• 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.
    • Oral Human Papillomavirus (HPV) Prevalence and Abundance in the UK Young Adult Population

      Marsh, Elizabeth; Knight, Gillian; Whitton, Aimee (University of DerbySchool of Biomedical & Forensic Science, 2021)
      BACKGROUND: The incidence rates of HPV positive oropharyngeal cancers (OPSCCs) are on the rise, yet oral HPV prevalence rates in clinically healthy populations are poorly understood. To determine the risk of healthy adults developing OPSCCs, first we must establish oral HPV prevalence, viral load, persistence, and clearance rates in healthy populations to understand the link between oral HPV and OPSCCs. This is even more pertinent within the young adult population as the HPV vaccination programme has been shown to reduce cervical cancer incidence but not OPSCC incidence. Therefore, other factors that could affect oral HPV contraction and OPSCC development need investigation such as population demographics, lifestyle risk behaviours, HPV screening methods and vaccination status. OBJECTIVES: The aims of this study were centred on establishing reproducible and sensitive HPV screening methods for the detection of oral HPV in clinically healthy young adults, for determining prevalence and abundance, and establishing if oral HPV was influenced by vaccination status, demographics, and lifestyle risk behaviours. METHODS: The study established a novel and sensitive real-time PCR HPV consensus screening method for the detection of multiple HPV subtypes in the oral cavity of 408 clinically healthy UK-based young adults (92.01%; 18-25 years old in 2016-17). HPV positive or undetermined samples were then screened using qPCR for HPV subtypes, HPV-6, HPV-11, HPV-16, HPV-18. All results were analysed alongside vaccination status, demographics and lifestyle risk behaviour data collected via questionnaire. RESULTS: An oral HPV prevalence rate of 22.79% was found, with HPV-16 being the most prevalent and abundant subtype at 19.12%; 1.08x105 copies/million cells, followed by HPV-18 at 1.72%; 1.89x104 copies/million cells, HPV-6 at 0.49%; 4.50x102 copies/million cells and HPV-11 at 0.25%; 1.06x102 copies/million cells. Unknown HPV subtypes were detected in 2.21% of the cohort. Oral HPV was found to be significantly associated with open-mouth kissing (p <.001), oral sex (p = .049), masturbation in males (p = .020), sexual intercourse (p = .026), sexual activity diversity (p = .043), frequent smoking (p = .024), wine drinking (p = .045) and drinking ≥2 types of alcohol per sitting (p = .015), especially in males (p = .023). HPV-16 was significantly associated with masturbation (p = .004), whilst there was a reduction in viral load in vaccinated individuals, but this was not statistically significant. CONCLUSIONS: Oral HPV is prevalent in the young adult UK population, especially HR-HPV subtype HPV-16, questioning the efficacy of the HPV vaccination on reducing oral prevalence. However, HPV vaccination may instead influence oral HPV viral load, but further research is required, demonstrating the importance of measuring both presence and abundance. Oral HPV prevalence did appear to be influenced by sexual practice, including open-mouth kissing and oral sex, but less so by smoking and alcohol consumption, reaffirming the link between oral HPV and OPSCCs.