Elucidation of the DNA-Binding Activity of VirF from Shigella flexneri for the icsA and rnaG Promoters and Characterization of the N-Terminal Domain To Identify Residues Crucial for Dimerization.

A novel approach to treat the highly virulent and infectious enteric pathogen Shigella flexneri, with the potential for reduced resistance development, is to target virulence pathways. One promising such target is the AraC-family transcription factor VirF, which activates downstream virulence factors. VirF harbors a conserved C-terminal DNA-binding domain (DBD) and an N-terminal dimerization domain (NTD). Previously, we studied the wild type (WT) and seven alanine DBD mutants of VirF binding to the virB promoter (N. J. Ragazzone, G. T. Dow, and A. Garcia, J Bacteriol 204:e00143-22, 2022, https://doi.org/10.1128/jb.00143-22). Here, we report studies of VirF binding to the icsA and rnaG promoters. Gel shift assays (electrophoretic mobility shift assays [EMSAs]) of WT VirF binding to these promoters revealed multiple bands at higher apparent molecular weights, indicating the likelihood of VirF dimerization when bound to DNA. For three of the mutants, we observed consistent effects on binding to the three promoters. For the four other mutants, we observed differential effects on promoter binding. Results of a cell-based, LexA monohybrid ß-galactosidase reporter assay [D. A. Daines, M. Granger-Schnarr, M. Dimitrova, and R. P. Silver, Methods Enzymol 358:153-161, 2002, https://doi.org/10.1016/s0076-6879(02)58087-3] indicated that WT VirF dimerizes in vivo and that alanine mutations to Y132, L137, and L147 significantly reduced dimerization. However, these mutations negatively impacted protein stability. We did purify enough of the Y132A mutant to determine that it binds in vitro to the virB and rnaG promoters, albeit with weaker affinities. Full-length VirF model structures, generated with I-TASSER, predict that these three amino acids are in a "dimerization" helix in the NTD, consistent with our results. IMPORTANCE Antimicrobial-resistant (AMR) infections continue to rise dramatically, and the lack of new approved antibiotics is not ameliorating this crisis. A promising route to reduce AMR is by targeting virulence. The Shigella flexneri virulence pathway is a valuable source for potential therapeutic targets useful to treat this infection. VirF, an AraC-family virulence transcription factor, is responsible for activating necessary downstream virulence genes that allow the bacteria to invade and spread within the human colon. Previous studies have identified how VirF interacts with the virB promoter and have even developed a lead DNA-binding inhibitor, but not much is known about VirF dimerization or binding to the icsA and rnaG promoters. Fully characterizing VirF can be a valuable resource for inhibitor discovery/design.

Impact of animal programming on children's attitudes toward local wildlife.

Attitudes toward wildlife can have direct implications on children's interest in conservation behaviors. Animal programs are an example of interactive, educational experiences that have the potential to change attitudes by providing individuals the opportunity to get close to animal ambassadors and participate in engaging conversations about them. We conducted an animal program assessment with summer camps at the Ohio Wildlife Center to quantify changes in children's affiliation with local wildlife and their willingness to live near local wildlife. Campers showed an overall increase in affiliation and willingness scores from before to after an animal program, although with a small effect size. Overall willingness scores were lower than affiliation scores, but there was a significantly larger increase in willingness following the program. We found a strong correlation between affiliation and willingness scores. Overall, the study found that these animal programs positively influenced children's attitudes toward local wildlife and increased their willingness to live near them, suggesting animal programming could be used to decrease human-wildlife conflict. Visually seeing animals in the programs improved attitude scores, even for those not seen in this study, which suggests that program animals can act as an ambassador for other species. This opens the potential for utilizing animal ambassadors as powerful tools in conservation education about threatened and endangered species.

Coevolution of relative brain size and life expectancy in parrots.

Previous studies have demonstrated a correlation between longevity and brain size in a variety of taxa. Little research has been devoted to understanding this link in parrots; yet parrots are well-known for both their exceptionally long lives and cognitive complexity. We employed a large-scale comparative analysis that investigated the influence of brain size and life-history variables on longevity in parrots. Specifically, we addressed two hypotheses for evolutionary drivers of longevity: the cognitivebuffer hypothesis, which proposes that increased cognitive abilities enable longer lifespans, and the expensive brain hypothesis, which holds that increases in lifespan are caused by prolonged developmental time of, and increased parental investment in, large-brained offspring. We estimated life expectancy from detailed zoo records for 133 818 individuals across 244 parrot species. Using a principled Bayesian approach that addresses data uncertainty and imputation of missing values, we found a consistent correlation between relative brain size and life expectancy in parrots. This correlation was best explained by a direct effect of relative brain size. Notably, we found no effects of developmental time, clutch size or age at first reproduction. Our results suggest that selection for enhanced cognitive abilities in parrots has in turn promoted longer lifespans.

Teaching animal behavior online: A primer for the pandemic and beyond.

Behavior courses face numerous challenges when moving to an online environment, as has been made necessary by the COVID-19 pandemic. These challenges occur largely because behavior courses, like most organismal biology courses, often stress experiential learning through laboratories that involve live animals, as well as a lecture component that emphasizes formative assessment, discussion, and critical thinking. Although online behavior courses may be remote, they can still be interactive and social, and designed with inclusive pedagogy. Here, we discuss some of the key decisions that instructors should consider, provide recommendations, and point out new opportunities for student learning that stem directly from the move to online instruction. Specific topics include challenges related to generating an inclusive and engaging online learning environment, synchronous versus asynchronous formats, assignments that enhance student learning, testing format and execution, grade schemes, design of laboratory experiences including opportunities for community science, design of synthetic student projects, and workload balance for students and instructors. We designed this primer both for animal behavior instructors who need to quickly transition to online teaching in the midst of a pandemic, and for those facing such transitions in upcoming terms. Much of the manuscript's content should also be of general interest and value to instructors from all areas of organismal biology who are attempting to quickly transition to online teaching.

Characterizing trends in human-wildlife conflicts in the American Midwest using wildlife rehabilitation records.

Human-wildlife conflict is difficult to measure, but the analysis of records from wildlife rehabilitation facilities has shown potential as a technique for characterizing human impacts on wildlife. To examine the value of wildlife rehabilitation records for characterizing local human-wildlife conflicts and prevalence of select wildlife diseases, we reviewed 45,668 records representing over 280 species admitted to a wildlife rehabilitation facility over a 10-year period (2005-2014). We identified the most frequently recorded causes of admission for commonly admitted species, and evaluated how causes of admission may vary across taxa throughout the year. Our analyses support the value of wildlife rehabilitation facility data for characterizing some pressures from human-wildlife conflict and select disease trends for certain taxa, as well as utility for informing topics to emphasize in local conservation education efforts. For example, orphaned neonatal wildlife accounted for the largest proportion of admissions to this facility, and highlights a opportunity for conservation education regarding when wildlife is truly orphaned and requires professional intervention. Additionally, domestic dog attack cases accounted for a proportion of admissions comparable to that of domestic cat attacks, demonstrating a need for the conversation surrounding the impact of domestic pets on local wildlife to expand to include dogs in addition to cats.

Polypharmacy, Opioid Use, and Fibromyalgia: A Secondary Analysis of Clinical Trial Data.

The major therapeutic approach for treating fibromyalgia (FM), a chronic widespread pain syndrome, is pharmacotherapy-centered symptom management. Complexity of treatment often leads to multiple medication prescriptions. While there is no current alternative to the probable need for polypharmacy in this patient population, there remains concern related to potential side effects and adverse drug events. In this secondary analysis of data on medications taken collected from two parent studies, all medications were broken down into the following categories: opioid, nonopioid, antidepressant, anticonvulsant, muscle relaxant, and benzodiazepine. The impact on pain severity and pain interference of these medication categories as well as perceived stress, fatigue, and depression scores was assessed. Baseline pain severity ( p = .0106) and pain interference ( p = .0002) were significantly correlated with opioid use as compared to nonopioid use. A multivariate regression with backward elimination resulted in a model for pain severity with one significant predictor variable, fatigue ( p < .0001); pain interference had three significant predictor variables: opioid use ( p = .04), fatigue ( p < .0001), and depression ( p = .04). While future studies should further address the utility of opioids and examine the role of polypharmacy as part of symptom management strategies for individuals with FM, study findings suggest that, for those who suffer chronic widespread pain as the predominant symptom experience, a challenge equally as perplexing for nurses and nursing research alike as managing the pain lies in addressing the fatigue and depression in this patient population.

Social calls provide novel insights into the evolution of vocal learning.

Learned song is among the best-studied models of animal communication. In oscine songbirds, where learned song is most prevalent, it is used primarily for intrasexual selection and mate attraction. Learning of a different class of vocal signals, known as contact calls, is found in a diverse array of species, where they are used to mediate social interactions among individuals. We argue that call learning provides a taxonomically rich system for studying testable hypotheses for the evolutionary origins of vocal learning. We describe and critically evaluate four nonmutually exclusive hypotheses for the origin and current function of vocal learning of calls, which propose that call learning (1) improves auditory detection and recognition, (2) signals local knowledge, (3) signals group membership, or (4) allows for the encoding of more complex social information. We propose approaches to testing these four hypotheses but emphasize that all of them share the idea that social living, not sexual selection, is a central driver of vocal learning. Finally, we identify future areas for research on call learning that could provide new perspectives on the origins and mechanisms of vocal learning in both animals and humans.

A test of multiple hypotheses for the function of call sharing in female budgerigars, Melopsittacus undulatus.

In many social species group, members share acoustically similar calls. Functional hypotheses have been proposed for call sharing, but previous studies have been limited by an inability to distinguish among these hypotheses. We examined the function of vocal sharing in female budgerigars with a two-part experimental design that allowed us to distinguish between two functional hypotheses. The social association hypothesis proposes that shared calls help animals mediate affiliative and aggressive interactions, while the password hypothesis proposes that shared calls allow animals to distinguish group identity and exclude nonmembers. We also tested the labeling hypothesis, a mechanistic explanation which proposes that shared calls are used to address specific individuals within the sender-receiver relationship. We tested the social association hypothesis by creating four-member flocks of unfamiliar female budgerigars (Melopsittacus undulatus) and then monitoring the birds' calls, social behaviors, and stress levels via fecal glucocorticoid metabolites. We tested the password hypothesis by moving immigrants into established social groups. To test the labeling hypothesis, we conducted additional recording sessions in which individuals were paired with different group members. The social association hypothesis was supported by the development of multiple shared call types in each cage and a correlation between the number of shared call types and the number of aggressive interactions between pairs of birds. We also found support for calls serving as a labeling mechanism using discriminant function analysis with a permutation procedure. Our results did not support the password hypothesis, as there was no difference in stress or directed behaviors between immigrant and control birds.

Validation of a fecal glucocorticoid metabolite assay to assess stress in the budgerigar (Melopsittacus undulatus).

The budgerigar (Melopsittacus undulatus) is a small parrot native to Australia that is commonly held in zoos, laboratories, and private homes. Assessment of budgerigar stress levels would aid welfare monitoring and improve our understanding of their biology. Analyzing fecal glucocorticoid metabolites provides a noninvasive method to measure stress levels in birds. For this method to be reliable, the antibody to be used in an immunoassay must be carefully selected for each species, and validation must be performed. A common limitation in many existing assays is the inability to accurately detect variable fecal glucocorticoid metabolites in minute quantities of feces, requiring small samples to be combined. We have developed a double antibody radioimmunoassay protocol based on a commercially available (125) I-corticosterone radioimmunoassay kit for use in detecting fecal glucocorticoid metabolites in small quantities (<20 mg) of budgerigar droppings. The assay was validated pharmacologically with an adrenocorticotropic hormone challenge and with oral administration of corticosterone. Our validation has demonstrated our assay is both sensitive and a reliable approach to noninvasive monitoring of stress in budgerigars.

Survival on the ark: life history trends in captive parrots.

Members of the order Psittaciformes (parrots and cockatoos) are among the most long-lived and endangered avian species. Comprehensive data on lifespan and breeding are critical to setting conservation priorities, parameterizing population viability models, and managing captive and wild populations. To meet these needs, we analyzed 83, 212 life history records of captive birds from the International Species Information System and calculated lifespan and breeding parameters for 260 species of parrots (71% of extant species). Species varied widely in lifespan, with larger species generally living longer than smaller ones. The highest maximum lifespan recorded was 92 years in Cacatua moluccensis, but only 11 other species had a maximum lifespan over 50 years. Our data indicate that while some captive individuals are capable of reaching extraordinary ages, median lifespans are generally shorter than widely assumed, albeit with some increase seen in birds presently held in zoos. Species that lived longer and bred later in life tended to be more threatened according to IUCN classifications. We documented several individuals of multiple species that were able to breed for more than two decades, but the majority of clades examined had much shorter active reproduction periods. Post-breeding periods were surprisingly long and in many cases surpassed the duration of active breeding. Our results demonstrate the value of the ISIS database to estimate life history data for an at-risk taxon that is difficult to study in the wild, and provide life history data that is crucial for predictive modeling of future species endangerment and proactively managing captive populations of parrots.