A rapid return to normal: temporal gene expression patterns following cold exposure in the bumble bee Bombus impatiens.

Bumble bees are common in cooler climates and many species likely experience periodic exposure to very cold temperatures, but little is known about the temporal dynamics of cold response mechanisms following chill exposure, especially how persistent effects of cold exposure may facilitate tolerance of future events. To investigate molecular processes involved in the temporal response by bumble bees to acute cold exposure, we compared mRNA transcript abundance in Bombus impatiens workers exposed to 0°C for 75 min (inducing chill coma) and control bees maintained at a constant ambient temperature (28°C). We sequenced the 3' end of mRNA transcripts (TagSeq) to quantify gene expression in thoracic tissue of bees at several time points (0, 10, 30, 120 and 720 min) following cold exposure. Significant differences from control bees were only detectable within 30 min after the treatment, with most occurring at the 10 min recovery time point. Genes associated with gluconeogenesis and glycolysis were most notably upregulated, while genes related to lipid and purine metabolism were downregulated. The observed patterns of expression indicate a rapid recovery after chill coma, suggesting an acute differential transcriptional response during recovery from chill coma and return to baseline expression levels within an hour, with no long-term gene expression markers of this cold exposure. Our work highlights the functions and pathways important for acute cold recovery, provides an estimated time frame for recovery from cold exposure in bumble bees, and suggests that cold hardening may be less important for these heterothermic insects.

Patient Characteristics During Early Transmission of SARS-CoV-2, Palau, January 13-February 24, 2022.

Palau had no reported evidence of COVID-19 community spread until January 2022. We chart reviewed hospitalized patients who had a positive SARS-CoV-2 test result during early community transmission. Booster vaccinations and early outpatient treatment decreased hospitalizations. Inadequate hospital infection control practices contributed to iatrogenic COVID-19 and preventable deaths.

High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within ∼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by ∼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in ∼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.

It is buzziness time: rearing, mating, and overwintering Bombus vosnesenskii (Hymenoptera: Apidae).

Bombus vosnesenskii Radowszkowski, 1862 is one of three bumble bee species commercially available for pollination services in North America; however, little is documented about B. vosnesenskii colony life cycle or the establishment of ex situ rearing, mating, and overwintering practices. In this study, we documented nest success, colony size, and gyne production; recorded the duration of mating events; assessed overwintering survival of mated gynes; and evaluated second-generation nest success for colonies established from low- and high-elevation wild-caught B. vosnesenskii gynes. Of the 125 gynes installed, 62.4% produced brood cells (nest initiation) and 43.2% had at least 1 worker eclose (nest establishment). High-elevation B. vosnesenskii gynes had significantly higher nest initiation and establishment success than low-elevation gynes. However, low-elevation colonies were significantly larger with queens producing more gynes on average. Mating was recorded for 200 low-elevation and 37 high-elevation gynes, resulting in a mean duration of 62 and 51 min, respectively. Mated gynes were then placed into cold storage for 54 days to simulate overwintering, which resulted in 59.1% of low-elevation gynes surviving and 91.9% of high-elevation gynes surviving. For second-generation low-elevation gynes, 26.4% initiated nesting and 14.3% established nesting. Second-generation high-elevation gynes did not initiate nesting despite CO2 narcosis treatments. Overall, these results increase our understanding of B. vosnesenskii nesting, mating, and overwintering biology from 2 elevations. Furthermore, this study provides information on successful husbandry practices that can be used by researchers and conservationists to address knowledge gaps and enhance the captive rearing of bumble bees.

Multiplex Lateral Flow Assay and the Sample Preparation Method for the Simultaneous Detection of Three Marine Toxins.

A multiplex lateral flow immunoassay (LFA) has been developed to detect the primary marine biotoxin groups: amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, and diarrhetic shellfish poisoning toxins. The performance characteristics of the multiplex LFA were evaluated for its suitability as a screening method for the detection of toxins in shellfish. The marine toxin-specific antibodies were class-specific, and there was no cross-reactivity between the three toxin groups. The test is capable of detecting all three marine toxin groups, with working ranges of 0.2-1.5, 2.5-65.0, and 8.2-140.3 ng/mL for okadaic acid, saxitoxin, and domoic acid, respectively. This allows the multiplex LFA to detect all three toxin groups at the EU regulatory limits, with a single sample extraction method and dilution volume. No matrix effects were observed on the performance of the LFA with mussel samples spiked with toxins. The developed LFA uses a simple and pocket-sized, portable Cube Reader to provide an accurate result. We also evaluated the use of this Cube Reader with commercially available monoplex lateral flow assays for marine toxins.

Supercooling points of freeze-avoiding bumble bees vary with caste and queen life stage.

Bumble bees thrive in cold climates including high latitude and high altitude regions around the world, yet cold tolerance strategies are largely unknown for most species. To determine bumble bee cold tolerance strategy, we exposed bumble bees to a range of low temperatures and measured survival 72 h post-exposure. All bees that froze died within 72 h while only one bee died without freezing, suggesting that bumble bees are generally freeze-avoiding insects and may be slightly chill susceptible. We then assessed whether temperatures that cause internal ice formation (supercooling points, SCP) varied among bumble bee castes (drones, workers, and queens), or across queen life stages, collection elevation, species, or season. Males froze at significantly lower temperatures than workers or queens. Queens in pre-overwintering or overwintering states froze at significantly lower temperatures than queens stimulated to initiate ovary development by CO2 narcosis (i.e., "spring" queens). We also tested whether the presence of water (i.e., wet or dry) or ramping rate affected SCP. As expected, queens inoculated with water froze at significantly higher temperatures than dry queens. SCP tended to be lower, but not significantly so, at faster ramping rates (0.5 °C/min vs 0.25 °C/min). We also found no differences in SCP between queen bumble bees collected in spring and fall, between queens collected at two sites differing in elevation by 1100 m, or between three field-caught bumble bee species. Bumble bees appear to have relatively high, invariable SCPs, likely making them highly susceptible to freezing across all seasons. As bumble bees are not freeze-tolerant and appear to lack the ability to prevent freezing at temperatures much below 0 °C, they may rely on season- and caste-specific micro-habitat selection to thrive in cold climates.

Multi-Scale Airborne Infectious Disease Transmission.

Airborne disease transmission is central to many scientific disciplines including agriculture, veterinary biosafety, medicine, and public health. Legal and regulatory standards are in place to prevent agricultural, nosocomial, and community airborne disease transmission. However, the overall importance of the airborne pathway is underappreciated, e.g.,, US National Library of Medicine's Medical Subjects Headings (MESH) thesaurus lacks an airborne disease transmission indexing term. This has practical consequences as airborne precautions to control epidemic disease spread may not be taken when airborne transmission is important, but unrecognized. Publishing clearer practical methodological guidelines for surveillance studies and disease outbreak evaluations could help address this situation.To inform future work, this paper highlights selected, well-established airborne transmission events - largely cases replicated in multiple, independently conducted scientific studies. Methodologies include field experiments, modeling, epidemiology studies, disease outbreak investigations and mitigation studies. Collectively, this literature demonstrates that airborne viruses, bacteria, and fungal pathogens have the capability to cause disease in plants, animals, and humans over multiple distances - from near range (< 5 m) to continental (> 500 km) in scale. The plausibility and implications of undetected airborne disease transmission are discussed, including the notable underreporting of disease burden for several airborne transmitted diseases.

Regional Relative Risk, a Physics-Based Metric for Characterizing Airborne Infectious Disease Transmission.

Airborne infectious disease transmission events occur over a wide range of spatial scales and can be an important means of disease transmission. Physics- and biology-based models can assist in predicting airborne transmission events, overall disease incidence, and disease control strategy efficacy. We describe a new theory that extends current approaches for the case in which an individual is infected by a single airborne particle, including the scenario in which numerous infectious particles are present in the air but only one causes infection. A single infectious particle can contain more than one pathogenic microorganism and be physically larger than the pathogen itself. This approach allows robust relative risk estimates even when there is wide variation in (i) individual exposures and (ii) the individual response to that exposure (the pathogen dose-response function can take any mathematical form and vary by individual). Based on this theory, we propose the regional relative risk-a new metric, distinct from the traditional relative risk metric, that compares the risk between two regions. In theory, these regions can range from individual rooms to large geographic areas. In this paper, we apply the regional relative risk metric to outdoor disease transmission events over spatial scales ranging from 50 m to 20 km, demonstrating that in many common cases minimal input information is required to use the metric. Also, we demonstrate that the model predictions are consistent with data from prior outbreaks. Future efforts could apply and validate this theory for other spatial scales, such as transmission within indoor environments. This work provides context for (i) the initial stages of an airborne disease outbreak and (ii) larger-scale disease spread, including unexpected low-probability disease "sparks" that potentially affect remote populations, a key practical issue in controlling airborne disease outbreaks. IMPORTANCE Airborne infectious disease transmission events occur over a wide range of spatial scales and can be important to disease outbreaks. We describe a new physics- and biology-based theory for the important case in which individuals are infected by a single airborne particle (even though numerous infectious particles can be emitted into the air and inhaled). Based on this theory, we propose a new epidemiological metric, regional relative risk, that compares the risk between two geographic regions (in theory, regions can range from individual rooms to large areas). Our modeling of transmission events predicts that for many scenarios of interest, minimal information is required to use this metric for locations 50 m to 20 km downwind. This prediction is consistent with data from prior disease outbreaks. Future efforts could apply and validate this theory for other spatial scales, such as indoor environments. Our results may be applicable to many airborne diseases a priori, as these results depend on the physics of airborne particulate dispersion.

Regulation of the global orthotist/prosthetist workforce, and what we might learn from allied health professions with international-level regulatory support: a narrative review.

BACKGROUND: By 2050, the global demand for orthotic and prosthetic services is expected to double. Unfortunately, the orthotic/prosthetic workforce is not well placed to meet this growing demand. Strengthening the regulation of orthotist/prosthetists will be key to meeting future workforce demands, however little is known about the extent of orthotist/prosthetist regulation nor the mechanisms through which regulation could best be strengthened. Fortunately, a number of allied health professions have international-level regulatory support that may serve as a model to strengthen regulation of the orthotic/prosthetic profession. The aims of this study were to describe the national-level regulation of orthotist/prosthetists globally, and the international-level regulatory support provided to allied health professions. METHOD: Two environmental scans benchmarked the national-level regulation of the orthotist/prosthetist workforce, and the regulatory support provided by international allied health professional bodies using a set of nine core practitioner standards (core standards) including: Minimum Training/Education, Entry-level Competency Standards, Scope of Practice, Code of Conduct and/or Ethics, Course Accreditation, Continuing Professional Development, Language Standard, Recency of Practice, and Return-to-Practice. Each identified country was categorised by income status (i.e. High-, Upper-Middle-, Lower-Middle-, and Low-Income countries). RESULTS: Some degree of regulation of the orthotist/prosthetist workforce was identified in 30 (15%) of the world's 197 countries. All core standards were present in 6 of these countries. Countries of higher economic status had more core standards in place than countries of lower economic status. International-level professional bodies were identified for 14 of 20 allied health professions. International bodies for the physical therapy (8 core standards) and occupational therapy (5 core standards) professions provided regulatory support to help national associations meet most of the core standards. CONCLUSION: Given the small proportion of countries that have national practitioner regulatory standards in place, most orthotist/prosthetists are working under little-to-no regulation. This presents an opportunity to develop rigorous national-level regulation that can support workforce growth to meet future workforce demands. Given the financial and expertise barriers that hinder the development of a more regulated orthotist/prosthetist workforce, particularly for Low- and Lower-Middle-Income countries, we recommend the establishment of an international professional body with the express purpose to support national-level regulation of orthotist/prosthetists, and thereby build the regulatory capacity of national orthotic/prosthetic associations.

The changing demographics of the orthotist/prosthetist workforce in Australia: 2007, 2012 and 2019.

BACKGROUND: Previous Australian workforce analyses revealed a small orthotist/prosthetist workforce with a low number of practitioners per 100,000 Australians. In recent years, initiatives were implemented to increase relative workforce size, including a government-led change in immigration policy to facilitate entry of experienced internationally trained orthotist/prosthetists into the Australian workforce. Given these changes, this project aimed to compare demographics of the orthotist/prosthetist workforce in Australia and each state/territory between 2007, 2012 and 2019. METHODS: This quasi-experiment analysed data from the Australian Orthotic Prosthetic Association (AOPA) database of certified orthotist/prosthetists, to compare changes in the absolute number of practitioners and the number of practitioners per 100,000 population, as well as practitioner age, gender and service location (i.e., metropolitan, regional/remote) across three time points, with a breakdown by each Australian state and territory. RESULTS: Between 2007 and 2019, the number of orthotist/prosthetists per 100,000 population increased 90%. Average age reduced significantly between 2007 (41.5 years) and 2019 (35 years) (p = 0.001). While the proportion of female practitioners increased significantly between 2007 (30%) and 2019 (49%), and between 2012 (38%) and 2019 (49%) (p < 0.05); only 22% of the female workforce is over 40 years of age. The proportion of practitioners servicing a regional/remote location did not change over time (range 13-14%). CONCLUSIONS: Between 2007 and 2019, the national orthotist/prosthetist workforce increased at a rate that exceeded Australia's population growth, became younger, and more female. However, the number of practitioners per 100,000 population remains below international recommendations; particularly in states outside of Victoria and Tasmania, and in regional/remote areas. In addition, low numbers of mid-late career female practitioners suggest challenges to retention of this particular cohort. These data can help inform workforce initiatives to retain a younger and more female workforce, and improve access to orthotic/prosthetic services.

Atrial Fibrillation Induced From Commotio Cordis.

CASE: A 20-year-old male collegiate basketball player was evaluated for sudden chest pain, shortness of breath, dizziness, and blurry vision, following an elbow to the anterior chest by another player. His symptoms improved over 10 minutes of observation, but rhythm strip performed onsite showed atrial fibrillation, and the athlete was transmitted to the emergency department for further evaluation. Electrocardiogram in the ER confirmed atrial fibrillation with a rate of 85 bpm. Electrocardioversion was being arranged when he spontaneously converted to normal sinus rhythm, 2.5 hours from the traumatic event. CONCLUSIONS: Our case illustrates an unusual example of atrial fibrillation induced by commotio cordis (AFCC). Although less acutely life threatening and much less frequently described than ventricular fibrillation induced by commotio cordis, AFCC should be considered in the differential after blunt chest wall trauma. Currently, there are little data regarding management of patients with AFCC.

Current Trends and Challenges for Rapid SMART Diagnostics at Point-of-Site Testing for Marine Toxins.

In the past twenty years marine biotoxin analysis in routine regulatory monitoring has advanced significantly in Europe (EU) and other regions from the use of the mouse bioassay (MBA) towards the high-end analytical techniques such as high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS). Previously, acceptance of these advanced methods, in progressing away from the MBA, was hindered by a lack of commercial certified analytical standards for method development and validation. This has now been addressed whereby the availability of a wide range of analytical standards from several companies in the EU, North America and Asia has enhanced the development and validation of methods to the required regulatory standards. However, the cost of the high-end analytical equipment, lengthy procedures and the need for qualified personnel to perform analysis can still be a challenge for routine monitoring laboratories. In developing regions, aquaculture production is increasing and alternative inexpensive Sensitive, Measurable, Accurate and Real-Time (SMART) rapid point-of-site testing (POST) methods suitable for novice end users that can be validated and internationally accepted remain an objective for both regulators and the industry. The range of commercial testing kits on the market for marine toxin analysis remains limited and even more so those meeting the requirements for use in regulatory control. Individual assays include enzyme-linked immunosorbent assays (ELISA) and lateral flow membrane-based immunoassays (LFIA) for EU-regulated toxins, such as okadaic acid (OA) and dinophysistoxins (DTXs), saxitoxin (STX) and its analogues and domoic acid (DA) in the form of three separate tests offering varying costs and benefits for the industry. It can be observed from the literature that not only are developments and improvements ongoing for these assays, but there are also novel assays being developed using upcoming state-of-the-art biosensor technology. This review focuses on both currently available methods and recent advances in innovative methods for marine biotoxin testing and the end-user practicalities that need to be observed. Furthermore, it highlights trends that are influencing assay developments such as multiplexing capabilities and rapid POST, indicating potential detection methods that will shape the future market.

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h-1 in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and 13C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from 13C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.

Local adaptation across a complex bioclimatic landscape in two montane bumble bee species.

Understanding evolutionary responses to variation in temperature and precipitation across species ranges is of fundamental interest given ongoing climate change. The importance of temperature and precipitation for multiple aspects of bumble bee (Bombus) biology, combined with large geographic ranges that expose populations to diverse environmental pressures, make these insects well-suited for studying local adaptation. Here, we analyzed genome-wide sequence data from two widespread bumble bees, Bombus vosnesenskii and Bombus vancouverensis, using multiple environmental association analysis methods to investigate climate adaptation across latitude and altitude. The strongest signatures of selection were observed in B. vancouverensis, but despite unique responses between species for most loci, we detected several shared responses. Genes relating to neural and neuromuscular function and ion transport were especially evident with respect to temperature variables, while genes relating to cuticle formation, tracheal and respiratory system development, and homeostasis were associated with precipitation variables. Our data thus suggest that adaptive responses for tolerating abiotic variation are likely to be complex, but that several parallels among species can emerge even for these complex traits and landscapes. Results provide the framework for future work into mechanisms of thermal and desiccation tolerance in bumble bees and a set of genomic targets that might be monitored for future conservation efforts.

Mountain stoneflies may tolerate warming streams: Evidence from organismal physiology and gene expression.

Rapid glacier recession is altering the physical conditions of headwater streams. Stream temperatures are predicted to rise and become increasingly variable, putting entire meltwater-associated biological communities at risk of extinction. Thus, there is a pressing need to understand how thermal stress affects mountain stream insects, particularly where glaciers are likely to vanish on contemporary timescales. In this study, we measured the critical thermal maximum (CTMAX ) of stonefly nymphs representing multiple species and a range of thermal regimes in the high Rocky Mountains, USA. We then collected RNA-sequencing data to assess how organismal thermal stress translated to the cellular level. Our focal species included the meltwater stonefly, Lednia tumana, which was recently listed under the U.S. Endangered Species Act due to climate-induced habitat loss. For all study species, critical thermal maxima (CTMAX  > 20°C) far exceeded the stream temperatures mountain stoneflies experience (<10°C). Moreover, while evidence for a cellular stress response was present, we also observed constitutive expression of genes encoding proteins known to underlie thermal stress (i.e., heat shock proteins) even at low temperatures that reflected natural conditions. We show that high-elevation aquatic insects may not be physiologically threatened by short-term exposure to warm temperatures and that longer-term physiological responses or biotic factors (e.g., competition) may better explain their extreme distributions.

Factors Associated With Health-Related Quality of Life in People Living With Partial Foot or Transtibial Amputation.

OBJECTIVES: To determine which demographic, amputation, and health-related factors were associated with health-related quality of life (HR-QoL) in people living with partial foot amputation (PFA) or transtibial amputation (TTA). DESIGN: Cross-sectional survey. SETTING: Community. PARTICIPANTS: Adults (N=123) with unilateral PFA (n=42) or TTA (n=81). INTERVENTION: Not applicable. MAIN OUTCOME MEASURE: Medical Outcome Short Form (SF-36) version 2. RESULTS: Variation in the SF-36 Physical or Mental Component Summary scores were associated with complex interactions between factors, including: time since amputation, fatigue, anxiety, depression, pain interference, and physical function. Level of amputation (ie, PFA or TTA) did not explain a significant part of the variation in either the SF-36 Physical or Mental Component Summary scores. CONCLUSIONS: Given the complex interactions between factors associated with the physical and mental health components of HR-QoL, there are opportunities to consider the long-term holistic care required by people living in the community with PFA or TTA.

Distance, elevation and environment as drivers of diversity and divergence in bumble bees across latitude and altitude.

Identifying drivers of dispersal limitation and genetic differentiation is a key goal in biogeography. We examine patterns of population connectivity and genetic diversity using restriction site-associated DNA sequencing (RADseq) in two bumble bee species, Bombus vosnesenskii and Bombus bifarius, across latitude and altitude in mountain ranges from California, Oregon and Washington, U.S.A. Bombus vosnesenskii, which occurs across a broader elevational range at most latitudes, exhibits little population structure while B. bifarius, which occupies a relatively narrow higher elevation niche across most latitudes, exhibits much stronger population differentiation, although gene flow in both species is best explained by isolation with environmental niche resistance. A relationship between elevational habitat breadth and genetic diversity is also apparent, with B. vosnesenskii exhibiting relatively consistent levels of genetic diversity across its range, while B. bifarius has reduced genetic diversity at low latitudes, where it is restricted to high-elevation habitat. The results of this study highlight the importance of the intersect between elevational range and habitat suitability in influencing population connectivity and suggest that future climate warming will have a fragmenting effect even on populations that are presently well connected, as they track their thermal niches upward in montane systems.

Critical thermal limits of bumblebees (Bombus impatiens) are marked by stereotypical behaviors and are unchanged by acclimation, age or feeding status.

Critical thermal limits often determine species distributions for diverse ectotherms and have become a useful tool for understanding past and predicting future range shifts in response to changing climates. Despite recently documented population declines and range shifts of bumblebees (genus Bombus), the few measurements of thermal tolerance available for the group have relied on disparate measurement approaches. We describe a novel stereotypical behavior expressed by bumblebee individuals during entry into chill coma. This behavioral indicator of minimum critical temperature (CTmin) occurred at ambient temperatures of 3-5°C (approximately 7-9°C core temperatures) and was accompanied by a pronounced CO2 pulse, indicative of loss of spiracle function. Maximum critical temperature (CTmax) was indicated by the onset of muscular spasms prior to entering an unresponsive state and occurred at ambient temperatures of approximately 52-55°C (42-44°C core temperatures). Measurements of CTmin and CTmax were largely unaffected by acclimation, age or feeding status, but faster ramping rates significantly increased CTmax and decreased CTmin This high-throughput approach allows rapid measurement of critical thermal limits for large numbers of individuals, facilitating large-scale comparisons among bumblebee populations and species - a key step in determining current and future effects of climate on these critical pollinators.

Cross-neutralization of influenza A viruses mediated by a single antibody loop.

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

Fatty acid composition in native bees: Associations with thermal and feeding ecology.

Fatty acid (FA) composition of lipids plays a crucial role in the functioning of lipid-containing structures in organisms and may be affected by the temperature an organism experiences, as well as its diet. We compared FA composition among four bee genera: Andrena, Bombus, Megachile, and Osmia which differ in their thermal ecology and diet. Fatty acid methyl esters (FAME) were prepared by direct transesterification with KOH and analyzed using gas-liquid chromatography with a flame ionization detector. Sixteen total FAs ranging in chain length from eight to 22 carbon atoms were identified. Linear discriminant analysis separated the bees based on their FA composition. Andrena was characterized by relatively high concentrations of polyunsaturated FAs, Bombus by high monounsaturated FAs and Megachilids (Megachile and Osmia) by relatively high amounts of saturated FAs. These differences in FA composition may in part be explained by variation in the diets of these bees. Because tongue (proboscis) length may be used as a proxy for the types of flowers bees may visit for nectar and pollen, we compared FA composition among Bombus that differed in proboscis length (but have similar thermal ecology). A clear separation in FA composition within Bombus with varying proboscis lengths was found using linear discriminant analysis. Further, comparing the relationship between each genus by cluster analysis revealed aggregations by genus that were not completely separated, suggesting potential overlap in dietary acquisition of FAs.