Fantastic beasts and how to study them: rethinking experimental animal behavior.

Humans have been trying to understand animal behavior at least since recorded history. Recent rapid development of new technologies has allowed us to make significant progress in understanding the physiological and molecular mechanisms underlying behavior, a key goal of neuroethology. However, there is a tradeoff when studying animal behavior and its underlying biological mechanisms: common behavior protocols in the laboratory are designed to be replicable and controlled, but they often fail to encompass the variability and breadth of natural behavior. This Commentary proposes a framework of 10 key questions that aim to guide researchers in incorporating a rich natural context into their experimental design or in choosing a new animal study system. The 10 questions cover overarching experimental considerations that can provide a template for interspecies comparisons, enable us to develop studies in new model organisms and unlock new experiments in our quest to understand behavior.

The CDC Foundation's COVID-19 emergency response pilot project: a case study in knowledge brokering for older adults and caregivers.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) public health emergency has disproportionately affected older adults and their caregivers, requiring evidence-based and coordinated efforts to meet their health and social needs. This paper describes the role of the CDC Foundation as a knowledge broker working with public health partners to rapidly meet the unmet health, social, and other needs of older adults and caregivers during the COVID-19 pandemic. Type of program or service: Qualitative case study using the Role Model for Knowledge Brokering framework to describe a project that translated public health research into practice during the COVID-19 pandemic response. METHODS: This case study documents the experiences of a US-based foundation serving as a knowledge broker, carrying out three roles: establishing research partnerships to study unmet health, social, and other needs of older adults and caregivers during COVID-19; coordinating with partners to identify evidence-based strategies; and rapidly implementing four emergency response pilot projects. RESULTS: The emergency response pilot projects created included: an online resource library - -SearchFindHelp.org - of public health programs and resources for organisations serving older adults and caregivers; digital literacy training for older adults and caregivers; multicultural caregiver tools to serve rural and Asian American and Pacific Islander older adults; and a grant program to expand local, direct services for older adults. SearchFindHelp.org had 46 781 new users and 101 908 total views from June 2021-March 2023. Older adults and caregivers who participated in digital literacy training from May-September 2021 were more likely to find health resources online and schedule and attend an online doctor's visit. A paid media campaign in December 2021 was launched to raise awareness of multicultural caregiver tools. Ten community organisations expanded direct, local services for older adults. LESSONS LEARNT: This project highlights the valuable role a foundation can play as a knowledge broker in rapidly translating research into practice during a public health emergency response, to address emerging community needs.

Establishment of a Labile Bound Copper Reference Interval in a Healthy Population via an Inductively Coupled Plasma Mass Spectrometry Dual Filtration-Based Assay.

CONTEXT.­: Clinical testing for Wilson disease (WD) is potentially challenging. Measuring the fraction of labile bound copper (LBC) to total copper may be a promising alternative diagnostic tool with better sensitivity and specificity than some current biomarker approaches. A dual filtration-based inductively coupled mass spectrometry (ICP-MS) assay to measure LBC in serum was developed. OBJECTIVE.­: To establish a reference interval for LBC and LBC to total copper (LBC fraction) in a healthy adult population, and to examine associations between total copper, LBC, and LBC fraction with age, sex, menopausal status, hormone replacement therapy, and supplement use. DESIGN.­: Serum samples were collected from healthy male (n = 110) and female (n = 104) patients between the ages of 19 and 80 years. Total copper and LBC were analyzed using ICP-MS. Results were used to calculate the LBC fraction. Reference intervals were calculated for the 2.5th and 97.5th percentiles for both LBC and LBC fraction. RESULTS.­: The reference intervals for LBC were determined to be 13 to 105 ng/mL and 12 to 107 ng/mL for female and male patients, respectively. The reference intervals for the LBC fraction were 1.0% to 8.1% and 1.2% to 10.5% for female and male patients, respectively. No significant associations were found regarding age, menopausal status, hormone replacement therapy, or vitamin and supplement use. CONCLUSIONS.­: Sex-specific reference intervals have now been established for LBC and LBC fraction. These data in conjunction with further testing of WD populations can be used to assess the sensitivity and specificity of LBC fraction in screening, monitoring, and diagnosis.

Insect neuroethology: Flight constructs a compass for monarch migration.

A new study shows that a brain map in the monarch butterfly can be re-drawn during flight. Migrating butterflies integrate efferent and visual signals to create an adaptable compass in their central brain.

Takeoff diversity in Diptera.

The order Diptera (true flies) are named for their two wings because their hindwings have evolved into specialized mechanosensory organs called halteres. Flies use halteres to detect body rotations and maintain stability during flight and other behaviours. The most recently diverged dipteran monophyletic subsection, the Calyptratae, is highly successful, accounting for approximately 12% of dipteran diversity, and includes common families like house flies. These flies move their halteres independently from their wings and oscillate their halteres during walking. Here, we demonstrate that this subsection of flies uses their halteres to stabilize their bodies during takeoff, whereas non-Calyptratae flies do not. We find that flies of the Calyptratae are able to take off more rapidly than non-Calyptratae flies without sacrificing stability. Haltere removal decreased both velocity and stability in the takeoffs of Calyptratae, but not other flies. The loss of takeoff velocity following haltere removal in Calyptratae (but not other flies) is a direct result of a decrease in leg extension speed. A closely related non-Calyptratae species (D. melanogaster) also has a rapid takeoff, but takeoff duration and stability are unaffected by haltere removal. Haltere use thus allows for greater speed and stability during fast escapes, but only in the Calyptratae clade.

Haltere and visual inputs sum linearly to predict wing (but not gaze) motor output in tethered flying Drosophila.

In the true flies (Diptera), the hind wings have evolved into specialized mechanosensory organs known as halteres, which are sensitive to gyroscopic and other inertial forces. Together with the fly's visual system, the halteres direct head and wing movements through a suite of equilibrium reflexes that are crucial to the fly's ability to maintain stable flight. As in other animals (including humans), this presents challenges to the nervous system as equilibrium reflexes driven by the inertial sensory system must be integrated with those driven by the visual system in order to control an overlapping pool of motor outputs shared between the two of them. Here, we introduce an experimental paradigm for reproducibly altering haltere stroke kinematics and use it to quantify multisensory integration of wing and gaze equilibrium reflexes. We show that multisensory wing-steering responses reflect a linear superposition of haltere-driven and visually driven responses, but that multisensory gaze responses are not well predicted by this framework. These models, based on populations, extend also to the responses of individual flies.

Evaluation of essential oils and a prebiotic for newborn dairy calves.

A blend of essential oils (EO) and a prebiotic were combined (EOC) to formulate a colostrum-based liquid birth supplement and a separate feeding supplement (Start Strong and Stay Strong, Ralco Inc., Marshall, MN). These products were designed to promote immunity and stimulate appetite to diminish health challenges and stresses experienced by newborn calves. The hypothesis was that calves supplemented with an oral dose of liquid EOC at birth (10-mL aliquot at birth and 10 mL at 12 h of age) when fed the EOC feeding supplement would result in improved growth performance, health, and immunity. The objective was to determine if an additional feeding of liquid EOC at birth in combination with EOC in the milk replacer (MR) would allow calves to demonstrate improved growth, health, and immunity compare with calves only offered EO in MR. Sixty-one Holstein calves (18 males and 43 females) from a commercial dairy operation were blocked by birth date and randomly assigned to 1 of 3 treatments. Treatments were 1) Control (CON): a 24% crude protein (CP):20% fat (as-fed basis) MR; 2) EP: a 24:20 MR with EOC mixed at 1.25 g/d; or 3) EPC: a 24:20 MR with EOC mixed at 1.25 g/d in addition to calves receiving one 10-mL oral dose of liquid EOC at birth and 10 mL again at 12 h. The 24:20 MR was fed via bucket 2 times per day at a rate of 0.57 kg/calf daily for 14 d, increased to 0.85 kg/calf at 2 times per day until 35 d and was reduced to 0.43 kg at 1 time per day at 36 d to facilitate weaning after 42 d. Decoquinate was added to the MR at 41.6 mg/kg for coccidiosis control. Calves were housed in individual hutches bedded with straw with ad libitum access to a 20% CP-pelleted calf starter and water. All data were analyzed using PROC MIXED as a randomized complete block design. Calves in this study had similar (P > 0.10) average daily gains, body weight, and growth measurements. Calves fed EPC had significantly (P < 0.05) higher IgA titers on day 0 of the trial compared with calves fed EP or CON, which was expected as calves were supplemented with liquid EOC at birth and 12 h later demonstrating an increase in immune response. The use of a liquid EOC product being administrated after birth can improve IgA titers to improve the immune status of the new born calf to fight off potential diseases and pathogens. A formulation error resulted in the EOC being fed at half the rate of the previous experiment of 2.5 g/d, which appears to be below an efficacious dosage.

AAV8 Ins1-Cre can produce efficient ß-cell recombination but requires consideration of off-target effects.

In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on ß-cell function and organism physiology. Cre-LoxP is a system wherein LoxP sites flanking a gene are recognized by Cre recombinase. Cre transgenic mice are the most prevalent technology used to deliver Cre but many models have caveats of off-target recombination, impaired ß-cell function, and high cost of animal production. Inducible estrogen receptor conjugated Cre models face leaky recombination and confounding effects of tamoxifen. As an alternative, we characterize an adeno associated virus (AAV) with a rat insulin 1 promoter driving Cre recombinase (AAV8 Ins1-Cre) that is economical and rapid to implement, and has limited caveats. Intraperitoneal AAV8 Ins1-Cre produced efficient ß-cell recombination, alongside some hepatic, exocrine pancreas, α-cell, δ-cell, and hypothalamic recombination. Delivery of lower doses via the pancreatic duct retained good rates of ß-cell recombination and limited rates of off-target recombination. Unlike inducible Cre in transgenic mice, AAV8 Ins1-Cre required no tamoxifen and premature recombination was avoided. We demonstrate the utility of this technology by inducing hyperglycemia in inducible insulin knockout mice (Ins1-/-;Ins2f/f). AAV-mediated expression of Cre in ß-cells provides an effective alternative to transgenic approaches for inducible knockout studies.

Giving credit to reforestation for water quality benefits.

While there is a general belief that reforesting marginal, often unprofitable, croplands can result in water quality benefits, to date there have been very few studies that have attempted to quantify the magnitude of the reductions in nutrient (N and P) and sediment export. In order to determine the magnitude of a credit for water quality trading, there is a need to develop quantitative approaches to estimate the benefits from forest planting in terms of load reductions. Here we first evaluate the availability of marginal croplands (i.e. those with low infiltration capacity and high slopes) within a large section of the Ohio River Basin (ORB) to assess the magnitude of the land that could be reforested. Next, we employ the Nutrient Tracking Tool (NTT) to study the reduction in N, P and sediment losses from converting corn or corn/soy rotations to forested lands, first in a case study and then for a large region within the ORB. We find that after reforestation, N losses can decrease by 40 to 80 kg/ha-yr (95-97% reduction), while P losses decrease by 1 to 4 kg/ha-yr (96-99% reduction). There is a significant influence of local conditions (soils, previous crop management practices, meteorology), which can be considered with NTT and must be taken into consideration for specific projects. There is also considerable interannual and monthly variability, which highlights the need to take the longer view into account in nutrient credit considerations for water quality trading, as well as in monitoring programs. Overall, there is the potential for avoiding 60 million kg N and 2 million kg P from reaching the streams and rivers of the northern ORB as a result of conversion of marginal farmland to tree planting, which is on the order of 12% decrease for TN and 5% for TP, for the entire basin. Accounting for attenuation, this represents a significant fraction of the goal of the USEPA Gulf of Mexico Hypoxia Task Force to reduce TN and TP reaching the dead zone in the Gulf of Mexico, the second largest dead zone in the world. More broadly, the potential for targeted forest planting to reduce nutrient loading demonstrated in this study suggests further consideration of this approach for managing water quality in waterways throughout the world. The study was conducted using computational models and there is a need to evaluate the results with empirical observations.

Representation of Haltere Oscillations and Integration with Visual Inputs in the Fly Central Complex.

The reduced hindwings of flies, known as halteres, are specialized mechanosensory organs that detect body rotations during flight. Primary afferents of the haltere encode its oscillation frequency linearly over a wide bandwidth and with precise phase-dependent spiking. However, it is not currently known whether information from haltere primary afferent neurons is sent to higher brain centers where sensory information about body position could be used in decision making, or whether precise spike timing is useful beyond the peripheral circuits that drive wing movements. We show that in cells in the central brain, the timing and rates of neural spiking can be modulated by sensory input from experimental haltere movements (driven by a servomotor). Using multichannel extracellular recording in restrained flesh flies (Sarcophaga bullata of both sexes), we examined responses of central complex cells to a range of haltere oscillation frequencies alone, and in combination with visual motion speeds and directions. Haltere-responsive units fell into multiple response classes, including those responding to any haltere motion and others with firing rates linearly related to the haltere frequency. Cells with multisensory responses showed higher firing rates than the sum of the unisensory responses at higher haltere frequencies. They also maintained visual properties, such as directional selectivity, while increasing response gain nonlinearly with haltere frequency. Although haltere inputs have been described extensively in the context of rapid locomotion control, we find haltere sensory information in a brain region known to be involved in slower, higher-order behaviors, such as navigation.SIGNIFICANCE STATEMENT Many animals use vision for navigation; however, these cues must be interpreted in the context of the body's position. In mammalian brains, hippocampal cells combine visual and vestibular information to encode head direction. A region of the arthropod brain, known as the central complex (CX), similarly encodes heading information, but it is unknown whether proprioceptive information is integrated here as well. We show that CX neurons respond to input from halteres, specialized proprioceptors in flies that detect body rotations. These neurons also respond to visual input, providing one of the few examples of multiple sensory modalities represented in individual CX cells. Haltere stimulation modifies neural responses to visual signals, providing a mechanism for integrating vision with proprioception.

Sensory Feedback and Animal Locomotion: Perspectives from Biology and Biorobotics: An Introduction to the Symposium.

The successful completion of many behaviors relies on sensory feedback. This symposium brought together researchers using novel techniques to study how different stimuli are encoded, how and where multimodal feedback is integrated, and how feedback modulates motor output in diverse modes of locomotion (aerial, aquatic, and terrestrial) in a diverse range of taxa (insects, fish, tetrapods), and in robots. Similar to biological organisms, robots can be equipped with integrated sensors and can rely on sensory feedback to adjust the output signal of a controller. Engineers often look to biology for inspiration on how animals have evolved solutions to problems similar to those experienced in robotic movement. Similarly, biologists too must proactively engage with engineers to apply computer and robotic models to test hypotheses and answer questions on the capacity and roles of sensory feedback in generating effective movement. Through a diverse group of researchers, including both biologists and engineers, the symposium attempted to catalyze new interdisciplinary collaborations and identify future research directions for the development of bioinspired sensory control systems, as well as the use of robots to test hypotheses in neuromechanics.

Single mechanosensory neurons encode lateral displacements using precise spike timing and thresholds.

During locomotion, animals rely on multiple sensory modalities to maintain stability. External cues may guide behaviour, but they must be interpreted in the context of the animal's own body movements. Mechanosensory cues that can resolve dynamic internal and environmental conditions, like those from vertebrate vestibular systems or other proprioceptors, are essential for guided movement. How do afferent proprioceptor neurons transform movement into a neural code? In flies, modified hindwings known as halteres detect forces produced by body rotations and are essential for flight. However, the mechanisms by which haltere neurons transform forces resulting from three-dimensional body rotations into patterns of neural spikes are unknown. We use intracellular electrodes to record from haltere primary afferent neurons during a range of haltere motions. We find that spike timing activity of individual neurons changes with displacement and propose a mechanism by which single neurons can encode three-dimensional haltere movements during flight.

Haltere removal alters responses to gravity in standing flies.

Animals detect the force of gravity with multiple sensory organs, from subcutaneous receptors at body joints to specialized sensors like the vertebrate inner ear. The halteres of flies, specialized mechanoreceptive organs derived from hindwings, are known to detect body rotations during flight, and some groups of flies also oscillate their halteres while walking. The dynamics of halteres are such that they could act as gravity detectors for flies standing on substrates, but their utility during non-flight behaviors is not known. We observed the behaviors of intact and haltere-ablated flies during walking and during perturbations in which the acceleration due to gravity suddenly changed. We found that intact halteres are necessary for flies to maintain normal walking speeds on vertical surfaces and to respond to sudden changes in gravity. Our results suggest that halteres can serve multiple sensory purposes during different behaviors, expanding their role beyond their canonical use in flight.

Inertial Sensing and Encoding of Self-Motion: Structural and Functional Similarities across Metazoan Taxa.

To properly orient and navigate, moving animals must obtain information about the position and motion of their bodies. Animals detect inertial signals resulting from body accelerations and rotations using a variety of sensory systems. In this review, we briefly summarize current knowledge on inertial sensing across widely disparate animal taxa with an emphasis on neuronal coding and sensory transduction. We outline systems built around mechanosensory hair cells, including the chordate vestibular complex and the statocysts seen in many marine invertebrates. We next compare these to schemes employed by flying insects for managing inherently unstable aspects of flapping flight, built around comparable mechanosensory cells but taking unique advantage of the physics of rotating systems to facilitate motion encoding. Finally, we highlight fundamental similarities across taxa with respect to the partnering of inertial senses with visual senses and conclude with a discussion of the functional utility of maintaining a multiplicity of encoding schemes for self-motion information.

Playing with fire: effects of negative mood induction and working memory on vocabulary acquisition.

We investigated the impact of emotions on learning vocabulary in an unfamiliar language to better understand affective influences in foreign language acquisition. Seventy native English speakers learned new vocabulary in either a negative or a neutral emotional state. Participants also completed two sets of working memory tasks to examine the potential mediating role of working memory. Results revealed that participants exposed to negative stimuli exhibited difficulty in retrieving and correctly pairing English words with Indonesian words, as reflected in a lower performance on the prompted recall tests and the free recall measure. Emotional induction did not change working memory scores from pre to post manipulation. This suggests working memory could not explain the reduced vocabulary learning in the negative group. We argue that negative mood can adversely affect language learning by suppressing aspects of native-language processing and impeding form-meaning mapping with second language words.

Novel associations of bile acid diarrhoea with fatty liver disease and gallstones: a cohort retrospective analysis.

BACKGROUND: Bile acid diarrhoea (BAD) is a common cause of chronic diarrhoea with a population prevalence of primary BAD around 1%. Previous studies have identified associations with low levels of the ileal hormone fibroblast growth factor 19 (FGF19), obesity and hypertriglyceridaemia. The aim of this study was to identify further associations of BAD. METHODS: A cohort of patients with chronic diarrhoea who underwent 75selenohomocholic acid taurate (SeHCAT) testing for BAD was further analysed retrospectively. Additional clinical details available from the electronic patient record, including imaging, colonoscopy, chemistry and histopathology reports were used to calculate the prevalence of fatty liver disease, gallstones, colonic neoplasia and microscopic colitis, which was compared for BAD, the primary BAD subset and control patients with diarrhoea. FINDINGS: Of 578 patients, 303 (52%) had BAD, defined as a SeHCAT 7d retention value <15%, with 179 (31%) having primary BAD. 425 had an alanine aminotransferase (ALT) recorded, 184 had liver imaging and 176 had both. Overall, SeHCAT values were negatively associated with ALT (rs=-0.19, p<0.0001). Patients with BAD had an OR of 3.1 for an ALT >31 ng/mL with imaging showing fatty liver (p<0.001); similar figures occurred in the primary BAD group. FGF19 was not significantly related to fatty liver but low levels were predictive of ALT >40 IU/L. In 176 subjects with gallbladder imaging, 27% had gallstones, 7% had a prior cholecystectomy and 34% either of these. The median SeHCAT values were lower in those with gallstones (3.8%, p<0.0001), or gallstones/cholecystectomy (7.2%, p<0.001), compared with normal gallbladder imaging (14%). Overall, BAD had an OR of 2.0 for gallstones/cholecystectomy (p<0.05). BAD was not significantly associated with colonic adenoma/carcinoma or with microscopic colitis. INTERPRETATION: The diagnosis of BAD is associated with fatty liver disease and with gallstones. The reasons for these associations require further investigation into potential metabolic causes.

Cross-modal influence of mechanosensory input on gaze responses to visual motion in Drosophila.

Animals typically combine inertial and visual information to stabilize their gaze against confounding self-generated visual motion, and to maintain a level gaze when the body is perturbed by external forces. In vertebrates, an inner ear vestibular system provides information about body rotations and accelerations, but gaze stabilization is less understood in insects, which lack a vestibular organ. In flies, the halteres, reduced hindwings imbued with hundreds of mechanosensory cells, sense inertial forces and provide input to neck motoneurons that control gaze. These neck motoneurons also receive input from the visual system. Head movement responses to visual motion and physical rotations of the body have been measured independently, but how inertial information might influence gaze responses to visual motion has not been fully explored. We measured the head movement responses to visual motion in intact and haltere-ablated tethered flies to explore the role of the halteres in modulating visually guided head movements in the absence of rotation. We note that visually guided head movements occur only during flight. Although halteres are not necessary for head movements, the amplitude of the response is smaller in haltereless flies at higher speeds of visual motion. This modulation occurred in the absence of rotational body movements, demonstrating that the inertial forces associated with straight tethered flight are important for gaze-control behavior. The cross-modal influence of halteres on the fly's responses to fast visual motion indicates that the haltere's role in gaze stabilization extends beyond its canonical function as a sensor of angular rotations of the thorax.

Haltere morphology and campaniform sensilla arrangement across Diptera.

One of the primary specializations of true flies (order Diptera) is the modification of the hind wings into club-shaped halteres. Halteres are complex mechanosensory structures that provide sensory feedback essential for stable flight control via an array of campaniform sensilla at the haltere base. The morphology of these sensilla has previously been described in a small number of dipteran species, but little is known about how they vary across fly taxa. Using a synoptic set of specimens representing 42 families from all of the major infraorders of Diptera, we used scanning electron microscopy to map the gross and fine structures of halteres, including sensillum shape and arrangement. We found that several features of haltere morphology correspond with dipteran phylogeny: Schizophora generally have smaller halteres with stereotyped and highly organized sensilla compared to nematoceran flies. We also found a previously undocumented high variation of haltere sensillum shape in nematoceran dipterans, as well as the absence of a dorsal sensillum field in multiple families. Overall, variation in haltere sensillar morphology across the dipteran phylogeny provides insight into the evolution of a highly specialized proprioceptive organ and a basis for future studies on haltere sensory function.

Dipteran Halteres: Perspectives on Function and Integration for a Unique Sensory Organ.

The halteres of dipteran insects (true flies) are essential mechanosensory organs for flight. These are modified hindwings with several arrays of sensory cells at their base, and they are one of the characteristic features of flies. Mechanosensory information from the halteres is sent with low latency to wing-steering and head movement motoneurons, allowing direct control of body position and gaze. Analyses of the structure and dynamics of halteres indicate that they experience very small aerodynamic forces but significant inertial forces, including Coriolis forces associated with body rotations. The sensory cells at the base of the haltere detect these forces and allow the fly to correct for perturbations during flight, but new evidence suggests that this may not be their only role. This review will examine our current understanding of how these organs move, encode forces, and transmit information about these forces to the nervous system to guide behavior.

Hypothyroidism Impairs Human Stem Cell-Derived Pancreatic Progenitor Cell Maturation in Mice.

Pancreatic progenitors derived from human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating diabetes and are currently being tested in clinical trials. Yet, how the milieu of pancreatic progenitor cells, including exposure to different factors after transplant, may influence their maturation remains unclear. Here, we examined the effect of thyroid dysregulation on the development of hESC-derived progenitor cells in vivo. Hypothyroidism was generated in SCID-beige mice using an iodine-deficient diet containing 0.15% propyl-2-thiouracil, and hyperthyroidism was generated by addition of L-thyroxine (T4) to drinking water. All mice received macroencapsulated hESC-derived progenitor cells, and thyroid dysfunction was maintained for the duration of the study ("chronic") or for 4 weeks posttransplant ("acute"). Acute hyperthyroidism did not affect graft function, but acute hypothyroidism transiently impaired human C-peptide secretion at 16 weeks posttransplant. Chronic hypothyroidism resulted in severely blunted basal human C-peptide secretion, impaired glucose-stimulated insulin secretion, and elevated plasma glucagon levels. Grafts from chronic hypothyroid mice contained fewer ß-cells, heterogenous MAFA expression, and increased glucagon(+) and ghrelin(+) cells compared to grafts from euthyroid mice. Taken together, these data suggest that long-term thyroid hormone deficiency may drive the differentiation of human pancreatic progenitor cells toward α- and ε-cell lineages at the expense of ß-cell formation.