The interplay between extrinsic and intrinsic factors in determining migration decisions in brown trout (Salmo trutta): An experimental study

Many species are capable of facultative migration, but the relative roles of extrinsic versus intrinsic factors in generating diverse migratory tactics remain unclear. Here we explore the proximate drivers of facultative migration in brown trout in an experimental laboratory setting. The effects of reduced food, as a putative environmental cue, were examined in two populations: one that exhibits high rates of anadromy (sea-migration) in nature, and one that does not exhibit anadromy in nature. Juveniles derived from wild-caught parents were reared for two years under four environmental treatments: low food in years 1 and 2 (Low-Low); high food in years 1 and 2 (High-High), low food in year 1 and high in year 2 (Low-High), and vice versa (High-Low). Food restriction had a significant effect on migratory tactics, with the frequency of smolts (juveniles choosing migration) highest in the Low-Low treatment in both populations. No individuals became smolts in the High-High treatment, and intermediate smolting rates were observed in the Low-High and High-Low treatments. Higher overall smolting rates in the naturally anadromous population suggested an inherited component to anadromy/migration decisions, but both populations showed variability in migratory tactics. Importantly, some fish from the naturally non-anadromous population became smolts in the experiment, implying the capacity for migration was lying ‘dormant’, but they exhibited lower hypo-osmoregulatory function than smolts from the naturally anadromous population. Tactic frequencies in the naturally anadromous population were more affected by food in the 2nd year, while food in the 1st year appeared more important for the naturally non-anadromous population. Migratory tactics were also related to sex, but underpinned in both sexes by growth in key periods, size and energetic state. Collectively these results reveal how migration decisions are shaped by a complex interplay between extrinsic and intrinsic factors, informing our ability to predict how facultatively migratory populations will respond to environmental change

Proteome-wide Changes in the mdx-4cv Spleen due to Pathophysiological Cross Talk with Dystrophin-Deficient Skeletal Muscle

Duchenne muscular dystrophy is primarily characterized by progressive muscle wasting due to deficiency in the membrane cytoskeletal protein dystrophin but is also associated with body-wide cellular disturbances in a variety of non-muscle tissues. In this study, we have focused on the comparative proteomic analysis of the spleen and established considerable changes in this crucial secondary lymphoid organ from the genetic mdx-4cv mouse model of dystrophinopathy. An apparent short isoform of dystrophin and associated glycoproteins were identified in spleen by mass spectrometry but appear not be affected in muscular dystrophy. In contrast, the mdx-4cv spleen showed significant proteome-wide changes in other protein species that are involved in metabolism, signaling, and cellular architecture. Since the spleen plays a key role in the immune response, these proteomic alterations may reflect pathophysiological cross talk between the lymphoid system and dystrophic muscles, which are affected by both fiber degeneration and inflammation

Protocol for the Bottom-Up Proteomic Analysis of Mouse Spleen

This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation. For complete details on the use and execution of this protocol, please refer to Dowling et al. (2020)

Metabolic traits in brown trout (Salmo trutta) vary in response to food restriction and intrinsic factors

Metabolic rates vary hugely within and between populations, yet we know relatively little about factors causing intraspecific variation. Since metabolic rate determines the energetic cost of life, uncovering these sources of variation is important to understand and forecast responses to environmental change. Moreover, few studies have examined factors causing intraspecific variation in metabolic flexibility. We explore how extrinsic environmental conditions and intrinsic factors contribute to variation in metabolic traits in brown trout, an iconic and polymorphic species that is threatened across much of its native range. We measured metabolic traits in offspring from two wild populations that naturally show life-history variation in migratory tactics (one anadromous, i.e. sea-migratory, one non-anadromous) that we reared under either optimal food or experimental conditions of long-term food restriction (lasting between 7 and 17 months). Both populations showed decreased standard metabolic rates (SMR—baseline energy requirements) under low food conditions. The anadromous population had higher maximum metabolic rate (MMR) than the non-anadromous population, and marginally higher SMR. The MMR difference was greater than SMR and consequently aerobic scope (AS) was higher in the anadromous population. MMR and AS were both higher in males than females. The anadromous population also had higher AS under low food compared to optimal food conditions, consistent with population-specific effects of food restriction on AS. Our results suggest different components of metabolic rate can vary in their response to environmental conditions, and according to intrinsic (population-background/sex) effects. Populations might further differ in their flexibility of metabolic traits, potentially due to intrinsic factors related to life history (e.g. migratory tactics). More comparisons of populations/individuals with divergent life histories will help to reveal this. Overall, our study suggests that incorporating an understanding of metabolic trait variation and flexibility and linking this to life history and demography will improve our ability to conserve populations experiencing global change

Food and temperature stressors have opposing effects in determining flexible migration decisions in brown trout (Salmo trutta)

With rapid global change, organisms in natural systems are exposed to a multitude of stressors that likely co‐occur, with uncertain impacts. We explored individual and cumulative effects of co‐occurring environmental stressors on the striking, yet poorly understood, phenomenon of facultative migration. We reared offspring of a brown trout population that naturally demonstrates facultative anadromy (sea migration), under different environmental stressor treatments and measured life history responses in terms of migratory tactics and freshwater maturation rates. Juvenile fish were exposed to reduced food availability, temperatures elevated to 1.8°C above natural conditions or both treatments in combination over 18 months of experimental tank rearing. When considered in isolation, reduced food had negative effects on the size, mass and condition of fish across the experiment. We detected variable effects of warm temperatures (negative effects on size and mass, but positive effect on lipids). When combined with food restriction, temperature effects on these traits were less pronounced, implying antagonistic stressor effects on morphological traits. Stressors combined additively, but had opposing effects on life history tactics: migration increased and maturation rates decreased under low food conditions, whereas the opposite occurred in the warm temperature treatment. Not all fish had expressed maturation or migration tactics by the end of the study, and the frequency of these ‘unassigned’ fish was higher in food deprivation treatments, but lower in warm treatments. Fish showing migration tactics were smaller and in poorer condition than fish showing maturation tactics, but were similar in size to unassigned fish. We further detected effects of food restriction on hypo‐osmoregulatory function of migrants that may influence the fitness benefits of the migratory tactic at sea. We also highlight that responses to multiple stressors may vary depending on the response considered. Collectively, our results indicate contrasting effects of environmental stressors on life history trajectories in a facultatively migratory species

Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles

The progressive loss of skeletal muscle mass and concomitant reduction in contractile strength plays a central role in frailty syndrome. Age-related neuronal impairments are closely associated with sarcopenia in the elderly, which is characterized by severe muscular atrophy that can considerably lessen the overall quality of life at old age. Mass-spectrometry-based proteomic surveys of senescent human skeletal muscles, as well as animal models of sarcopenia, have decisively improved our understanding of the molecular and cellular consequences of muscular atrophy and associated fiber-type shifting during aging. This review outlines the mass spectrometric identification of proteome-wide changes in atrophying skeletal muscles, with a focus on contractile proteins as potential markers of changes in fiber-type distribution patterns. The observed trend of fast-to-slow transitions in individual human skeletal muscles during the aging process is most likely linked to a preferential susceptibility of fast-twitching muscle fibers to muscular atrophy. Studies with senescent animal models, including mostly aged rodent skeletal muscles, have confirmed fiber-type shifting. The proteomic analysis of fast versus slow isoforms of key contractile proteins, such as myosin heavy chains, myosin light chains, actins, troponins and tropomyosins, suggests them as suitable bioanalytical tools of fiber-type transitions during aging

Identification of marker proteins of muscular dystrophy in the urine proteome from the mdx-4cv model of dystrophinopathy

Since the protein constituents of urine present a dynamic proteome that can reflect a variety of disease-related alterations in the body, the mass spectrometric survey of proteome-wide changes in urine promises new insights into pathogenic mechanisms. Urine can be investigated in a completely non-invasive way and provides valuable biomedical information on body-wide changes. In this report, we have focused on the urine proteome in X-linked muscular dystrophy using the established mdx-4cv mouse model of dystrophinopathy. In order to avoid potential artefacts due to the manipulation of the biofluid proteome prior to mass spectrometry, crude urine specimens were analyzed without the prior usage of centrifugation steps or concentration procedures. Comparative proteomics revealed 21 increased and 8 decreased proteins out of 870 identified urinary proteoforms using 50 μl of biofluid per investigated sample, i.e. 14 wild type versus 14 mdx-4cv specimens. Promising marker proteins that were almost exclusively found in mdx-4cv urine included nidogen, parvalbumin and titin. Interestingly, the mass spectrometric identification of urine-associated titin revealed a wide spread of peptides over the sequence of this giant muscle protein. The newly established urinomic signature of dystrophinopathy might be helpful for the design of non-invasive assays to improve diagnosis, prognosis, therapy-monitoring and evaluation of potential harmful side effects of novel treatments in the field of muscular dystrophy research

Protocol for the Bottom-Up Proteomic Analysis of Mouse Spleen

This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation. For complete details on the use and execution of this protocol, please refer to Dowling et al. (2020)

Proteomic Identification of Markers of Membrane Repair, Regeneration and Fibrosis in the Aged and Dystrophic Diaphragm

Deficiency in the membrane cytoskeletal protein dystrophin is the underlying cause of the progressive muscle wasting disease named Duchenne muscular dystrophy. In order to detect novel disease marker candidates and confirm the complexity of the pathobiochemical signature of dystrophinopathy, mass spectrometric screening approaches represent ideal tools for comprehensive biomarker discovery studies. In this report, we describe the comparative proteomic analysis of young versus aged diaphragm muscles from wild type versus the dystrophic mdx-4cv mouse model of X-linked muscular dystrophy. The survey confirmed the drastic reduction of the dystrophin-glycoprotein complex in the mdx-4cv diaphragm muscle and concomitant age-dependent changes in key markers of muscular dystrophy, including proteins involved in cytoskeletal organization, metabolite transportation, the cellular stress response and excitation-contraction coupling. Importantly, proteomic markers of the regulation of membrane repair, tissue regeneration and reactive myofibrosis were detected by mass spectrometry and changes in key proteins were confirmed by immunoblotting. Potential disease marker candidates include various isoforms of annexin, the matricellular protein periostin and a large number of collagens. Alterations in these proteoforms can be useful to evaluate adaptive, compensatory and pathobiochemical changes in the intracellular cytoskeleton, myofiber membrane integrity and the extracellular matrix in dystrophin-deficient skeletal muscle tissues

Histopathology of Duchenne muscular dystrophy in correlation with changes in proteomic biomarkers

Duchenne muscular dystrophy is an inherited disorder of early childhood that affects multiple systems in the body. Besides late-onset cardio-respiratory syndrome and various body-wide pathophysiological changes, X-linked muscular dystrophy is primarily classified as a disorder of the skeletal musculature. This is reflected by severe histopathological alterations in voluntary contractile tissues, including progressive fibre degeneration, fat substitution, reactive myofibrosis and chronic inflammation. The underlying cause for dystrophinopathy are genetic abnormalities in the DMD gene, which can result in the almost complete loss of the membrane cytoskeletal protein dystrophin, which triggers the collapse of the dystrophin-associated glycoprotein complex and disintegration of sarcolemmal integrity. This in turn results in an increased frequency of membrane micro-rupturing and abnormal calcium ion fluxes through the impaired plasmalemma, which renders muscle fibres more susceptible to enhanced proteolytic degradation and necrosis. This review focuses on the complexity of skeletal muscle changes in X-linked muscular dystrophy and outlines cell biological and histological alterations in correlation to proteomewide variations as judged by mass spectrometric analyses. This includes a general outline of sample handling, subcellular fraction protocols and modern proteomic approaches using gel electrophoretic and liquid chromatographic methods for efficient protein separation prior to mass spectrometry. The proteomic profiling of the dystrophic and highly fibrotic diaphragm muscle is described as an example to swiftly identify novel proteomic markers of complex histopathological changes during skeletal muscle degeneration. The potential usefulness of new protein markers is examined in relation to key histopathological hallmarks for establishing improved diagnostic, prognostic and therapy-monitoring approaches in the field of dystrophinopathy