season every year.

South Georgia is renowned for the abundance of Antarctic krill (Euphausia superba) and a range of krill predators. Variability in krill availability at a range of scales, and the consequences of this for predator-prey interactions, mean that quantifying the spatially explicit demand for krill by those predators is essential to understanding the mechanisms underlying ecosystem changes in the region. In this thesis demand within a distinct study box to the northwest of the island has been assessed. The thesis has three sections; (1) the number of predators; (2) the distribution of predators; and (3) the demand for krill by those predators. (1) Predator densities with confidence intervals were determined from appropriately designed shipboard transect survey; counts of Antarctic fur seals (Arctocephalus gazella), macaroni penguins (Eudyptes chrysolophus), gentoo penguins (Pygoscelis papua), and Antarctic prions (Pachyptila desolata) were adjusted for sea state, distance from observer and dive behaviour. Providing the first at-sea predator density estimates for the region. (2) A comparison of the distribution of female Antarctic fur seals engaged in pup-rearing (using satellite telemetry) and the whole population that were not restricted to a single part of the population (from shipboard transect survey) was undertaken. Using two general additive models based on the relationship between seal distribution (one derived from transect and the other from telemetry) and the physical environment indicated that the spatial distribution of lactating females is representative of the general population. (3) Using the derived predator density, the local krill demand estimate was 2581 tonnes krill per day, a consumption rate of 0.45% per day of the concurrently estimated krill biomass (using shipboard acoustics). Antarctic fur seals accounted for 75% of this demand. This level of demand was less than the increase in biomass resulting from krill growth. However, based on the length-specific demand, determined from concurrent predator diet samples demand exceeded growth for krill >48mm.

Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described. In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied. In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I. Key words:Proteins, peptides, proteases, Antarctickrill,Euphausia superba, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.

<p>Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described.</p><p>In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (<i>Euphausia superba</i>) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied.</p><p>In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I.</p><p><b>Key words:</b>Proteins, peptides, proteases, Antarctickrill,<i>Euphausia superba</i>, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.</p>