In vitro production of naked mole-rats' blastocysts from non-breeding females using in vitro maturation and intracytoplasmic sperm injection.

The African naked mole-rat (Heterocephalus glaber) is an attractive model for cancer and aging research due to its peculiar biological traits, such as unusual long life span and resistance to cancer. The establishment of induced pluripotent stem cells (iPSCs) would be a useful tool for in vitro studies but, in this species, the reprogramming of somatic cells is problematic because of their stable epigenome. Therefore, an alternative approach is the derivation of embryonic stem cells from in vitro-produced embryos. In this study, immature oocytes, opportunistically retrieved from sexually inactive females, underwent first in vitro maturation (IVM) and then in vitro fertilization via piezo-intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultivated with two different approaches: (i) in an in vitro culture and (ii) in an isolated mouse oviduct organ culture system. The second approach led to the development of blastocysts, which were fixed and stained for further analysis.

Mating without intromission in a bat.

Copulatory behaviours stand as cornerstones of sexual selection, yet they remain mysterious in many species. Because of their nocturnal and elusive lifestyle, the copulatory behaviours of bats have been mostly overlooked1. Several aspects of bat reproduction differ from other mammals (e.g. prolonged sperm storage2, delayed development3). Here, we show that in serotine bats (Eptesicus serotinus) the penis is used as a 'copulatory arm' rather than an intromittent organ, revealing a novel copulatory behaviour in mammals.

In vitro fertilization program in white rhinoceros.

In brief: To save endangered rhinoceros species, assisted reproductive technologies are warranted. We here report in vitro blastocyst generation of the Near-Threatened Southern white rhinoceros and, for the first time, also of the technically Extinct Northern white rhinoceros. Abstract: The Anthropocene is marked by a dramatic biodiversity decline, particularly affecting the family Rhinocerotidae. Three of five extant species are listed as Critically Endangered (Sumatran, Javan, black rhinoceros), one as Vulnerable (Indian rhinoceros), and only one white rhino (WR) subspecies, the Southern white rhinoceros (SWR), after more than a century of successful protection is currently classified as Near Threatened by the IUCN, while numbers again are declining. Conversely, in 2008, the SWR's northern counterpart and second WR subspecies, the Northern white rhinoceros (NWR), was declared extinct in the wild. Safeguarding these vanishing keystone species urgently requires new reproductive strategies. We here assess one such strategy, the novel in vitro fertilization program in SWR and - for the first-time NWR - regarding health effects, donor-related, and procedural factors. Over the past 8 years, we performed 65 procedures in 22 white rhinoceros females (20 SWR and 2 NWR) comprising hormonal ovarian stimulation, ovum pick-up (OPU), in vitro oocyte maturation, fertilization, embryo culture, and blastocyst cryopreservation, at an efficiency of 1.0 ± 1.3 blastocysts per OPU, generating 22 NWR, 19 SWR and 10 SWR/NWR hybrid blastocysts for the future generation of live offspring.

The neonatal southern white rhinoceros ovary contains oogonia in germ cell nests.

The northern white rhinoceros is functionally extinct with only two females left. Establishing methods to culture ovarian tissues, follicles, and oocytes to generate eggs will support conservation efforts using in vitro embryo production. To the best of our knowledge, this is the first description of the structure and molecular signature of any rhinoceros, more specifically, we describe the neonatal and adult southern white rhinoceros (Ceratotherium simum simum) ovary; the closest relation of the northern white rhinoceros. Interestingly, all ovaries contain follicles despite advanced age. Analysis of the neonate reveals a population of cells molecularly characterised as mitotically active, pluripotent with germ cell properties. These results indicate that unusually, the neonatal ovary still contains oogonia in germ cell nests at birth, providing an opportunity for fertility preservation. Therefore, utilising ovaries from stillborn and adult rhinoceros can provide cells for advanced assisted reproductive technologies and investigating the neonatal ovaries of other endangered species is crucial for conservation.

Mammalian maxilloturbinal evolution does not reflect thermal biology.

The evolution of endothermy in vertebrates is a major research topic in recent decades that has been tackled by a myriad of research disciplines including paleontology, anatomy, physiology, evolutionary and developmental biology. The ability of most mammals to maintain a relatively constant and high body temperature is considered a key adaptation, enabling them to successfully colonize new habitats and harsh environments. It has been proposed that in mammals the anterior nasal cavity, which houses the maxilloturbinal, plays a pivotal role in body temperature maintenance, via a bony system supporting an epithelium involved in heat and moisture conservation. The presence and the relative size of the maxilloturbinal has been proposed to reflect the endothermic conditions and basal metabolic rate in extinct vertebrates. We show that there is no evidence to relate the origin of endothermy and the development of some turbinal bones by using a comprehensive dataset of µCT-derived maxilloturbinals spanning most mammalian orders. Indeed, we demonstrate that neither corrected basal metabolic rate nor body temperature significantly correlate with the relative surface area of the maxilloturbinal. Instead, we identify important variations in the relative surface area, morpho-anatomy, and complexity of the maxilloturbinal across the mammalian phylogeny and species ecology.

The functional anatomy of elephant trunk whiskers.

Behavior and innervation suggest a high tactile sensitivity of elephant trunks. To clarify the tactile trunk periphery we studied whiskers with the following findings. Whisker density is high at the trunk tip and African savanna elephants have more trunk tip whiskers than Asian elephants. Adult elephants show striking lateralized whisker abrasion caused by lateralized trunk behavior. Elephant whiskers are thick and show little tapering. Whisker follicles are large, lack a ring sinus and their organization varies across the trunk. Follicles are innervated by ~90 axons from multiple nerves. Because elephants don't whisk, trunk movements determine whisker contacts. Whisker-arrays on the ventral trunk-ridge contact objects balanced on the ventral trunk. Trunk whiskers differ from the mobile, thin and tapered facial whiskers that sample peri-rostrum space symmetrically in many mammals. We suggest their distinctive features-being thick, non-tapered, lateralized and arranged in specific high-density arrays-evolved along with the manipulative capacities of the trunk.

Macrophages from naked mole-rat possess distinct immunometabolic signatures upon polarization.

The naked mole-rat (NMR) is a unique long-lived rodent which is highly resistant to age-associated disorders and cancer. The immune system of NMR possesses a distinct cellular composition with the prevalence of myeloid cells. Thus, the detailed phenotypical and functional assessment of NMR myeloid cell compartment may uncover novel mechanisms of immunoregulation and healthy aging. In this study gene expression signatures, reactive nitrogen species and cytokine production, as well as metabolic activity of classically (M1) and alternatively (M2) activated NMR bone marrow-derived macrophages (BMDM) were examined. Polarization of NMR macrophages under pro-inflammatory conditions led to expected M1 phenotype characterized by increased pro-inflammatory gene expression, cytokine production and aerobic glycolysis, but paralleled by reduced production of nitric oxide (NO). Under systemic LPS-induced inflammatory conditions NO production also was not detected in NMR blood monocytes. Altogether, our results indicate that NMR macrophages are capable of transcriptional and metabolic reprogramming under polarizing stimuli, however, NMR M1 possesses species-specific signatures as compared to murine M1, implicating distinct adaptations in NMR immune system.

Comparative analysis of thyroid hormone systems in rodents with subterranean lifestyle.

African mole-rats are subterranean rodents inhabiting underground burrows. This habitat entails risks of overheating, hypoxia, and scarce food availability. Consequently, many subterranean species have evolved low basal metabolism and low body temperature, but the regulation of these traits at the molecular level were unknown. Measurements of serum thyroid hormone (TH) concentrations in African mole-rats have revealed a unique TH phenotype, which deviates from the typical mammalian pattern. Since THs are major regulators of metabolic rate and body temperature, we further characterised the TH system of two African mole-rat species, the naked mole-rat (Heterocephalus glaber) and the Ansell's mole-rat (Fukomys anselli) at the molecular level in a comparative approach involving the house mouse (Mus musculus) as a well-studied laboratory model in TH research. Most intriguingly, both mole-rat species had low iodide levels in the thyroid and naked mole-rats showed signs of thyroid gland hyperplasia. However, contrary to expectations, we found several species-specific differences in the TH systems of both mole-rat species, although ultimately resulting in similar serum TH concentrations. These findings indicate a possible convergent adaptation. Thus, our study adds to our knowledge for understanding adaptations to the subterranean habitat.

Pup Recruitment in a Eusocial Mammal-Which Factors Influence Early Pup Survival in Naked Mole-Rats?

In eusocial insects, offspring survival strongly depends on the quality and quantity of non-breeders. In contrast, the influence of social factors on offspring survival is more variable in cooperatively breeding mammals since maternal traits also play an important role. This difference between cooperative insects and mammals is generally attributed to the difference in the level of sociality. Examining offspring survival in eusocial mammals should, therefore, clarify to what extent social organization and taxonomic differences determine the relative contribution of non-breeders and maternal effects to offspring survival. Here, we present the first in-depth and long-term study on the influence of individual, maternal, social and environmental characteristics on early offspring survival in a eusocial breeding mammal, the naked mole-rat (Heterocephalus glaber). Similarly to other mammals, pup birth mass and maternal characteristics such as body mass and the number of mammae significantly affected early pup survival. In this eusocial species, the number of non-breeders had a significant influence on early pup survival, but this influence was negative-potentially an artifact of captivity. By contrasting our findings with known determinants of survival in eusocial insects we contribute to a better understanding of the origin and maintenance of eusociality in mammals.

Application of decision tools to ethical analysis in biodiversity conservation.

Achieving ethically responsible decisions is crucial for the success of biodiversity conservation projects. We adapted the ethical matrix, decision tree, and Bateson's cube to assist in the ethical analysis of complex conservation scenarios by structuring these tools so that they can implement the different value dimensions (environmental, social, and animal welfare) involved in conservation ethics. We then applied them to a case study relative to the decision-making process regarding whether or not to continue collecting biomaterial on the oldest of the two remaining northern white rhinoceroses (Ceratotherium simum cottoni), a functionally extinct subspecies of the white rhinoceros. We used the ethical matrix to gather ethical pros and cons and as a starting point for a participatory approach to ethical decision-making. We used decision trees to compare the different options at stake on the basis of a set of ethical desiderata. We used Bateson's cube to establish a threshold of ethical acceptability and model the results of a simple survey. The application of these tools proved to be pivotal in structuring the decision-making process and in helping reach a shared, reasoned, and transparent decision on the best option from an ethical point of view among those available.

Que se logren decisiones éticamente responsables es crucial para el éxito de los proyectos de conservación de la biodiversidad. Adaptamos la matriz ética, el árbol de decisión y el cubo de Bateson para apoyar con el análisis ético de escenarios de conservación compleja mediante la estructuración de estas herramientas de tal manera que puedan ejecutar las diferentes dimensiones de valor (ambiental, social y bienestar animal) involucradas en la ética de la conservación. Después aplicamos las herramientas a un estudio de caso relacionado con el proceso de toma de decisiones respecto a si se debe seguir o no recolectando material biológico del rinoceronte blanco del norte (Ceratotherium simum cottoni) más viejo (una subespecie funcionalmente extinta) de los dos que existen. Usamos la matriz ética como un punto de partida para una estrategia participativa para la toma ética de decisiones y para recopilar los pros y contras éticos. Usamos el árbol de decisión para comparar las diferentes opciones en juego con base en un conjunto de deseos éticos. Usamos el cubo de Bateson para establecer un umbral de aceptación ética y modelar los resultados de una encuesta simple. La aplicación de estas herramientas demostró ser central en la estructuración del proceso de toma de decisiones y en el apoyo para lograr una decisión compartida, razonada y transparente sobre la mejor opción a partir de un punto de vista ético entre aquellos disponibles.

Robust induction of primordial germ cells of white rhinoceros on the brink of extinction.

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

Induced pluripotent stem cells and cerebral organoids from the critically endangered Sumatran rhinoceros.

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting the rescue of this critically endangered species.

The role of colour patterns for the recognition of flowers by bees.

Bees discriminate between many different colours of flower petals, but it is not well understood how they perceive and learn patterns frequently found in flowers with colourful structures. We used multi-spectral imaging to explore chromatic cues in concentric flower patterns as they are seen through the low-resolution eyes of the honeybee. We find a diversity of colour combinations, which suggests that plants might exploit the sensory capabilities of pollinators, like bees, that learn colours easily. A consistent feature is that the surround of the pattern has a stronger chromatic contrast to the foliage background than the centre. This can potentially facilitate the fast identification of floral objects within colourful scenes when a foraging bee moves through a flower patch. In behavioural experiments we trained and tested bees with three types of concentric patterns. They recognized and discriminated patterns accurately in most tests, relying flexibly on both chromatic and spatial cues. Only rarely, depending on the training stimulus, chromatic cues determined their choices whilst pattern cues were ignored. The variability of floral designs and the bees' flexibility in recalling colour and spatial information suggest a role for colour vision in pattern processing. Implications for the signalling strategies of flowers are discussed. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Unique Features of the Tissue Structure in the Naked Mole Rat (Heterocephalus glaber): Hypertrophy of the Endoplasmic Reticulum and Spatial Mitochondrial Rearrangements in Hepatocytes.

The reason for the exceptional longevity of the naked mole rat (Heterocephalus glaber) remains a mystery to researchers. We assumed that evolutionarily, H. glaber acquired the ability to quickly stabilize the functioning of mitochondria and endoplasmic reticulum (ER) to adjust metabolism to external challenges. To test this, a comparison of the hepatic mitochondria and ER of H. glaber and C57BL/6 mice was done. Electron microscopy showed that 2-months-old mice have more developed rough ER (RER) than smooth ER (SER), occupying ~17 and 2.5% of the hepatocytic area correspondingly, and these values do not change with age. On the other hand, in 1-week-old H. glaber, RER occupies only 13% constantly decreasing with age, while SER occupies 35% in a 1-week-old animal, constantly rising with age. The different localization of mitochondria in H. glaber and mouse hepatocytes was confirmed by confocal and electron microscopy: while in H. glaber, mitochondria were mainly clustered around the nucleus and on the periphery of the cell, in mouse hepatocytes they were evenly distributed throughout the cell. We suggest that the noted structural and spatial features of ER and mitochondria in H. glaber reflect adaptive rearrangements aimed at greater tolerance of the cellular system to challenges, primarily hypoxia and endogenous and exogenous toxins. Different mechanisms of adaptive changes including an activated hepatic detoxification system as a hormetic response, are discussed considering the specific metabolic features of the naked mole rat.

Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures.

Originally applied on domestic and lab animals, assisted reproduction technologies (ARTs) have also found application in conservation breeding programs, where they can make the genetic management of populations more efficient, and increase the number of individuals per generation. However, their application in wildlife conservation opens up new ethical scenarios that have not yet been fully explored. This study presents a frame for the ethical analysis of the application of ART procedures in conservation based on the Ethical Matrix (EM), and discusses a specific case study-ovum pick-up (OPU) procedures performed in the current conservation efforts for the northern white rhinoceros (Ceratotherium simum cottoni)-providing a template for the assessment of ART procedures in projects involving other endangered species.

Global response of conservationists across mass media likely constrained bat persecution due to COVID-19.

Most people lack direct experience with wildlife and form their risk perception primarily on information provided by the media. The way the media frames news may substantially shape public risk perception, promoting or discouraging public tolerance towards wildlife. At the onset of the COVID-19 pandemic, bats were suggested as the most plausible reservoir of the virus, and this became a recurrent topic in media reports, potentially strengthening a negative view of this ecologically important group. We investigated how media framed bats and bat-associated diseases before and during the COVID-19 pandemic by assessing the content of 2651 online reports published across 26 countries, to understand how and how quickly worldwide media may have affected the perception of bats. We show that the overabundance of poorly contextualized reports on bat-associated diseases likely increased the persecution towards bats immediately after the COVID-19 outbreak. However, the subsequent interventions of different conservation communication initiatives allowed pro-conservation messages to resonate across the global media, likely stemming an increase in bat persecution. Our results highlight the modus operandi of the global media regarding topical biodiversity issues, which has broad implications for species conservation. Knowing how the media acts is pivotal for anticipating the propagation of (mis)information and negative feelings towards wildlife. Working together with journalists by engaging in dialogue and exchanging experiences should be central in future conservation management.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Trigeminal ganglion and sensory nerves suggest tactile specialization of elephants.

Sensory nerves are information bottlenecks giving rise to distinct sensory worlds across animal species.1 Here, we investigate trigeminal ganglion2,3 and sensory nerves4 of elephants. The elephant trigeminal ganglion is very large. Its maxillary branch, which gives rise to the infraorbital nerve innervating the trunk, has a larger diameter than the animal's spinal cord, i.e., trunk innervation is more substantive than connections of the brain to the rest of the body. Hundreds of satellite cells surround each trigeminal neuron, an indication of exceptional glial support to these large projection neurons.5-7 Fiber counts of Asian elephant infraorbital nerves of averaged 4,00,000 axons. The infraorbital nerve consists of axons that are ∼10 µm thick and it has a large diameter of 17 mm, roughly 3 times as thick as the optic and 6 times as thick as the vestibulocochlear nerve. In most mammals (including tactile specialists) optic nerve fibers8-10 greatly outnumber infraorbital nerve fibers,11,12 but in elephants the infraorbital nerve fiber count is only slightly lower than the optic nerve fiber count. Trunk innervation (nerves and ganglia) weighs ∼1.5 kg in elephant cows. Our findings characterize the elephant trigeminal ganglion as one of the largest known primary sensory structures and point to a high degree of tactile specialization in elephants.

Progressive reorganization of mitochondrial apparatus in aging skeletal muscle of naked mole rats (Heterocephalus glaber) as revealed by electron microscopy: potential role in continual maintenance of muscle activity.

The authors examined the ultrastructure of mitochondrial apparatus of skeletal muscles of naked mole rats (Heterocephalus glaber) from the age of 6 months to 11 years. The obtained results have demonstrated that the mitochondria in skeletal muscles of naked mole rats aged below 5 years is not well-developed and represented by few separate small mitochondria. Mitochondrial reticulum is absent. Starting from the age of 5 years, a powerful mitochondrial structure is developed. By the age of 11 years, it become obvious that the mitochondrial apparatus formed differs from that in the skeletal muscle of adult rats and mice, but resembles that of cardiomyocytes of rats or naked mole rats cardiomyocytes. From the age of 6 months to 11 years, percentage area of mitochondria in the skeletal muscle of naked mole rat is increasing by five times. The growth of mitochondria is mainly driven by increased number of organelles. Such significant growth of mitochondria is not associated with any abnormal changes in mitochondrial ultrastructure. We suppose that specific structure of mitochondrial apparatus developed in the skeletal muscle of naked mole rats by the age of 11 years is necessary for continual skeletal muscle activity of these small mammals burrowing very long holes in stony earth, resembling continual activity of heart muscle. In any case, ontogenesis of naked mole rat skeletal muscles is much slower than of rats and mice (one more example of neoteny).

Alternative Animal Models of Aging Research.

Most research on mechanisms of aging is being conducted in a very limited number of classical model species, i.e., laboratory mouse (Mus musculus), rat (Rattus norvegicus domestica), the common fruit fly (Drosophila melanogaster) and roundworm (Caenorhabditis elegans). The obvious advantages of using these models are access to resources such as strains with known genetic properties, high-quality genomic and transcriptomic sequencing data, versatile experimental manipulation capabilities including well-established genome editing tools, as well as extensive experience in husbandry. However, this approach may introduce interpretation biases due to the specific characteristics of the investigated species, which may lead to inappropriate, or even false, generalization. For example, it is still unclear to what extent knowledge of aging mechanisms gained in short-lived model organisms is transferable to long-lived species such as humans. In addition, other specific adaptations favoring a long and healthy life from the immense evolutionary toolbox may be entirely missed. In this review, we summarize the specific characteristics of emerging animal models that have attracted the attention of gerontologists, we provide an overview of the available data and resources related to these models, and we summarize important insights gained from them in recent years. The models presented include short-lived ones such as killifish (Nothobranchius furzeri), long-lived ones such as primates (Callithrix jacchus, Cebus imitator, Macaca mulatta), bathyergid mole-rats (Heterocephalus glaber, Fukomys spp.), bats (Myotis spp.), birds, olms (Proteus anguinus), turtles, greenland sharks, bivalves (Arctica islandica), and potentially non-aging ones such as Hydra and Planaria.