Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold.

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1ß (IL-1ß). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1ß. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1ß. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1ß by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1ß, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold.

Noninsect-Based Diet Leads to Structural and Functional Changes of Acidic Chitinase in Carnivora.

Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys residues to an active form and by introducing five meerkat Chia residues. Mutations affecting the Chia conformation and activity after pseudogenization have accumulated in the common ancestor of Felidae due to functional constraints. Evolutionary analysis indicates that Chia genes are under relaxed selective constraint in species with noninsect-based diets except for Canidae. These results suggest that there are two types of inactivating processes in Carnivora and that dietary changes affect the structure and activity of Chia.

Spectral tuning and deactivation kinetics of marine mammal melanopsins.

In mammals, the photopigment melanopsin (Opn4) is found in a subset of retinal ganglion cells that serve light detection for circadian photoentrainment and pupil constriction (i.e., mydriasis). For a given species, the efficiency of photoentrainment and length of time that mydriasis occurs is determined by the spectral sensitivity and deactivation kinetics of melanopsin, respectively, and to date, neither of these properties have been described in marine mammals. Previous work has indicated that the absorbance maxima (λmax) of marine mammal rhodopsins (Rh1) have diversified to match the available light spectra at foraging depths. However, similar to the melanopsin λmax of terrestrial mammals (~480 nm), the melanopsins of marine mammals may be conserved, with λmax values tuned to the spectrum of solar irradiance at the water's surface. Here, we investigated the Opn4 pigments of 17 marine mammal species inhabiting diverse photic environments including the Infraorder Cetacea, as well as the Orders Sirenia and Carnivora. Both genomic and cDNA sequences were used to deduce amino acid sequences to identify substitutions most likely involved in spectral tuning and deactivation kinetics of the Opn4 pigments. Our results show that there appears to be no amino acid substitutions in marine mammal Opn4 opsins that would result in any significant change in λmax values relative to their terrestrial counterparts. We also found some marine mammal species to lack several phosphorylation sites in the carboxyl terminal domain of their Opn4 pigments that result in significantly slower deactivation kinetics, and thus longer mydriasis, compared to terrestrial controls. This finding was restricted to cetacean species previously found to lack cone photoreceptor opsins, a condition known as rod monochromacy. These results suggest that the rod monochromat whales rely on extended pupillary constriction to prevent photobleaching of the highly photosensitive all-rod retina when moving between photopic and scotopic conditions.

Evolutionary loss of inflammasomes in the Carnivora and implications for the carriage of zoonotic infections.

Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1ß. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora.

The steroid metabolome of pregnancy, insights into the maintenance of pregnancy and evolution of reproductive traits.

Modes of mammalian reproduction are diverse and not always conserved among related species. Progesterone is universally required to supports pregnancy but sites of synthesis and metabolic pathways vary widely. The steroid metabolome of mid-to late gestation was characterized, focusing on 5α-reduced pregnanes in species representing the Perissodactyla, Cetartiodactyla and Carnivora using mass spectrometry. Metabolomes and steroidogenic enzyme ortholog sequences were used in heirarchial analyses. Steroid metabolite profiles were similar within orders, whales within cetartiodactyls for instance, but with notable exceptions such as rhinoceros clustering with goats, and tapirs with pigs. Steroidogenic enzyme sequence clustering reflected expected evolutionary relationships but once again with exceptions. Human sequences (expected outgroups) clustered with perissodactyl CYP11A1, CYP17A1 and SRD5A1 gene orthologues, forming outgroups only for HSD17B1 and SRD5A2. Spotted hyena CYP19A1 clustered within the Perissodactyla, between rhinoceros and equid orthologues, whereas CYP17A1 clustered within the Carnivora. This variability highlights the random adoption of divergent physiological strategies as pregnancy evolved among genetically similar species.

The Great American Biotic Interchange revisited: a new perspective from the stable isotope record of Argentine Pampas fossil mammals.

This study aims at assessing resource and habitat use, niche occupation and trophic interactions from a stable isotope perspective on fossil mammals from the Argentine Pampas during the Great American Biotic Interchange (GABI). We present stable isotope data of more than 400 samples belonging to 10 mammalian orders and spanning a temporal range from ~9.5 Ma to ~12 ky. Rodents, notoungulates and pilosians record an increase in the consumption of C4 plants, whereas litopterns and cingulates show δ13C values that remain mostly within a C3-dominated diet. Our stable isotope data indicates that the expansion of C4 vegetation opened up new niche opportunities, probably alleviating resource competition among endemic taxa. Gomphothere, equid and camelid δ13C records show a broad variability pointing to consumption of C3 and mixed C3-C4 vegetation. This flexible dietary behavior may have facilitated the successful settlement of immigrant groups in South America. In the case of carnivorous taxa, Late Miocene pre-GABI endemic sparassodonts consumed prey from C3 environments, whereas immigrant carnivorans preferred prey from mixed C3-C4 areas. Our research contributes to the study of the GABI from a different perspective as stable isotope records permit to characterize, from a (semi)quantitative standpoint, ecological traits within extinct fauna.

Semen Cryopreservation and Banking for the Conservation of Neotropical Carnivores.

Neotropical carnivores include a large number of threatened and endangered species. It is critical to develop conservation efforts to ensure the sustainability of populations in situ and ex situ. The highest priorities are to protect natural habitats and better understand the biology of rare species. Conservation efforts also are directed toward the implementation of breeding programs and the development of reproductive biotechnologies in which the cryopreservation of male gametes plays a major role. It also is fundamental to create semen banks that contribute to maintaining genetic diversity in small and endangered populations. The present article aims at reviewing the state of the art in cryopreservation of semen from neotropical carnivores and discuss the development of systematic banking for the conservation of these understudied species.

Analysis of ZP1 gene reveals differences in zona pellucida composition in carnivores.

The zona pellucida (ZP) is an extracellular envelope that surrounds mammalian oocytes. This coat participates in the interaction between gametes, induction of the acrosome reaction, block of polyspermy and protection of the oviductal embryo. Previous studies suggested that carnivore ZP was formed by three glycoproteins (ZP2, ZP3 and ZP4), with ZP1 being a pseudogene. However, a recent study in the cat found that all four proteins were expressed. In the present study, in silico and molecular analyses were performed in several carnivores to clarify the ZP composition in this order of mammals. The in silico analysis demonstrated the presence of the ZP1 gene in five carnivores: cheetah, panda, polar bear, tiger and walrus, whereas in the Antarctic fur seal and the Weddell seal there was evidence of pseudogenisation. Molecular analysis showed the presence of four ZP transcripts in ferret ovaries (ZP1, ZP2, ZP3 and ZP4) and three in fox ovaries (ZP2, ZP3 and ZP4). Analysis of the fox ZP1 gene showed the presence of a stop codon. The results strongly suggest that all four ZP genes are expressed in most carnivores, whereas ZP1 pseudogenisation seems to have independently affected three families (Canidae, Otariidae and Phocidae) of the carnivore tree.

Muscle mercury and selenium in fishes and semiaquatic mammals from a selenium-deficient area.

The aim of this study was to investigate and compare total mercury (Hg), selenium (Se), and Se:Hg molar ratios in fish muscles (phytophages n=3; benthophages n=32; predators n=5) and semiaquatic carnivores, including piscivores (the European otter n=8, the feral American mink n=7) and the omnivorous raccoon (n=37) from a riverine European ecosystem in a Se-deficient area. The Hg concentration in fish reached 0.337µg/g dry weight, dw (0.084µg/g wet weight, ww). We found significant differences among Hg levels in tested vertebrate groups (predators vs benthophages: 0.893 vs 0.281µg/g; piscivores vs omnivores: 6.085 vs 0.566µg/g dw). Fish groups did not differ in Se concentrations, with a mean value of 0.653µg/g dw. Significant differences were revealed between Se levels in piscivorous and omnivorous carnivores (0.360 vs 0.786µg/g dw, respectively). Fish Se:Hg molar ratio values were >2.2. Benthophages had higher the ratio than predators but similar to phytophages. Among carnivores, piscivores had much lower the ratio than raccoon (0.14 vs 3.75) but raccoon and fish medians did not significantly differ. We found almost two times higher Se levels in fish and raccoons compared to piscivores, possibly resulting from lower fish Se digestibility by piscivores in contrast to higher absorption of plant Se by many fish and omnivorous raccoons. Considering that a tissue Se:Hg molar ratio <1 may be connected with a Hg toxicity potential increase, we assume that piscivores in Se-deficient area are in worse situation and more exposed to Hg than fish and omnivores.

Brains of Native and Alien Mesocarnivores in Biomonitoring of Toxic Metals in Europe.

Mercury (Hg), lead (Pb) and cadmium (Cd) are involved in mammalian brain damage. However, little is known about Pb and Cd brain levels in wildlife that reflect the geochemical background. The aims of the study include the estimation of Hg, Pb and Cd concentrations, and the determination of relationships between these elements in the brains of 94 mesocarnivores. Road-killed or hunted animals were obtained from north-western Poland near the Polish-German border. The investigation covered the native Eurasian otter Lutra lutra, badger Meles meles, pine marten Martes martes, beech marten M. foina, European polecat Mustela putorius, red fox Vulpes vulpes, and alien species: feral and ranch American mink Neovison vison, raccoon Procyon lotor and raccoon dog Nyctereutes procyonoides. Depending on the diet and environmental pollution, the carnivore brains accumulated toxic metals in varying amounts. The highest median Hg levels (in mg/kg dry weight, dw) were found in the piscivorous Eurasian otter and feral mink (2.44 and 3.96), Pb in the omnivorous raccoon (0.47), while Cd in minks (~0.06). We indicated that Pb-based ammunition is a significant source of the element in scavengers from hunting area, and we also found a significant correlation between Pb and Cd levels in the fox brain. Finally, this study is the first to suggest background levels for brain Pb and Cd in mesocarnivores (<0.50 and <0.04 mg/kg dw, respectively).

Nutritional regulation of the anabolic fate of amino acids within the liver in mammals: concepts arising from in vivo studies.

At the crossroad between nutrient supply and requirements, the liver plays a central role in partitioning nitrogenous nutrients among tissues. The present review examines the utilisation of amino acids (AA) within the liver in various physiopathological states in mammals and how the fates of AA are regulated. AA uptake by the liver is generally driven by the net portal appearance of AA. This coordination is lost when demands by peripheral tissues is important (rapid growth or lactation), or when certain metabolic pathways within the liver become a priority (synthesis of acute-phase proteins). Data obtained in various species have shown that oxidation of AA and export protein synthesis usually responds to nutrient supply. Gluconeogenesis from AA is less dependent on hepatic delivery and the nature of nutrients supplied, and hormones like insulin are involved in the regulatory processes. Gluconeogenesis is regulated by nutritional factors very differently between mammals (glucose absorbed from the diet is important in single-stomached animals, while in carnivores, glucose from endogenous origin is key). The underlying mechanisms explaining how the liver adapts its AA utilisation to the body requirements are complex. The highly adaptable hepatic metabolism must be capable to deal with the various nutritional/physiological challenges that mammals have to face to maintain homeostasis. Whereas the liver responds generally to nutritional parameters in various physiological states occurring throughout life, other complex signalling pathways at systemic and tissue level (hormones, cytokines, nutrients, etc.) are involved additionally in specific physiological/nutritional states to prioritise certain metabolic pathways (pathological states or when nutritional requirements are uncovered).

The estimation of tree posterior probabilities using conditional clade probability distributions.

In this article I introduce the idea of conditional independence of separated subtrees as a principle by which to estimate the posterior probability of trees using conditional clade probability distributions rather than simple sample relative frequencies. I describe an algorithm for these calculations and software which implements these ideas. I show that these alternative calculations are very similar to simple sample relative frequencies for high probability trees but are substantially more accurate for relatively low probability trees. The method allows the posterior probability of unsampled trees to be calculated when these trees contain only clades that are in other sampled trees. Furthermore, the method can be used to estimate the total probability of the set of sampled trees which provides a measure of the thoroughness of a posterior sample.

Trace elements in tissues of wild carnivores and omnivores in Croatia.

The differences in metal exposure (As, Cd, Cu, Pb and Hg) in the muscle, liver and kidney tissues of brown bears (Ursus arctos), grey wolfs (Canis lupus), Eurasian lynxs (Lynx lynx), Eurasian badgers (Meles meles) and pine martens (Martes martes) from Croatia were observed. The highest mean Cd levels were found in kidney and liver of Eurasian badger (3.05 and 0.537 mg/kg). The highest Cu concentrations (mg/kg) measured in liver tissue were obtained in order: Eurasian badger (15.2) > brown bear (12.1) > pine marten (10.3) > Eurasian lynx (8.43) > grey wolf (6.44). Result presented that Eurasian badger accumulated the highest levels of elements: As, Cu and Pb in muscle; As, Cd, Cu and Pb in liver; Cd and Pb in kidney. Kidney of pine marten accumulated the highest concentrations of As, Cu and Hg. Omnivorous species observed present an important bioindicator for the accumulation of toxic elements indicating an enhanced vulnerability for response to ecological changes in forested terrain. Generally, element concentrations found in five species observed were lower in comparison to levels reported in previous studies and below levels related to toxicosis in mammals.

Hypercarnivory and the brain: protein requirements of cats reconsidered.

The domestic hypercarnivores cat and mink have a higher protein requirement than other domestic mammals. This has been attributed to adaptation to a hypercarnivorous diet and subsequent loss of the ability to downregulate amino acid catabolism. A quantitative analysis of brain glucose requirements reveals that in cats on their natural diet, a significant proportion of protein must be diverted into gluconeogenesis to supply the brain. According to the model presented here, the high protein requirement of the domestic cat is the result of routing of amino acids into gluconeogenesis to supply the needs of the brain and other glucose-requiring tissues, resulting in oxidation of amino acid in excess of the rate predicted for a non-hypercarnivorous mammal of the same size. Thus, cats and other small hypercarnivores do not have a high protein requirement per se, but a high endogenous glucose demand that is met by obligatory amino acid-based gluconeogenesis. It is predicted that for hypercarnivorous mammals with the same degree of encephalisation, endogenous nitrogen losses increase with decreasing metabolic mass as a result of the allometric relationships of brain mass and brain metabolic rate with body mass, possibly imposing a lower limit for body mass in hypercarnivorous mammals.

Does encephalization correlate with life history or metabolic rate in Carnivora?

A recent analysis of brain size evolution reconstructed the plesiomorphic brain-body size allometry for the mammalian order Carnivora, providing an important reference frame for comparative analyses of encephalization (brain volume scaled to body mass). I performed phylogenetically corrected regressions to remove the effects of body mass, calculating correlations between residual values of encephalization with basal metabolic rate (BMR) and six life-history variables (gestation time, neonatal mass, weaning time, weaning mass, litter size, litters per year). No significant correlations were recovered between encephalization and any life-history variable or BMR, arguing against hypotheses relating encephalization to maternal energetic investment. However, after correcting for clade-specific adaptations, I recovered significant correlations for several variables, and further analysis revealed a conserved carnivoran reproductive strategy, linking degree of encephalization to the well-documented mammalian life-history trade-off between neonatal mass and litter size. This strategy of fewer, larger offspring correlating with increased encephalization remains intact even after independent changes in encephalization allometries in the evolutionary history of this clade.

Out of Africa: modern human origins special feature: isotopic evidence for the diets of European Neanderthals and early modern humans.

We report here on the direct isotopic evidence for Neanderthal and early modern human diets in Europe. Isotopic methods indicate the sources of dietary protein over many years of life, and show that Neanderthals had a similar diet through time (approximately 120,000 to approximately 37,000 cal BP) and in different regions of Europe. The isotopic evidence indicates that in all cases Neanderthals were top-level carnivores and obtained all, or most, of their dietary protein from large herbivores. In contrast, early modern humans (approximately 40,000 to approximately 27,000 cal BP) exhibited a wider range of isotopic values, and a number of individuals had evidence for the consumption of aquatic (marine and freshwater) resources. This pattern includes Oase 1, the oldest directly dated modern human in Europe (approximately 40,000 cal BP) with the highest nitrogen isotope value of all of the humans studied, likely because of freshwater fish consumption. As Oase 1 was close in time to the last Neanderthals, these data may indicate a significant dietary shift associated with the changing population dynamics of modern human emergence in Europe.

Truncation of TRIM5 in the Feliformia explains the absence of retroviral restriction in cells of the domestic cat.

TRIM5alpha mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5alpha. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

Running, swimming and diving modifies neuroprotecting globins in the mammalian brain.

The vulnerability of the human brain to injury following just a few minutes of oxygen deprivation with submergence contrasts markedly with diving mammals, such as Weddell seals (Leptonychotes weddellii), which can remain underwater for more than 90 min while exhibiting no neurological or behavioural impairment. This response occurs despite exposure to blood oxygen levels concomitant with human unconsciousness. To determine whether such aquatic lifestyles result in unique adaptations for avoiding ischaemic-hypoxic neural damage, we measured the presence of circulating (haemoglobin) and resident (neuroglobin and cytoglobin) oxygen-carrying globins in the cerebral cortex of 16 mammalian species considered terrestrial, swimming or diving specialists. Here we report a striking difference in globin levels depending on activity lifestyle. A nearly 9.5-fold range in haemoglobin concentration (0.17-1.62 g Hb 100 g brain wet wt(-1)) occurred between terrestrial and deep-diving mammals; a threefold range in resident globins was evident between terrestrial and swimming specialists. Together, these two globin groups provide complementary mechanisms for facilitating oxygen transfer into neural tissues and the potential for protection against reactive oxygen and nitrogen groups. This enables marine mammals to maintain sensory and locomotor neural functions during prolonged submergence, and suggests new avenues for averting oxygen-mediated neural injury in the mammalian brain.

Elephants and human color-blind deuteranopes have identical sets of visual pigments.

Being the largest land mammals, elephants have very few natural enemies and are active during both day and night. Compared with those of diurnal and nocturnal animals, the eyes of elephants and other arrhythmic species, such as many ungulates and large carnivores, must function in both the bright light of day and dim light of night. Despite their fundamental importance, the roles of photosensitive molecules, visual pigments, in arrhythmic vision are not well understood. Here we report that elephants (Loxodonta africana and Elephas maximus) use RH1, SWS1, and LWS pigments, which are maximally sensitive to 496, 419, and 552 nm, respectively. These light sensitivities are virtually identical to those of certain "color-blind" people who lack MWS pigments, which are maximally sensitive to 530 nm. During the day, therefore, elephants seem to have the dichromatic color vision of deuteranopes. During the night, however, they are likely to use RH1 and SWS1 pigments and detect light at 420-490 nm.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20ºC until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37ºC for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 ± 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350 percent above baseline). On one occasion, we did not detect any variation. Mean (± SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 ± 26.7, after: 213.7 ± 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.