Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24‐25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox – Chinese raccoon dog) to 99.5% (red fox – arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3′‐flanking sequence, showed a significant association (P < 0.01) with body weight.     

Chromosomal evolution of the Canidae. II. Divergence from the primitive carnivore karyotype

The Giemsa-banding patterns of chromosomes from the arctic fox (Alopex lagopus), the red fox (Vulpes vulpes), the kit fox (Vulpes macrotis), and the raccoon dog (Nyctereutes procyonoides) are compared. Despite their traditional placement in different genera, the arctic fox and the kit fox have an identical chromosome morphology and G-banding pattern. The red fox has extensive chromosome arm homoeology with these two species, but has only two entire chromosomes in common. All three species share some chromosomes with the raccoon dog, as does the high diploid-numbered grey wolf (Canis lupus, 2n = 78). Moreover, some chromosomes of the raccoon dog show partial or complete homoeology with metacentric feline chromosomes which suggests that these are primitive canid chromosomes. We present the history of chromosomal rearrangements within the Canidae family based on the assumption that a metacentric-dominated karyotype is primitive for the group.     

Use of RAPD technique in evolution studies of four species in the family Canidae

The RAPD-PCR technique was applied to identify genetic markers able to distinguish between four canid species: the arctic fox (Alopex lagopus), red fox (Vulpes vulpes), Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and six breeds of the domestic dog (Canis familiaris). A total of 29 ten-nucleotide arbitrary primers were screened for their potential use in the differentiation of these species. Ten primers amplified RAPD profiles that made it possible to distinguish between the investigated taxa. A number of species-specific bands was scored within RAPD profiles produced by these primers: 35.6% of all the polymorphic bands were unique to the Chinese raccoon dog, 29.6% were unique to the domestic dog, 21.2% were diagnostic for the red fox and 13.6% for the arctic fox. No breed-specific fragments were amplified from canine DNA; however, three primers produced bands characteristic for the dog, but not present in all of the investigated breeds. A Neighbor-Joining tree constructed on the basis of the analysis of RAPD profiles amplified by six primers revealed that the phylogenetic distance between the dog and the arctic fox is larger than the distance between the dog and the red fox. The phylogenetic branch of the Chinese raccoon dog was the most distinct on the dendrogram, suggesting that this species belongs to a different phylogenetic lineage. Obtained results make it possible to conclude that RAPD analysis can be a powerful tool for developing molecular markers useful in distinguishing between species of the family Canidae and for studying their phylogenetic relations.     

赤狐线粒体全基因组及系统发育分析

测定了赤狐的线粒体基因组全序列,总长度为16 723 bp,碱基组成为：31.3% A、26.1% C、14.8% G、27.8% T。和大多数哺乳动物一样,赤狐的线粒体全基因组包含13个蛋白质编码基因、2个核糖体RNA基因、22个转运RNA基因和1个控制区。除ND3基因起始密码子为不常见的ATT外,赤狐与北极狐、狼、家犬、郊狼的线粒体蛋白质编码遵循相同模式。在控制区的保守序列区段1和2之间发现一段较长的富含AC的随机重复序列。为了验证赤狐与其他犬科动物的系统发育关系,利用12个重链蛋白质编码基因,分别通过邻接法和最大简约法构建了系统发育树。结果表明：赤狐与北极狐是姐妹群,它们在犬科中都属于赤狐型分支,而灰狼、家犬和郊狼则属于狼型分支,与现有的系统进化研究结果一致。     

Variability of CAG tandem repeats in exon 1 of the androgen receptor gene is not related with dog intersexuality

Numerous mutations of the human androgen receptor (AR) gene cause an intersexual phenotype, called the androgen insensitivity syndrome. The intersexual phenotype is also quite often diagnosed in dogs. The aim of this study was to conduct a comparative analysis of the entire coding sequence (eight exons) of the AR gene in healthy and four intersex dogs, as well as in three other canids (the red fox, arctic fox and Chinese raccoon dog). The coding sequence of the studied species appeared to be conserved (similarity above 97%) and polymorphism was found in exon 1 only. Altogether, 2 SNPs were identified in healthy dogs, 14 in red foxes, 16 in arctic foxes and 6 were found in Chinese raccoon dogs, respectively. Moreover, a variable number of tandem repeats (CAG and CAA), encoding an array of glutamines, was also observed in this exon. The CAA codon numbers were invariable within species, but the CAG repeats were polymorphic. The highest number of the CAG and CAA repeats was found in dogs (from 40 to 42) and the observed variability was similar in intersex and healthy dogs. In the other canids the variability fell within the following ranges: 29–37 (red fox), 37–39 (arctic fox) and 29–32 (Chinese raccoon dog). In addition, a polymorphic microsatellite marker in intron 2 was found in the dog, red fox and Chinese raccoon dog. It was concluded that the polymorphism level of the AR gene in the dog was lower than in the other canids and none of the detected polymorphisms, including variability of the CAG tandem repeats, could be related with the intersexual phenotype of the studied dogs.     

Development of a cytogenetic map for the Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and the arctic fox (Alopex lagopus) genomes, using canine-derived microsatellite probes

New chromosomal assignments of canine-derived cosmid clones containing microsatellites to the Chinese raccoon dog and arctic fox genomes are presented in the study. The localizations are in agreement with data obtained from comparative chromosome painting experiments between the dog and arctic fox genomes. However, paracentric inversions have been detected by comparing the loci order in canid karyotypes. The number of physically mapped loci increased to thirty-five both in the Chinese raccoon dog and in the arctic fox. Furthermore, the present status of the cytogenetic map of the Chinese raccoon dog and arctic fox is presented in this study.     

Comparative cytogenetic analysis of sex chromosomes in several Canidae species using zoo-FISH

Sex chromosome differentiation began early during mammalian evolution. The karyotype of almost all placental mammals living today includes a pair of heterosomes: XX in females and XY in males. The genomes of different species may contain homologous synteny blocks indicating that they share a common ancestry. One of the tools used for their identification is the Zoo-FISH technique. The aim of the study was to determine whether sex chromosomes of some members of the Canidae family (the domestic dog, the red fox, the arctic fox, an interspecific hybrid: arctic fox x red fox and the Chinese raccoon dog) are evolutionarily conservative. Comparative cytogenetic analysis by Zoo-FISH using painting probes specific to domestic dog heterosomes was performed. The results show the presence of homologous synteny covering the entire structures of the X and the Y chromosomes. This suggests that sex chromosomes are conserved in the Canidae family. The data obtained through Zoo-FISH karyotype analysis append information obtained using other comparative genomics methods, giving a more complete depiction of genome evolution.     

Prevalence of Toxoplasma gondii infection diagnosed by PCR in farmed red foxes, arctic foxes and raccoon dogs

The aim of this study was to compare Toxoplasma gondii infection in three canid species: red fox Vulpes vulpes, arctic fox Vulpes lagopus and raccoon dog Nyctereutesprocyonoides kept at the same farm. Anal swabs were taken from 24 adult and 10 juvenile red foxes, 12 adult arctic foxes, three adult and seven juvenile raccoon dogs. Additionally, muscle samples were taken from 10 juvenile red foxes. PCR was used to detect T. gondii DNA. T. gondii infection was not detected in any of the arctic foxes; 60% ofraccoon dogs were infected; the prevalence of the parasite in material from red fox swabs was intermediate between the prevalence observed in arctic foxes and raccoon dogs. It is possible that susceptibility and immune response to the parasite differ between the three investigated canid species. T. gondii DNA was detected in muscle tissue of five young foxes. The results of this study suggest that T. gondii infection is not rare in farmed canids.     

Concluding Remarks

Rabies seems to persist throughout most arctic regions, and the northern parts of Norway, Sweden and Finland, is the only part of the Arctic where rabies has not been diagnosed in recent time. The arctic fox is the main host, and the same arctic virus variant seems to infect the arctic fox throughout the range of this species. The epidemiology of rabies seems to have certain common characteristics in arctic regions, but main questions such as the maintenance and spread of the disease remains largely unknown. The virus has spread and initiated new epidemics also in other species such as the red fox and the racoon dog. Large land areas and cold climate complicate the control of the disease, but experimental oral vaccination of arctic foxes has been successful. This article summarises the current knowledge and the typical characteristics of arctic rabies including its distribution and epidemiology.     

Strength of asymmetric competition between predators in food webs ruled by fluctuating prey: the case of foxes in tundra

In food webs heavily influenced by multi‐annual population fluctuations of key herbivores, predator species may differ in their functional and numerical responses as well as their competitive ability. Focusing on red and arctic fox in tundra with cyclic populations of rodents as key prey, we develop a model to predict how population dynamics of a dominant and versatile predator (red fox) impacted long‐term growth rate of a subdominant and less versatile predator (arctic fox). We compare three realistic scenarios of red fox performance: (1) a numerical response scenario where red fox acted as a resident rodent specialist exhibiting population cycles lagging one year after the rodent cycle, (2) an aggregative response scenario where red fox shifted between tundra and a nearby ecosystem (i.e. boreal forest) so as to track rodent peaks in tundra without delay, and (3) a constant subsidy scenario in which the red fox population was stabilized at the same mean density as in the other two scenarios. For all three scenarios it is assumed that the arctic fox responded numerically as a rodent specialist and that the mechanisms of competition is of a interference type for space, in which the arctic fox is excluded from the most resource rich patches in tundra. Arctic fox is impacted most by the constant subsidy scenario and least by the numerical response scenario. The differential effects of the scenarios stemmed from cyclic phase‐dependent sensitivity to competition mediated by changes in temporal mean and variance of available prey to the subdominant predator. A general implication from our result is that external resource subsidies (prey or habitats), monopolized by the dominant competitor, can significantly reduce the likelihood for co‐existence within the predator guild. In terms of conservation of vulnerable arctic fox populations this means that the likelihood of extinction increases with increasing amount of subsidies (e.g. carcasses of large herbivores or marine resources) in tundra and nearby forest areas, since it will act to both increase and stabilize populations of red fox.     

Natal den selection by sympatric arctic and red foxes on Herschel Island,Yukon, Canada

In the twentieth century, red fox (Vulpes vulpes) expanded into the Canadian Arctic, where it competes with arctic fox (Vulpes lagopus) for food and shelter. Red fox dominates in physical interactions with the smaller arctic fox, but little is known about competition between them on the tundra. On Hershel Island, north Yukon, where these foxes are sympatric, we focused on natal den choice, a critical aspect of habitat selection. We tested the hypothesis that red fox displaces arctic fox from dens in prey-rich habitats. We applied an approach based on model comparisons to analyse a 10-year data set and identify factors important to den selection. Red fox selected dens in habitats that were more prey-rich in spring. When red foxes reproduced, arctic fox selected dens with good springtime access, notably many burrows unblocked by ice and snow. These provided the best refuge early in the reproductive season. In the absence of red foxes, arctic foxes selected dens offering good shelter (i.e. large isolated dens). Proximity to prey-rich habitats was consistently less important than the physical aspects of dens for arctic fox. Our study shows for the first time that red foxes in the tundra select dens associated primarily with prey-rich areas, while sympatric arctic foxes do not. These results fit a model of red fox competitively interfering with arctic fox, the first detailed study of such competition in a true arctic setting.     

The arctic fox Alopex lagopus in Fennoscandia: a victim of human-induced changes in interspecific competition and predation?

After a marked decline at the beginning of the 1900s, the arctic fox Alopex lagopus population in Fennoscandia has remained at a very low level. We suggest that the main cause for the population crash was winter starvation caused by (1) over-hunting of reindeer Rangifer tarandus populations, and thus reduced carcass availability in the mountains, and (2) increased interspecific competition for these carcasses because of increased invasion of red foxes Vulpes vulpes from lower altitudes. The failure of arctic fox populations to recover, despite increasing reindeer populations in the mid 1900s, can be explained by a concurrent strong increase in red fox numbers. Analyses of countywide hunting statistics from Norway 1891–1920 suggest that there actually was an increase in red fox numbers in the period of arctic fox decline, and that the increase in reindeer populations from the 1920s to the 1950s was accompanied by a new increase in red fox numbers. We conclude that restoring arctic fox populations most likely will require a substantial and lasting reduction of red fox populations.     

Red fox takeover of arctic fox breeding den: an observation from Yamal Peninsula, Russia

Here, we report from the first direct observation of a red fox (Vulpes vulpes) intrusion on an arctic fox (Vulpes lagopus) breeding den from the southern Arctic tundra of Yamal Peninsula, Russia in 2007. At the same time, as a current range retraction of the original inhabitant of the circumpolar tundra zone the arctic fox is going on, the red fox is expanding their range from the south into arctic habitats. Thus, within large parts of the northern tundra areas the two species are sympatric which gives opportunities for direct interactions including interference competition. However, direct first-hand observations of such interactions are rare, especially in the Russian Arctic. In the present study, we observed one red fox taking over an arctic fox breeding den which resulted in den abandonment by the arctic fox. On July 19, eight arctic fox pups were observed on the den before the red fox was observed on the same den July 22. The pups were never seen at the den or elsewhere after the red fox was observed on the den for as long as we stayed in the area (until August 10). Our observation supports the view that direct interference with red fox on breeding dens may contribute to the range retraction of arctic foxes from the southern limits of the Arctic tundra in Russia.     

A comparative analysis of MC4R gene sequence, polymorphism, and chromosomal localization in Chinese raccoon dog and Arctic fox

Numerous mutations of the human melanocortin receptor type 4 (MC4R) gene are responsible for monogenic obesity, and some of them appear to be associated with predisposition or resistance to polygenic obesity. Thus, this gene is considered a functional candidate for fat tissue accumulation and body weight in domestic mammals. The aim of the study was comparative analysis of chromosome localization, nucleotide sequence, and polymorphism of the MC4R gene in two farmed species of the Canidae family, namely the Chinese raccoon dog (Nycterutes procyonoides procyonoides) and the arctic fox (Alopex lagopus). The whole coding sequence, including fragments of 3'UTR and 5'UTR, shows 89% similarity between the arctic fox (1276 bp) and Chinese raccoon dog (1213 bp). Altogether, 30 farmed Chinese raccoon dogs and 30 farmed arctic foxes were searched for polymorphisms. In the Chinese raccoon dog, only one silent substitution in the coding sequence was identified; whereas in the arctic fox, four InDels and two single-nucleotide polymorphisms (SNPs) in the 5'UTR and six silent SNPs in the exon were found. The studied gene was mapped by FISH to the Chinese raccoon dog chromosome 9 (NPP9q1.2) and arctic fox chromosome 24 (ALA24q1.2-1.3). The obtained results are discussed in terms of genome evolution of species belonging to the family Canidae and their potential use in animal breeding.     

Arctic fox versus red fox in the warming Arctic: four decades of den surveys in north Yukon

During the last century, the red fox (Vulpes vulpes) has expanded its distribution into the Arctic, where it competes with the arctic fox (Vulpes lagopus), an ecologically similar tundra predator. The red fox expansion correlates with climate warming, and the ultimate determinant of the outcome of the competition between the two species is hypothesized to be climate. We conducted aerial and ground fox den surveys in the northern Yukon (Herschel Island and the coastal mainland) to investigate the relative abundance of red and arctic foxes over the last four decades. This region has undergone the most intense warming observed in North America, and we hypothesized that this climate change led to increasing dominance of red fox over arctic fox. Results of recent surveys fall within the range of previous ones, indicating little change in the relative abundance of the two species. North Yukon fox dens are mostly occupied by arctic fox, with active red fox dens occurring sympatrically. While vegetation changes have been reported, there is no indication that secondary productivity and food abundance for foxes have increased. Our study shows that in the western Arctic of North America, where climate warming was intense, the competitive balance between red and arctic foxes changed little in 40?years. Our results challenge the hypotheses linking climate to red fox expansion, and we discuss how climate warming’s negative effects on predators may be overriding positive effects of milder temperatures and longer growing seasons.     

Two candidate genes (FTO and INSIG2) for fat accumulation in four canids: chromosome mapping, gene polymorphisms and association studies of body and skin weight of red foxes

Fat accumulation is a polygenic trait which has a significant impact on human health and animal production. Obesity is also an increasingly serious problem in dog breeding. The FTO and INSIG2 are considered as candidate genes associated with predisposition for human obesity. In this report we present a comparative genomic analysis of these 2 genes in 4 species belonging to the family Canidae - the dog and 3 species which are kept in captivity for fur production, i.e. red fox, arctic fox and Chinese raccoon dog. We cytogenetically mapped these 2 loci by FISH and compared the entire coding sequence of INSIG2 and a fragment of the coding sequence of FTO. The FTO gene was assigned to the following chromosomes: CFA2q25 (dog), VVU2q21 (red fox), ALA8q25 (arctic fox) and NPP10q24-25 (Chinese raccoon dog), while the INSIG2 was mapped to CFA19q17, VVU5p14, ALA24q15 and NPP9q22, respectively. Altogether, 29 SNPs were identified (16 in INSIG2 and 13 in FTO) and among them 2 were missense substitutions in the dog (23C/T, Thr>Met in the FTO gene and 40C/A, Arg>Ser in INSIG2). The distribution of these 2 SNPs was studied in 14 dog breeds. Two synonymous SNPs, one in the FTO gene (-28T>C in the 5'-flanking region) and one in the INSIG2 (10175C>T in intron 2), were used for the association studies in red foxes (n = 390) and suggestive evidence was observed for their association with body weight (FTO, p < 0.08) and weight of raw skin (INSIG2, p < 0.05). These associations indicate that both genes are potential candidates for growth or adipose tissue accumulation in canids. We also suggest that the 2 missense substitutions found in dogs should be studied in terms of genetic predisposition to obesity.     

Canine-derived cosmid probes containing microsatellites can be used in physical mapping of Arctic fox (Alopex lagopus) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides) genomes

Rapid development of the canine marker genome map facilitates genome mapping of other Canidae species. In this study we present chromosomal localization of 18 canine-derived cosmid probes containing microsatellites in the arctic fox (Alopex lagopus) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides) genomes by the use of fluorescence in situ hybridization (FISH). The chromosome localizations in the arctic fox are in general agreement with data obtained from comparative genome maps of the dog and the fox. However, our studies showed that the order of the loci on some chromosomes was changed during karyotype evolution. Therefore, we suggest that small intrachromosomal rearrangements took place.     

Molecular evolution of the leptin exon 3 in some species of the family Canidae

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.     

Fox colors in relation to colors in mice and sheep

Color inheritance in foxes is explained in terms of homology between color loci in foxes, mice, and sheep. The hypothesis presented suggests that the loci A (agouti), B (black/chocolate brown pigment) and E (extension of eumelanin vs. phaeomelanin) all occur in foxes, both the red fox, Vulpes vulpes, and the arctic fox, Alopex lagopus. Two alleles are postulated at each locus in each species. At the A locus, the (top) dominant allele in the red fox, Ar, produces red color and the corresponding allele in the arctic fox, Aw, produces the winter-white color. The bottom recessive allele in both species is a, which results in the black color of the silver fox and a rare black color in the Icelandic arctic fox when homozygous. The B alleles are assumed to be similar in both species: B, dominant, producing black eumelanin, and b, recessive, producing chocolate brown eumelanin when homozygous. The recessive E allele at the E locus in homozygous form has no effect on the phenotype determined by alleles at the A locus, while Ed, the dominant allele is epistatic to the A alleles and results in Alaska black in the red fox and the dark phase in the arctic fox. Genetic formulae of various color forms of red and arctic fox and their hybrids are presented.     

Exclusion by interference competition? The relationship between red and arctic foxes

The distribution of many predators may be limited by interactions with larger predator species. The arctic fox in mainland Europe is endangered, while the red fox is increasing its range in the north. It has been suggested that the southern distribution limit of the arctic fox is determined by interspecific competition with the red fox. This has been criticised, on the basis that the species co-exist on a regional scale. However, if the larger red fox is superior and interspecific competition important, the arctic fox should avoid close contact, especially during the breeding season. Consequently, the distribution of breeding dens for the two species would be segregated on a much smaller spatial and temporal scale, in areas where they are sympatric. We tested this hypothesis by analysing den use of reproducing arctic and red foxes over 9 years in Sweden. High quality dens were inhabited by reproducing arctic foxes more often when no red foxes bred in the vicinity. Furthermore, in two out of three cases when arctic foxes did reproduce near red foxes, juveniles were killed by red foxes. We also found that breeding arctic foxes occupied dens at higher altitudes than red foxes did. In a large-scale field experiment, red foxes were removed, but the results were not conclusive. However, we conclude that on the scale of individual territories, arctic foxes avoid areas with red foxes. Through interspecific interference competition, the red fox might thus be excluding the arctic fox from breeding in low altitude habitat, which is most important in years when food abundance is limited and competition is most fierce. With high altitude refuges being less suitable, even small-scale behavioural effects could scale up to significant effects at the population level.