| Summary: | © 2015 Dr. Fletcher William Warren-Myers Domestication of the world’s oceans has resulted in many new environmental and ecological interactions between farmed and wild fish. For aquaculture, a particular concern is farm fish escapees, which can have negative effects on wild conspecifics thorough the transfer of diseases, genetic mixing, and competition for resources. In contrast to concerns associated with escapees, a second major area of anthropogenic influence is the process of restocking wild fish populations through the intentional release of hatchery-bred fish. Assessing the impacts of farmed fish, whether because of unintended escapees, or through intended release from restocking, can be difficult due to the vast volumes of fish being farmed (> 60 million tonnes) and restocked (> 5 billion fish) annually. Currently, fish used for sea cage farming are not marked. For restocking, most fish are marked, yet the methods used to identify restocked fish post release, for example otolith thermal marking, adipose fin clipping, coded wire tags, fluorescent staining, or chemical marking, all have some limitations in terms of ease and cost of application, mark retention and detectability, or fish welfare. In this thesis I investigate an alternate potential mass marking technique, known as enriched stable isotope marking, to develop delivery methods for mass marking farmed fish for the purposes of monitoring and tracing escapees, and as an alternative mass marking method to those currently used in restocking programs. Marking the otoliths of fish with enriched stable isotopes has been validated in many freshwater and marine species, but never before tested as an industry applicable mass marking tool. Using farmed Atlantic salmon, a common escapee from aquaculture, I tested three potential stable isotope mark delivery methods: vaccination, egg immersion, and transgenerational marking, with combinations of seven enriched isotopes (86Sr, 87Sr, 134Ba, 135Ba, 136Ba, 137Ba, and 26Mg). Marks were detected using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Each delivery method was designed to have high ease of application, be cost effective, have high retention and detection success, and complement current hatchery practices. Furthermore, I monitored fish for potential welfare effects on growth, condition or survival for each delivery method. The first method tested, marking during vaccination, takes advantage of the situation that all farmed Atlantic salmon in Norway are vaccinated. To validate stable isotope marking during vaccination, I combined enriched 86Sr, 137Ba and 26Mg with three carrier solutions, a vaccine, vaccine mimic, or water, and injected in two locations, either the abdominal cavity or muscle of Atlantic salmon parr. Upon confirming the method is a potentially viable mass-marking technique irrespective of carry solution or injection location, I then tested the method in a large scale experiment and showed that marking during vaccination is feasible for industrial application, and demonstrated there are 63 combinations of Sr and Ba isotopes available to mark at a cost of 0.0002 - 1.72 $US per fish. Assessing re-stocking success requires the ability to confidently identify and quantify the survival of hatchery fish after release. The second method I tested, egg immersion, uses the egg swelling stage in salmonids to deliver a combination of Ba, Sr and Mg isotopes. One hundred percent mark success was achieved with the isotopes 135Ba, 136Ba, and 137Ba, giving 7 possible combinations at a cost of 0.0001 - 0.0017 $US per egg to mark. There were no effects on egg survival, larval deformity or larval mortality rates, indicating that marking via egg immersion as an effective, low cost, and accurate alternative marking method to otolith thermal marking for marking hatchery reared salmonids destined for restocking purposes. Transgenerational marking has been validated in a variety of fish species using combinations of 1 or 2 isotopes. For farm Atlantic salmon, I increased the number of possible isotope combinations and optimised the technique by injecting 4-year old female broodfish with seven enriched isotopes at four concentrations (2, 0.2, 0.02, or 0.002 μg of isotope per g broodfish). I found that six of the isotopes tested (86Sr, 87Sr, 134Ba, 135Ba, 136Ba, & 137Ba) can be used to produce 63 mark combinations, and that mark success is dependent on the concentration of isotope injected and the length of time between injection date and spawning date. Material cost to mark was 0.0002 - 0.13 $US per embryo. The methods I developed for mass marking salmon are easy to apply, cost effective, have high retention and detection success, and complement current hatchery practices. To differentiate between farmed and wild fish, any one method could be used to cost effectively mass mark the entire Norwegian annual salmon production. I show that by combining delivery methods, differentiating between companies, or individual farm sites is feasible, for example by combining transgenerational marking with vaccination, 1023 unique fingerprint codes are possible. For restocking, egg immersion is an ideal technique for mass marking salmonids due to its ease of application, low cost, permanency, accuracy, and welfare friendliness. Globally, if applied correctly, the mass marking methods developed in this thesis provided an excellent tool box for monitoring and identifying a wide range of finfish species, whether escaped from aquaculture, or released for restocking purposes.
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