The scattering and attenuation caused by fish schools has been extensively studied for applications in fisheries management and naval sonar. The literature contains extensive in situ measurements of scattering by fish schools, however significant uncertainties exist with respect to characterizing the size, quantity, and distribution of fish within the schools, that confounds accurate measurement-model comparison. Hence there is a need for application of measurement techniques that can more precisely characterize the acoustic properties of fish schools and the variations intrinsic to live subjects in continual motion. To begin to address this deficiency, measurements of the sound speed through collections of live fish were conducted in a laboratory setting. The species chosen for measurement were zebrafish (Danio rerio). The sound speed was investigated using a resonator technique which yielded inferences of the phase speed within the fish school though measurements of the resonances of a one-dimensional waveguide. The waveguide was calibrated to compensate for finite wall impedance and for finite reflections from the ends of the waveguide. Fish densities were investigated ranging from 8.6 to 1.7 fish lengths per mean free path. Measurements agree well with a predictive model that is based on shell-free spherical bubbles, which indicates that the phase speed is not significantly affected by the fish flesh or swimbladder morphology for the species studied. The variation in phase speed due to individual fish motion within the model school was measured to be up to ± 5.6 %. This indicates that precise knowledge of the fish position is required to achieve greater model accuracy. To compliment the phase speed measurements, the attenuation through a cloud of encapsulated bubbles was evaluated through insertion loss measurements. Multiple arrangements of balloons of radius 4.68 cm were used to surround a projector. The insertion loss measurements indicated an amplification of around 10 dB at frequencies below the individual balloon resonance frequency and an insertion loss of around 40 dB above the individual balloon resonance frequency. Analytical modeling of the bubble collection predicted both the amplification and loss effect, but failed to accurately predict the level of amplification and insertion loss. Effective medium models and full scattering models (requiring knowledge of bubble size and position) were evaluated for a model fish school. The two models agree for forward scattering for all frequencies except those immediately around the individual bubble resonance frequency. Back scattered results agree at low frequencies, however as soon as the wavelength becomes smaller than four mean free paths between fish the models diverge. Ramifications of these findings and potential future research directions are discussed. / text

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

It is now well established that there are a wide variety of known EDCs (Endocrine Disrupting Chemicals) in the aquatic environment, which include natural (e.g. oestrogen) and synthetic hormones (e.g. ethinyloestradiol), weak environmental oestrogens (e.g. bisphenol A and nonylphenol) and pesticides, fungicides and herbicides, that are able to alter the physiology of exposed wildlife and humans and have become a focus of increasing concern for human and environmental health over the past two last decades. However, the functional implications and potential significance of these findings has yet to be established. In this thesis, a transgenic fish system approach (using a new plasmid) was employed to investigate the effect pathways of EDCs in fish, using transgenic UAS-GFP zebrafish (Danio rerio) as a model species. One of the most critical steps in this approach is the development of an effective construct for the development of sensitive transgenic zebrafish. The required ERE was cloned and three copies were incorporated into a construct. Three vector systems (pKS+, pCS2+ and pBR322-Tol2) were selected for making new plasmids, and investigated to improve the efficiency for sensitivity and tissue specificity. Three constructs were made and tested progressively in the development process. The plasmids contain three oestrogen response elements (3ERE), a TATA box and GAL4ff. The three constructs, pKS-ERE-Gal4ff, pCS2-ERE-Galf4ff and pBR-Tol2-ERE, were examined by injecting into zebrafish in a transient assay system. The pBR-Tol2-ERE-Gal4ff construct was selected for generating transgenic zebrafish because it showed tissue specific manner (i.e. reduce mosaicism) and Tol2 transposon has the capacity to improving greatly the chances for generating TG fish such as high germline transmission frequency. The construct pBR-Tol2-ERE-Gal4ff required to examine the functional capability of using transient expression assay before generating transgenic fish. The Gal4-UAS/GFP method was adopted and produced a two step amplification of the oestrogenic signal. A novel transient expression assay system was developed using a synthetic oestrogen responsive element, the Tol2 mediated Gal4-UAS systems and the GFP reporter gene, which are responsive to environmental oestrogens using green fluorescent microscopy in zebrafish (Danio rerio) and medaka (Oryzias latipes). The construct worked well and vector system in transient expression assays proved a rapid, sensitive, tissue specific system for the detection of oestrogenic EDCs in both the zebrafish and medaka, indicating the likely wide suitability for application to other fish species. These data indicate that this system would be a particularly powerful technique for use in species with long generation times and/or where there are other difficulties for generating transgenic lines in those species. After the transient expression assay, the construct pBR-Tol2-ERE-Gal4ff with transposase mRNA was co-injected into early one cell stage and bred for 3 months. The embryos were collected from the founders (a male and a female) and exposed to EE2 to identify the transgenic zebrafish. An oestrogen responsive transgenic zebrafish (ERE-TG fish) was established under an oestrogen inducible promoter derived from multiple tandem oestrogen responsive elements. Gal4ff-UAS system was adopted to generate ERE-TG fish to enhance the sensitivity, which has not been used for generating biosensor zebrafish yet. Oestrogen exposures were shown to induce specific GFP expression in the heart, muscle, otic vesicle, brain, neuromasts, eye/ear ganglions and fin, in addition to that in the liver and gonad, which has not been shown previously in other oestrogen responsive transgenic zebrafish, illustrating both the enhanced sensitivity of our detection system and the potential for these tissues as target sites for wider health effects for exposure to environmental oestrogens. Furthermore, oestrogen chemicals are converted to an oestrogenic signal in different tissues preference. Oestrogen receptors (ER α, β1 and β2) morpholinos (MOs) and exposures with oestrogen receptor antagonist were carried out to assess whether the GFP expression was mediated through the ER, regardless of the subtypes. GFP expression was inhibited through injection of the mixture of ERs MOs compared with uninjected transgenic fish larvae. This result indicated that GFP expression was mediated through the ERs. The lowest detectable concentration of EE2, E2, BPA and NP was 1 ngEE2/L, 5 ngE2/L, 100 µgBPA/L and 1 µgNP/L in ERE-TG fish embryos/larvae. These findings provide that ERE-TG fish embryos/larvae systems are useful both for studying physiological mechanisms and for detecting biological target sites of environmental oestrogens. As a preliminary experiment, immature (40 day old) and mature (3 month old) ERE-TG fish were exposed to low concentrations of EE2 for 7 days to prove its potential for developing tissue-specific models of EDCs at different life stages. The lowest detectable EE2 effect concentration was 5 ngEE2/L in the liver and gonad and 10 ngEE2/L in the muscle in both immature and mature ERE-TG fish. These data demonstrate that muscle is as susceptible as liver and gonad to EE2 and ERE-TG fish (both immature and mature fish) would appear to be a useful system for both detecting target sites for oestrogen chemicals in these life stages fish. In summary, the work undertaken in this thesis has developed transiently and stably transfected zebrafish as a tool for screening oestrogen chemicals to assess for the potential health impacts of oestrogens in the environment, provided novel insights into many tissue target tissues by oestrogen exposure, and has established different patterns of tissue responses for different environmental oestrogens tested suggesting differing functional implications.

Anthropogenic influences on riverine ecosystems have led to numerous impacts that have compromised fish communities. Large European lowland rivers have been amongst the most impacted aquatic environments globally. However, paradoxically, these environments have also been amongst the least studied, due the inherent difficulties of studying fish in large, turbid, dynamic environments. Common bream Abramis brama (L.) populations are amongst those impacted by reduced lateral and longitudinal connectivity that are a consequence of modifications to large lowland rivers in Europe. Thus common bream in the lower River Witham, Lincolnshire, UK, were selected as a case study to examine how fish utilize these river systems. A literature review was carried out to assess the influence of human intervention on riverine ecosystems and their impacts on fish communities. In addition the use of telemetry techniques for studying fish populations was reviewed. An ecological assessment of the lower River Witham was carried out using data available from The Environment Agency monitoring and hydrological systems and data specifically collected during this study. The history of this highly modified lowland river was reviewed to give perspective, timescale and context to the degree of modification that has been undertaken on the river and its consequences. Chemical and biological water quality indicators showed improvements in the last twenty years. Fisheries data identified three species that had become locally extirpated in the lower river since the 1800s. The river is now roach dominated, but common bream are still present, possibly due to available lateral spawning habitats that provide surrogates for, and are functionally similar to, the lentic floodplain waterbodies of natural riverine ecosystems. Hydroacoustic surveys show that fish communities are aggregated and favour the upstream half of the lower river where the channel is more heterogeneous. Recent changes in river character have made traditional fishery assessments by seine netting less effective. Data collected during fish tracking studies are most valuable when the tagged fish are behaving naturally, thus the effects of tag attachment should not impact on the behaviour or well being of the fish. Acoustic telemetry was used to investigate the impacts of tagging, surgical inter-peritoneal implantation, and translocation of common bream. Fish were recaptured following surgery and the incision site photographed to assess healing, which was clean in all cases. All recaptured fish appeared to be behaving normally as they were part of large shoals of fish. On five separate occasions fish were tagged when other previously tagged fish were present allowing their behaviour to be compared in terms of the distances moved and linear range; there were no significant differences between the two groups indicating the tagging procedure to have no detectable impact on the short-term behaviour of the fish at the resolution of the tracking undertaken. Three recaptured fish were translocated ~35 km downstream; these fish appeared to exhibit homing behaviour, returning to the capture site between 6-24 days. Their level of activity (in terms of distance moved) did not significantly differ from non-translocated fish, but their linear range was larger. The distribution and habitat use of adult common bream was assessed by tracking their longitudinal and lateral movements over forty-three months, producing a dataset of over 3.1 million detections. A positive relationship between activity and temperature was revealed, with common bream moving greater cumulative distances during the warmer months and occupying a greater longitudinal proportion of the main channel. The occupancy of tributaries was related to temperature, with common bream entering shallow tributaries during rising temperatures in the spring, whilst a deeper, slow-flowing tributary was used more frequently during the cooler autumn/winter months. During the autumn, occupancy of this deeper tributary was positively related to flow rate, suggesting that common bream use this as a refuge from high main channel flow. The home range concept is at the centre of theoretical models to explain the spatio-temporal behaviour of a wide range of animals including fish. There are lots of different ways of expressing home ranges, including areas, but since rivers are principally linear systems, most range is reflected linearly. Home ranges were calculated for 100% and 90% of locations. Seasonal differences were identified with fish occupying larger home ranges during the spring and summer opposed to autumn and winter. Artificial water level manipulations, management actions for the control of flood risk, affected home range size with fish occupying smaller ranges at reduced winter levels than at increased summer levels. Analysis of when and how often individuals within a population interact with one another provides a method to study the social organisation of animals with the potential to reveal ecologically significant aspects that would otherwise have remained hidden. Network analysis and social network theory were used to examine the social interactions of the tracked fish. The extent of sociality appears to follow a normal distribution, such that there was no evidence of discrete classes of social/non-social fish. Fish were more social immediately after tagging and less social as time progressed, as such little evidence could be found to support the hypothesis that these groups of fish remained in each others’ company for extended periods. However, more active individuals were found to be more socially connected, but home-range size did not significantly affect sociality. The functions of these behaviours are discussed along with potential management and rehabilitation strategies for the lower River Witham and other heavily modified lowland rivers in order to meet good ecological potential/status under the Water Framework Directive (2000/60/EEC).

The peripheral blood of O.mossambicus was examined using light and electron microscopy and was found to contain four forms of leucocytes: lymphocytes, thrombocytes, monocytes and three types of granulocytes. The monocyte and two types of granulocyte were found to be phagocytic and ingest colloidal carbon and bacteria. The alimentary tract was found to contain a number of leucocytes, some showing a morphological similarity to those in the peripheral blood, while others were unique to the gut tissue. These intestinal leucocytes were found mainly as a diffuse cell population in the epithelium and lamina propria, and only occasionally as discrete lymphoid accumulations within the gut tissue. Ontogenic studies showed that a limited number of leucocytes were found in the gut tissue after hatching, however, there was a gradual increase in these numbers once exogenous feeding began. The intestinal enterocytes of both the anterior and posterior intestine were found to take up intubated macromolecules. An electron microscopical investigation revealed that these macromolecules were absorbed by pinocytosis and were found within large intraepithelial macrophages. These macromolecules were also absorbed and transported into the systemic circulation. In juvenile fish macromolecules were detected in the plasma following both oral and anal intubation, however, in adult fish they were detected in the plasma only after anal intubation, and in smaller quantities. Macromolecular absorption in O.mossambicus was compared to that in two other fish species, Cyprinus carpio and Sa1mo gairdneri, and it was found that higher levels of absorbed macromolecules were found in the plasma of O.mossambicus. Bovine serum albumin absorption by the gut of the three species revealed that both the 'intact' macromolecule and smaller antigenic fragments, probably resulting from enzymatic modification, were ansorbed and transported into the plasma.

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