Access to services related to reproductive and sexual health is critical to the health of women but has been threatened in recent years. Family physicians are trained to provide a range of women’s health care services and are an essential part of the health care workforce in rural and underserved areas, where access to these services may be limited.

Family physician researchers continue to provide assistance to improve family medicine care. Commentaries on social determinants of health lead off this issue. Next, we have several papers on successful interventions by clinicians and/or patients to improve diabetes control, and then other provide information on other practice interventions that make a difference in overall care. Drug advertising continues to mislead. There is costly and nonproductive overuse of specific types of care. Herein is also a Scoping Review of possible indices for determining timely initiation of advance care planning. The issue’s clinical reviews on use of transgender care, cervical myelopathy, and inhaled steroids for chronic obstructive pulmonary disease are pertinent, thorough, and timely.

Kathryn Knight

Flora Borne, Alexander Kovalev, Stanislav Gorb, and Virginie Courtier-Orgogozo

Insects produce a variety of adhesives for diverse functions such as locomotion, mating, and egg or pupal anchorage to substrates. Although they are important for the biology of organisms and potentially represent a great resource for developing new materials, insect adhesives have been little studied so far. Here, we examined the adhesive properties of the larval glue of Drosophila melanogaster. This glue is made of glycosylated proteins and allows the animal to adhere to a substrate during metamorphosis. We designed an adhesion test to measure the pull-off force required to detach a pupa from a substrate and to evaluate the contact area covered by the glue. We found that the pupa adheres with similar forces to a variety of substrates (with distinct roughness, hydrophilic and charge properties). We obtained an average pull-off force of 217 mN, corresponding to 15,500 times the weight of a pupa and an adhesion strength of 137–244 kPa. Surprisingly, the pull-off forces did not depend on the contact area. Our study paves the way for a genetic dissection of the components of D. melanogaster glue that confer its particular adhesive properties.

Fredrik Andreasson, Arne Hegemann, Andreas Nord, and Jan-Ake Nilsson

The capacity to get rid of excess heat produced during hard work is a possible constraint on parental effort during reproduction [heat dissipation limit (HDL) theory]. We released hard-working blue tits (Cyanistes caeruleus) from this constraint by experimentally removing ventral plumage. We then assessed whether this changed their reproductive effort (feeding rate and nestling size) and levels of self-maintenance (change in body mass and innate immune function). Feather-clipped females reduced the number of feeding visits and increased levels of constitutive innate immunity compared with unclipped females but did not fledge smaller nestlings. Thus, they increased self-maintenance without compromising current reproductive output. In contrast, feather clipping did not affect the number of feeding visits or innate immune function in males, despite increased heat loss rate. Our results show that analyses of physiological parameters, such as constitutive innate immune function, can be important when trying to understand sources of variation in investment in self-maintenance versus reproductive effort and that risk of overheating can influence innate immune function during reproduction.

Ana Gabriela Jimenez, Emily Cornelius Ruhs, Kailey J. Tobin, Katie N. Anderson, Audrey Le Pogam, Lyette Regimbald, and Francois Vezina

Seasonal changes in maximal thermogenic capacity (M_sum) in wild black-capped chickadees suggests that adjustments in metabolic performance are slow and begin to take place before winter peaks. However, when mean minimal ambient temperature (T_a) reaches –10°C, the chickadee phenotype appears to provide enough spare capacity to endure days with colder T_a, down to –20°C or below. This suggests that birds could also maintain a higher antioxidant capacity as part of their cold-acclimated phenotype to deal with sudden decreases in temperature. Here, we tested how environmental mismatch affected oxidative stress by comparing cold-acclimated (–5°C) and transition (20°C) phenotypes in chickadees exposed to an acute 15°C drop in temperature with that of control individuals. We measured superoxide dismutase, catalase and glutathione peroxidase activities, as well as lipid peroxidation damage and antioxidant scavenging capacity in pectoralis muscle, brain, intestine and liver. We generally found differences between seasonal phenotypes and across tissues, but no differences with respect to an acute cold drop treatment. Our data suggest oxidative stress is closely matched to whole-animal physiology in cold-acclimated birds compared with transition birds, implying that changes to the oxidative stress system happen slowly.

Peter J. Edmunds and Scott C. Burgess

Experiments with coral fragments (i.e. nubbins) have shown that net calcification is depressed by elevated P_CO2. Evaluating the implications of this finding requires scaling of results from nubbins to colonies, yet the experiments to codify this process have not been carried out. Building from our previous research demonstrating that net calcification of Pocillopora verrucosa (2–13 cm diameter) was unaffected by P_CO2 (400 and 1000 µatm) and temperature (26.5 and 29.7°C), we sought generality to this outcome by testing how colony size modulates P_CO2 and temperature sensitivity in a branching acroporid. Together, these taxa represent two of the dominant lineages of branching corals on Indo-Pacific coral reefs. Two trials conducted over 2 years tested the hypothesis that the seasonal range in seawater temperature (26.5 and 29.2°C) and a future P_CO2 (1062 µatm versus an ambient level of 461 µatm) affect net calcification of an ecologically relevant size range (5–20 cm diameter) of colonies of Acropora hyacinthus. As for P. verrucosa, the effects of temperature and P_CO2 on net calcification (mg day^–1) of A. verrucosa were not statistically detectable. These results support the generality of a null outcome on net calcification of exposing intact colonies of branching corals to environmental conditions contrasting seasonal variation in temperature and predicted future variation in P_CO2. While there is a need to expand beyond an experimental culture relying on coral nubbins as tractable replicates, rigorously responding to this need poses substantial ethical and logistical challenges.

Vanessa Corby-Harris, Megan E. Deeter, Lucy Snyder, Charlotte Meador, Ashley C. Welchert, Amelia Hoffman, and Bethany T. Obernesser

Recent widespread honey bee (Apis mellifera) colony loss is attributed to a variety of stressors, including parasites, pathogens, pesticides and poor nutrition. In principle, we can reduce stress-induced declines in colony health by either removing the stressor or increasing the bees' tolerance to the stressor. This latter option requires a better understanding than we currently have of how honey bees respond to stress. Here, we investigated how octopamine, a stress-induced hormone that mediates invertebrate physiology and behavior, influences the health of young nurse-aged bees. Specifically, we asked whether octopamine induces abdominal lipid and hypopharyngeal gland (HG) degradation, two physiological traits of stressed nurse bees. Nurse-aged workers were treated topically with octopamine and their abdominal lipid content, HG size and HG autophagic gene expression were measured. Hemolymph lipid titer was measured to determine whether tissue degradation was associated with the release of nutrients from these tissues into the hemolymph. The HGs of octopamine-treated bees were smaller than control bees and had higher levels of HG autophagy gene expression. Octopamine-treated bees also had higher levels of hemolymph lipid compared with control bees. Abdominal lipids did not change in response to octopamine. Our findings support the hypothesis that the HGs are a rich source of stored energy that can be mobilized during periods of stress.

Ayelen M. Blanco, Juan I. Bertucci, Jose L. Soengas, and Suraj Unniappan

This research assessed the direct effects of insulin on nutrient-sensing mechanisms in the brain of rainbow trout (Oncorhynchus mykiss) using an in vitro approach. Cultured hypothalamus and hindbrain were exposed to 1 µmol l^–1 insulin for 3 h, and signals involved in appetite regulation and nutrient-sensing mechanisms were measured. Additionally, the involvement of the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the actions of insulin was studied by using the inhibitor wortmannin. Treatment with insulin alone did not elicit many changes in the appetite regulators and nutrient-sensing-related genes and enzymes tested in the hypothalamus and hindbrain. However, we found that, when insulin and nutrients were added together, insulin reversed most of the effects exerted by nutrients alone, suggesting that insulin changes responsiveness to nutrients at the central level. Effects reversed by insulin included expression levels of genes related to the sensing of both glucose (slc2a2, slc5a1, gck, pck1, pklr, g6pcb, gys1, tas1r3 and nr1h3 in the hindbrain, and slc2a2, pklr and pck1 in the hypothalamus) and fatty acid (cd36 in the hindbrain, and cd36 and acly in the hypothalamus). Nutrient-induced changes in the activity of Acly and Cpt-1 in the hindbrain and of Pepck, Acly, Fas and Hoad in the hypothalamus were also reversed by insulin. Most of the insulin effects disappeared in the presence of wortmannin, suggesting the PI3K/Akt pathway is a mediator of the effects of insulin reported here. This study adds new information to our knowledge of the mechanisms regulating nutrient sensing in fish.

Daniel P. Small, Piero Calosi, Samuel P. S. Rastrick, Lucy M. Turner, Stephen Widdicombe, and John I. Spicer

Regulation of extracellular acid–base balance, while maintaining energy metabolism, is recognised as an important aspect when defining an organism's sensitivity to environmental changes. This study investigated the haemolymph buffering capacity and energy metabolism (oxygen consumption, haemolymph [l-lactate] and [protein]) in early benthic juveniles (carapace length <40 mm) of the European lobster, Homarus gammarus, exposed to elevated temperature and P_CO2. At 13°C, H. gammarus juveniles were able to fully compensate for acid–base disturbances caused by the exposure to elevated seawater P_CO2 at levels associated with ocean acidification and carbon dioxide capture and storage (CCS) leakage scenarios, via haemolymph [HCO₃^–] regulation. However, metabolic rate remained constant and food consumption decreased under elevated P_CO2, indicating reduced energy availability. Juveniles at 17°C showed no ability to actively compensate haemolymph pH, resulting in decreased haemolymph pH particularly under CCS conditions. Early benthic juvenile lobsters at 17°C were not able to increase energy intake to offset increased energy demand and therefore appear to be unable to respond to acid–base disturbances due to increased P_CO2 at elevated temperature. Analysis of haemolymph metabolites suggests that, even under control conditions, juveniles were energetically limited. They exhibited high haemolymph [l-lactate], indicating recourse to anaerobic metabolism. Low haemolymph [protein] was linked to minimal non-bicarbonate buffering and reduced oxygen transport capacity. We discuss these results in the context of potential impacts of ongoing ocean change and CCS leakage scenarios on the development of juvenile H. gammarus and future lobster populations and stocks.

Karen L. Carleton, Daniel Escobar-Camacho, Sara M. Stieb, Fabio Cortesi, and N. Justin Marshall

Among vertebrates, teleost eye diversity exceeds that found in all other groups. Their spectral sensitivities range from ultraviolet to red, and the number of visual pigments varies from 1 to over 40. This variation is correlated with the different ecologies and life histories of fish species, including their variable aquatic habitats: murky lakes, clear oceans, deep seas and turbulent rivers. These ecotopes often change with the season, but fish may also migrate between ecotopes diurnally, seasonally or ontogenetically. To survive in these variable light habitats, fish visual systems have evolved a suite of mechanisms that modulate spectral sensitivities on a range of timescales. These mechanisms include: (1) optical media that filter light, (2) variations in photoreceptor type and size to vary absorbance and sensitivity, and (3) changes in photoreceptor visual pigments to optimize peak sensitivity. The visual pigment changes can result from changes in chromophore or changes to the opsin. Opsin variation results from changes in opsin sequence, opsin expression or co-expression, and opsin gene duplications and losses. Here, we review visual diversity in a number of teleost groups where the structural and molecular mechanisms underlying their spectral sensitivities have been relatively well determined. Although we document considerable variability, this alone does not imply functional difference per se. We therefore highlight the need for more studies that examine species with known sensitivity differences, emphasizing behavioral experiments to test whether such differences actually matter in the execution of visual tasks that are relevant to the fish.

Kei Jokura, Junko M. Nishino, Michio Ogasawara, and Atsuo Nishino

Ciliary movement is a fundamental process to support animal life, and the movement pattern may be altered in response to external stimuli under the control of nervous systems. Juvenile and adult ascidians have ciliary arrays around their pharyngeal gill slits (stigmata), and continuous beating is interrupted for seconds by mechanical stimuli on other parts of the body. Although it has been suggested that neural transmission to evoke ciliary arrest is cholinergic, its molecular basis has not yet been elucidated in detail. We herein attempted to clarify the molecular mechanisms underlying this neurociliary transmission in the model ascidian Ciona. Acetylcholinesterase histochemical staining showed strong signals on the laterodistal ciliated cells of stigmata, hereafter referred to as trapezial cells. The direct administration of acetylcholine (ACh) and other agonists of nicotinic ACh receptors (nAChRs) onto ciliated cells reliably evoked ciliary arrest that persisted for seconds in a dose-dependent manner. Only one isoform among all nAChR subunits encoded in the Ciona genome, called nAChR-A7/8-1, a relative of vertebrate α7 nAChRs, was expressed by trapezial cells. Exogenously expressed nAChR-A7/8-1 on Xenopus oocytes responded to ACh and other agonists with consistent pharmacological traits to those observed in vivo. Further efforts to examine signaling downstream of this receptor revealed that an inhibitor of phospholipase C (PLC) hampered ACh-induced ciliary arrest. We herein propose that homomeric α7-related nAChR-A7/8-1 mediates neurociliary transmission in Ciona stigmata to elicit persistent ciliary arrest by recruiting intracellular Ca²⁺ signaling.

Christopher T. Richards and Enrico A. Eberhard

Muscle force-length dynamics are governed by intrinsic contractile properties, motor stimulation and mechanical load. Although intrinsic properties are well-characterised, physiologists lack in vitro instrumentation accounting for combined effects of limb inertia, musculoskeletal architecture and contractile dynamics. We introduce in vitro virtual-reality (in vitro-VR) which enables in vitro muscle tissue to drive a musculoskeletal jumping simulation. In hardware, muscle force from a frog plantaris was transmitted to a software model where joint torques, inertia and ground reaction forces were computed to advance the simulation at 1 kHz. To close the loop, simulated muscle strain was returned to update in vitro length. We manipulated 1) stimulation timing and, 2) the virtual muscle's anatomical origin. This influenced interactions among muscular, inertial, gravitational and contact forces dictating limb kinematics and jump performance. We propose that in vitro-VR can be used to illustrate how neuromuscular control and musculoskeletal anatomy influence muscle dynamics and biomechanical performance.

Krishnamoorthy Krishnan, Asif Shahriar Nafi, Roi Gurka, and Roi Holzman

Fish larvae are the smallest self-sustaining vertebrates. As such, they face multiple challenges that stem from their minute size, and from the hydrodynamic regime in which they dwell. This regime, of intermediate Reynolds numbers, was shown to affect the swimming of larval fish and impede their ability to capture prey. Prey capture is impeded because smaller larvae produce weaker suction flows, exerting weaker forces on the prey. Previous observations on feeding larvae also showed prey exiting the mouth after initially entering it (hereafter "in-and-out"), although the mechanism causing such failures had been unclear. In this study, we used numerical simulations to investigate the hydrodynamic mechanisms responsible for the failure to feed caused by this in-and-out prey movement. Detailed kinematics of the expanding mouth during prey capture by larval Sparus aurata were used to parameterize age-specific numerical models of the flows inside the mouth. These models revealed that for small larvae which expand their mouth slowly, fluid entering the mouth cavity is expelled through the mouth before it is closed, resulting in flow reversal at the orifice. This relative efflux of water through the mouth was >8% of the influx through the mouth for younger ages. However similar effluxes were found when we simulated slow strikes by larger fish. The simulations can explain the observations of larval fish failing to fish due to the in-and-out movement of the prey. These results further highlight the importance of transporting the prey from the gape deeper into the mouth cavity in determining suction-feeding success.

Qingxiang Guo, Yang Liu, Yanhua Zhai, and Zemao Gu

Disassembling the parasitic spores and acquiring the main subunits is a prerequisite for deep understanding of the basic biology of parasites. Herein we present a fast and efficient method to dissect the myxospores in a few steps, which mainly involved sonication, sucrose density gradient and Percoll density gradient. We tested our method on three myxozoans species and demonstrated this method allows the dismembering of myxospores, isolation of intact and clean nematocysts and shell valves within 2h by low-cost. This new tool will facilitate subsequent analyses and enable a better understanding of the ecological and evolutionary significance of parasitic spores.

Rohini Singh and Timothy A. Linksvayer

Wolbachia is a widespread group of maternally-transmitted endosymbiotic bacteria that often manipulates the reproductive strategy and life history of its hosts to favor its own transmission. Wolbachia mediated phenotypic effects are well characterized in solitary hosts, but effects in social hosts are unclear. The invasive pharaoh ant, Monomorium pharaonis, shows natural variation in Wolbachia infection between colonies and can be readily bred under laboratory conditions. We previously showed that Wolbachia-infected pharaoh ant colonies had more queen-biased sex ratios than uninfected colonies, which is expected to favor the spread of maternally-transmitted Wolbachia. Here, we further characterize the effects of Wolbachia on the short- and longer-term reproductive and life history traits of pharaoh ant colonies. First, we characterized the reproductive differences between naturally infected and uninfected colonies at three discrete time points and found that infected colonies had higher reproductive investment (i.e. infected colonies produced more new queens), particularly when existing colony queens were three months old. Next, we compared the long-term growth and reproduction dynamics of infected and uninfected colonies across their whole life cycle. Infected colonies had increased colony-level growth and early colony reproduction, resulting in a shorter colony life cycle, when compared to uninfected colonies.

Marc E. Deetjen, Diana D. Chin, and David Lentink

Animal flight requires aerodynamic power, which is challenging to determine accurately in vivo. Existing methods rely on approximate calculations based on wake flow field measurements, inverse dynamics approaches, or invasive muscle physiological recordings. In contrast, the external mechanical work required for terrestrial locomotion can be determined more directly by using a force platform as an ergometer. Based on an extension of the recent invention of the aerodynamic force platform, we now present a more direct method to determine the in vivo aerodynamic power by taking the dot product of the aerodynamic force vector on the wing with the representative wing velocity vector based on kinematics and morphology. We demonstrate this new method by studying a slowly flying dove, but it can be applied more generally across flying and swimming animals as well as animals that locomote over water surfaces. Finally, our mathematical framework also works for power analyses based on flow field measurements.

S. Ghods, S. Waddell, E. Weller, C. Renteria, H.-Y. Jiang, J. M. Janak, S. S. Mao, T. J. Linley, and D. Arola

Fish scales serve as a dermal armor that provides protection from physical injury. Due to a number of outstanding properties, fish scales are inspiring new concepts for layered engineered materials and next-generation flexible armors. While past efforts have primarily focused on the structure and mechanical behavior of ontogenetic scales, the structure-property relationships of regenerated scales have received limited attention. In the present study, common carp (Cyprinus carpio) acquired from the wild were held live in an aquatic laboratory at 10° and 20°C. Ontogenetic scales were extracted from the fish for analysis, as well as regenerated scales after approximately 1 year of development and growth. Their microstructure was characterized using microscopy and Raman spectroscopy, and the mechanical properties were evaluated in uniaxial tension to failure under hydrated conditions. The strength, strain to fracture and toughness of the regenerated scales were significantly lower than those of ontogenetic scales from the same fish, regardless of the water temperature. Scales that regenerated at 20°C exhibited significantly higher strength, strain to fracture and toughness than those regenerated at 10°C. The regenerated scales exhibited a highly mineralized outer layer, but no distinct limiting layer or external elasmodine; they also possessed a significantly lower number of plies in the basal layer than in the ontogenetic scales. The results suggest that a mineralized layer develops preferentially during scale regeneration with the topology needed for protection, prior to the development of other qualities.

Thomas J. Lisney, Shaun P. Collin, and Jennifer L. Kelley

Ecological factors such as spatial habitat complexity and diet can explain variation in visual morphology, but few studies have sought to determine whether visual specialisation can occur among populations of the same species. We used a small Australian freshwater fish (the western rainbowfish, Melanotaenia australis) to determine whether populations showed variation in eye size and eye position, and whether this variation could be explained by environmental (light availability, turbidity) and ecological (predation risk, habitat complexity, invertebrate abundance) variables. We investigated three aspects of eye morphology, (1) eye size relative to body size, (2) pupil size relative to eye size, and (3) eye position in the head, for fish collected from 14 sites in a major river catchment in northwest Western Australia. We found significant variation among populations in all three measures of eye morphology, but no effect of sex on eye size or eye position. Variation in eye diameter and eye position was best explained by the level of habitat complexity. Specifically, fish occurring in habitats with low complexity (i.e. open water) tended to have smaller, more dorsally-located eyes, than those occurring in more complex habitats (i.e. vegetation present). The size of the pupil relative to the size of the eye was most influenced by the presence of surrounding rock formations; fish living in gorge habitats had significantly smaller pupils (relative to eye size) than those occupying semi-gorge sites or open habitats. Our findings reveal that different ecological and environmental factors contribute to habitat-specific visual specialisations within a species.

C. M. Lind, J. Agugliaro, and T. M. Farrell

Mounting an immune response may be energetically costly and require the diversion of resources away from other physiological processes. Yet, both the metabolic cost of immune responses and the factors that impact investment priorities remain poorly described in many vertebrate groups. For example, although viviparity has evolved many times in vertebrates, the relationship between immune function and pregnancy has been disproportionately studied in placental mammals. To examine the energetic costs of immune activation and the modulation of immune function during pregnancy in a non-mammalian vertebrate, we elicited an immune response in pregnant and non-pregnant pygmy rattlesnakes, Sistrurus miliarius, using lipopolysaccharide (LPS). Resting metabolic rate (RMR) was measured using flow-through respirometry. Immune function was examined using bactericidal assays and leukocyte counts. The RMR of pygmy rattlesnakes increased significantly in response to LPS injection. There was no statistically significant difference in the metabolic response of non-reproductive and pregnant snakes to LPS. Mean metabolic increments for pregnant females, non-reproductive females, and males were 13%, 18%, and 26%, respectively. The ratio of heterophils to lymphocytes was elevated in response to LPS across reproductive categories; however, LPS did not impact plasma bactericidal ability in non-reproductive snakes. Although pregnant females had significantly higher plasma bactericidal ability compared to non-reproductive snakes prior to manipulation, their bactericidal ability declined in response to LPS. LPS administration also significantly reduced several litter characteristics, particularly when administrated relatively early in pregnancy. Our results indicate that immune performance is energetically costly, altered during pregnancy, and that immune activation during pregnancy may result in tradeoffs that affect offspring in a viviparous reptile.

Jing Wen, Tingbei Bo, Xueying Zhang, Zuoxin Wang, and Dehua Wang

Ambient temperature and food composition can affect energy metabolism of the host. Thermal transient receptor potential (thermo-TRPs) ion channels can detect temperature signals and are involved in the regulation of thermogenesis and energy homeostasis. Further, the gut microbiota has also been implicated in thermogenesis and obesity. In the present study, we tested the hypothesis that thermo-TRPs and gut microbiota are involved in reducing diet-induced obesity (DIO) during low temperature exposure. C57BL/6J mice in obese (body mass gain >45%), lean (body mass gain <15%), and control (body mass gain<1%) groups were exposed to high (23±1°C) or low (4±1°C) ambient temperature for 28 days. Our data showed that low temperature exposure attenuated DIO, but enhanced brown adipose tissue (BAT) thermogenesis. Low temperature exposure also resulted in increased norepinephrine (NE) concentrations in the hypothalamus, decreased TRP melastatin 8 (TRPM8) expression in the small intestine, and altered composition and diversity of gut microbiota. In DIO mice, there was a decrease in overall energy intake along with a reduction in TRP ankyrin 1 (TRPA1) expression and an increase in NE concentration in the small intestine. DIO mice also showed increases in Oscillospira, [Ruminococcus], Lactococcus, and Christensenella and decreases in Prevotella, Odoribacter, and Lactobacillus at the genus level in fecal samples. Together, our data suggest that thermos-TRPs and gut microbiota are involved in thermogenesis and energy metabolism during low temperature exposure in DIO mice.

Kelsey L. Jones, Rahmatollah Rajabzadeh, Guncha Ishangulyyeva, Nadir Erbilgin, and Maya L. Evenden

Flight polyphenisms naturally occur as discrete or continuous traits in insects. Discrete flight polyphenisms include winged and wingless morphs, whereas continuous flight polyphenisms can take the form of short- or long-distance fliers. The mountain pine beetle (Dendroctonus ponderosae) exhibits polyphenic variation in flight distance but the consequences of this flight variation on life history strategies of beetles is unknown. This study assessed the effect of flight on two particular aspects of beetle biology: (1) an energetic trade-off between flight distance and host colonisation capacity; and (2) the relationship between flight distance and pheromone production. A 23-h flight treatment was applied to a subset of beetles using computer. After flight treatment, both flown and unflown (control) beetles were given the opportunity to colonise bolts of host trees, and beetles that entered hosts were aerated to collect pheromone. A trade-off occurred between initiation of host colonisation and percent body weight lost during flight, which indicates energy-use during flight affects host acceptance in female mountain pine beetles. Furthermore, production of the aggregation pheromone trans-verbenol by female beetles was influenced by both percent weight lost during flight and flight distance. Male production of exo-brevicomin was affected by beetle condition following flight but not by the energy used during flight. These novel results give new insight into the polyphenic flight behaviour of mountain pine beetles. Flight variation is adaptive by acting to maintain population levels through safe and risky host colonisation strategies. These findings suggest mechanisms that facilitate the extremities of the continuous flight polyphenism spectrum. These opposing mechanisms appear to maintain the high variation in flight exhibited by this species.

Jorge de Costa, Gustavo Barja, and Pedro F. Almaida-Pagan

Lipid composition of cell membranes is linked to metabolic rate and lifespan in mammals and birds but very little information is available for fishes. In this study, three fish species of the short-lived annual genus Nothobranchius with different maximum lifespan potentials (MLSP) and the longer-lived outgroup species Aphyosemion australe were studied to test whether they conform to the predictions of the longevity-homeoviscous adaptation (LHA) theory of aging. Lipid analyses were performed in whole fish samples and peroxidation indexes (PIn) for every PL class and for the whole membrane, were calculated. Total PL content was significantly lower in A. australe and N. korthausae, the two species with the highest MLSP, and a negative correlation between membrane total PIn and fish MLSP was found, this meaning that the longer-lived fish species have more saturated membranes and therefore, a lower susceptibility to oxidative damage, as the LHA theory posits.

Jack Nguyen and Meir M. Barak

Cortical bone remodeling is an ongoing process triggered by microdamage, where osteoclasts resorb existing bone and osteoblasts deposit new bone in the form of secondary osteons (Haversian systems). Previous studies revealed regional variance in Haversian systems structure and possibly material, between opposite cortices of the same bone. As bone mechanical properties depend on tissue structure and material, it is predicted that bone mechanical properties will vary in accordance with structural and material regional heterogeneity. To test this hypothesis, we analyzed the structure, mineral content and compressive stiffness of secondary bone from the cranial and caudal cortices of the white-tailed deer proximal humerus. We found significantly larger Haversian systems and canals in the cranial cortex but no significant difference in mineral content between the two cortices. Accordingly, we found no difference in compressive stiffness between the two cortices and thus our working hypothesis was rejected. Seeing that the deer humerus is curved and thus likely subjected to bending during habitual locomotion, we expect that similar to other curved long bones, the cranial cortex of the deer humerus is likely subjected primarily to tensile strains and the caudal cortex is likely subject primarily to compressive strains. Consequently, our results suggest that strain magnitude (larger in compression) and sign (compression vs. tension) affect differently the osteoclasts and osteoblasts in the BMU. Our results further suggest that osteoclasts are inhibited in regions of high compressive strains (creating smaller Haversian systems) while osteoblasts’ osteoid deposition and mineralization is not affected by strain magnitude and sign.

Flemming Dahlke, Magnus Lucassen, Ulf Bickmeyer, Sylke Wohlrab, Velmurugu Puvanendran, Atle Mortensen, Melissa Chierici, Hans-Otto Pörtner, and Daniela Storch

The vulnerability of fish embryos and larvae to environmental factors is often attributed to a lack of adult-like organ systems (gills) and thus insufficient homeostatic capacity. However, experimental data supporting this hypothesis are scarce. Here, by using Atlantic cod (Gadus morhua) as a model, the relationship between embryo vulnerability (to projected ocean acidification and warming) and homeostatic capacity was explored through parallel analyses of stage-specific mortality and in vitro activity and expression of major ion pumps (ATP-Synthase, Na⁺/K⁺-ATPase, H⁺-ATPase) and co-transporters (NBC1, NKCC1). Immunolocalization of these transporters was used to study ionocyte morphology in newly-hatched larvae. Treatment-related embryo mortality until hatch (+20% due to acidification and warming) occurred primarily during an early period (gastrulation) characterized by extremely low ion transport capacities. Thereafter, embryo mortality decreased in parallel with an exponential increase in activity and expression of all investigated ion transporters. Significant changes in transporter activity and expression in response to acidification (+15% activity) and warming (-30% expression) indicate some potential for short-term acclimatization, although likely associated with energetic trade-offs. Interestingly, whole-larvae enzyme capacities (supported by abundant epidermal ionocytes) reached levels similar to those previously measured in gill tissue of adult cod, suggesting that early-life stages without functional gills are better equipped in terms of ion homeostasis than previously thought. This study implies that the gastrulation period represents a critical transition from inherited (maternal) defenses to active homeostatic regulation, which facilitates enhanced resilience of later stages to environmental factors.

Brad A. Seibel and Curtis Deutsch

The capacity to extract oxygen from the environment and transport it to respiring tissues in support of metabolic demand reportedly has implications for species’ thermal tolerance, body-size, diversity and biogeography. Here we derive a quantifiable linkage between maximum and basal metabolic rate and their oxygen, temperature and size dependencies. We show that, regardless of size or temperature, the physiological capacity for oxygen supply precisely matches the maximum evolved demand at the highest persistently available oxygen pressure and this is the critical PO₂ for the maximum metabolic rate. For most terrestrial and shallow-living marine species, this "P_crit-max" is the current atmospheric pressure, 21 kPa. Any reduction in oxygen partial pressure from current values will result in a calculable decrement in maximum metabolic performance. However, oxygen supply capacity has evolved to match demand across temperatures and body sizes and so does not constrain thermal tolerance or cause the well-known reduction in mass-specific metabolic rate with increasing body mass. The critical oxygen pressure for resting metabolic rate, typically viewed as an indicator of hypoxia tolerance, is, instead, simply a rate-specific reflection of the oxygen supply capacity. A compensatory reduction in maintenance metabolic costs in warm-adapted species constrains factorial aerobic scope and the critical PO₂ to a similar range, between ~2 and 6, across each species’ natural temperature range. The simple new relationship described here redefines many important physiological concepts and alters their ecological interpretation.

Background

Canagliflozin reduced renal and cardiovascular events in people with type 2 diabetes in the CREDENCE trial. We assessed efficacy and safety of canagliflozin by initial estimated glomerular filtration rate (eGFR).

Background

Clinically significant CKD following surgery for kidney cancer is associated with increased morbidity and mortality, but identifying patients at increased CKD risk remains difficult. Simple methods to stratify risk of clinically significant CKD after nephrectomy are needed.

Background

Fluid overload in patients undergoing hemodialysis contributes to cardiovascular morbidity and mortality. There is a global trend to lower dialysate sodium with the goal of reducing fluid overload.

Background

Kidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C- (PKC) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKC is involved in ischemic or transplantation-associated kidney injury is unknown.

Background

Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue.

Background

Water and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient.

Background

Previous studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown.

Background

The serine/threonine kinases MST1 and MST2 are core components of the Hippo pathway, which has been found to be critically involved in embryonic kidney development. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the pathway’s main effectors. However, the biologic functions of the Hippo/YAP pathway in adult kidneys are not well understood, and the functional role of MST1 and MST2 in the kidney has not been studied.

Background

The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell–derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation.

Growing evidence indicates that oxidative and endoplasmic reticular stress, which trigger changes in ion channels and inflammatory pathways that may undermine cellular homeostasis and survival, are critical determinants of injury in the diabetic kidney. Cells are normally able to mitigate these cellular stresses by maintaining high levels of autophagy, an intracellular lysosome-dependent degradative pathway that clears the cytoplasm of dysfunctional organelles. However, the capacity for autophagy in both podocytes and renal tubular cells is markedly impaired in type 2 diabetes, and this deficiency contributes importantly to the intensity of renal injury. The primary drivers of autophagy in states of nutrient and oxygen deprivation—sirtuin-1 (SIRT1), AMP-activated protein kinase (AMPK), and hypoxia-inducible factors (HIF-1α and HIF-2α)—can exert renoprotective effects by promoting autophagic flux and by exerting direct effects on sodium transport and inflammasome activation. Type 2 diabetes is characterized by marked suppression of SIRT1 and AMPK, leading to a diminution in autophagic flux in glomerular podocytes and renal tubules and markedly increasing their susceptibility to renal injury. Importantly, because insulin acts to depress autophagic flux, these derangements in nutrient deprivation signaling are not ameliorated by antihyperglycemic drugs that enhance insulin secretion or signaling. Metformin is an established AMPK agonist that can promote autophagy, but its effects on the course of CKD have been demonstrated only in the experimental setting. In contrast, the effects of sodium-glucose cotransporter–2 (SGLT2) inhibitors may be related primarily to enhanced SIRT1 and HIF-2α signaling; this can explain the effects of SGLT2 inhibitors to promote ketonemia and erythrocytosis and potentially underlies their actions to increase autophagy and mute inflammation in the diabetic kidney. These distinctions may contribute importantly to the consistent benefit of SGLT2 inhibitors to slow the deterioration in glomerular function and reduce the risk of ESKD in large-scale randomized clinical trials of patients with type 2 diabetes.

Understanding fructose metabolism might provide insights to renal pathophysiology. To support systemic glucose concentration, the proximal tubular cells reabsorb fructose as a substrate for gluconeogenesis. However, in instances when fructose intake is excessive, fructose metabolism is costly, resulting in energy depletion, uric acid generation, inflammation, and fibrosis in the kidney. A recent scientific advance is the discovery that fructose can be endogenously produced from glucose under pathologic conditions, not only in kidney diseases, but also in diabetes, in cardiac hypertrophy, and with dehydration. Why humans have such a deleterious mechanism to produce fructose is unknown, but it may relate to an evolutionary benefit in the past. In this article, we aim to illuminate the roles of fructose as it relates to gluconeogenesis and fructoneogenesis in the kidney.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5'–3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'–3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.

Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA).

Endogenous viral elements (EVEs) are found in many eukaryotic genomes. Despite considerable knowledge about genomic elements such as transposons (TEs) and retroviruses, we still lack information about nonretroviral EVEs. Aedes aegypti mosquitoes have a highly repetitive genome that is covered with EVEs. Here, we identified 129 nonretroviral EVEs in the AaegL5 version of the A. aegypti genome. These EVEs were significantly associated with TEs and preferentially located in repeat-rich clusters within intergenic regions. Genome-wide transcriptome analysis showed that most EVEs generated transcripts although only around 1.4% were sense RNAs. The majority of EVE transcription was antisense and correlated with the generation of EVE-derived small RNAs. A single genomic cluster of EVEs located in a 143 kb repetitive region in chromosome 2 contributed with 42% of antisense transcription and 45% of small RNAs derived from viral elements. This region was enriched for TE-EVE hybrids organized in the same coding strand. These generated a single long antisense transcript that correlated with the generation of phased primary PIWI-interacting RNAs (piRNAs). The putative promoter of this region had a conserved binding site for the transcription factor Cubitus interruptus, a key regulator of the flamenco locus in Drosophila melanogaster. Here, we have identified a single unidirectional piRNA cluster in the A. aegypti genome that is the major source of EVE transcription fueling the generation of antisense small RNAs in mosquitoes. We propose that this region is a flamenco-like locus in A. aegypti due to its relatedness to the major unidirectional piRNA cluster in Drosophila melanogaster.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K_1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI–piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI–piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI–piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI–piRNA pathway.

The importance of noncoding RNA sequences has become increasingly clear over the past decade. New RNA families are often detected and analyzed using comparative methods based on multiple sequence alignments. Accordingly, a number of programs have been developed for aligning and deriving secondary structures from sets of RNA sequences. Yet, the best tools for these tasks remain unclear because existing benchmarks contain too few sequences belonging to only a small number of RNA families. RNAconTest (RNA consistency test) is a new benchmarking approach relying on the observation that secondary structure is often conserved across highly divergent RNA sequences from the same family. RNAconTest scores multiple sequence alignments based on the level of consistency among known secondary structures belonging to reference sequences in their output alignment. Similarly, consensus secondary structure predictions are scored according to their agreement with one or more known structures in a family. Comparing the performance of 10 popular alignment programs using RNAconTest revealed that DAFS, DECIPHER, LocARNA, and MAFFT created the most structurally consistent alignments. The best consensus secondary structure predictions were generated by DAFS and LocARNA (via RNAalifold). Many of the methods specific to noncoding RNAs exhibited poor scalability as the number or length of input sequences increased, and several programs displayed substantial declines in score as more sequences were aligned. Overall, RNAconTest provides a means of testing and improving tools for comparative RNA analysis, as well as highlighting the best available approaches. RNAconTest is available from the DECIPHER website (http://DECIPHER.codes/Downloads.html).