Balancing Act: an Interdisciplinary Exploration of Trade-offs in Reproducing Females.

Trade-offs resulting from the high demand of offspring production are a central focus of many subdisciplines within the field of biology. Yet, despite the historical and current interest on this topic, large gaps in our understanding of whole-organism trade-offs that occur in reproducing individuals remain, particularly as it relates to the nuances associated with female reproduction. This volume of Integrative and Comparative Biology (ICB) contains a series of papers that focus on reviewing trade-offs from the female-centered perspective of biology (i.e., a perspective that places female reproductive biology at the center of the topic being investigated or discussed). These papers represent some of the work showcased during our symposium held at the 2024 meeting of the Society for Integrative and Comparative Biology (SICB) in Seattle, Washington. In this roundtable discussion, we use a question-and-answer format to capture the diverse perspectives and voices involved in our symposium. We hope that the dialogue featured in this discussion will be used to motivate researchers interested in understanding trade-offs in reproducing females and provide guidance on future research endeavors.

Let's talk about sex: instructor views and hesitancies related to sex and gender in the biology classroom.

There is much current debate in the US regarding how sex and gender are approached in science and medical classrooms. There does not seem to be sufficient consensus around why it must be taught and how it should be implemented. State-enacted restrictions to both education and healthcare in recent years demonstrate the relevance and importance of sex and gender in the college classroom, not only including but especially in the biology classroom. Given the areas comprising the Society for Integrative and Comparative Biology (SICB), these topics of sex and gender in biology instruction are incredibly salient to our members. Thus, this survey aimed to determine instructors' views of and experiences with sex-diverse gender-inclusive biology. College-level biology instructors who are members of SICB were surveyed about their views of science, views of sex and gender, teaching philosophy, and their experiences with inclusive teaching and with sex-diverse gender-inclusive teaching. The resulting data lead us to implore academic biology to provide more sex-diverse and gender-inclusive teaching tools and resources to educators, while minimizing potential fear of retaliation and backlash to instructors who utilize these teaching methods.

Incorporating Sex-Diverse and Gender-Inclusive Perspectives in Higher Education Biology Courses.

Inclusive teaching is teaching in a way that reaches all students in the classroom; this is beneficial for everyone and particularly for those with minoritized identities. Instructors play a critical role in scaffolding how students are exposed to and learn science content in the classroom. In this manuscript we discuss how biology instructors can make their classrooms more inclusive with regard to sex and gender diversity content. Many topics in biology are based on androcentric, heteronormative, and oppressive framing, even though those lenses are more reflective of our own history and culture than they are of the diversity we see in nature. Here, we summarize information presented in the SICB 2024 workshop titled "Incorporating sex diversity and gender inclusivity in biology undergraduate classrooms" and provide instructors with a) rationale for why inclusive teaching matters, b) guidance on how to challenge unscientific views and make their curricula more sex-diverse and gender inclusive, and c) practical and easy-to-implement strategies for discussing "contentious" topics in the classroom. Incorporation of this material will be beneficial for students, for science and medicine, and for accurately representing the diversity found across the tree of life.

The desert woodrat (Neotoma lepida) induces a diversity of biotransformation genes in response to creosote bush resin.

Liver biotransformation enzymes have long been thought to enable animals to feed on diets rich in xenobiotic compounds. However, despite decades of pharmacological research in humans and rodents, little is known about hepatic gene expression in specialized mammalian herbivores feeding on toxic diets. Leveraging a recently identified population of the desert woodrat (Neotoma lepida) found to be highly tolerant to toxic creosote bush (Larrea tridentata), we explored the expression changes of suites of biotransformation genes in response to diets enriched with varying amounts of creosote resin. Analysis of hepatic RNA-seq data indicated a dose-dependent response to these compounds, including the upregulation of several genes encoding transcription factors and numerous phase I, II, and III biotransformation families. Notably, elevated expression of five biotransformation families - carboxylesterases, cytochromes P450, aldo-keto reductases, epoxide hydrolases, and UDP-glucuronosyltransferases - corresponded to species-specific duplication events in the genome, suggesting that these genes play a prominent role in N. lepida's adaptation to creosote bush. Building on pharmaceutical studies in model rodents, we propose a hypothesis for how the differentially expressed genes are involved in the biotransformation of creosote xenobiotics. Our results provide some of the first details about how these processes likely operate in the liver of a specialized mammalian herbivore.

Science abhors a surveillance vacuum: Detection of ticks and tick-borne pathogens in southern New Mexico through passive surveillance.

Robust tick surveillance enhances diagnosis and prevention of tick-borne pathogens, yet surveillance efforts in the United States are highly uneven, resulting in large surveillance vacuums, one of which spans the state of New Mexico. As part of a larger effort to fill this vacuum, we conducted both active and passive tick sampling in New Mexico, focusing on the southern portion of the state. We conducted active tick sampling using dragging and CO2 trapping at 45 sites across Hidalgo, Doña Ana, Otero, and Eddy counties between June 2021 to May 2022. Sampling occurred intermittently, with at least one sampling event each month from June to October 2021, pausing in winter and resuming in March through May 2022. We also conducted opportunistic, passive tick sampling in 2021 and 2022 from animals harvested by hunters or captured or collected by researchers and animals housed in animal hospitals, shelters, and farms. All pools of ticks were screened for Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Ehrlichia ewingii, and Ehrlichia chaffeensis. Active sampling yielded no ticks. Passive sampling yielded 497 ticks comprising Carios kelleyi from pallid bats, Rhipicephalus sanguineus from dogs, mule deer, and Rocky Mountain elk, Otobius megnini from dogs, cats, horses, and Coues deer, Dermacentor parumapertus from dogs and black-tailed jackrabbits, Dermacentor albipictus from domesticated cats, mule deer and Rocky Mountain elk, and Dermacentor spp. from American black bear, Rocky Mountain elk, and mule deer. One pool of D. parumapterus from a black-tailed jackrabbit in Luna County tested positive for R. parkeri, an agent of spotted fever rickettsiosis. Additionally, a spotted fever group Rickettsia was detected in 6 of 7 C. kelleyi pools. Two ticks showed morphological abnormalities; however, these samples did not test positive for any of the target pathogens, and the cause of the abnormalities is unknown. Passive surveillance yielded five identified species of ticks from three domestic and six wild mammal species. Our findings update tick distributions and inform the public, medical, and veterinary communities of the potential tick-borne pathogens present in southern New Mexico.

Testing Morphological Relationships Between Female and Male Copulatory Structures in Bats.

The lower reproductive tract of female mammals has several competing functions including mating, tract health maintenance, and parturition. Diverse vaginal anatomy suggests interactions between natural and sexual selection, yet despite its importance, female copulatory morphology remains under-studied. We undertook a comparative study across the species-rich mammalian order Chiroptera (bats) with a focus on the suborder Yangochiroptera (Vespertilioniformes) to examine how female vaginal features may have coevolved with male penis morphology to minimize mechanical damage to their tissues during copulation. The penis morphology is diverse, presenting great potential for post-copulatory sexual selection and coevolution with the female morphology, but vaginas have not been carefully examined. Here we test the hypotheses that vaginal thickness and collagen density have coevolved with features of the male penis including the presence of spines and a baculum. We present histological data from females of 24 species from 7 families of bats, and corresponding data on male penis anatomy. We also examine the role of phylogenetic history in the morphological patterns we observe. We found evidence that female vaginal thickness has coevolved with the presence of penile spines, but not with baculum presence or width. Collagen density did not appear to covary with male penile features. Our findings highlight the importance of considering interactions between the sexes in influencing functional reproductive structures and examine how these structures have been under selection in bats.

Trio-binned genomes of the woodrats Neotoma bryanti and Neotoma lepida reveal novel gene islands and rapid copy number evolution of xenobiotic metabolizing genes.

The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third-generation sequencing and novel analytical toolsets. Applying these techniques to woodrats (Neotoma spp.) provides a unique opportunity to study how herbivores respond to environmental change. Neotoma bryanti and N. lepida independently achieved a major dietary feat in the aftermath of a natural climate change event: switching to the novel, toxic food source creosote bush (Larrea tridentata). To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti × N. lepida F1 hybrid, allowing the simultaneous assembly of genomes representing each parental species. The resulting phased, chromosome-level, highly complete haploid references enabled us to explore the genomic architecture of several gene families-cytochromes P450, UDP-glucuronosyltransferases (UGTs), and ATP-binding cassette (ABC) transporters-known to play key roles in the metabolism of naturally occurring toxic dietary compounds. In addition to duplication events in the ABCG and UGT2B subfamilies, we found expansions in three P450 gene families (2A, 2B, 3A), including the evolution of multiple novel gene islands within the 2B and 3A subfamilies, which may have provided the crucial substrate for dietary adaptation. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago.

Toxin tolerance across landscapes: Ecological exposure not a prerequisite.

Little is known about the tolerances of mammalian herbivores to plant specialized metabolites across landscapes.We investigated the tolerances of two species of herbivorous woodrats, Neotoma lepida (desert woodrat) and Neotoma bryanti (Bryant's woodrat) to creosote bush (Larrea tridentata), a widely distributed shrub with a highly toxic resin. Woodrats were sampled from 13 locations both with and without creosote bush across a 900 km transect in the US southwest. We tested whether these woodrat populations consume creosote bush using plant metabarcoding of feces and quantified their tolerance to creosote bush through feeding trials using chow amended with creosote resin.Toxin tolerance was analyzed in the context of population structure across collection sites with microsatellite analyses. Genetic differentiation among woodrats collected from different locations was minimal within either species. Tolerance differed substantially between the two species, with N. lepida persisting 20% longer than N. bryanti in feeding trials with creosote resin. Furthermore, in both species, tolerance to creosote resin was similar among woodrats near or within creosote bush habitat. In both species, woodrats collected greater than 25 km from creosote had markedly lower tolerances to creosote resin compared to animals from within the range of creosote bush.The results imply that mammalian herbivores are adapted to the specialized metabolites of plants in their diet, and that this tolerance can extend several kilometers outside of the range of dietary items. That is, direct ecological exposure to the specialized chemistry of particular plant species is not a prerequisite for tolerance to these compounds. These findings lay the groundwork for additional studies to investigate the genetic mechanisms underlying toxin tolerance and to identify how these mechanisms are maintained across landscape-level scales in mammalian herbivores.

Microbiome stability and structure is governed by host phylogeny over diet and geography in woodrats (Neotoma spp.).

The microbiome is critical for host survival and fitness, but gaps remain in our understanding of how this symbiotic community is structured. Despite evidence that related hosts often harbor similar bacterial communities, it is unclear whether this pattern is due to genetic similarities between hosts or to common ecological selection pressures. Here, using herbivorous rodents in the genus Neotoma, we quantify how geography, diet, and host genetics, alongside neutral processes, influence microbiome structure and stability under natural and captive conditions. Using bacterial and plant metabarcoding, we first characterized dietary and microbiome compositions for animals from 25 populations, representing seven species from 19 sites across the southwestern United States. We then brought wild animals into captivity, reducing the influence of environmental variation. In nature, geography, diet, and phylogeny collectively explained ∼50% of observed microbiome variation. Diet and microbiome diversity were correlated, with different toxin-enriched diets selecting for distinct microbial symbionts. Although diet and geography influenced natural microbiome structure, the effects of host phylogeny were stronger for both wild and captive animals. In captivity, gut microbiomes were altered; however, responses were species specific, indicating again that host genetic background is the most significant predictor of microbiome composition and stability. In captivity, diet effects declined and the effects of host genetic similarity increased. By bridging a critical divide between studies in wild and captive animals, this work underscores the extent to which genetics shape microbiome structure and stability in closely related hosts.

It Takes Two to Tango: Including a Female Perspective in Reproductive Biology.

Like many scientific disciplines, the field of reproductive biology is subject to biases in terminology and research foci. For example, females are often described as coy and passive players in reproductive behaviors and are termed "promiscuous" if they engage in extra-pair copulations. Males on the other hand are viewed as actively holding territories and fighting with other males. Males are termed "multiply mating" if they mate with multiple females. Similarly, textbooks often illustrate meiosis as it occurs in males but not females. This edition of Integrative and Comparative Biology (ICB) includes a series of papers that focus on reproduction from the female perspective. These papers represent a subset of the work presented in our symposium and complementary sessions on female reproductive biology. In this round table discussion, we use a question and answer format to leverage the diverse perspectives and voices involved with the symposium in an exploration of theoretical, cultural, pedagogical, and scientific issues related to the study of female biology. We hope this dialog will provide a stepping-stone toward moving reproductive science and teaching to a more inclusive and objective framework.

The Female Snark Is Still a Boojum: Looking toward the Future of Studying Female Reproductive Biology.

Philosophical truths are hidden in Lewis Carroll's nonsense poems, such as "The hunting of the snark." When the poem is used as a scientific allegory, a snark stands for the pursuit of scientific truth, while a boojum is a spurious discovery. In the study of female biology, boojums have been the result of the use of cultural stereotypes to frame hypotheses and methodologies. Although female reproduction is key for the continuation of sexually reproducing species, not only have females been understudied in many regards, but also data have commonly been interpreted in the context of now-outdated social mores. Spurious discoveries, boojums, are the result. In this article, we highlight specific gaps in our knowledge of female reproductive biology and provide a jumping-off point for future research. We discuss the promise of emerging methodologies (e.g., micro-CT scanning, high-throughput sequencing, proteomics, big-data analysis, CRISPR-Cas9, and viral vector technology) that can yield insights into previously cryptic processes and features. For example, in mice, deoxyribonucleic acid sequencing via chromatin immunoprecipitation followed by sequencing is already unveiling how epigenetics lead to sex differences in brain development. Similarly, new explorations, including microbiome research, are rapidly debunking dogmas such as the notion of the "sterile womb." Finally, we highlight how understanding female reproductive biology is well suited to the National Science Foundation's big idea, "Predicting Rules of Life." Studies of female reproductive biology will enable scholars to (1) traverse levels of biological organization from reproductive proteins at the molecular level, through anatomical details of the ovum and female reproductive tract, into physiological aspects of whole-organism performance, leading to behaviors associated with mating and maternal care, and eventually reaching population structure and ecology; (2) discover generalizable rules such as the co-evolution of maternal-offspring phenotypes in gestation and lactation; and (3) predict the impacts of changes to reproductive timing when the reliability of environmental cues becomes unpredictable. Studies in these key areas relative to female reproduction are sure to further our understanding across a range of diverse taxa.

Strategies in herbivory by mammals revisited: The role of liver metabolism in a juniper specialist (Neotoma stephensi) and a generalist (Neotoma albigula).

Although herbivory is widespread among mammals, few species have adopted a strategy of dietary specialization. Feeding on a single plant species often exposes herbivores to high doses of plant secondary metabolites (PSMs), which may exceed the animal's detoxification capacities. Theory predicts that specialists will have unique detoxification mechanisms to process high levels of dietary toxins. To evaluate this hypothesis, we compared liver microsomal metabolism of a juniper specialist, Neotoma stephensi (diet >85% juniper), to a generalist, N. albigula (diet ≤30% juniper). Specifically, we quantified the concentration of a key detoxification enzyme, cytochrome P450 2B (CYP2B) in liver microsomes, and the metabolism of α-pinene, the most abundant terpene in the juniper species consumed by the specialist woodrat. In both species, a 30% juniper diet increased the total CYP2B concentration (2-3×) in microsomes and microsomal α-pinene metabolism rates (4-fold). In N. stephensi, higher levels of dietary juniper (60% and 100%) further induced CYP2B and increased metabolism rates of α-pinene. Although no species-specific differences in metabolism rates were observed at 30% dietary juniper, total microsomal CYP2B concentration was 1.7× higher in N. stephensi than in N. albigula (p < .01), suggesting N. stephensi produces one or more variant of CYP2B that is less efficient at processing α-pinene. In N. stephensi, the rates of α-pinene metabolism increased with dietary juniper and were positively correlated with CYP2B concentration. The ability of N. stephensi to elevate CYP2B concentration and rate of α-pinene metabolism with increasing levels of juniper in the diet may facilitate juniper specialization in this species.

Natural diets promote retention of the native gut microbiota in captive rodents.

Wild animals entering captivity experience radical lifestyle changes resulting in microbiome alterations. However, little is known about the factors that drive microbial community shifts in captivity, and what actions could mitigate microbial changes. Using white-throated woodrats (Neotoma albigula), we tested whether offering natural diets in captivity facilitates retention of native microbial communities of captive animals. Wild-caught woodrats were brought to laboratory conditions. Woodrats received either a natural diet of Opuntia cactus or an artificial diet of commercial chow over three weeks. Microbial inventories from woodrat feces at the time of capture and in captivity were generated using Illumina 16S rRNA sequencing. We found that providing woodrats with wild-natural diets significantly mitigated alterations in their microbiota, promoting a 90% retention of native microbial communities across the experiment. In contrast, the artificial diet significantly impacted microbial structure to the extent that 38% of the natural microflora was lost. Core bacteria including Bifidobacterium and Allobaculum were lost, and abundances of microbes related to oxalate degradation decreased in individuals fed artificial but not natural diets. These results highlight the importance of supplementing captive diets with natural foods to maintain native microbiomes of animals kept in artificial conditions for scientific or conservation purposes.

Metagenomic sequencing provides insights into microbial detoxification in the guts of small mammalian herbivores (Neotoma spp.).

Microbial detoxification of plant toxins influences the use of plants as food sources by herbivores. Stephen's woodrats (Neotoma stephensi) specialize on juniper, which is defended by oxalate, phenolics and monoterpenes, while closely related N. albigula specialize on cactus, which only contains oxalate. Woodrats maintain two gut chambers harboring dense microbial communities: a foregut chamber proximal to the major site of toxin absorption, and a cecal chamber in their hindgut. We performed several experiments to investigate the location and nature of microbial detoxification in the woodrat gut. First, we measured toxin concentrations across gut chambers of N. stephensi. Compared to food material, oxalate concentrations were immediately lower in the foregut, while concentrations of terpenes remained high in the foregut, and were lowest in the cecal chamber. We conducted metagenomic sequencing of the foregut chambers of both woodrat species and cecal chambers of N. stephensi to compare microbial functions. We found that most genes associated with detoxification were more abundant in the cecal chambers of N. stephensi. However, some genes associated with degradation of oxalate and phenolic compounds were more abundant in the foregut chambers. Thus, microbial detoxification may take place in various chambers depending on the class of chemical compound.

Role of cytochrome P450 2B sequence variation and gene copy number in facilitating dietary specialization in mammalian herbivores.

Theory postulates that dietary specialization in mammalian herbivores is enabled by a specialized set of liver enzymes that process the high concentrations of similar plant secondary metabolites (PSMs) in the diets of specialists. To investigate whether qualitative and quantitative differences in detoxification mechanisms distinguish dietary specialists from generalists, we compared the sequence diversity and gene copy number of detoxification enzymes in two woodrat species: a generalist, the white-throated woodrat (Neotoma albigula) and a juniper specialist, Stephens' woodrat (N. stephensi). We focused on enzymes in the cytochrome P450 subfamily 2B (CYP2B), because previous research suggests this subfamily plays a key role in the processing of PSMs. For both woodrat species, we obtained and sequenced CYP2B cDNA, generated CYP2B phylogenies, estimated CYP2B gene copy number and created a homology model of the active site. We found that the specialist possessed on average ~5 more CYP2B gene copies than the generalist, but the specialist's CYP2B sequences were less diverse. Phylogenetic analysis of putative CYP2B homologs resolved woodrat species as reciprocally monophyletic and suggested evolutionary convergence of distinct homologs on similar key amino acid residues in both species. Homology modelling of the CYP2B enzyme suggests that interspecific differences in substrate preference and function likely result from amino acid differences in the enzyme active site. The characteristics of CYP2B in the specialist, that is greater gene copy number coupled with less sequence variation, are consistent with specialization to a narrow range of dietary toxins.

All Features Great and Small-the Potential Roles of the Baculum and Penile Spines in Mammals.

Mammalian penises are morphologically diverse, including a highly variable and taxonomically informative baculum (os penis), and variable penile spines, both of which are possessed by many-but not all-species. To understand the evolution of genital morphologies, as well as the potential role of both the baculum, and penile spines that directly interact with female reproductive tract, we undertook a comparative study of male penile spines and their relationship with the baculum across all mammalian orders. Specifically, we investigated several factors that may explain the presence or absence of penile spines in mammals, including mating system, risk of sperm competition, female reproductive physiology, presence and width of the baculum, and phylogenetic history. We observed that the presence of both the baculum and penile spines is correlated with residual testes size, suggesting a potential role in sexual selection for these traits. We found no association between the presence of spines and mating system, or with the presence/width of the baculum, although relative testes mass was marginally associated with baculum width. We found no relationship between baculum presence or width and mating system. We also noted that spines presence or absence have an order-level distribution, and clear phylogenetic patterns of distribution across mammals. It is likely that spine morphology and distribution, not just presence, play an important role in genital evolution in mammals, but these features are poorly described in most groups. Quantitative data collection in most mammalian taxa would be useful to further elucidate the evolution of the complex genital morphology of this group.

Reproductive mode and the shifting arenas of evolutionary conflict.

In sexually reproducing organisms, the genetic interests of individuals are not perfectly aligned. Conflicts among family members are prevalent since interactions involve the transfer of limited resources between interdependent players. Intrafamilial conflict has traditionally been considered along three major axes: between the sexes, between parents and offspring, and between siblings. In these interactions, conflict is expected over traits in which the resulting phenotypic value is determined by multiple family members who have only partially overlapping fitness optima. We focus on four major categories of animal reproductive mode (broadcast spawning, egg laying, live bearing, and live bearing with matrotrophy) and identify the shared phenotypes or traits over which conflict is expected, and then review the empirical literature for evidence of their occurrence. Major transitions among reproductive mode, such as a shift from external to internal fertilization, an increase in egg-retention time, modifications of embryos and mothers for nutrient transfer, the evolution of postnatal parental care, and increased interaction with the kin network, mark key shifts that both change and expand the arenas in which conflict is played out.

Sperm storage: distinguishing selective processes and evaluating criteria.

Sperm storage, the extended maintenance of viable sperm, probably occurs in most internally fertilizing animals. Because it temporally separates mating from conception, sperm storage can be adaptive in ecologically diverse habitats and affect life histories, mating systems, cryptic female choice, sperm competition, and sexual conflict. Sperm storage can result from different selective forces acting on females and/or males, sometimes resulting in coevolution. The various criteria often used to determine the presence of sperm storage in any given taxon can result from the action of any one or all of these selective forces. Here we discuss the criteria used to study sperm storage and how we can use these to better understand the evolution of diversity in sperm-storage adaptations.

Cacti supply limited nutrients to a desert rodent community.

In the Sonoran Desert, cacti represent a potentially important source of nutrients and water for consumers. Columnar cacti, in particular, produce a large pulse of flowers and succulent fruit during hot summer months. The importance of cactus stems, flowers and fruit to the small mammal community has not been quantified. We exploited natural variation in the carbon isotope (δ(13)C) values of cacti (CAM) versus C3 plants to quantify the relative use of these resources by a diverse desert small mammal community. We also estimated trophic level by measuring nitrogen isotope (δ(15)N) values. We hypothesized that (H1) granivorous heteromyids (kangaroo rats, pocket mice) would exploit the summer pulse of seeds and pulp; (H2) folivorous and omnivorous cactus mice, wood rats, and ground squirrels would exploit cacti stems year-round and seeds when available; and (H3) kangaroo rats and pocket mice would shift from seeds to insects during hot dry months. We found that heteromyids made minimal use of seeds during the period of heavy seed rain. Of the cricetids, only the folivore Neotoma albigula made continuous but highly variable use of cacti resources (annual mean = 32%, range 0-81%), whereas the omnivore Peromyscus eremicus ignored cacti except during the summer, when it exploited seeds and/or fruit pulp (June-July mean = 39%, range 20-64%). We also found little evidence for a shift to greater consumption of insects by heteromyids during the hot dry months. Overall, use of cactus resources by the small mammal community is very limited and highly variable among species.