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Bats (order Chiroptera) are hosts for highly virulent zoonotic pathogens. Many bats demonstrate seasonally varying antiviral responses, including antibody responses which have been observed to peak during the nutritionally depleted dry-season and female gestation periods, suggesting some impact of resource deficits on bat virus immunity. Given the frequent overlap in these energetically demanding periods, it is likely that endocrinological changes associated with pregnancy might partially explain the aforementioned pattern in antibody dynamics. Regardless, we know little about the seasonality of reproduction in many fruit bat species, despite the importance of reproductive biology to informing conservation management (e.g. population viability) and disease dynamics. Here, we aimed to elucidate the reproductive biology of three species of endemic fruit bat native to the island of Madagascar: Pteropus rufus, Eidolon dupreanum, and Rousettus madagascarensis. To do so, we leveraged plasma samples collected in part with a longitudinal field study, from 2018 to 2020. We adapted three standard reproductive assays previously validated in humans to quantify seasonal changes in reproductive hormones for bats and applied a mixture model approach to determine hormone cutoffs for pregnancy. As expected, we found that pregnant females showed the highest levels of estradiol and progesterone and adult males the highest levels of testosterone. Additionally, female P. rufus and R. madagascariensis showed clear seasonality in reproduction with peaks in estradiol and progesterone in August and October, respectively. Seasonality was less clearly discernible in the female E. dupreanum and male data. In general, we found that the commercially available assays were successful in quantifying endocrinological hormones for bats; when paired with histological embryo sections or field data, these offer a powerful tool to elucidate bat reproductive calendars.

Flying birds and bats have simplified gastrointestinal tracts (GITs) and low intestinal mass to facilitate flight. Previous work showed reduced GIT transit times in birds relative to other vertebrates-but GIT transit has never been collectively quantified for bats. Unique among mammals, bat GIT microbiomes are dominated by Pseudomonadota bacteria (previously Proteobacteria), which also dominate the microbiomes of flying birds - we hypothesized this convergence to result from rapid GIT transit times for both volant taxa. We conducted a meta-analysis of vertebrate GIT transit times which showed that bats and flying birds have significantly faster transit times relative to nonvolant vertebrates. Additionally, within the bat order (Chiroptera), we demonstrated decreasing transit times associated with increasing body mass, a pattern contrasting other vertebrates (including volant birds) and possibly influencing GIT microbiome composition. This inverted mass-transit association is likely driven by diet as fruit- and nectar-consuming Pteropodids are the largest of all bats.

Empirical data relating body mass to immune defence against infections remain limited. Although the metabolic theory of ecology predicts that larger organisms would have weaker immune responses, recent studies have suggested that the opposite may be true. These discoveries have led to the safety factor hypothesis, which proposes that larger organisms have evolved stronger immune defences because they carry greater risks of exposure to pathogens and parasites. In this study, we simulated sepsis by exposing blood from nine primate species to a bacterial lipopolysaccharide (LPS), measured the relative expression of immune and other genes using RNAseq, and fitted phylogenetic models to determine how gene expression was related to body mass. In contrast to non-immune-annotated genes, we discovered hypermetric scaling in the LPS-induced expression of innate immune genes, such that large primates had a disproportionately greater increase in gene expression of immune genes compared to small primates. Hypermetric immune gene expression appears to support the safety factor hypothesis, though this pattern may represent a balanced evolutionary mechanism to compensate for lower per-transcript immunological effectiveness. This study contributes to the growing body of immune allometry research, highlighting its importance in understanding the complex interplay between body size and immunity over evolutionary timescales.

Aquatic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) formulations, are commonly used for invasive species management throughout the United States. Ecologically relevant concentrations of 2,4-D can impair essential behaviors, reduce survival, and act as an endocrine disruptor; however, there is limited knowledge of its effects on the health of non-target organisms. Here, we investigate the acute and chronic exposure impacts of 2,4-D on adult male and female fathead minnow (Pimephales promelas) innate immune function. We exposed both adult male and female fathead minnows to three different ecologically relevant concentrations of 2,4-D (0.00, 0.40, and 4.00 mg/L) and took blood samples at three acute time points (6, 24, and 96 h) and one chronic time point (30 days). We found that male fatheads had higher total white blood cell concentrations when exposed to 2,4-D at the acute time points. For the females, only proportions of specific cell types were altered when exposed to 2,4-D at the acute time points. However, we did not observe any significant impacts of chronic exposure to 2,4-D on any innate immune responses for either males or females. Overall, this study is the first step in answering an important question for game fisheries and management agencies while providing insight to future studies that investigate the impacts of herbicide exposure to freshwater fish health and immunity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Powered flight has evolved several times in vertebrates and constrains morphology and physiology in ways that likely have shaped how organisms cope with infections. Some of these constraints probably have impacts on aspects of immunology, such that larger fliers might prioritize risk reduction and safety. Addressing how the evolution of flight may have driven relationships between body size and immunity could be particularly informative for understanding the propensity of some taxa to harbor many virulent and sometimes zoonotic pathogens without showing clinical disease. Here, we used a comparative framework to quantify scaling relationships between body mass and the proportions of two types of white blood cells - lymphocytes and granulocytes (neutrophils/heterophils) - across 63 bat species, 400 bird species and 251 non-volant mammal species. By using phylogenetically informed statistical models on field-collected data from wild Neotropical bats and from captive bats, non-volant mammals and birds, we show that lymphocyte and neutrophil proportions do not vary systematically with body mass among bats. In contrast, larger birds and non-volant mammals have disproportionately higher granulocyte proportions than expected for their body size. Our inability to distinguish bat lymphocyte scaling from birds and bat granulocyte scaling from all other taxa suggests there may be other ecological explanations (i.e. not flight related) for the cell proportion scaling patterns. Future comparative studies of wild bats, birds and non-volant mammals of similar body mass should aim to further differentiate evolutionary effects and other aspects of life history on immune defense and its role in the tolerance of (zoonotic) infections., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Phenotypic flexibility has received considerable attention in the last decade; however, whereas many studies have reported amplitude of variation in phenotypic traits, much less attention has focused on the rate at which traits can adjust in response to sudden changes in the environment. We investigated whole animal and muscle phenotypic changes occurring in black-capped chickadees ( Poecile atricapillus ) acclimated to cold (-5°C) and warm (20°C) temperatures in the first 3 h following a 15°C temperature drop (over 3 h). Before the temperature change, cold-acclimated birds were consuming 95% more food, were carrying twice as much body fat, and had 23% larger pectoralis muscle fiber diameters than individuals kept at 20°C. In the 3 h following the temperature drop, these same birds altered their pectoralis muscle ultrastructure by increasing the number of capillaries per fiber area and the number of nuclei per millimeter of fiber by 22%, consequently leading to a 22% decrease in myonuclear domain (amount of cytoplasm serviced per nucleus), whereas no such changes were observed in the warm-acclimated birds. To our knowledge, this is the first demonstration of such a rapid adjustment in muscle fiber ultrastructure in vertebrates. These results support the hypothesis that chickadees maintaining a cold phenotype are better prepared than warm-phenotype individuals to respond to a sudden decline in temperature, such as what may be experienced in their natural wintering environment.

Food availability might sometimes be unpredictable for wild birds. To alleviate this possible food limitation, millions of households in North America provide food supplementation to bird populations. However, the ecoimmunological impacts of this supplementation on free-living birds are largely unclear. Therefore, we compared immune function and body composition of three groups of free-living black-capped chickadees (Poecile atricapillus) that were provided either constant food supplementation ("supplemented"), interrupted food supplementation ("interrupted"), or no food supplementation ("unsupplemented"). At capture, all three groups had similar body mass and fat scores. All three groups also had similar levels of circulating immunoglobulin Y antibodies and complement lysis ability, two measures of constitutive immune function. Supplemented and interrupted groups mounted a somewhat similar body mass and temperature response to injection with lipopolysaccharide; however, the supplemented group had a higher haptoglobin (acute-phase protein) response to lipopolysaccharide injection compared to the interrupted group. This study demonstrates that birds maintained similar levels of fat despite their level of food supplementation; however, sudden removal of supplemental food might elicit a short-term decline in aspects of immunity. Future studies should investigate how food supplementation might impact induced or adaptive aspects of immune function to add to our understanding of immunology in free-living animals.