The effects of noise exposure and genetic background on auditory-evoked behaviors in larval zebrafish

Thesis (Ph.D.)--University of Washington, 2016-06

Interspecific variation in the use of carotenoid-based coloration in birds: diet, life history and phylogeny

Birds show striking interspecific variation in their use of carotenoid-based coloration. Theory predicts that the use of carotenoids for coloration is closely associated with the availability of carotenoids in the diet but, although this prediction has been supported in single-species studies and those using small numbers of closely related species, there have been no broad-scale quantitative tests of the link between carotenoid coloration and diet. Here we test for such a link using modern comparative methods, a database on 140 families of birds and two alternative avian phylogenies. We show that carotenoid pigmentation is more common in the bare parts (legs, bill and skin) than in plumage, and that yellow coloration is more common than red. We also show that there is no simple, general association between the availability of carotenoids in the diet and the overall use of carotenoid-based coloration. However, when we look at plumage coloration separately from bare part coloration, we find there is a robust and significant association between diet and plumage coloration, but not between diet and bare part coloration. Similarly, when we look at yellow and red plumage colours separately, we find that the association between diet and coloration is typically stronger for red coloration than it is for yellow coloration. Finally, when we build multivariate models to explain variation in each type of carotenoid-based coloration we find that a variety of life history and ecological factors are associated with different aspects of coloration, with dietary carotenoids only being a consistent significant factor in the case of variation in plumage. All of these results remain qualitatively unchanged irrespective of the phylogeny used in the analyses, although in some cases the precise life history and ecological variables included in the multivariate models do vary. Taken together, these results indicate that the predicted link between carotenoid coloration and diet is idiosyncratic rather than general, being strongest with respect to plumage colours and weakest for bare part coloration. We therefore suggest that, although the carotenoid-based bird plumage may a good model for diet-mediated signalling, the use of carotenoids in bare part pigmentation may have a very different functional basis and may be more strongly influenced by genetic and physiological mechanisms, which currently remain relatively understudied.

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development

The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass. notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tall and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tall. Imo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

A study of the regulatory role of retinoic acid receptor gamma in Zebrafish development

Retinoic acid (RA) is thought to signal through retinoic acid receptors (RARs), i.e. RARα, β, and γ to play important roles in embryonic development and tissue regeneration. In this thesis, the zebrafish (Danio rario) was used as a vertebrate model organism to examine the role of RARγ. Treatment of zebrafish embryos with a RARγ specific agonist reduced the axial length of developing embryos, associated with reduced somite number and loss of hoxb13a expression. There were no clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist disrupted the formation of anterior structures of the head, the cranial bones and the anterior lateral line ganglia, associated with a loss of sox9 immunopositive cells in the same regions. Pectoral fin outgrowth was blocked by treatment with the RARγ agonist; however, this was not associated with loss of tbx5a immunopositive lateral plate cells and was reversed by wash out of the RARγ agonist or co-treatment with a RARγ antagonist. Regeneration of the transected caudal fin was also blocked by RARγ agonist treatment and restored by agonist washout or antagonist co-treatment; this phenotype was associated with a localised reduction in canonical Wnt signalling. Conversely, elevated canonical Wnt signalling after RARγ treatment was seen in other tissues, including ectopically in the notochord. Furthermore, some phenotypes seen in the RARγ treated embryos were present in mutant zebrafish embryos in which canonical Wnt signalling was constitutively increased. These data suggest that RARγ plays an essential role in maintaining neural crest and mesodermal stem/progenitor cells during normal embryonic development and tissue regeneration when the receptor is in its non-ligated state. In addition, this work has provided evidence that the activation status of RARγ may regulate hoxb13a gene expression and canonical Wnt signalling. Further research is required to confirm such novel regulatory roles.

Arsenic transport by zebrafish aquaglyceroporins

Background Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized. Results In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were namedaqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic. Conclusion This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

Efeitos da privação do sono em tarefas cognitivas

Learning and memory are important mechanism for species, since its allows to recognize conspecifics, routes and food place. Sleep is one of behaviors known by facilitate learning, it is a widespread phenomenon, present in most of vertebrates lives and highly investigated in many aspects. It is known that sleep deprivation modifies physiologic behavioral processes in animals, however, sleep function in organism is still debatable. Hypothesis range from energy conservation to memory consolidation, with different roles in animal’s evolution. The zebrafish (Danio rerio) emerg e in the last years as vertebrate model in genetics and developmental biology and quickly become popular in behavioral studies, as learning and memory. Despite the fact that zebrafish is a diurnal animal and have well characterized sleep behavior, zebrafish fish still has advantages due to its small size and low cost of maintenance, whichestablishes this species as interesting model for research on sleep. In this study we aimed to analyze the effects of partial and total sleep deprivation on learning acquisition, as well the concomitant administration of alcohol and melatonin. For this, the research was divided in three phases, each one with a different kind of conditioning: (1) object Recognition, (2) avoidance conditioning and (3) appetitive conditioning. The results showed the fish partially sleep deprived and totally sleep deprived + et hanol could perform the tasks just like the control group, however, fish totally sleep deprived and totally sleep deprived + melatonin showed impairments in attention and memory during the tests. Our results suggest that only one night of sleep deprivation is enough to harm the zebrafish performance in cognitive tasks. In addition, ethanol exposure on the night previously the test seems to suppress the negative effects of sleep deprivation, while the melatonin treatment seems not to be enough to promote sleep state, at least on the protocol applied here.

Efeitos da privação do sono em tarefas cognitivas

Learning and memory are important mechanism for species, since its allows to recognize conspecifics, routes and food place. Sleep is one of behaviors known by facilitate learning, it is a widespread phenomenon, present in most of vertebrates lives and highly investigated in many aspects. It is known that sleep deprivation modifies physiologic behavioral processes in animals, however, sleep function in organism is still debatable. Hypothesis range from energy conservation to memory consolidation, with different roles in animal’s evolution. The zebrafish (Danio rerio) emerg e in the last years as vertebrate model in genetics and developmental biology and quickly become popular in behavioral studies, as learning and memory. Despite the fact that zebrafish is a diurnal animal and have well characterized sleep behavior, zebrafish fish still has advantages due to its small size and low cost of maintenance, whichestablishes this species as interesting model for research on sleep. In this study we aimed to analyze the effects of partial and total sleep deprivation on learning acquisition, as well the concomitant administration of alcohol and melatonin. For this, the research was divided in three phases, each one with a different kind of conditioning: (1) object Recognition, (2) avoidance conditioning and (3) appetitive conditioning. The results showed the fish partially sleep deprived and totally sleep deprived + et hanol could perform the tasks just like the control group, however, fish totally sleep deprived and totally sleep deprived + melatonin showed impairments in attention and memory during the tests. Our results suggest that only one night of sleep deprivation is enough to harm the zebrafish performance in cognitive tasks. In addition, ethanol exposure on the night previously the test seems to suppress the negative effects of sleep deprivation, while the melatonin treatment seems not to be enough to promote sleep state, at least on the protocol applied here.

Aprendizagem espaço-temporal e efeitos das condições luminosas no aprendizado do peixe paulistinha

Para processar a informação ambiental e perceber o tempo, os indivíduos utilizam-se de pistas ambientais, como luz e temperatura, que servem como guias para o relógio interno. O mecanismo temporizador endógeno é chamado relógio circadiano, o qual comanda uma grande variedade de ritmos diários bioquímicos, fisiológicos e comportamentais presentes nos organismos. Com isso, os animais podem antecipar eventos espaço-temporalmente distribuídos e usar essa informação para organizar as atividades diárias, o que é uma vantagem adaptativa para os indivíduos, já que muitos fatores ambientais apresentam variação circadiana. Aprendizagem espaço-temporal (do inglês: "time-place learning’’-TPL) é a habilidade de associar lugares com importantes eventos biológicos em diferentes horas do dia. Em nosso estudo utilizamos como modelo o peixe paulistinha (Danio rerio), conhecido por ser altamente social, para testar aprendizagem espaço-temporal baseada em reforço social. Além disso, objetivamos averiguar os efeitos das condições de claro constante e escuro constante na aprendizagem espaço-temporal, e se nessas condições, a atividade do peixe paulistinha é alterada. Para isso, testamos três diferentes condições (n=10): grupo claro-escuro (CE), grupo claro constante (CC) e grupo escuro constante (EE) durante 30 dias da seguinte maneira: diariamente, um grupo de 5 peixes paulistinha foi introduzido em um recipiente localizado no compartimento da manhã (um dos lados do aquário), às 8:00h e retirado às 9:00h, e em outro recipiente do compartimento da tarde (lado oposto do aquário), às 17:00h e removido às 18:00h, servindo como estímulo para que o peixe experimental ocupasse o compartimento onde o grupo fosse colocado. O comportamento foi filmado nos dois horários, 15 minutos antes e durante os 60 minutos de exposição ao estímulo, no 15º e no 30ª dia, porém neste último, os peixes foram filmados sem a presença do estímulo a fim de averiguarmos a aprendizagem espaço-temporal. Por fim, para saber a influência das três condições luminosas na atividade dos peixes, filmamos os últimos 6 dias de teste, para registrar o padrão de atividade. Nossos resultados mostraram que em ciclo claro-escuro (CE) o peixe paulistinha apresenta TPL, bem como é capaz de antecipar a hora e local do estímulo (grupo de coespecíficos), enfatizando a importância do estímulo social para a aprendizagem. Em condições de claro constante e escuro constante, o peixe paulistinha não apresentou aprendizagem espaço-temporal. Ademais, após 30 dias em condições luminosas constantes (claro constante e escuro constante), o peixe paulistinha mantém ritmo circadiano, porém em claro constante sua atividade é aumentada e seu ritmo atividade-repouso é alterado, através de um padrão de atividade distribuída homogeneamente ao longo das 24h, ao invés de concentrada na subjetiva fase clara, como nos grupos de ciclo claro-escuro e escuro constante, os quais conservam o padrão de atividade diurno da espécie.

PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.

Evaluation of semi-passive treatment technologies for septic lagoon capacity expansion

In Canada, increases in rural development has led to a growing need to effectively manage the resulting municipal and city sewage without the addition of significant cost- and energy- expending infrastructure. Storring Septic Service Limited is a family-owned, licensed wastewater treatment facility located in eastern Ontario. It makes use of a passive waste stabilization pond system to treat and dispose of waste and wastewater in an environmentally responsible manner. Storring Septic, like many other similar small-scale wastewater treatment facilities across Canada, has the potential to act as a sustainable eco-engineered facility that municipalities and service providers could utilize to manage and dispose of their wastewater. However, it is of concern that the substantial inclusion of third party material could be detrimental to the stability and robustness of the pond system. In order to augment the capacity of the current facility, and ensure it remains a self-sustaining system with the capacity to safely accept septage from other sewage haulers, it was hypothesized that pond effluent treatment could be further enhanced through the incorporation of one of three different technology solutions, which would allow the reduction of wastewater quality parameters below existing regulatory effluent discharge limits put in place by Ontario’s Ministry of the Environment and Climate Change (MOECC). Two of these solutions make use of biofilm technologies in order to enhance the removal of wastewater parameters of interest, and the third utilizes the natural water filtration capabilities of zebra mussels. Pilot-scale testing investigated the effects of each of these technologies on treatment performance under both cold and warm weather operation. This research aimed to understand the important mechanisms behind biological filtration methods in order to choose and optimize the best treatment strategy for full-scale testing and implementation. In doing so, a recommendation matrix was elaborated provided with the potential to be used as a universal operational strategy for wastewater treatment facilities located in environments of similar climate and ecology.

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

A new method for resolving uncertainty of energy requirements in large water breathers: the ‘mega-flume’ seagoing swim-tunnel respirometer

Body size is a key determinant of metabolic rate, but logistical constraints have led to a paucity of energetics measurements from large water-breathing animals. As a result, estimating energy requirements of large fish generally relies on extrapolation of metabolic rate from individuals of lower body mass using allometric relationships that are notoriously variable. Swim-tunnel respirometry is the ‘gold standard’ for measuring active metabolic rates in water-breathing animals, yet previous data are entirely derived from body masses <10 kg – at least one order of magnitude lower than the body masses of many top-order marine predators. Here, we describe the design and testing of a new method for measuring metabolic rates of large water-breathing animals: a c. 26 000 L seagoing ‘mega-flume’ swim-tunnel respirometer. We measured the swimming metabolic rate of a 2·1-m, 36-kg zebra shark Stegostoma fasciatum within this new mega-flume and compared the results to data we collected from other S. fasciatum (3·8–47·7 kg body mass) swimming in static respirometers and previously published measurements of active metabolic rate measurements from other shark species. The mega-flume performed well during initial tests, with intra- and interspecific comparisons suggesting accurate metabolic rate measurements can be obtained with this new tool. Inclusion of our data showed that the scaling exponent of active metabolic rate with mass for sharks ranging from 0·13 to 47·7 kg was 0·79; a similar value to previous estimates for resting metabolic rates in smaller fishes. We describe the operation and usefulness of this new method in the context of our current uncertainties surrounding energy requirements of large water-breathing animals. We also highlight the sensitivity of mass-extrapolated energetic estimates in large aquatic animals and discuss the consequences for predicting ecosystem impacts such as trophic cascades.

A new method for resolving uncertainty of energy requirements in large water breathers: the ‘mega-flume’ seagoing swim-tunnel respirometer

Body size is a key determinant of metabolic rate, but logistical constraints have led to a paucity of energetics measurements from large water-breathing animals. As a result, estimating energy requirements of large fish generally relies on extrapolation of metabolic rate from individuals of lower body mass using allometric relationships that are notoriously variable. Swim-tunnel respirometry is the ‘gold standard’ for measuring active metabolic rates in water-breathing animals, yet previous data are entirely derived from body masses <10 kg – at least one order of magnitude lower than the body masses of many top-order marine predators. Here, we describe the design and testing of a new method for measuring metabolic rates of large water-breathing animals: a c. 26 000 L seagoing ‘mega-flume’ swim-tunnel respirometer. We measured the swimming metabolic rate of a 2·1-m, 36-kg zebra shark Stegostoma fasciatum within this new mega-flume and compared the results to data we collected from other S. fasciatum (3·8–47·7 kg body mass) swimming in static respirometers and previously published measurements of active metabolic rate measurements from other shark species. The mega-flume performed well during initial tests, with intra- and interspecific comparisons suggesting accurate metabolic rate measurements can be obtained with this new tool. Inclusion of our data showed that the scaling exponent of active metabolic rate with mass for sharks ranging from 0·13 to 47·7 kg was 0·79; a similar value to previous estimates for resting metabolic rates in smaller fishes. We describe the operation and usefulness of this new method in the context of our current uncertainties surrounding energy requirements of large water-breathing animals. We also highlight the sensitivity of mass-extrapolated energetic estimates in large aquatic animals and discuss the consequences for predicting ecosystem impacts such as trophic cascades.

Investigation of the chemical origin and evidential value of differences in the SERS spectra of blue gel inks

Highly swellable polymer films doped with Ag nanoparticle aggregates (poly-SERS films) have been used to record very high signal:noise ratio, reproducible surface-enhanced (resonance) Raman (SER(R)S) spectra of in situ dried ink lines and their constituent dyes using both 633 and 785 nm excitation. These allowed the chemical origins of differences in the SERRS spectra of different inks to be determined. Initial investigation of pure samples of the 10 most common blue dyes showed that the dyes which had very similar chemical structures such as Patent Blue V and Patent Blue VF (which differ only by a single OH group) gave SERRS spectra in which the only indications that the dye structure had been changed were small differences in peak positions or relative intensities of the bands. SERRS studies of 13 gel pen inks were consistent with this observation. In some cases inks from different types of pens could be distinguished even though they were dominated by a single dye such as Victoria Blue B (Zebra Surari) or Victoria Blue BO (Pilot Acroball) because their predominant dye did not appear in other inks. Conversely, identical spectra were also recorded from different types of pens (Pilot G7, Zebra Z-grip) because they all had the same dominant Brilliant Blue G dye. Finally, some of the inks contained mixtures of dyes which could be separated by TLC and removed from the plate before being analysed with the same poly-SERS films. For example, the Pentel EnerGel ink pen was found to give TLC spots corresponding to Erioglaucine and Brilliant Blue G. Overall, this study has shown that the spectral differences between different inks which are based on chemically similar, but nonetheless distinct dyes, are extremely small, so very close matches between SERRS spectra are required for confident identification. Poly-SERS substrates can routinely provide the very stringent reproducibility and sensitivity levels required. This, coupled with the awareness of the reasons underlying the observed differences between similarly coloured inks allows a more confident assessment of the evidential value of inks SERS and should underpin adoption of this approach as a routine method for the forensic examination of inks.