Conservation genomics of natural and managed populations: building a conceptual and practical framework

The boom of massive parallel sequencing (MPS) technology and its applications in conservation of natural and managed populations brings new opportunities and challenges to meet the scientific questions that can be addressed. Genomic conservation offers a wide range of approaches and analytical techniques, with their respective strengths and weaknesses that rely on several implicit assumptions. However, finding the most suitable approaches and analysis regarding our scientific question are often difficult and time‐consuming. To address this gap, a recent workshop entitled ‘ConGen 2015’ was held at Montana University in order to bring together the knowledge accumulated in this field and to provide training in conceptual and practical aspects of data analysis applied to the field of conservation and evolutionary genomics. Here, we summarize the expertise yield by each instructor that has led us to consider the importance of keeping in mind the scientific question from sampling to management practices along with the selection of appropriate genomics tools and bioinformatics challenges.     

Genetic Color Morphs in the Eastern Mosquitofish Experience Different Social Environments in the Wild and Laboratory

The social environment of an animal is an especially interesting component of its environment because it can be shaped by both genetic and non‐genetic variation among social partners. Indirect genetic effects (IGEs) are those created when genetic variation in social partners contributes to variation in an individual's phenotype; a potentially common form of IGE occurs when the expression of a behavioral phenotype depends on the particular genotypic combination of interacting individuals. Although IGEs can profoundly affect individual‐ and group‐level fitness, population dynamics, and even community structure, understanding their importance is complicated by two inherent challenges: (1) identifying individuals with genetic differences in social interactions that can contribute to IGEs and (2) characterizing natural social interactions that potentially involve IGEs. As a first step toward addressing both these challenges in the same system, we investigated social interactions involving genetically distinct male color morphs in the poeciliid fish Gambusia holbrooki under natural and laboratory conditions. Previous work indicates that melanic (M) and silver (S) males differ in social behavior and in how conspecifics respond to them, suggesting the potential for IGEs. We used a combination of live and video recording of social groups in two natural populations and in the laboratory to determine the potential for IGEs to contribute to behavioral variation in this species. We found that M males had more social partners, and especially more female social partners than did S males, in nature and in the laboratory. These results suggest that both direct and indirect genetic effects have the potential to play a role in the expression and evolution of social behavior in G. holbrooki.     

A Model to Inform Management Actions as a Response to Chytridiomycosis-Associated Decline

Decision-analytic models provide forecasts of how systems of interest will respond to management. These models can be parameterized using empirical data, but sometimes require information elicited from experts. When evaluating the effects of disease in species translocation programs, expert judgment is likely to play a role because complete empirical information will rarely be available. We illustrate development of a decision-analytic model built to inform decision-making regarding translocations and other management actions for the boreal toad (Anaxyrus boreas boreas), a species with declines linked to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd). Using the model, we explored the management implications of major uncertainties in this system, including whether there is a genetic basis for resistance to pathogenic infection by Bd, how translocation can best be implemented, and the effectiveness of efforts to reduce the spread of Bd. Our modeling exercise suggested that while selection for resistance to pathogenic infection by Bd could increase numbers of sites occupied by toads, and translocations could increase the rate of toad recovery, efforts to reduce the spread of Bd may have little effect. We emphasize the need to continue developing and parameterizing models necessary to assess management actions for combating chytridiomycosis-associated declines.

     

Increased Early Processing of Task-Irrelevant Auditory Stimuli in Older Adults

The inhibitory deficit hypothesis of cognitive aging posits that older adults’ inability to adequately suppress processing of irrelevant information is a major source of cognitive decline. Prior research has demonstrated that in response to task-irrelevant auditory stimuli there is an age-associated increase in the amplitude of the N1 wave, an ERP marker of early perceptual processing. Here, we tested predictions derived from the inhibitory deficit hypothesis that the age-related increase in N1 would be 1) observed under an auditory-ignore, but not auditory-attend condition, 2) attenuated in individuals with high executive capacity (EC), and 3) augmented by increasing cognitive load of the primary visual task. ERPs were measured in 114 well-matched young, middle-aged, young-old, and old-old adults, designated as having high or average EC based on neuropsychological testing. Under the auditory-ignore (visual-attend) task, participants ignored auditory stimuli and responded to rare target letters under low and high load. Under the auditory-attend task, participants ignored visual stimuli and responded to rare target tones. Results confirmed an age-associated increase in N1 amplitude to auditory stimuli under the auditory-ignore but not auditory-attend task. Contrary to predictions, EC did not modulate the N1 response. The load effect was the opposite of expectation: the N1 to task-irrelevant auditory events was smaller under high load. Finally, older adults did not simply fail to suppress the N1 to auditory stimuli in the task-irrelevant modality; they generated a larger response than to identical stimuli in the task-relevant modality. In summary, several of the study’s findings do not fit the inhibitory-deficit hypothesis of cognitive aging, which may need to be refined or supplemented by alternative accounts.     

Membrane Potentials,Synaptic Responses,Neuronal Circuitry,Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell''s membrane potential and which we address in these experiments are: (1) Ion concentration of K⁺ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.     

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution^1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions ^7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin ¹⁸, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH < 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.     

Phosphoramidate derivates as controlled-release prodrugs of l-Dopa

l-Dopa has continued to be a mainstay in the symptomatic treatment of Parkinson’s disease (PD). However, extensive peripheral metabolism, a short systemic circulation half-life and development of motor complications called dyskinesia prevents its long-term utilization as a PD therapeutic. Herein, we report a series of phosphoramidate derivatives of l-Dopa and controlled release of l-Dopa at pH 7.4 and 3. The kinetic data for the release of l-Dopa support our hypothesis that a proximal carboxylic acid can promote the pH-triggered hydrolysis of the phosphoramidate PN bond. As expected, esterification of the proximal carboxylic acid protects the scaffold from rapid release at low pH. This latter observation is particularly noteworthy as it suggests that the phosphoramidate-based l-Dopa?conjugate scaffold can be adapted for oral administration as an ester prodrug.     

Peripheral T-cell lymphoma, unspecified--the analysis of the data from the Czech Lymphoma Study Group (CLSG) registry

BACKGROUND: Peripheral T-cell lymphoma, unspecified (PTCL-US) is one of the entities from the infrequent family of nodal mature T-cell lymphomas. The clinical course is aggressive, and despite multiagent chemotherapy, the median survival is about 2 years. Published data are limited to retrospective, mostly single-center studies or reviews and usually include more lymphoma subtypes. AIM: To evaluate the current treatment modalities, clinical outcome and prognostic factors in unselected, new diagnosed patients with PTCL-US in the population of the central european region (Czech Republic). METHOD: Czech Lymphoma Study Group is a national scientific organization which provides an on-line database registry which collects a data about almost all new diagnosed lymphoma patients since year 2000. All diagnostic biopsies were reviewed by a reference pathologist. RESULTS: We analyzed 63 patients with new diagnosis of PTCL-US. The median age was 59 years (25-81), chemotherapy (CHT) was administered in 56 of the 63 patients: anthracyclin-based CHT in 51%, intensive CHT in 21% and non-anthracyclin regimen was applied in 13% of the patients. The overall response rate was 74.4%, (CR in 57.4%). After a median follow-up of 19.6 months, 41% of the patients were in CR, 3.4% in PR or stable disease and 55% of the patients died. The estimated survival probability in 3 years was 36%. Clinical stage (IV) and CR achievement were found to be independent survival predictors in a multivariate analysis. CONCLUSIONS: Although the current treatment modalities are mostly ineffective in PTCL-US, appropriate intensive treatment may lead to prolonged remission but not survival.