Reporting guidelines for terrestrial respirometry: Building openness, transparency of metabolic rate and evaporative water loss data.

Respirometry is an important tool for understanding whole-animal energy and water balance in relation to the environment. Consequently, the growing number of studies using respirometry over the last decade warrants reliable reporting and data sharing for effective dissemination and research synthesis. We provide a checklist guideline on five key sections to facilitate the transparency, reproducibility, and replicability of respirometry studies: 1) materials, set up, plumbing, 2) subject conditions/maintenance, 3) measurement conditions, 4) data processing, and 5) data reporting and statistics, each with explanations and example studies. Transparency in reporting and data availability has benefits on multiple fronts. Authors can use this checklist to design and report on their study, and reviewers and editors can use the checklist to assess the reporting quality of the manuscripts they review. Improved standards for reporting will enhance the value of primary studies and will greatly facilitate the ability to carry out higher quality research syntheses to address ecological and evolutionary theories.

Cyclone: an accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data.

In the past decade, high-dimensional single-cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation, which are computationally intense and difficult to evaluate and optimize. Here, we present Cytometry Clustering Optimization and Evaluation (Cyclone), an analysis pipeline integrating dimensionality reduction, clustering, evaluation, and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification but also enables the unsupervised identification of lymphocytes and mononuclear phagocyte subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on a variety of cytometry datasets, which will further power immunology research and provide a scaffold for biological discovery.

Physiological Characterization of the Coral Holobiont Using a New Micro-Respirometry Tool.

Metabolic activity, defined as the sum of organismal processes that involve energy, is of critical importance in understanding the function and evolution of life on earth. Measuring organismal metabolic rates is, therefore, at the center of explaining the physiological states of organisms, their ecological roles, and the impact of environmental change on species within terrestrial and aquatic ecosystems. On coral reefs, measures of metabolism have been used to quantify symbiosis functioning between corals and their obligate algal symbionts (Symbiodiniaceae), as well as assess how environmental stressors, including climate change, will impact coral health. Despite this significance, there is a lack of methods, and therefore data, relating to metabolic rate measurements in coral offspring, likely due to their small size. To address this gap, this study aimed to develop a custom setup for measuring the respiration of small (millimeter size range) marine animal ecologies. This low cost and easy setup should allow for the improved measurement of metabolic rate. This will be essential for applied ecological research utilizing the sexual production of corals for reef restoration.

Cyclone: an accessible pipeline to analyze, evaluate and optimize multiparametric cytometry data.

In the past decade, high-dimensional single cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation which are computationally intense and difficult to evaluate and optimize. Here we present Cyclone, an analysis pipeline integrating dimensionality reduction, clustering, evaluation and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification, but also enables the unsupervised identification of lymphocytes and mononuclear phagocytes subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on variety of cytometry datasets which will further power immunology research and provide a scaffold for biological discovery.

LTßR overexpression promotes plasma cell accumulation.

Multiple myeloma (MM), a malignancy of plasma cells (PCs), has diverse genetic underpinnings and in rare cases these include amplification of the lymphotoxin b receptor (Ltbr) locus. LTßR has well defined roles in supporting lymphoid tissue development and function through actions in stromal and myeloid cells, but whether it is functional in PCs is unknown. Here we showed that Ltbr mRNA was upregulated in mouse PCs compared to follicular B cells, but deficiency in the receptor did not cause a reduction in PC responses to a T-dependent or T-independent immunogen. However, LTßR overexpression (OE) enhanced PC formation in vitro after LPS or anti-CD40 stimulation. In vivo, LTßR OE led to increased antigen-specific splenic and bone marrow (BM) plasma cells responses. LTßR OE PCs had increased expression of Nfkb2 and of the NF-kB target genes Bcl2 and Mcl1, factors involved in the formation of long-lived BM PCs. Our findings suggest a pathway by which Ltbr gene amplifications may contribute to MM development through increased NF-kB activity and induction of an anti-apoptotic transcriptional program.

The limits of convergence in the collective behavior of competing marine taxa.

Collective behaviors in biological systems such as coordinated movements have important ecological and evolutionary consequences. While many studies examine within-species variation in collective behavior, explicit comparisons between functionally similar species from different taxonomic groups are rare. Therefore, a fundamental question remains: how do collective behaviors compare between taxa with morphological and physiological convergence, and how might this relate to functional ecology and niche partitioning? We examined the collective motion of two ecologically similar species from unrelated clades that have competed for pelagic predatory niches for over 500 million years-California market squid, Doryteuthis opalescens (Mollusca) and Pacific sardine, Sardinops sagax (Chordata). We (1) found similarities in how groups of individuals from each species collectively aligned, measured by angular deviation, the difference between individual orientation and average group heading. We also (2) show that conspecific attraction, which we approximated using nearest neighbor distance, was greater in sardine than squid. Finally, we (3) found that individuals of each species explicitly matched the orientation of groupmates, but that these matching responses were less rapid in squid than sardine. Based on these results, we hypothesize that information sharing is a comparably important function of social grouping for both taxa. On the other hand, some capabilities, including hydrodynamically conferred energy savings and defense against predators, could stem from taxon-specific biology.

Rapid Range Expansion of a Marine Ectotherm Reveals the Demographic and Ecological Consequences of Short-Term Variability in Seawater Temperature and Dissolved Oxygen.

AbstractThe distributions of marine ectotherms are governed by physiological sensitivities to long-term trends in seawater temperature and dissolved oxygen. Short-term variability in these parameters has the potential to facilitate rapid range expansions, and the resulting ecological and socioeconomic consequences may portend those of future marine communities. Here, we combine physiological experiments with ecological and demographic surveys to assess the causes and consequences of sudden but temporary poleward range expansions of a marine ectotherm with considerable life history plasticity (California market squid, Doryteuthis opalescens). We show that sequential factors related to resource accessibility in the core range-the buildup of large populations as a result of competitive release and climate-associated temperature increase and oxygen loss that constrain aerobic activity-may drive these expansions. We also reveal that poleward range expansion alters the body size-and therefore trophic role-of invading populations, with potential negative implications for socioeconomically valuable resident species. To help forecast rapid range expansions of marine ectotherms, we advocate that research efforts focus on factors impacting resource accessibility in core ranges. Determining how environmental conditions in receiving ecosystems affect body size and how body size is related to trophic role will help refine estimates of the impacts of future marine communities.

Comparing Resident Procedures in Rural Versus Urban Emergency Departments.

BACKGROUND: Rural rotations can be a valuable experience for emergency medicine (EM) residents. To date, there has not been a retrospective cohort study comparing procedures performed at urban versus rural emergency departments (EDs). OBJECTIVES: The purpose of this study was to compare procedures performed by EM residents in urban versus rural EDs, with the hypothesis that there will be no significant difference in the procedures performed. METHODS: A retrospective cohort study was conducted comparing procedures performed by second- and third-year EM residents based on medical chart review. The procedures were counted at three locations in West Virginia, including a small rural ED, a large rural ED, and a tertiary care ED. Procedure notes were collected from September 2018 to September 2019. The final analysis included nine months, as three months did not have residents at all locations. Eight procedures were standardized based on the number of procedures performed per 100 hours worked by residents. A comparison of total procedures and complex versus simple procedures was performed. A Kruskal-Wallis H test was performed to compare resident hours for procedures between each of the three locations. To compare each of the hospitals to one another separately, Mann-Whitney U tests were performed. RESULTS: The total resident hours worked included 1,800 at the small rural ED, 13,725.5 at the tertiary care ED, and 5,319 at the large rural ED. A p-value of 0.0311 for the Kruskal-Wallis H Test indicated a difference between at least two of the ED sites. A statistically significant difference exists (p-value = 0.0135) between the urban ED (95% CI: 0.15-0.62) and the large rural ED (95% CI: 0.54-1.53). There was no significant difference in complex versus simple procedures among the three locations (p-value = 0.4159). CONCLUSIONS: When compared with the tertiary care ED, residents performed more total procedures at the large rural ED and similar total procedure numbers at the small rural ED when standardized for hours worked. There was no significant difference when comparing complex and simple procedures among the three locations.

Predator-informed looming stimulus experiments reveal how large filter feeding whales capture highly maneuverable forage fish.

The unique engulfment filtration strategy of microphagous rorqual whales has evolved relatively recently (<5 Ma) and exploits extreme predator/prey size ratios to overcome the maneuverability advantages of swarms of small prey, such as krill. Forage fish, in contrast, have been engaged in evolutionary arms races with their predators for more than 100 million years and have performance capabilities that suggest they should easily evade whale-sized predators, yet they are regularly hunted by some species of rorqual whales. To explore this phenomenon, we determined, in a laboratory setting, when individual anchovies initiated escape from virtually approaching whales, then used these results along with in situ humpback whale attack data to model how predator speed and engulfment timing affected capture rates. Anchovies were found to respond to approaching visual looming stimuli at expansion rates that give ample chance to escape from a sea lion-sized predator, but humpback whales could capture as much as 30-60% of a school at once because the increase in their apparent (visual) size does not cross their prey's response threshold until after rapid jaw expansion. Humpback whales are, thus, incentivized to delay engulfment until they are very close to a prey school, even if this results in higher hydrodynamic drag. This potential exaptation of a microphagous filter feeding strategy for fish foraging enables humpback whales to achieve 7× the energetic efficiency (per lunge) of krill foraging, allowing for flexible foraging strategies that may underlie their ecological success in fluctuating oceanic conditions.

TCO, a Putative Transcriptional Regulator in Arabidopsis, Is a Target of the Protein Kinase CK2.

As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.

Kinematics of ram filter feeding and beat-glide swimming in the northern anchovy Engraulis mordax.

In the dense aquatic environment, the most adept swimmers are streamlined to reduce drag and increase the efficiency of locomotion. However, because they open their mouth to wide gape angles to deploy their filtering apparatus, ram filter feeders apparently switch between diametrically opposite swimming modes: highly efficient, streamlined 'beat-glide' swimming, and ram filter feeding, which has been hypothesized to be a high-cost feeding mode because of presumed increased drag. Ram filter-feeding forage fish are thought to play an important role in the flux of nutrients and energy in upwelling ecosystems; however, the biomechanics and energetics of this feeding mechanism remain poorly understood. We quantified the kinematics of an iconic forage fish, the northern anchovy, Engraulis mordax, during ram filter feeding and non-feeding, mouth-closed beat-glide swimming. Although many kinematic parameters between the two swimming modes were similar, we found that swimming speeds and tailbeat frequencies were significantly lower during ram feeding. Rather than maintain speed with the school, a speed which closely matches theoretical optimum filter-feeding speeds was consistently observed. Beat-glide swimming was characterized by high variability in all kinematic parameters, but variance in kinematic parameters was much lower during ram filter feeding. Under this mode, body kinematics are substantially modified, and E. mordax swims more slowly and with decreased lateral movement along the entire body, but most noticeably in the anterior. Our results suggest that hydrodynamic effects that come with deployment of the filtering anatomy may limit behavioral options during foraging and result in slower swimming speeds during ram filtration.

The role of AUXIN RESPONSE FACTORs in the development and differential growth of inflorescence stems.

Plants react to environmental cues by altering their growth and development, which can include organ tropic responses. These differential growth responses are triggered by the hormone auxin, and AUXIN RESPONSE FACTORs (ARFs) have been implicated in numerous organ tropisms in Arabidopsis thaliana. Surprisingly, despite being critical for light capture and overall plant morphology, inflorescence stem tropic responses remain relatively understudied, with presumed direct links to ARF function yet to be established. Here, we show that the expression patterns of ARF5/MONOPTEROS and ARF7/NONPHOTOTROPIC HYPOCOTYL4 are consistent with roles in inflorescence stem tropisms. Mutation of these factors does not alter inflorescence stem responses to gravity or unilateral auxin application, meaning their participation in these processes is presumably masked by functional redundancies. Future resolution of these redundancies will likely require higher order arf mutant combinations, guided by detailed expression analyses of ARFs in the inflorescence stem.

Sea Hare Aplysia punctata (Mollusca: Gastropoda) Can Maintain Shell Calcification under Extreme Ocean Acidification.

Ocean acidification is expected to cause energetic constraints upon marine calcifying organisms such as molluscs and echinoderms, because of the increased costs of building or maintaining shell material in lower pH. We examined metabolic rate, shell morphometry, and calcification in the sea hare Aplysia punctata under short-term exposure (19 days) to an extreme ocean acidification scenario (pH 7.3, ∼2800 µatm pCO2), along with a group held in control conditions (pH 8.1, ∼344 µatm pCO2). This gastropod and its congeners are broadly distributed and locally abundant grazers, and have an internal shell that protects the internal organs. Specimens were examined for metabolic rate via closed-chamber respirometry, followed by removal and examination of the shell under confocal microscopy. Staining using calcein determined the amount of new calcification that occurred over 6 days at the end of the acclimation period. The width of new, pre-calcified shell on the distal shell margin was also quantified as a proxy for overall shell growth. Aplysia punctata showed a 30% reduction in metabolic rate under low pH, but calcification was not affected. This species is apparently able to maintain calcification rate even under extreme low pH, and even when under the energetic constraints of lower metabolism. This finding adds to the evidence that calcification is a largely autonomous process of crystallization that occurs as long as suitable haeomocoel conditions are preserved. There was, however, evidence that the accretion of new, noncalcified shell material may have been reduced, which would lead to overall reduced shell growth under longer-term exposures to low pH independent of calcification. Our findings highlight that the chief impact of ocean acidification upon the ability of marine invertebrates to maintain their shell under low pH may be energetic constraints that hinder growth of supporting structure, rather than maintenance of calcification.

Sea urchins in a high-CO2 world: partitioned effects of body size, ocean warming and acidification on metabolic rate.

Body size and temperature are the major factors explaining metabolic rate, and the additional factor of pH is a major driver at the biochemical level. These three factors have frequently been found to interact, complicating the formulation of broad models predicting metabolic rates and hence ecological functioning. In this first study of the effects of warming and ocean acidification, and their potential interaction, on metabolic rate across a broad range in body size (two to three orders of magnitude difference in body mass), we addressed the impact of climate change on the sea urchin ITALIC! Heliocidaris erythrogrammain context with climate projections for southeast Australia, an ocean warming hotspot. Urchins were gradually introduced to two temperatures (18 and 23°C) and two pH levels (7.5 and 8.0), at which they were maintained for 2 months. Identical experimental trials separated by several weeks validated the fact that a new physiological steady state had been reached, otherwise known as acclimation. The relationship between body size, temperature and acidification on the metabolic rate of ITALIC! H. erythrogrammawas strikingly stable. Both stressors caused increases in metabolic rate: 20% for temperature and 19% for pH. Combined effects were additive: a 44% increase in metabolism. Body size had a highly stable relationship with metabolic rate regardless of temperature or pH. None of these diverse drivers of metabolism interacted or modulated the effects of the others, highlighting the partitioned nature of how each influences metabolic rate, and the importance of achieving a full acclimation state. Despite these increases in energetic demand there was very limited capacity for compensatory modulating of feeding rate; food consumption increased only in the very smallest specimens, and only in response to temperature, and not pH. Our data show that warming, acidification and body size all substantially affect metabolism and are highly consistent and partitioned in their effects, and for ITALIC! H. erythrogramma, near-future climate change will incur a substantial energetic cost.

Size matters: plasticity in metabolic scaling shows body-size may modulate responses to climate change.

Variability in metabolic scaling in animals, the relationship between metabolic rate ( R: ) and body mass ( M: ), has been a source of debate and controversy for decades. R: is proportional to MB: , the precise value of B: much debated, but historically considered equal in all organisms. Recent metabolic theory, however, predicts B: to vary among species with ecology and metabolic level, and may also vary within species under different abiotic conditions. Under climate change, most species will experience increased temperatures, and marine organisms will experience the additional stressor of decreased seawater pH ('ocean acidification'). Responses to these environmental changes are modulated by myriad species-specific factors. Body-size is a fundamental biological parameter, but its modulating role is relatively unexplored. Here, we show that changes to metabolic scaling reveal asymmetric responses to stressors across body-size ranges; B: is systematically decreased under increasing temperature in three grazing molluscs, indicating smaller individuals were more responsive to warming. Larger individuals were, however, more responsive to reduced seawater pH in low temperatures. These alterations to the allometry of metabolism highlight abiotic control of metabolic scaling, and indicate that responses to climate warming and ocean acidification may be modulated by body-size.

Grazing under experimental hypercapnia and elevated temperature does not affect the radula of a chiton (Mollusca, Polyplacophora, Lepidopleurida).

Chitons (class Polyplacophora) are benthic grazing molluscs with an eight-part aragonitic shell armature. The radula, a serial tooth ribbon that extends internally more than half the length of the body, is mineralised on the active feeding teeth with iron magnetite apparently as an adaptation to constant grazing on rocky substrates. As the anterior feeding teeth are eroded they are shed and replaced with a new row. The efficient mineralisation and function of the radula could hypothetically be affected by changing oceans in two ways: changes in seawater chemistry (pH and pCO2) may impact the biomineralisation pathway, potentially leading to a weaker or altered density of the feeding teeth; rising temperatures could increase activity levels in these ectothermic animals, and higher feeding rates could increase wear on the feeding teeth beyond the animals' ability to synthesise, mineralise, and replace radular rows. We therefore examined the effects of pH and temperature on growth and integrity in the radula of the chiton Leptochiton asellus. Our experiment implemented three temperature (∼10, 15, 20 °C) and two pCO2 treatments (∼400 µatm, pH 8.0; ∼2000 µatm, pH 7.5) for six treatment groups. Animals (n = 50) were acclimated to the treatment conditions for a period of 4 weeks. This is sufficient time for growth of ca. 7-9 new tooth rows or 20% turnover of the mineralised portion. There was no significant difference in the number of new (non-mineralised) teeth or total tooth row count in any treatment. Examination of the radulae via SEM revealed no differences in microwear or breakage on the feeding cusps correlating to treatment groups. The shell valves also showed no signs of dissolution. As a lineage, chitons have survived repeated shifts in Earth's climate through geological time, and at least their radulae may be robust to future perturbations.