Implementing code review in the scientific workflow: Insights from ecology and evolutionary biology.

Code review increases reliability and improves reproducibility of research. As such, code review is an inevitable step in software development and is common in fields such as computer science. However, despite its importance, code review is noticeably lacking in ecology and evolutionary biology. This is problematic as it facilitates the propagation of coding errors and a reduction in reproducibility and reliability of published results. To address this, we provide a detailed commentary on how to effectively review code, how to set up your project to enable this form of review and detail its possible implementation at several stages throughout the research process. This guide serves as a primer for code review, and adoption of the principles and advice here will go a long way in promoting more open, reliable, and transparent ecology and evolutionary biology.

Impacts of ocean warming on echinoderms: A meta-analysis.

Rising ocean temperatures are threatening marine species and populations worldwide, and ectothermic taxa are particularly vulnerable. Echinoderms are an ecologically important phylum of marine ectotherms and shifts in their population dynamics can have profound impacts on the marine environment. The effects of warming on echinoderms are highly variable across controlled laboratory-based studies. Accordingly, synthesis of these studies will facilitate the better understanding of broad patterns in responses of echinoderms to ocean warming. Herein, a meta-analysis incorporating the results of 85 studies (710 individual responses) is presented, exploring the effects of warming on various performance predictors. The mean responses of echinoderms to all magnitudes of warming were compared across multiple biological responses, ontogenetic life stages, taxonomic classes, and regions, facilitated by multivariate linear mixed effects models. Further models were conducted, which only incorporated responses to warming greater than the projected end-of-century mean annual temperatures at the collection sites. This meta-analysis provides evidence that ocean warming will generally accelerate metabolic rate (+32%) and reduce survival (-35%) in echinoderms, and echinoderms from subtropical (-9%) and tropical (-8%) regions will be the most vulnerable. The relatively high vulnerability of echinoderm larvae to warming (-20%) indicates that this life stage may be a significant developmental bottleneck in the near-future, likely reducing successful recruitment into populations. Furthermore, asteroids appear to be the class of echinoderms that are most negatively affected by elevated temperature (-30%). When considering only responses to magnitudes of warming representative of end-of-century climate change projections, the negative impacts on asteroids, tropical species and juveniles were exacerbated (-51%, -34% and -40% respectively). The results of these analyses enable better predictions of how keystone and invasive echinoderm species may perform in a warmer ocean, and the possible consequences for populations, communities and ecosystems.

Coral adaptation to climate change: Meta-analysis reveals high heritability across multiple traits.

Anthropogenic climate change is a rapidly intensifying selection pressure on biodiversity across the globe and, particularly, on the world's coral reefs. The rate of adaptation to climate change is proportional to the amount of phenotypic variation that can be inherited by subsequent generations (i.e., narrow-sense heritability, h2 ). Thus, traits that have higher heritability (e.g., h2  > 0.5) are likely to adapt to future conditions faster than traits with lower heritability (e.g., h2  < 0.1). Here, we synthesize 95 heritability estimates across 19 species of reef-building corals. Our meta-analysis reveals low heritability (h2 < 0.25) of gene expression metrics, intermediate heritability (h2  = 0.25-0.50) of photochemistry, growth, and bleaching, and high heritability (h2  > 0.50) for metrics related to survival and immune responses. Some of these values are higher than typically observed in other taxa, such as survival and growth, while others were more comparable, such as gene expression and photochemistry. There was no detectable effect of temperature on heritability, but narrow-sense heritability estimates were generally lower than broad-sense estimates, indicative of significant non-additive genetic variation across traits. Trait heritability also varied depending on coral life stage, with bleaching and growth in juveniles generally having lower heritability compared to bleaching and growth in larvae and adults. These differences may be the result of previous stabilizing selection on juveniles or may be due to constrained evolution resulting from genetic trade-offs or genetic correlations between growth and thermotolerance. While we find no evidence that heritability decreases under temperature stress, explicit tests of the heritability of thermal tolerance itself-such as coral thermal reaction norm shape-are lacking. Nevertheless, our findings overall reveal high trait heritability for the majority of coral traits, suggesting corals may have a greater potential to adapt to climate change than has been assumed in recent evolutionary models.

The mechanism underlying transient weakness in myotonia congenita.

In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane potential to -25 to -35 mV in the genetic and pharmacologic models of Becker disease. Both Na+ and Ca2+ currents contribute to plateau potentials. Na+ persistent inward current (NaPIC) through NaV1.4 channels is the key trigger of plateau potentials and current through CaV1.1 Ca2+ channels contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo. These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita.

Myotonia is a neuromuscular condition that causes problems with the relaxation of muscles following voluntary movements. One type of myotonia is Becker disease, also called recessive myotonia congenita. This is a genetic condition that causes muscle stiffness as a result of involuntary muscle activity. Patients may also suffer transient weakness for a few seconds or as long as several minutes after initiating a movement. The cause of these bouts of temporary weakness is still unclear, but there are hints that it could be linked to the muscle losing its excitability, the ability to respond to the stimuli that make it contract. However, this is at odds with findings that show that muscles in Becker disease are hyperexcitable. Muscle excitability depends on the presence of different concentrations of charged ions (positively charged sodium, calcium and potassium ions and negatively charged chloride ions) inside and outside of each muscle cells. These different concentrations of ions create an electric potential across the cell membrane, also called the 'membrane potential'. When a muscle cell gets stimulated, proteins on the cell membrane known as ion channels open. This allows the flow of ions between the inside and the outside of the cell, which causes an electrical current that triggers muscle contraction. To better understand the causes behind this muscle weakness, Myers et al. used mice that had either been genetically manipulated or given drugs to mimic Becker disease. By measuring both muscle force and the electrical currents that drive contraction, Myers et al. found that the mechanism underlying post-movement weakness involved a transient change in the concentrations of positively charged ions inside and outside the cells. Further experiments showed that proteins that regulate the passage of both sodium and calcium in and out of the cell ­ called sodium and calcium channels ­ contributed to this change in concentration. In addition, Myers et al. discovered that using a drug called ranolazine to stop sodium ions from entering the cell eliminated transient weakness in live mice. These findings suggest that in Becker disease, muscles cycle rapidly between being hyperexcited or not able to be excited, and that targeting the flow of sodium ions into the cell could be an effective treatment to prevent transient weakness in myotonia congenita. This study paves the way towards the development of new therapies to treat Becker disease as well as other muscle ion channel diseases with transient weakness such as periodic paralysis.

Gone girl: Richardson's ground squirrel offspring and neighbours are resilient to female removal.

Within matrilineal societies, the presence of mothers and female kin can greatly enhance survival and reproductive success owing to kin-biased alarm calling, cooperation in territory defence, protection from infanticidal conspecifics, joint care of young and enhanced access to resources. The removal of mothers by predators or disease is expected to increase the stress experienced by offspring via activation of their hypothalamic-pituitary-adrenal axis, increasing circulating glucocorticoids and reducing offspring survival and reproductive success. Yet, few studies have removed mothers in the post-weaning period to examine the assumed physiological and fitness consequences associated with these mortality events. We examined how the loss of a mother affects juvenile Richardson's ground squirrels' (Urocitellus richardsonii) faecal glucocorticoid metabolites and their survival. Given that neighbours are often close kin, we further hypothesized that conspecific removal would similarly diminish the fitness of neighbouring individuals. Upon removing the mother, we detected no impact on offspring or neighbouring conspecific faecal glucocorticoid metabolites in the removal year, or on overwinter survival in the following year. Furthermore, no impact on neighbour reproductive success was detected. Given the high predation rates of ground squirrels in wild populations, resilience to a changing social environment would prove adaptive for both surviving kin and non-kin.

A novel alarm signal in aquatic prey: Familiar minnows coordinate group defences against predators through chemical disturbance cues.

Animal signalling systems outside the realm of human perception remain largely understudied. These systems consist of four main components: a signalling context, a voluntary signal, receiver responses and resulting fitness benefits to both the signaller and receiver(s). It is often most difficult to determine incidental cues from voluntary signals. One example is chemical disturbance cues released by aquatic prey during predator encounters that may serve to alert conspecifics of nearby risk and initiate tighter shoaling. We aimed to test whether disturbance cues are released incidentally (i.e. as a cue) or are produced voluntarily depending on a specific signalling context such as the audience surrounding the individual, and thus constitute a signal. We hypothesized that if receivers use disturbance cues to communicate risk among themselves, they would produce more (or more potent) disturbance cues when present in a group of conspecifics rather than when they are isolated (presence/absence of an audience) and use disturbance cues more when present alongside familiar rather than unfamiliar conspecifics (audience composition effect). We placed fathead minnows (Pimephales promelas) in groups with familiar fish, unfamiliar fish or as isolated individuals with no audience present, and then simulated a predator chase to evoke disturbance cues. We used bioassays with independent receivers to assess whether the disturbance cues produced differed depending on the signallers' audience. We found evidence of voluntary signalling, as minnows responded to disturbance cues from groups of fish with tighter shoaling while disturbance cues from isolated minnows did not evoke a significant shoaling response (presence/absence audience effect). Receivers also increased shoaling, freezing and dashing more in response to disturbance cues from familiar groups compared to disturbance cues from unfamiliar groups or isolated minnows (audience composition effect). Together, these findings support our hypothesis that disturbance cues are used as an antipredator signal to initiate coordinated group defences among familiar conspecifics involving shoaling, freezing and dashing. This study represents the strongest evidence to date that chemicals released by aquatic prey upon disturbance by predators serve as voluntary signals rather than simply cues that prey have evolved to detect when assessing their risk of predation.

Like mother, like daughter: heritability of female Richardson's ground squirrel Urocitellus richardsonii cortisol stress responses.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis liberates glucocorticoids, which provides an acute indication of an individual's response to stressors. The heritability of the stress response in wild mammals, however, remains poorly documented. We quantified the cortisol stress response of female Richardson's ground squirrels (RGSs) to handling and physical restraint, testing for: (1) the effects of individual age, time of day, and sample latency; (2) repeatability within individuals; (3) narrow-sense heritability; and (4) differences among individuals owing to potential genetic and/or environmental effects. We detected a positive linear relationship between baseline plasma cortisol (BL-cortisol) concentration and stress-induced plasma cortisol (SI-cortisol) concentration that defined each individual's cortisol stress response. BL-cortisol, SI-cortisol, and stress response did not differ according to the time the sample was taken, or by subject age. Cortisol stress response was highly repeatable within individuals, had a mother-offspring heritability of h 2 = 0.40 ± 0.24 (mean ± SE), full-sibling heritability of h FS 2 = 0.37 ± 0.71 , and half-sibling heritability of h HS 2 = 0.75 ± 1.41 . Stress responses of sibling groups, immediate-family groups, and squirrels within a given area did not differ, whereas those of individuals from more distantly related matrilines did. Our results highlight the natural variability in HPA axis reactivity among individuals by quantifying both BL- and SI-cortisol levels, demonstrate partial heritability of the stress response that is not attributable to environmental variation, and suggest that at least part of an individual's stress response can be accounted for by differences in matrilineal history.

The socially mediated recovery of a fearful fish paired with periodically replaced calm models.

Social learning is an important mechanism for acquiring knowledge about environmental risk. However, little work has explored the learning of safety and how such learning outcomes are shaped by the social environment. Here, we exposed minnows, Pimephales promelas, to a high-risk environment to induce behavioural responses associated with fear (e.g. neophobia). We then used the presence of calm conspecific models (low-risk individuals) to weaken these responses. When observers (individuals from the high-risk environment) and models were paired consistently in a one-on-one setting, the observers showed no recovery (i.e. no weakening of the fear responses), and instead the models indirectly acquired those responses (i.e. a socially transmitted state of fear). However, observers paired with models that were periodically replaced with new calm models showed a significant recovery, and each new model showed diminished socially transmitted fear. We argue that our understanding of predation-related fear and social information transfer can prove fruitful in understanding problems with fear and stress across animal taxa, including among humans who experience post-traumatic stress and secondary trauma. Our findings indicate that the periodic replacement of models can promote fear recovery in observers and reduce socially transmitted fear in models.

Trust thy neighbour in times of trouble: background risk alters how tadpoles release and respond to disturbance cues.

In aquatic environments, uninjured prey escaping a predator release chemical disturbance cues into the water. However, it is unknown whether these cues are a simple physiological by-product of increased activity or whether they represent a social signal that is under some control by the sender. Here, we exposed wood frog tadpoles (Lithobates sylvaticus) to either a high or low background risk environment and tested their responses to disturbance cues (or control cues) produced by tadpoles from high-risk or low-risk backgrounds. We found an interaction between risk levels associated with the cue donor and cue recipient. While disturbance cues from low-risk donors did not elicit an antipredator response in low-risk receivers, they did in high-risk receivers. In addition, disturbance cues from high-risk donors elicited a marked antipredator response in both low- and high-risk receivers. The response of high-risk receivers to disturbance cues from high-risk donors was commensurate with other treatments, indicating an all-or-nothing response. Our study provides evidence of differential production and perception of social cues and provides insights into their function and evolution in aquatic vertebrates. Given the widespread nature of disturbance cues in aquatic prey, there may exist a social signalling system that remains virtually unexplored by ecologists.

Open-label trial of ranolazine for the treatment of myotonia congenita.

OBJECTIVE: To determine open-label, pilot study whether ranolazine could improve signs and symptoms of myotonia and muscle stiffness in patients with myotonia congenita (MC). METHODS: Thirteen participants were assessed at baseline and 2, 4, and 5 weeks. Ranolazine was started after baseline assessment (500 mg twice daily), increased as tolerated after week 2 (1,000 mg twice daily), and maintained until week 4. Outcomes included change from baseline to week 4 in self-reported severity of symptoms (stiffness, weakness, and pain), Timed Up and Go (TUG), hand grip and eyelid myotonia, and myotonia on EMG. RESULTS: Self-reported severity of stiffness (p < 0.0001) and weakness (p < 0.01) was significantly improved compared with baseline. TUG and grip myotonia times were reduced (p = 0.03, p = 0.01). EMG of the abductor digiti minimi and tibialis anterior showed significantly reduced myotonia duration (p < 0.001, p < 0.01) at week 4. No participant discontinued ranolazine because of side effects. CONCLUSIONS: Ranolazine appeared to be well tolerated over a period of 4 weeks in individuals with MC, and ranolazine resulted in improvement of signs and symptoms of muscle stiffness. The findings of this study suggest that ranolazine should be investigated in a larger controlled study. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that ranolazine improves myotonia in myotonia congenita.

Mechanisms underlying the control of responses to predator odours in aquatic prey.

In aquatic systems, chemical cues are a major source of information through which animals are able to assess the current state of their environment to gain information about local predation risk. Prey use chemicals released by predators (including cues from a predator's diet) and other prey (such as alarm cues and disturbance cues) to mediate a range of behavioural, morphological and life-history antipredator defences. Despite the wealth of knowledge on the ecology of antipredator defences, we know surprisingly little about the physiological mechanisms that control the expression of these defensive traits. Here, we summarise the current literature on the mechanisms known to specifically mediate responses to predator odours, including dietary cues. Interestingly, these studies suggest that independent pathways may control predator-specific responses, highlighting the need for greater focus on predator-derived cues when looking at the mechanistic control of responses. Thus, we urge researchers to tease apart the effects of predator-specific cues (i.e. chemicals representing a predator's identity) from those of diet-mediated cues (i.e. chemicals released from a predator's diet), which are known to mediate different ecological endpoints. Finally, we suggest some key areas of research that would greatly benefit from a more mechanistic approach.

Sodium channel slow inactivation as a therapeutic target for myotonia congenita.

OBJECTIVE: Patients with myotonia congenita have muscle hyperexcitability due to loss-of-function mutations in the chloride channel in skeletal muscle, which causes spontaneous firing of muscle action potentials (myotonia), producing muscle stiffness. In patients, muscle stiffness lessens with exercise, a change known as the warmup phenomenon. Our goal was to identify the mechanism underlying warmup and to use this information to guide development of novel therapy. METHODS: To determine the mechanism underlying warmup, we used a recently discovered drug to eliminate muscle contraction, thus allowing prolonged intracellular recording from individual muscle fibers during induction of warmup in a mouse model of myotonia congenita. RESULTS: Changes in action potentials suggested slow inactivation of sodium channels as an important contributor to warmup. These data suggested that enhancing slow inactivation of sodium channels might offer effective therapy for myotonia. Lacosamide and ranolazine enhance slow inactivation of sodium channels and are approved by the US Food and Drug Administration for other uses in patients. We compared the efficacy of both drugs to mexiletine, a sodium channel blocker currently used to treat myotonia. In vitro studies suggested that both lacosamide and ranolazine were superior to mexiletine. However, in vivo studies in a mouse model of myotonia congenita suggested that side effects could limit the efficacy of lacosamide. Ranolazine produced fewer side effects and was as effective as mexiletine at a dose that produced none of mexiletine's hypoexcitability side effects. INTERPRETATION: We conclude that ranolazine has excellent therapeutic potential for treatment of patients with myotonia congenita.

Altered sodium channel-protein associations in critical illness myopathy.

BACKGROUND: During the acute phase of critical illness myopathy (CIM) there is inexcitability of skeletal muscle. In a rat model of CIM, muscle inexcitability is due to inactivation of sodium channels. A major contributor to this sodium channel inactivation is a hyperpolarized shift in the voltage dependence of sodium channel inactivation. The goal of the current study was to find a biochemical correlate of the hyperpolarized shift in sodium channel inactivation. METHODS: The rat model of CIM was generated by cutting the sciatic nerve and subsequent injections of dexamethasone for 7 days. Skeletal muscle membranes were prepared from gastrocnemius muscles, and purification and biochemical analyses carried out. Immunoprecipitations were performed with a pan-sodium channel antibody, and the resulting complexes probed in Western blots with various antibodies. RESULTS: We carried out analyses of sodium channel glycosylation, phosphorylation, and association with other proteins. Although there was some loss of channel glycosylation in the disease, as assessed by size analysis of glycosylated and de-glycosylated protein in control and CIM samples, previous work by other investigators suggest that such loss would most likely shift channel inactivation gating in a depolarizing direction; thus such loss was viewed as compensatory rather than causative of the disease. A phosphorylation site at serine 487 was identified on the NaV 1.4 sodium channel α subunit, but there was no clear evidence of altered phosphorylation in the disease. Co-immunoprecipitation experiments carried out with a pan-sodium channel antibody confirmed that the sodium channel was associated with proteins of the dystrophin associated protein complex (DAPC). This complex differed between control and CIM samples. Syntrophin, dystrophin, and plectin associated strongly with sodium channels in both control and disease conditions, while ß-dystroglycan and neuronal nitric oxide synthase (nNOS) associated strongly with the sodium channel only in CIM. Recording of action potentials revealed that denervated muscle in mice lacking nNOS was more excitable than control denervated muscle. CONCLUSION: Taken together, these data suggest that the conformation/protein association of the sodium channel complex differs in control and critical illness myopathy muscle membranes; and suggest that nitric oxide signaling plays a role in development of muscle inexcitability.

Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy.

The processes that trigger severe muscle atrophy and loss of myosin in critical illness myopathy (CIM) are poorly understood. It has been reported that muscle disuse alters Ca(2+) handling by the sarcoplasmic reticulum. Since inactivity is an important contributor to CIM, this finding raises the possibility that elevated levels of the proteins involved in Ca(2+) handling might contribute to development of CIM. CIM was induced in 3- to 5-mo-old rats by sciatic nerve lesion and infusion of dexamethasone for 1 wk. Western blot analysis revealed increased levels of ryanodine receptor (RYR) isoforms-1 and -2 as well as the dihydropyridine receptor/voltage-gated calcium channel type 1.1 (DHPR/Ca(V) 1.1). Immunostaining revealed a subset of fibers with elevation of RYR1 and Ca(V) 1.1 that had severe atrophy and disorganization of sarcomeres. These findings suggest increased Ca(2+) release from the sarcoplasmic reticulum may be an important contributor to development of CIM. To assess the endogenous functional effects of increased intracellular Ca(2+) in CIM, proteolysis of α-fodrin, a well-known target substrate of Ca(2+)-activated proteases, was measured and found to be 50% greater in CIM. There was also selective degradation of myosin heavy chain relative to actin in CIM muscle. Taken together, our findings suggest that increased Ca(2+) release from the sarcoplasmic reticulum may contribute to pathology in CIM.

Inactivation of sodium channels underlies reversible neuropathy during critical illness in rats.

Neuropathy and myopathy can cause weakness during critical illness. To determine whether reduced excitability of peripheral nerves, rather than degeneration, is the mechanism underlying acute neuropathy in critically ill patients, we prospectively followed patients during the acute phase of critical illness and early recovery and assessed nerve conduction. During the period of early recovery from critical illness, patients recovered from neuropathy within days. This rapidly reversible neuropathy has not to our knowledge been previously described in critically ill patients and may be a novel type of neuropathy. In vivo intracellular recordings from dorsal root axons in septic rats revealed reduced action potential amplitude, demonstrating that reduced excitability of nerve was the mechanism underlying neuropathy. When action potentials were triggered by hyperpolarizing pulses, their amplitudes largely recovered, indicating that inactivation of sodium channels was an important contributor to reduced excitability. There was no depolarization of axon resting potential in septic rats, which ruled out a contribution of resting potential to the increased inactivation of sodium channels. Our data suggest that a hyperpolarized shift in the voltage dependence of sodium channel inactivation causes increased sodium inactivation and reduced excitability. Acquired sodium channelopathy may be the mechanism underlying acute neuropathy in critically ill patients.