Phenoloxidase activity and organic carbon dynamics in historic Anthrosols in Scotland, UK

Phenolic compounds are chemical precursor building blocks of soil organic matter. Their occurrence can be inhibitory to certain enzymes present in soil, thereby influencing the rate of decomposition of soil organic matter. Microbe-derived phenoloxidases (laccases) are extracellular enzymes capable of degrading recalcitrant polyphenolic compounds. In this study, our aim was to investigate the relationships between phenoloxidase enzyme activity, organic carbon content and microbial abundance in the context of long-term anthropogenically amended soils. To achieve this, we used a series of complementary biochemical analytical methods including gas chromatography, enzyme assays and solid-state Carbon-13 Cross Polarisation Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy (13C CPMAS NMR). Using several anthrosols found in St Andrews (Scotland, UK) that had been subjected to intense anthropogenic modification since the medieval period (11th century AD) to present-day, we were able to scope the impact of past waste disposal on soils. The long-term anthropogenic impact led to organic matter-rich soils. Overall, phenoloxidase activity increased by up to 2-fold with soil depth (up to 100 cm) and was inversely correlated with microbial biomass. Solid-state 13C NMR characterisation of carbon species revealed that the observed decline in soil organic matter with depth corresponded to decreases in the labile organic carbon fractions as evidenced by changes in the O/N-alkyl C region of the spectra. The increase in phenoloxidase activity with depth would appear to be a compensatory mechanism for the reduced quantities of organic carbon and lower overall nutrient environment in subsoils. By enzymatically targeting phenolic compounds, microbes can better utilise recalcitrant carbon when other labile soil carbon sources become limited, thereby maintaining metabolic processes

The stress–immunity axis in shellfish

It is a difficult task to describe what constitutes a ‘healthy’ shellfish (e.g., crustacean, bivalve). Visible defects such as discolouration, missing limbs or spines, fouling, lesions, and exoskeletal fractures can be indicative of underlying issues, senescence, or a ‘stressed’ animal. The absence of such symptoms is not evidence of a disease-free or a stress-free state. Now, more than ever, aquatic invertebrates must cope with acute and chronic environmental perturbations, such as, heatwaves and cold shocks, xenobiotic contaminants, intoxication events, and promiscuous pathogens expanding their host and geographic ranges. With that in mind, how does one determine the extent to which shellfish become stressed in situ (natural) or in cultured (artificial) settings to enhance disease susceptibility? Many biomarkers – predominantly biochemical and cellular measures of shellfish blood (haemolymph) – are considered to gauge immunosuppression and immunocompetence. Such measures range from immune cell (haemocyte) counts to enzymic activities and metabolite quantitation. Stressed invertebrates often reflect degraded conditions of their ecosystems, referred to as environmental indicators. We audit briefly the broad immune functions of shellfish, how they are modulated by known and emerging stressors, and discuss these concepts with respect to neuroendocrinology and immunotoxicology. We assert that chronic stress, alone or in combination with microbial, chemical and abiotic factors, increases the risk of infectious disease in shellfish, exacerbates idiopathic morbidity, and reduces the likelihood of recovery. Acute stress events can lead to immunomodulation, but these effects are largely transient. Enhancing our understanding of shellfish health and immunity is imperative for tackling losses at each stage of the aquatic food cycle and disease outbreaks in the wild

Hemocyanin-derived Phenoloxidase; Biochemical and Cellular Investigations of Innate Immunity

Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates

Preferences, Perceptions, and Veto Players: Explaining Devolution Negotiation Outcomes in the Canadian Territorial North

Since the early part of the 20th century, the federal government has engaged in a long and slow process of devolution in the Canadian Arctic. Although the range of powers devolved to the territorial governments has been substantial over the years, the federal government still maintains control over the single most important jurisdiction in the region, territorial lands and resources, which it controls in two of the three territories, the Northwest Territories and Nunavut. This fact is significant for territorial governments because gaining jurisdiction over their lands and resources is seen as necessary for dramatically improving the lives of residents and governments in the Canadian north. Relying on archival materials, secondary sources, and 33 elite interviews, this paper uses a rational choice framework to explain why the Yukon territorial government was able to complete a final devolution agreement relating to lands and resources in 2001 and why the governments of the Northwest Territories and Nunavut have not. It finds that the nature and distance of federal-territorial preferences, combined with government perceptions of aboriginal consent and federal perceptions of territorial capacity and maturity, explain the divergent outcomes experienced by the three territorial governments in the Canadian arctic. The following acronyms are employed: AIP: Agreement-in-Principle; DTA: Devolution Transfer Agreement; GEB: gross expenditure base; GN: Government of Nunavut; GNWT: Government of Northwest Territories; NCLA: Nunavut Land Claims Agreement; NTI: Nunavut Tunngavik Incorporated; NWT; Northwest Territories; ON: Ontario; TFF: Territorial Formula Financing; UFA: Umbrella Final Agreement; YDTA: Yukon Devolution Transfer Agreement; YTG: Yukon Territorial Government; YK: Yukon

The diarrhetic shellfish-poisoning toxin, okadaic acid, provokes gastropathy, dysbiosis and susceptibility to bacterial infection in a non-rodent bioassay, Galleria mellonella

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80–400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 105 Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays