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A diversity of U1 small nuclear RNAs in the silk moth Bombyx mori.

Variants of U1 small nuclear RNAs (snRNAs) have been previously detected in a permanent cell line (BmN) of the silk moth Bombyx mori. In this study, the existence of U1 snRNA isoforms in the silk gland (SG) of the organism is investigated. The polyploidy (approximately 200,000X the 2N somatic value) state of the B. mori silk gland cells represents a unique system to explore the potential presence and differential expression of multiple U1 variants in a normal tissue. B. mori U1-specific RT-PCR libraries from the silk gland were generated and five U1 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were examined in these variants and compared to the previously reported isoforms from the transformed BmN cell line. In all these SG U1 variants, variant sites and inter-species differences are located in moderately conserved regions. Substitutional or compensatory changes were found in the double stranded areas and clustered in moderately conserved regions. Some of the changes generate stronger base pairing. Calculated free energy (DeltaG) values for the entire U1 snRNA secondary structures and for the individual stem/loops (I, II, III and IV) domains of the isoforms were generated and compared to determine their structural stability. Using phylogenetic analysis, an evolutionary parallelism is observed between the polymorphic sites in B. mori and variant locations found among animal and plant species.

Animals↗

Phylogenetic position of Mediterranean Astereae and character evolution of daisies (Bellis, Asteraceae) inferred from nrDNA ITS sequences.

Phylogenetic analyses of nrITS sequences of Asteraceae revealed that the Bellis group is a natural assemblage comprising all the species of Bellis and Bellium, but not Rhynchospermum. In contrast, we propose to include the genera Bellis, Bellium, and Bellidastrum in the subtribe Bellidinae in the interest of circumscribing natural groups. Our results also suggest an early diversification in the western Mediterranean Basin of two monophyletic lineages, Bellis and Bellium. Three major groups can be distinguished within BELLIS: (1) the B. perennis group, containing five annual and perennial species with three ploidy levels (diploid, octoploid, and decaploid), which are distributed throughout the Mediterranean Basin despite lack of pappus; (2) the Bellis sylvestris group, with five annual and perennial species primarily from the western Mediterranean, in which there are five ploidy levels (diploid, tetraploid, hexaploid, octoploid, and decaploid); and (3) a basal grade consisting of three diploid, perennial species which displays remarkable diversification of morphologies. Striking characteristics, such as an annual life form, polyploidy, and loss of pappus, seem to have occurred in parallel several times and in different geographical areas during the early diversification of Bellis species in the western Mediterranean. Character evolution reconstructions allow us to describe a putative ancestor of the genus Bellis (proto-Bellis).

Asteraceae↗

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae).

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S-26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the "glaucescens" clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.

Base Sequence↗

Regulation and functions of myogenic regulatory factors in lower vertebrates.

The transcription factors of the MyoD family have essential functions in myogenic lineage determination and muscle differentiation. These myogenic regulatory factors (MRFs) activate muscle-specific transcription through binding to a DNA consensus sequence known as the E-box present in the promoter of numerous muscle genes. Four members, MyoD, myogenin, myf5 and MRF4/herculin/myf6, have been identified in higher vertebrates and have been shown to exhibit distinct but overlapping functions. Homologues of these four MRFs have also been isolated in a variety of lower vertebrates, including amphibians and fish. Differences have been observed, however, in both the expression patterns of MRFs during muscle development and the function of individual MRFs between lower and higher vertebrates. These differences reflect the variety of body muscle formation patterns among vertebrates. Furthermore, as a result of an additional polyploidy that occurred during the evolution of some amphibians and fish, MyoD, myogenin, myf5 and MRF4 may exist in lower vertebrates in two distinct copies that have evolved separately, acquiring specific roles and resulting in increased complexity of the myogenic regulatory network. Evidence is now accumulating that many of the co-factors (E12, Id, MEF2 and CRP proteins) that regulate MRF activity in mammals are also present in lower vertebrates. The inductive signals controlling the initial expression of MRFs within the developing somite of lower vertebrate proteins are currently being elucidated.

Animals↗

Plant snoRNAs: functional evolution and new modes of gene expression.

Small nucleolar RNAs (snoRNAs) are a well-characterized family of non-coding RNAs whose main function is rRNA modification. The diversity and complexity of this gene family continues to expand with the discovery of snoRNAs with non-rRNA or unknown targets. Plants contain more snoRNAs than other eukaryotes and have developed novel expression and processing strategies. The increased number of modifications, which will influence ribosome function, and the novel modes of expression might reflect the environmental conditions to which plants are exposed. Polyploidy and chromosomal rearrangements have generated multiple copies of snoRNA genes, allowing the generation of new snoRNAs for selection. The large snoRNA family in plants is an ideal model for investigation of mechanisms of evolution of gene families in plants.

Base Sequence↗

Plant evolutionary genomics.

Evolutionary genomics combines functional and evolutionary analyses of genome conservation and differentiation. Gene duplication and polyploidy have fundamentally shaped the genomes of Arabidopsis and all angiosperms. Recent comparative studies have focussed on gene regulation, the function of untranscribed genomic regions, and the effects of natural selection on protein function. A large fraction of interspecific protein divergence is probably adaptive, and may be useful for experimental studies of genes and proteins.

Animals↗

Meiosis: vive la difference!

The application of molecular and modern cell biological techniques is beginning to show that the classical picture of the meiotic process is an oversimplification. The comparison of different species and analysis of mutants have recently demonstrated that many of the features previously thought to be an integral part of meiosis can be altered in their timing or even entirely dispensed with. In plants, differences in the meiotic pathway have been observed, by using methods for 3-D optical imaging, between the polyploids maize and wheat. The fact that two such closely related species differ may be the result of different mechanisms for dealing with polyploidy or may be species differences, but shows that a detailed understanding of the process in the particular species of interest is necessary.

Animals↗

Chromosomal effects of newly identified water pollutants PBTA-1 and PBTA-2 and their possible mother compounds (azo dyes) and intermediates (non-ClPBTAs) in two Chinese hamster cell lines.

We performed the in vitro micronucleus (MN) test on 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)-ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), which are newly identified water pollutants from the Nishitakase river in Kyoto, Japan, and on their possible mother compounds (AZO DYE) and intermediates (non-ClPBTAs). We tested these compounds in the absence and presence of S9 mix in two Chinese hamster cell lines CHL and V79-MZ and scored MN, polynuclear and karyorrhectic (PN), and mitotic (M) cells. PBTA-2 in the absence of S9 mix induced the strongest responses in both cell lines. It was also a strong inducer of binucleate cells in PN cells in both cell lines, which suggested that it induced polyploidy. PBTA-1 showed clear positive results only in the absence of S9 mix and only in V79-MZ cells, inducing aneuploidy. In CHL cells AZO DYE-1 significantly induced MN cells in the presence of S9 mix, and AZO DYE-2 induced MN and PN cells, including binucleate cells and cells with a multilobed nucleus, in the absence of S9 mix. In V79-MZ cells, AZO DYE-1 and -2 induced primarily M cells in the presence of S9 mix. 9% of the M cells treated with 50 microg/ml AZO DYE-1 showed endoreduplication. AZO DYE-2 at 200 microg/ml condensed the chromatin in 100% of the cells. The non-ClPBTAs were a bit more cytotoxic than the other compounds and induced a slight increase in MN cells in both cell lines. Some of the chemicals tested induced a characteristic karyomorphology that might reflect abnormal cell division. Abnormalities of cell division could be detected in PN and M cells as well as in MN cells. Structure-activity relationships have also been discussed.

Animals↗

Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate.

Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.

Animals↗

Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro.

Fasinex (triclabendazole) has been reported to be an active fasciolocidal agent used in humans and in farm animals. The clastogenic effects of fasinex were tested in lymphocyte cultures of the river buffalo at three final concentrations: 25, 50 and 100 microg/ml. Chromosomal aberrations, sister chromatid exchanges and micronucleus formation are the three cytogenetic parameters used in this study. The results demonstrated that the number of cells with different types of chromosomal aberrations, including chromatid breaks and gaps, isochromatid breaks and gaps and polyploidy, was increased significantly in cultures treated with different doses of fasinex compared to the control. This increase was dose-dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations. The frequency of sister chromatid exchanges and the formation of micronuclei in all lymphocyte cultures treated with different doses of fasinex were increased significantly compared to the control; these increases were also dose-dependent. In conclusion, the three cytogenetic parameters used to evaluate the effect of fasinex revealed that the drug has a strong clastogenic effect on river buffalo lymphocytes in vitro.

Animals↗

Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization.

Threshold mechanisms of activity for mutagenic agents have been debated for some time, especially for those substances which induce aneuploidy by inhibiting mitotic spindle function. No observed effect levels (NOELs) or "practical thresholds" have been demonstrated for several aneugens both in vitro and in vivo generally by either counting chromosomes in metaphase preparations or by observing micronuclei. Recently, fluorescence in situ hybridization (FISH) has proven to be a sensitive and useful technique for the assessment of aneuploidy at low concentrations. Using binucleate human lymphocytes coupled with FISH, we have been able to characterize a threshold mechanism of action for two spindle inhibitors, benomyl and its active metabolite, carbendazim. Test chemicals were added 24 h following culture initiation. After a further 20 h, cytochalasin B was added, and cells were harvested 28 h later (72 h post initiation). The distribution of chromosomes between the nuclei of binucleate cells was evaluated by fluorescence microscopy for the simultaneous detection of centromeres labeled with FITC (green) or Cy-3 (red). Six human chromosomes were investigated in pairs (1 and 8, 11 and 18, and X and 17). Abnormalities were classified as chromosome loss (including centromeric positive micronuclei), chromosome gain, non-disjunction, or polyploidy. Dose-response data were generated over a range of closely spaced concentrations at 100 ng/ml intervals. The threshold, defined as the lowest "effect" concentration using statistical methods, was determined for each chromosome. Non-disjunction proved to be the most sensitive endpoint for the detection of aneuploidy occurring at higher frequencies and lower concentrations. Results for the six chromosomes demonstrated similar dose-response data which included a series of concentrations with no statistically significant increase above background, followed by a second range of higher concentrations with a statistically significant, concentration-dependent increase. Nearly equimolar threshold concentrations were determined for benomyl- and carbendazim-induced non-disjunction.

Adult↗

In vitro and in vivo extrapolations of genotoxin exposures: consideration of factors which influence dose-response thresholds.

The concept of a threshold of activity of a genotoxic agent is primarily based upon considerations of protective mechanisms and multiple cellular targets, which require inactivation before a toxic response is produced. In this paper, we have considered and evaluated the influences of compound metabolism, DNA lesion formation, mutation induction and sequence content, aneuploidy induction and the influence of repair enzymes upon genetic endpoints produced by both DNA reactive chemicals and by those chemicals which modify non-DNA cellular targets. Thresholds of activity have been evaluated by critical analysis of the published literature and original data analysing both the role of sequence context upon point mutation induction and DNA repair mechanisms upon the sensitivity of cultured cells to the induction of aneuploidy. In the case of DNA reactive chemicals, the presence of a threshold of chemical activity will be dependent upon cellular activities such as those of the Phase II enzymes reducing the activity of chemicals before lesion formation takes place and/or those of the DNA repair enzymes which reduce the proportion of DNA lesions which are processed into DNA sequence changes. Under such conditions, a given exposure of a DNA reactive chemical does not produce a linear or semi-linear increase in DNA lesions or in mutation frequency. However, even when these protective mechanisms are overwhelmed by the high exposures of genotoxic chemicals the biological effects of a genotoxin may be influenced by the sequence context of the gene under consideration. Here, we demonstrate that point mutations are detected at relatively higher frequencies in the non-coding introns compared with the coding exons. Many of the base changes detected in the exons do not produce amino acid changes in the proteins coded for by the genes being monitored for mutation induction. Both sequence context and the types of base changes induced may provide a "buffering" effect reducing the biological consequences of mutation induction. Spindle damaging chemicals, such as colcemid and vinblastine, induce aneuploidy by modifying the numbers of spindle fibres which regulate the segregation of chromosomes during mitosis and meiosis. The redundancy of spindle fibres in the dividing mammalian cell leads to the prediction that only chemical exposures which damage most, if not all, of the fibres will lead to the induction of polyploidy and/or aneuploidy. Such predicted thresholds of chemical activity can be observed when both chromosome loss and non-disjunction are measured in wild type cultures. However, we observed a substantial increase in sensitivity to aneugenic chemicals when measurements were made in primary cell cultures derived from xerodoma pigmentosum and trichothiodystrophy patients. Further studies are necessary to evaluate the consequences of the genetic background of tester strains upon the nature of the dose-response curve of aneugenic chemicals.

1,2-Dimethylhydrazine↗

Chromosome abnormalities and their relationship to morphology and development of human embryos.

This review covers the relationship between chromosome abnormalities, morphological abnormalities and embryonic development. The baseline of chromosome abnormalities in human embryos produced by assisted reproduction is higher than 50%, regardless of maternal age. While aneuploidy increases with maternal age, abnormalities arising post-meiotically, such as mosaicism, chaoticism, polyploidy and haploidy, have similar incidence in all age groups (about 33%). Post-meiotic abnormalities do increase with dysmorphism. The most common dysmorphisms found in cleavage-stage embryos are multinucleation, fragmentation and uneven cells, among others. All dysmorphisms are associated with an increase in post-meiotic chromosome abnormalities and a decreased implantation potential. Similarly, embryos developing slowly or with arrested development have higher incidence of post-meiotic abnormalities than normally developing ones. Chromosome studies in blastocysts indicate that mosaicism is the most common abnormality but that the load of abnormal cells decreases with increasing blastocyst quality. Regardless of blastocyst quality, more than 40% of mosaics are still chromosomally abnormal and will not implant or will spontaneously abort. Because aneuploidy is not related to cleavage stage dysmorphism and trisomies can reach blastocyst stage and beyond, morphological analysis is not enough to select against chromosome abnormalities, and thus preimplantation genetic diagnosis should be recommended in patients 35 and older.

Adult↗

Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis.

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. Here, we show that MgcRacGAP colocalizes with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. We also report that Aurora B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, previously known as a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B.

Aurora Kinase B↗

The COP9 signalosome promotes degradation of Cyclin E during early Drosophila oogenesis.

The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. Here we link the CSN to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, we show that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes similar defects as mutations in CSN or SCF(Ago) subunits: extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation.

Animals↗

Limitations to the development of humanized antibody producing Chinese hamster ovary cells using glutamine synthetase-mediated gene amplification.

Recombinant Chinese hamster ovary (CHO) cells expressing a humanized antibody were obtained by transfection of an antibody expression vector (pKC-GS-HC-huS) into CHO-K1 cells and subsequent glutamine synthetase (GS)-mediated gene amplification in media containing different concentrations of methionine sulfoximine (MSX). Concentrations consisted of 25, 200, 500, and 1000 microM of MSX. The highest producer (HP) subclones were isolated from each MSX level by the limiting dilution method and were characterized with respect to antibody production. No positive relationship was observed between specific antibody productivity (q(Ab)) and MSX concentration. Furthermore, it was found that the antibody production stability of these subclones was very poor even in the presence of selection pressure. During long-term cultures in the presence of the corresponding concentrations of MSX, q(Ab) of all HP subclones significantly decreased for the first six passages and thereafter stabilized. Southern and slot blot analyses showed that the loss of antibody gene copies was only partially responsible for the decreased q(Ab). Fluorescence in situ hybridization (FISH) analysis revealed some cytogenetic features indicative of antibody production instability. Unstable chromosomal structures including dicentrics, rings, and extremely long chromosomes were observed. Amplified sequences enclosed in nuclear projections were often observed. The telomeric repeat sequence, which may be involved in the stabilization of amplified arrays, was found to be absent at the ends of most marker chromosomes. Furthermore, FISH analysis revealed that the overall chromosome content was duplicated in some HP subclones. When metaphase of 12 high producing parental clones was examined, the frequency of occurrence of the polyploidy was 25%. Taken together, the data obtained here suggests that instability could be a concern in the development of CHO cells with GS-mediated gene amplification.

Animals↗

Heterogeneity of chemosensitivity in six clonal cell lines derived from a spontaneous murine astrocytoma and its relationship to genotypic and phenotypic characteristics.

Heterogeneity in drug sensitivity must, in part, account for the relative lack of success with single agent chemotherapy for glioblastoma multiforme (GBM). In order to develop in vitro model systems to investigate this, clones derived from the VM spontaneous murine astrocytoma have been characterised with regard to drug sensitivity. Six clonal cell lines have been tested for sensitivity to a panel of cytotoxic drugs using an intermediate duration 35S-methionine uptake assay. These lines have previously been extensively characterised with regard to morphological, antigenic, kinetic, tumourigenic potential in syngeneic animals and chromosomal properties and display considerable heterogeneity. The present study indicates that heterogeneity extends to sensitivity to all classes of cytotoxic drugs. The greatest difference in sensitivity between the clones was seen in response to cell cycle-specific drugs like the Vinca alkaloids (14-fold and 20-fold for vincristine (VCR) and vindesine (VIND) respectively), while the nitrosoureas, CCNU and BCNU displayed a smaller fold difference in sensitivity (4.3 and 3.6-fold difference respectively). All the clones were considerably more resistant to the adriamycin (ADM), cis-platinum (C-PLAT) and the Vinca alkaloids than the parental cell line although the difference in sensitivity between the clones and parental cell line were less marked for the nitrosoureas and procarbazine (PCB). It has also been possible to examine the relationship between drug sensitivity and the phenotypic and genotypic properties of these clonal cell lines. There is a relationship between chromosome number and sensitivity of a wide variety of cytotoxic drugs including the nitrosoureas, Vinca alkaloids, PCB, C-PLAT, BLEO but not ADR or 5-FU. Clones with small numbers of chromosomes were more resistant than clones with gross polyploidy. Similarly, sensitivity to Vinca alkaloids and ADM, but not other classes of drugs, was greatest in cells with numerous cytoplasmic processes and which did not express large amounts of cell surface fibronectin. Preliminary experiments have been conducted on reconstituting clonal mixtures of cells with different sensitivity to Vinca alkaloids and results from these studies indicate that the drug resistance phenotype is dominant, with clonal mixtures of sensitive and resistant cell adopting the sensitivity of the more resistant partner. These cell lines should prove to be useful models for examining the cell biological basis of drug resistance in glioma and may lead to the identification and exploitation of novel cellular targets in new therapies for GBM.

Animals↗

Selachian cytogenetics: a review.

The karyotype of Chondrichthyes is still the least investigated among vertebrates. Over the last 40 years, the karyotypes of 63 out of the 1100 known species (5.73%) have been described in literature, namely seven squalomorph, one squatinomorph, 20 galeomorph, 33 batoid and two holocephalian species. Generally, the diploid number ranges from a minimum of 28 to a maximum of 106 elements, with more frequent values observed between 50 and 100 chromosomes. None of the four superorders is characterized by a peculiar chromosome set or morphology; the number of uniarmed and biarmed elements is variable in all the karyotypes, and microchromosomes are often present. The general trend in all groups seems to be a progressive reduction of the telocentric chromosome number in the most specialized species, followed by the loss of the microchromosomes. Polyploidy, followed by diploidization events and Robertsonian rearrangements, might have played a key role in the karyological evolution of elasmobranch fish. Chondrichthyes have the largest genome sizes among vertebrates, with the exception of dipnoans and urodeles. In the whole class, the species examined vary greatly in size, from 3 to 34pg/N: the lowest values have been observed in holocephalians, while galeoids and batoids have a DNA amount ranging from 5 to 15 pg/N. Squaloids show heterogeneous DNA amounts, ranging from 8 to 34 pg/N. In more recent years, karyological studies have provided new data on the characterization of selachian karyotypes by C-banding, NOR staining, restriction enzymes in situ digestion and FISH with specific DNA probes, such as telomeric and SINE sequences.

Animals↗