• Alternative methods for assessing habitat quality in freshwater systems

      Sweet, Michael; Ramsey, Andrew; Brys, Rein; Mauvisseau, Quentin (University of DerbyAquatic Research Facility, Environmental Sustainability Research Centre, University of Derby, 2020-06-03)
      “Water, water, everywhere…”. 71% of the earth’s surface is covered by water, freshwater representing 2.5% of it, and only 1% being accessible. Due, largely to a number of anthropogenic activities (pollution, habitats modification) coupled with the impacts of climate change, a dramatic decline in biodiversity is occurring across all earth’s ecosystems. Surprisingly, freshwater ecosystems receive considerably less attention than many other habitats and therefore, effective biodiversity monitoring programs are urgently needed to assess the health and state of the endangered and threatened species in these aquatic systems. Further, current techniques utilised to survey freshwater ecosystems are often considered ineffective, invasive, time consuming and biased. As a result, the implementation of molecular-based detection tools are attractive options as they are often shown to be more sensitive and cost effective. The use of environmental DNA (eDNA) detection is one such molecular tool which is showing promising results, due to its high reliability, sensitivity and non-invasiveness characters. However, recent studies have highlighted potential limitations associated with eDNA-based detection. Such limitations may lead to a decrease in the confidence of this method. The aim of this thesis was to investigate the use of eDNA-based detection across a number of species and a number of systems, all as a proxy of habitat quality. Stringent laboratory practices and validation guidelines were adhered to, allowing for reliable quality assessments of newly designed eDNA assays outlined in this thesis. Moreover, distinct controlled mesocosm experiments allowed the investigation of critical factors, part of the sampling method or analysis processes leading to an optimisation of eDNA collection and decreasing the rates of false negative results. Several comparison between traditional monitoring techniques and the novel assays were also performed aiding in the confidence of these new methods. Interestingly, the results obtained in this thesis shows a similar efficiency between traditional and eDNA-based methods for monitoring invasive species, but a higher efficiency of eDNA detection when detecting rare or low abundant organisms (i.e. those that are endangered or threatened). Furthermore, this thesis reports an extreme example where a species was found at a number of locations within a stretch of a river, yet undetected with the eDNA assay. In this chapter eDNA detection was only possible when I utilised ddPCR rather than qPCR (the more standard technique for assessing eDNA in any given system). Overall, eDNA detection was found to be an effective tool for assessing the presence of invasive and/or endangered species, increasing theknowledge on their distribution and the impact of future management plans. In this thesis, chapters 2, 3, 4, 5 and 6 are organised as case studies, aiming to highlight benefits and limitations of species-specific detection using eDNA.
    • The cryptic and transboundary nature of ghost gear in the Maldivian Archipelago

      Sweet, Michael; Huck, Maren; Beaumont, Nel; Stelfox, Martin (University of Derby, 2019-06-11)
      Abandoned, lost or discarded fishing gear (ALDFG), more commonly referred to as ghost gear, is a global issue that impacts many marine organisms worldwide. In the Maldivian archipelago a large number of olive ridley sea turtles (Lepidochelys olivacea) are found entangled in these nets (more commonly referred to as ghost nets) each year. However, the origin of these nets or turtles are unknown considering fishing with the use of nets is restricted to the bait fisheries within the exclusive economic zone of the Maldives. Therefore, ghost gear has a transboundary and cryptic nature, making it difficult to assess the environmental impact and origin of the gear. This thesis aimed to develop new tools and techniques which could be utilised to examine these unknowns. I revealed in a literature review (Chapter 1) that research in ghost gear entanglements amongst marine megafauna are predominantly focussed in the Atlantic and Pacific Ocean. However, Indian, Arctic and southern Oceans are far less studied. Additionally, the majority of strategies to tackle ghost gear were centred around curative measures, such as ghost gear retrieval. I advise that future solutions, best practices and research should favour preventative rather than curative methods in ghost gear management and research. Statistical classifies (Chapter 2) were built in R to predict the probability of a net entangling a turtle. It was highlighted that nets with larger mesh sizes and the absence of floats were major gear characteristics that increased the likelihood of turtle entanglement. In addition, the time of year was an important variable with a higher chance of turtle entanglements in nets found during the northeast monsoon (November – April). Unfortunately, grouping of the nets by fisheries was not possible, beyond a broad classification. This was likely a result of the wide variety of nets used in the region. However, gill and trawl nets were recognised as high-risk fisheries. Regardless of the difficulties of assigning an origin of the nets, I was able to estimate the scale of the problem. Between 3,400 and 12,200 turtles could have become entangled in ghost nets over the 51-month study period, meaning this region has the highest turtle entanglement rate in ghost nets worldwide (0.17). Nesting and sightings of free-swimming individuals are rare and therefore the majority of entangled turtles do not originate from the Maldives. To discover the source population of these entangled olive ridleys we utilised a mixed stock analyses of mtDNA from samples of turtles entangled in nets in the Maldives (n = 38) and compared them to nesting stocks from published literature (Sri Lanka, east India and northern Australia). We were able to fill in data gaps in phylogenetics by including samples from previously undescribed nesting populations, such as those in Oman and improved resolution by including longer sequences from east India in our analyses (Chapter 3). Results suggest that the majority of entangled olive ridleys originate from east Indian (73%) and Sri Lankan (23%) genetic stocks when no population estimates were included in model design. This meant we could estimate the impact of ghost nets on these populations. Recorded ghost net entanglements may impact yearly recruitment of east Indian populations by 0.48% however a staggering 41% of the Sri Lankan population are thought to be negatively affected by the drifting nets. I then attempted to age ghost gear found drifting in the Maldives, and provided additional evidence to locate possible sites of origin. Percentage cover of biofouling communities and capitulum length of the goose barnacle (Lepas anatifera) provided the most robust metrics to estimate minimum drift times (Chapter 4). Lagrangian simulations (forced by Ocean Surface Current Analyses Realtime OSCAR) could then be utilised to backtrack drifting ghost gear to a putative origin. This analysis highlighted that the origin of these nets overlapped with purse seine (predominantly from Spain and France) and gill net fisheries operating in the area. Moreover, the models show that some of the nets originate close to the Indian and Sri Lanka shorelines, suggesting that small scale artisanal fisheries may provide additional high risk, contributing to ghost nets drifting into the Maldives and entangling turtles. In summary it is hoped that this thesis advances our knowledge on ghost gear significantly. Moreover, this thesis provides the information and tools necessary for the Olive Ridley Project (a British registered charity, tackling this issue face on), along with other stakeholders (government and non-government) in order to better manage resources and combat the ghost gear issue within the Indian Ocean.
    • Developing protocols and methods to predictably induce ex situ broadcast coral spawning and increase post settlement survivorship.

      Sweet, Michael; Bulling, Mark; Guest, James; Craggs, Jamie (University of Derby, 2020-04-22)
      The production of broadcast spawning gamete material ex situ has great potential in developing areas for coral research and/or to support initiatives aimed at rebuilding damaged reefs utilising sexually produced spat. Current effectiveness of such restoration practises are limited due to the high mortality rates post settlement and therefore methods aimed at increasing survival, and therefore productivity, are required and vital in order to further support upscaling of such practices. Therefore, this thesis focuses on developing methodology to predictably induce broadcast reef building corals to spawn ex situ and investigate ways to maximise post settlement survivorship. Acquisition of broodstock for any ex situ breeding project is essential. Chapter two describes the methodology designed and implemented in order for me to carry out long distance transportation (a journey time of ~34 hrs) of large (16-37 cm) gravid Acropora hyacinthus (Dana, 1846) colonies from fringing reefs south of Singapore to the Horniman Museum and Gardens, London. Collection was purposefully timed to occur just before the predicted annual mass spawning event and on the day of transportation 12 of the 14 genotypes contained large visible oocytes, which spawned ex situ within the same lunar month as those in the wild. A closed system mesocosm aquarium was designed at the same time, as described in chapter 3 that utilises microprocessor technology to accurately replicate environmental conditions associated with stimulating broadcast spawning events (photoperiod, seasonal solar irradiance, lunar cycles and seasonal temperature) from two synchronous spawning locations, Singapore and the Great Barrier Reef, Australia. Coupled with appropriate coral husbandry, four species (A. hyacinthus, Acropora millepora (Ehrenberg 1834), Acropora tenuis (Dana 1846) and Acropora microclados (Ehrenberg 1834)) completed full gametogenic cycles and spawned in a fully closed artificial ex situ environment (in synchrony with the wild). The effects of spawning broadcast corals ex situ is currently unknown, therefore following gamete release embryological development stages of three acroporids (A. millepora, A. tenuis and Acropora anthocercis (Brook 1893)) was assessed utilising scanning electron microscopy and confocal laser scanning microscopy techniques (Chapter 4). No abnormal developmental effects (as result of the ex situ environment) were observed, but the study built on previous works to provide increased detail of fertilisation and early cell stages. Reef building corals typically undergo a type III survival curve in their early life stages, with high mortality rates during early ontogeny. Increased post settlement survival can occur due to size mediated multi-genotype settlement aggregations and species hybridisation. These two factors were empirically tested (Chapter 5) in pure and interspecific hybrid crosses of A. millepora and Acropora anthocercis. Increased survival, and to a lesser extent growth, were observed in post settlement entities with >2 genotypes compared with single genotype primary polyps and in interspecific hybrid crosses compared to pure species crosses, highlighting the role of hybridisation vigour. Reef herbivory may enhance coral settlement and recruitment success however the influence of herbivory size classes on survival benefits are not ubiquitous. In order to assess the positive role that microherbivory may contribute to maximising coral survival and growth ex situ two species, the Tuxedo sea urchin, Mespilia globulus (Linnaeus, 1758) and the reef building coral, A. millepora, were co-cultured at varying densities. Increasing density of microherbivory significantly enhanced coral survival and growth, highlighting this as a potentially beneficial practise in improving productivity of coral produced via sexual reproduction. Finally closing the life cycle of a target organism marks an important milestone in any ex situ breeding programme or aquaculture method. Chapter 7 describes the production of the first F2 generation of A. millepora in a fully closed aquarium environment. In summary, it is therefore hoped this thesis will, in part, make a contribution to coral sexual reproductive research and the important work of reef restoration, particularly in light of the global decline in coral reef ecosystems.
    • Transforming a Research Concept into Commercial Practice: Addressing the ‘Hurdles’ of Single-Species eDNA-based Detection

      Sweet, Michael; Robinson, Louise; Burian, Alfred; Troth, Christopher (University of Derby, 2020-01-15)
      The use of environmental DNA (eDNA) for measuring and monitoring biodiversity has been identified as a novel molecular based method to complement more commonly utilised traditional ecological sampling techniques. It is a time and cost-efficient technique, which is rapidly advancing due to the capabilities of low eDNA detection levels. As the efficiency of the technique has increased, commercial organisations and end-users have gained a greater interest in its application. Despite this, the technique is currently only commercially available from a select few service providers. In the UK, the main target species for commercial scale eDNA-based detection is the great crested newt (Triturus cristatus). Interest has now been sparked for the development of eDNA assays to detect various other species, both for use as a regulated informative tool and a conservation aid. However, many recent studies have highlighted various limitations associated with the use of eDNA-based detection and this appears to be hampering commercialisation of this tool. eDNA-based detection methods remain relatively underdeveloped and un-validated for use as reliable and accurate widespread monitoring programs and other such applications. Here, the so called ‘hurdles’ associated with the development and validation of eDNA-based methods and its use as a fully available commercial service are reviewed and addressed, in order to develop and validate a commercially applicable eDNA assay for the endangered white-clawed crayfish, Austropotamobius pallipes, as a target organism. When designing novel species-specific assays, detailed validation steps need to be undertaken, ensuring they perform under various conditions, habitats, and which sampling methods should be utilised. Currently, more traditional methods used to asses populations of white-clawed crayfish (such as trapping and hand searching) are becoming increasingly more difficult to undertake as the species become rarer and populations more fragmented. Such techniques are therefore expensive (with regard to time spent surveying) and often result in low probability of detection. A new species-specific qPCR assay to detect white-clawed crayfish was developed and tested under various conditions both ex-situ (laboratory and mesocosms) and in-situ (ponds and rivers) to explore the optimum sampling strategy giving the most reliable results. Experiments were also conducted on a wider scale to determine the impact of DNA degradation and seasonal influence on eDNA persistence. Interestingly, this thesis illustrates that sample collection choice is not simple, and the ‘best’ methodology was shown to vary between habitat type. This indicates that great care should be taken when designing any such assays and implementing them in the field. Furthermore, this study highlights that a ‘standard operating procedure’ for eDNA-based detection in the commercial sector may not be possible and this will have to be explored on an assay by assay basis. Alongside case studies from real-world application of the technique, recommendations are made on how this novel eDNA assay can be used for the commercial practice of white-clawed crayfish assessment.