A Subset of Mouse Colonic Goblet Cells Expresses the Bitter Taste Receptor Tas2r131

The concept that gut nutrient sensing involves taste receptors has been fueled by recent reports associating the expression of taste receptors and taste-associated signaling molecules in the gut and in gut-derived cell lines with physiological responses induced by known taste stimuli. However, for bitter taste receptors (Tas2rs), direct evidence for their functional role in gut physiology is scarce and their cellular expression pattern remained unknown. We therefore investigated Tas2r expression in mice. RT-PCR experiments assessed the presence of mRNA for Tas2rs and taste signaling molecules in the gut. A gene-targeted mouse strain was established to visualize and identify cell types expressing the bitter receptor Tas2r131. Messenger RNA for various Tas2rs and taste signaling molecules were detected by RT-PCR in the gut. Using our knock-in mouse strain we demonstrate that a subset of colonic goblet cells express Tas2r131. Cells that express this receptor are absent in the upper gut and do not correspond to enteroendocrine and brush cells. Expression in colonic goblet cells is consistent with a role of Tas2rs in defense mechanisms against potentially harmful xenobiotics.     

Evolutionary insights into umami,sweet, and bitter taste receptors in amphibians

Umami and sweet sensations provide animals with important dietary information for detecting and consuming nutrients, whereas bitter sensation helps animals avoid potentially toxic or harmful substances. Enormous progress has been made toward animal sweet/umami taste receptor (Tas1r) and bitter taste receptor (Tas2r). However, information about amphibians is mainly scarce. This study attempted to delineate the repertoire of Tas1r/Tas2r genes by searching for currently available genome sequences in 14 amphibian species. This study identified 16 Tas1r1, 9 Tas1r2, and 9 Tas1r3 genes to be intact and another 17 Tas1r genes to be pseudogenes or absent in the 14 amphibians. According to the functional prediction of Tas1r genes, two species have lost sweet sensation and seven species have lost both umami and sweet sensations. Anurans possessed a large number of intact Tas2rs, ranging from 39 to 178. In contrast, caecilians possessed a contractive bitter taste repertoire, ranging from 4 to 19. Phylogenetic and reconciling analysis revealed that the repertoire of amphibian Tas1rs and Tas2rs was shaped by massive gene duplications and losses. No correlation was found between feeding preferences and the evolution of Tas1rs in amphibians. However, the expansion of Tas2rs may help amphibians adapt to both aquatic and terrestrial habitats. Bitter detection may have played an important role in the evolutionary adaptation of vertebrates in the transition from water to land.     

Functional Analyses of Bitter Taste Receptors in Domestic Cats (Felis catus)

Cats are obligate carnivores and under most circumstances eat only animal products. Owing to the pseudogenization of one of two subunits of the sweet receptor gene, they are indifferent to sweeteners, presumably having no need to detect plant-based sugars in their diet. Following this reasoning and a recent report of a positive correlation between the proportion of dietary plants and the number of Tas2r (bitter receptor) genes in vertebrate species, we tested the hypothesis that if bitter perception exists primarily to protect animals from poisonous plant compounds, the genome of the domestic cat (Felis catus) should have lost functional bitter receptors and they should also have reduced bitter receptor function. To test functionality of cat bitter receptors, we expressed cat Tas2R receptors in cell-based assays. We found that they have at least 7 functional receptors with distinct receptive ranges, showing many similarities, along with some differences, with human bitter receptors. To provide a comparative perspective, we compared the cat repertoire of intact receptors with those of a restricted number of members of the order Carnivora, with a range of dietary habits as reported in the literature. The numbers of functional bitter receptors in the terrestrial Carnivora we examined, including omnivorous and herbivorous species, were roughly comparable to that of cats thereby providing no strong support for the hypothesis that a strict meat diet influences bitter receptor number or function. Maintenance of bitter receptor function in terrestrial obligate carnivores may be due to the presence of bitter compounds in vertebrate and invertebrate prey, to the necessary role these receptors play in non-oral perception, or to other unknown factors. We also found that the two aquatic Carnivora species examined had fewer intact bitter receptors. Further comparative studies of factors driving numbers and functions of bitter taste receptors will aid in understanding the forces shaping their repertoire.     

Genetic diversity of bitter taste receptor gene family in Sichuan domestic and Tibetan chicken populations

The sense of bitter taste plays a critical role in animals as it can help them to avoid intake of toxic and harmful substances. Previous research had revealed that chicken has only three bitter taste receptor genes (Tas2r1, Tas2r2 and Tas2r7). To better understand the genetic polymorphisms and importance of bitter taste receptor genes (Tas2rs) in chicken, here, we sequenced Tas2rs of 30 Sichuan domestic chickens and 30 Tibetan chickens. Thirteen single-nucleotide polymorphisms (SNPs) including three nonsynonymous mutations (m.359G >C, m.503C >A and m.583A >G) were detected in Tas2r1 (m. is the abbreviation for mutation); three SNPs were detected in Tas2r2, but none of them were missense mutation; eight SNPs were detected in Tas2r7 including six nonsynonymous substitutions (m.178G >A, m.421A >C, m.787C >T, m.832G >T, m.907A >T and m.943G >A). Tajima’s D neutral test indicates that there is no population expansion in both populations, and the size of the population is relatively stable. All the three networks indicate that red jungle fowls share haplotypes with domestic chickens. In addition, we found that haplotypes H1 and HE1 were positively associated with high-altitude adaptation, whereas haplotypes H4 and HE4 showed a negative correlation with high-altitude adaptation in Tas2rs. Although, chicken has only three Tas2rs, our results showed that both Sichuan domestic chickens and Tibetan chickens have abundant haplotypes in Tas2rs, especially in Tas2r7, which might help chickens to recognize a wide variety of bitter-tasting compounds.     

Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations

Large scale isolation of small preantral follicles (40–90 μm) from nondomestic cats (lion, puma, cheetah, jaguar, and three kinds of tigers) is described and compared with domestic cats. The viability of preantral follicles was estimated by trypan blue staining of granulosa cells and/or by 5-bromo-2′-deoxy-uridine (BrdU) incorporation into oocytes and granulosa cells during short term culture. Native and isolated preantral follicles were compared ultrastructurally. In nondomestic cats the mechanical dissection of ovaries provided 0–12 500 follicles per ovary with a viability of 20–50%, estimated by trypan blue staining. Even the follicles recovered from domestic cats, whose ovaries are considerable smaller than ovaries from all other felids, are characterised by only 28.7% viable follicles. The follicles from one Siberian tiger and three Indian lions were cultivated and their in vitro viability assessed by BrdU labelling. Lion follicles were comparable to domestic cat follicles with respect to BrdU incorporation. Tiger follicles were characterised by a decreased staining of granulosa cells.The ultrastructure of feline oocytes appears similar to that of most mammalian species and was only slightly affected during the isolation procedure. A central vesicular body was only observed in tiger oocytes.     

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3^tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.     

Allelic variation of the Tas1r3 taste receptor gene selectively affects taste responses to sweeteners: evidence from 129.B6-Tas1r3 congenic mice.

The Tas1r3 gene encodes the T1R3 receptor protein, which is involved in sweet taste transduction. To characterize ligand specificity of the T1R3 receptor and the genetic architecture of sweet taste responsiveness, we analyzed taste responses of 129.B6-Tas1r3 congenic mice to a variety of chemically diverse sweeteners and glucose polymers with three different measures: consumption in 48-h two-bottle preference tests, initial licking responses, and responses of the chorda tympani nerve. The results were generally consistent across the three measures. Allelic variation of the Tas1r3 gene influenced taste responsiveness to nonnutritive sweeteners (saccharin, acesulfame-K, sucralose, SC-45647), sugars (sucrose, maltose, glucose, fructose), sugar alcohols (erythritol, sorbitol), and some amino acids (D-tryptophan, D-phenylalanine, L-proline). Tas1r3 genotype did not affect taste responses to several sweet-tasting amino acids (L-glutamine, L-threonine, L-alanine, glycine), glucose polymers (Polycose, maltooligosaccharide), and nonsweet NaCl, HCl, quinine, monosodium glutamate, and inosine 5'-monophosphate. Thus Tas1r3 polymorphisms affect taste responses to many nutritive and nonnutritive sweeteners (all of which must interact with a taste receptor involving T1R3), but not to all carbohydrates and amino acids. In addition, we found that the genetic architecture of sweet taste responsiveness changes depending on the measure of taste response and the intensity of the sweet taste stimulus. Variation in the T1R3 receptor influenced peripheral taste responsiveness over a wide range of sweetener concentrations, but behavioral responses to higher concentrations of some sweeteners increasingly depended on mechanisms that could override input from the peripheral taste system.     

The repertoire of bitter taste receptor genes in Ovalentaria fish

Bitter taste perception is important for vertebrates to select food and avoid toxic substances. A large number of Tas2r genes have been identified from vertebrate species previously; however, few studies have been conducted on the Tas2r genes of Ovalentaria species that have various dietary niches and are widely distributed, ranging from the sea to freshwater environments. Several genomes of Ovalentaria species have been released recently, allowing us to study Tas2r genes in these fishes. Thus, we explored the genomes of these fishes and identified 34 Tas2r genes in 21 species, including 27 intact Tas2r genes and seven pseudogenes. The results suggest that Ovalentaria species generally carry a small repertoire of Tas2r genes. To determine the phylogenetic relationship of Tas2r genes among 21 fishes, we constructed neighbor-joining (NJ) trees. The results showed that gene duplication may not occur in these fishes. Phylogenetic independent contrast (PIC) analysis showed that the fish Tas2r gene repertoire size was not positively correlated with diet, indicating that the food swallowing behavior might reduce the importance of bitter taste sense.     

Expression of taste receptors in Solitary Chemosensory Cells of rodent airways

Background

Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs.

Methods

We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP).

Results

Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways.

Conclusions

Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.     

Umami taste receptor functions as an amino acid sensor via Gαs subunit in N1E-115 neuroblastoma cells

The sensing of the nutritional level of the body fluid is pivotal for maintaining homeostasis in animals. However, it is not yet understood how the cells detect nutritional levels. In the present study, we examined the function of umami taste receptor, which has a dimeric protein structure composed of Tas1r1 and Tas1r3, as amino acid sensor in the cells. We found that deprivation of amino acids induced neurite outgrowth in N1E-115 cells. The neurite outgrowth was inhibited by almost all of the amino acids tested. To investigate the involvement of the umami taste receptor, siRNA against each of Tas1r1 or Tas1r3 was administered, resulting in suppression of the inhibitory effects of amino acids on neurite outgrowth. In addition, inosine 5'-monophosphate, which potentiates the response to amino acids in the taste cells, enhanced the inhibitory effect of glutamine on neurite outgrowth. These results suggest that Tas1r1 + 3 functions as an amino acid sensor in N1E-115 cells. Because glutamine increased intracellular cAMP concentration, we investigated the involvement of the Gαs subunit of the heterotrimeric G protein in signal transduction. The treatments to inhibit the Gαs subunit significantly suppressed the increase of intracellular cAMP concentration induced by glutamine and the inhibitory effect of amino acids on neurite outgrowth. In addition, the reagents for increasing intracellular cAMP concentration inhibited neurite outgrowth induced by deprivation of amino acids. We concluded that Tas1r1 + 3 functions as an amino acid sensor and activates the intracellular signaling pathway through the Gαs subunit in N1E-115 cells.     

Regional expression patterns of taste receptors and gustducin in the mouse tongue

In order to understand differences in taste sensitivities of taste bud cells between the anterior and posterior part of tongue, it is important to analyze the regional expression patterns of genes related to taste signal transduction on the tongue. Here we examined the expression pattern of a taste receptor family, the T1r family, and gustducin in circumvallate and fungiform papillae of the mouse tongue using double-labeled in situ hybridization. Each member of the T1r family was expressed in both circumvallate and fungiform papillae with some differences in their expression patterns. The most striking difference between fungiform and circumvallate papillae was observed in their co-expression patterns of T1r2, T1r3, and gustducin. T1r2-positive cells in fungiform papillae co-expressed T1r3 and gustducin, whereas T1r2 and T1r3 double-positive cells in circumvallate papillae merely expressed gustducin. These results suggested that in fungiform papillae, gustducin might play a role in the sweet taste signal transduction cascade mediated by a sweet receptor based on the T1r2 and T1r3 combination, in fungiform papillae.     

Positional cloning of the mouse saccharin preference (Sac) locus

Differences in sweetener intake among inbred strains of mice are partially determined by allelic variation of the saccharin preference (Sac) locus. Genetic and physical mapping limited a critical genomic interval containing Sac to a 194 kb DNA fragment. Sequencing and annotation of this region identified a gene (Tas1r3) encoding the third member of the T1R family of putative taste receptors, T1R3. Introgression by serial backcrossing of the 194 kb chromosomal fragment containing the Tas1r3 allele from the high-sweetener-preferring C57BL/6ByJ strain onto the genetic background of the low-sweetener-preferring 129P3/J strain rescued its low-sweetener-preference phenotype. Polymorphisms of Tas1r3 that are likely to have functional significance were identified using analysis of genomic sequences and sweetener-preference phenotypes of genealogically distant mouse strains. Tas1r3 has two common haplotypes, consisting of six single nucleotide polymorphisms: one haplotype was found in mouse strains with elevated sweetener preference and the other in strains relatively indifferent to sweeteners. This study provides compelling evidence that Tas1r3 is equivalent to the Sac locus and that the T1R3 receptor responds to sweeteners.     

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.     

Probing cochlear tuning and tonotopy in the tiger using otoacoustic emissions

Otoacoustic emissions (sound emitted from the ear) allow cochlear function to be probed noninvasively. The emissions evoked by pure tones, known as stimulus-frequency emissions (SFOAEs), have been shown to provide reliable estimates of peripheral frequency tuning in a variety of mammalian and non-mammalian species. Here, we apply the same methodology to explore peripheral auditory function in the largest member of the cat family, the tiger (Panthera tigris). We measured SFOAEs in 9 unique ears of 5 anesthetized tigers. The tigers, housed at the Henry Doorly Zoo (Omaha, NE), were of both sexes and ranged in age from 3 to 10 years. SFOAE phase-gradient delays are significantly longer in tigers--by approximately a factor of two above 2 kHz and even more at lower frequencies--than in domestic cats (Felis catus), a species commonly used in auditory studies. Based on correlations between tuning and delay established in other species, our results imply that cochlear tuning in the tiger is significantly sharper than in domestic cat and appears comparable to that of humans. Furthermore, the SFOAE data indicate that tigers have a larger tonotopic mapping constant (mm/octave) than domestic cats. A larger mapping constant in tiger is consistent both with auditory brainstem response thresholds (that suggest a lower upper frequency limit of hearing for the tiger than domestic cat) and with measurements of basilar-membrane length (about 1.5 times longer in the tiger than domestic cat).     

No relationship between sequence variation in protein coding regions of the Tas1r3 gene and saccharin preference in rats

Nearly all mammalian species like sweet-tasting foods and drinks, but there are differences in the degree of 'sweet tooth' both between species and among individuals of the same species. Some individual differences can be explained by genetic variability. Polymorphisms in a sweet taste receptor (Tas1r3) account for a large fraction of the differences in consumption of sweet solutions among inbred mouse strains. We wondered whether mice and rats share the same Tas1r3 alleles, and whether this gene might explain the large difference in saccharin preference among rats. We conducted three experiments to test this. We examined DNA sequence differences in the Tas1r3 gene among rats that differed in their consumption of saccharin in two-bottle choice tests. The animals tested were from an outbred strain (Sprague-Dawley; experiment 1), selectively bred to be high- or low-saccharin consumers (HiS and LoS; experiment 2), or from inbred strains with established differences in saccharin preference (FH/Wjd and ACI; experiment 3). Although there was considerable variation in saccharin preference among the rats there was no variation in the protein-coding regions of the Tas1r3 gene. DNA variants in intronic regions were detected in 1 (of 12) outbred rat with lower-than-average saccharin preference and in the ACI inbred strain, which also has a lower saccharin preference than the FH/Wjd inbred partner strain. Possible effects of these intronic nucleotide variants on Tas1r3 gene expression or the presence of T1R3 protein in taste papillae were evaluated in the ACI and FH/Wjd strains. Based upon the results of these studies, we conclude that polymorphisms in the protein-coding regions of the sweet receptor gene Tas1r3 are uncommon and do not account for individual differences in saccharin preference for these strains of rats. DNA variants in intron 4 and 5 are more common but appear to be innocuous.     

Female reproductive cycles of wild female felids

Many felid species are endangered because of destructive human activities. As a result, zoos are being tasked with sustaining genetically healthy populations in case of catastrophic extinctions. Unfortunately, with the exception of a few species, most felids do not reproduce well in captivity. The ability to track reproductive activity via hormones is key to developing successful ex situ breeding programs. Through the development of noninvasive fecal hormone monitoring techniques, a high degree of variability in estrous cycle characteristics has been found to exist across the taxon, including the type of ovulation. For example, although all felids have induced ovulations, the occurrence of spontaneous ovulations varies across species, and even between individuals within a species. Clouded leopards, fishing cats and margays frequently have spontaneous ovulations, whereas these are rarely observed in the cheetah, tigrina and ocelot. There are marked species differences in the impact of season on reproductive function, with some being exquisitely sensitive to photoperiod (e.g., Pallas' cat), some moderately affected (tiger, clouded leopard, snow leopard), and others that are not influenced at all (e.g., ocelot, tigrina, margay, lion, leopard, fishing cat). One of the greatest challenges remaining is overcoming the problems associated with highly variable ovarian responses to ovulation induction therapies used with assisted reproductive procedures, like artificial insemination (AI). Success is relatively high in the cheetah and ocelot, but few pregnancies have resulted after AI in clouded leopard, fishing cat and tiger. Current knowledge of the reproductive physiology of nondomestic felids, including aspects of the anatomy, behavior and ovarian cycles will be presented, and how the rapidly growing endocrine database is aiding ex situ management efforts.     

Embryo technology in conservation efforts for endangered felids

Most of the 36 species of wild cats are classified as threatened, vulnerable or endangered due to poaching and habitat loss. The important role of assisted reproduction techniques (ART) as part of a multifaceted captive breeding program for selected wild cat species is gradually gaining acceptance. This recognition is a result of the progress made during the last decade in which the feasibility of oocyte recovery from gonadotropin-treated females, in vitro fertilization, embryo cryopreservation and embryo transfer (ET) was demonstrated in the domestic cat (Felis catus). Additionally, embryos have been produced in vitro from oocytes matured in vitro after recovery from ex situ ovaries of both domestic and non-domestic cat species and domestic kittens have been born following transfer of these embryos. In vitro fertilization has been successful in at least one-third of wild cat species and kittens were born after transfer of Indian desert cat (Felis sylvestris ornata) embryos into a domestic cat and con-specific transfer of tiger (Panthera tigris) embryos. The domestic cat is not only a valuable model for development of in vitro techniques but may serve as a recipient of embryos from several species of small wild cats.