Genetic diversity in wild (Sus scrofa scrofa) and domestic (Sus scrofa domestica) pigs and their hybrids based on polymorphism of a fragment of the D-loop region in the mitochondrial DNA

We examined the variation in mitochondrial DNA by sequencing the D-loop region in wild and domestic (large-white breed) pigs, in hybrids between domestic and wild pigs, and in Monteiro pigs. A D-loop fragment of approximately 330 bp was amplified by PCR. Sequencing of DNA amplicons identified haplotypes previously described as European and Asian types. Monteiro pigs and wild pigs had European haplotypes and domestic pigs had both European and Asian haplotypes.     

Introgressive hybridisation between domestic pigs (Sus scrofa domesticus) and endemic Corsican wild boars (S. s. meridionalis): effects of human-mediated interventions

Owing to the intensified domestication process with artificial trait selection, introgressive hybridisation between domestic and wild species poses a management problem. Traditional free-range livestock husbandry, as practiced in Corsica and Sardinia, is known to facilitate hybridisation between wild boars and domestic pigs (Sus scrofa). Here, we assessed the genetic distinctness and genome-wide domestic pig ancestry levels of the Corsican wild boar subspecies S. s. meridionalis, with reference to its Sardinian conspecifics, employing a genome-wide single nucleotide polymorphism (SNP) assay and mitochondrial control region (mtCR) haplotypes. We also assessed the reliance of morphological criteria and the melanocortin-1 receptor (MC1R) coat colour gene to identify individuals with domestic introgression. While Corsican wild boars showed closest affinity to Sardinian and Italian wild boars compared to other European populations based on principal component analysis, the observation of previously undescribed mtCR haplotypes and high levels of nuclear divergence (Weir’s θ > 0.14) highlighted the genetic distinctness of Corsican S. s. meridionalis. Across three complementary analyses of mixed ancestry (i.e., STRUCTURE, PCADMIX, and ELAI), proportions of domestic pig ancestry were estimated at 9.5% in Corsican wild boars, which was significantly higher than in wild boars in Sardinia, where free-range pig keeping was banned in 2012. Comparison of morphologically pure- and hybrid-looking Corsican wild boars suggested a weak correlation between morphological criteria and genome-wide domestic pig ancestry. The study highlights the usefulness of molecular markers to assess the direct impacts of management practices on gene flow between domestic and wild species.Subject terms: Conservation genomics, Genetic hybridization, Structural variation     

Estimating body mass of wild pigs (Sus scrofa) using body morphometrics

Wild pigs (Sus scrofa) are invading many areas globally and impacting biodiversity and economies in their non‐native range. Thus, wild pigs are often targeted for eradication efforts. Age‐ and sex‐specific body measurements are important for informing these eradication efforts because they reflect body condition, resource availability, and fecundity, which are common indicators of population trajectory. However, body mass is often difficult to collect, especially on large individuals that require specialized equipment or multiple people to weigh. Measurements that can be rapidly taken by a single land or wildlife manager on any size wild pig without aid from specialized equipment would be beneficial if they accurately infer wild pig body mass. Our goals were to assess whether morphometric measurements could accurately predict wild pig body mass, and to provide tools to directly input these measures and estimate wild pig body mass. Using linear models, we quantified the relationship between body mass and morphometric measurements (i.e., body length, chest girth, ear length, eye to snout length, hindfoot length, shoulder length, and tail length) from a subset (n = 102) of wild pigs culled at the Mississippi Alluvial Valley, Mississippi, USA. We evaluated separate models for each individual morphometric measurement. We then used the model coefficients to develop equations to predict wild pig body mass. We validated these equations predicting body mass of 1592 individuals collected across eight areas in Australia, Guam, and the USA for cross‐validation. Each developed equation remained accurate when cross‐validated across regions. Body length, chest girth, and shoulder length were the morphometrics that best predicted wild pig body mass. Our analyses indicated it is possible to use the presented equations to infer wild pig body mass from simple metrics.     

Melanocortin receptor 1 (MC1R) mutations and coat color in pigs.

The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.     

The effect of age on the morphometric sperm traits of domestic pigs (Sus scrofa domestica)

The experiment was conducted using 20 male domestic pigs, which were in 2 (equal-sized) age groups: under 14 months old and over 18 months old. At least 5 ejaculates from each male were taken, and in each ejaculate, morphometric measurements of 50 spermatozoa were made. The measured parameters were head area, head length and width, and flagellum length. For each ejaculate, the basic physical traits and the frequency of occurrence of developmental anomalies of the spermatozoa were examined. It was found that sperm dimensions tended to increase along with the boars' age. Considerable and statistical differences in head area and flagellum length were proved. Spermatozoa collected from older boars (above 18 months of age) had a head area larger by 0.49 microm(2)(P< or = 0.01) and a flagellum longer by 0.67 microm (P< or =0.01) than spermatozoa collected from younger boars (under 14 months of age). The differences in head length and width between the spermatozoa of the tested boars were considerably smaller and were not statistically provable. Significant correlations between the head area of spermatozoa and the head length (r = 0.56, P< or =0.05) and head width (r = 0.36, P< or =0.05) were found. However, the head length was not significantly directly correlated with its width (r = 0.18, P< or =0.05), and flagellum length was negatively correlated with spermatozoan head width (r = -0.71, P< or =0.05). Slight correlations between the morphometric traits of the sperm and the physical traits of the ejaculates (r = -0.11 to 0.16) were found, although in most cases, the correlations were not statistically provable.     

Genetic evidence for introgression between domestic pigs and wild boars (Sus scrofa) in Belgium and Luxembourg: a comparative approach with multiple marker systems

Hybridization between wild species and their domestic relatives can be an important conservation and management problem. Genetic purity of the wild species is desirable per se and the phenomenon can have unpredictable evolutionary consequences. Declining European wild boar populations were frequently restocked with farmed wild boars that sometimes had been crossed with domestic pigs. We used simple polymerase chain reaction‐based diagnostic tests to detect the presence of mitochondrial DNA and coat colour alleles of domestic origin in wild boars from Belgium, Luxembourg, and western Germany. Microsatellite genotypes were used to test for genetic admixture between the wild boars and domestic pigs. Although almost one‐third of all Luxembourg wild boars carried Asian mitochondrial DNA haplotypes originating from domestic pigs, microsatellite‐based clustering only identified four putatively admixed individuals in Luxembourg. By contrast, clustering identified wild boar × domestic hybrids in most sampling locations in Belgium. We interpret these results as evidence of releases of hybrid captive‐reared wild boars. Our results emphasize the need (if working with classical markers) to use different systems to obtain an understanding as to whether hybridization between wild and domestic relatives might have affected the genetic make‐up of a local population. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 104–115.     

Serosurveillance for selected infectious disease agents in wild boars (Sus scrofa) and outdoor pigs in Switzerland

The large abundance of free-ranging wild boars (Sus scrofa) and a trend towards animal friendly outdoor management of domestic pigs lead to an increasing probability of disease transmission between those animal populations. In 2001, an active monitoring was started for classical swine fever (CSF), Aujeszky’s disease (AD) and porcine brucellosis (PB) in wild boars in Switzerland. The objective of this programme was to document the serological status of wild boars regarding the selected pathogens. To continue this serosurveillance, 1,060 wild boar samples were collected during two regular hunting seasons in 2004–2005. Furthermore, in a pilot study, 61 outdoor pigs from 14 farms located in areas with high wild boar densities were sampled in 2004 and serologically tested for AD and PB. All wild boar samples were negative for CSF. Seroprevalence for AD was 2.83% (95% CI 1.91–4.02%). Seroprevalence for PB was 13.5% (95% CI 10.7–16.7%) for the Rose Bengal test and 11.05% (95% CI 8.82–13.61%) for the indirect ELISA. There was no serological evidence for AD in domestic pigs. All tested animals from 13 piggeries were seronegative for PB, but three pigs from the same farm showed doubtful results. Further investigations on the farm did not indicate the presence of PB in the herd. These findings urge the need for better diagnostic tools to obtain reliable results concerning PB prevalence. Since contact and following transmission of infectious agents between infected wild boars and outdoor pigs might occur in the future, it is advisable to include outdoor pigs in areas at risk in routine surveillance programmes.     

Prevalence of Leptospira and Brucella antibodies in wild boars (Sus scrofa) in Tuscany, Italy

Five hundred sixty-two blood samples were collected from wild boars (Sus scrofa) shot in six districts of Tuscany, central Italy, between 1997 and 2000. Sera were examined for antibodies specific for Leptospira interrogans by microagglutination test and Brucella spp. by the Rose Bengal test and indirect enzyme-linked immunosorbent assay. Thirty-four (6.0%) samples tested positive for anti-Leptospira antibodies, 29 (5.1%) sera were positive for anti-L. interrogans serovar bratislava antibodies (titres ranging from 1:100-1:400), and 5 (0.9%) sera were positive for anti-L. interrogans serovar icterohaemorrhagiae antibodies (titres 1:100). All the examined sera were negative for anti-Brucella antibodies.     

DNA extraction from skins of wild (Hydrochoerus hydrochaeris and Pecari tajacu) and domestic (Sus scrofa domestica) species using a novel protocol

Sometimes, commercial products obtained from wild animals are sold as if they were from domestic animals and vice versa. At this point of the productive chain, legal control of possible wildlife products is difficult. Common in the commerce of northern Argentina, skins of two wild species, the carpincho and the collared peccary, look very similar to each other and to those of the domestic pig; it is extremely difficult to differentiate them after they have been tanned. Because there was no an adequate methodology to discriminate between leather of these three species, we developed a new methodology of DNA extraction from skin and leather. This new method involves digesting a leather sample using proteinase K, followed by precipitation of proteins with 5 M NaCl, cleaning with absolute isopropanol and DNA precipitation with 70% ethanol. DNA is hydrated in Tris-EDTA buffer. This protocol provided good-quality DNA suitable for analysis with molecular markers. This new protocol has potential for use in identifying leather products of these species using molecular markers based on RAPDs.     

Estimation of the fertility rates of Japanese wild boars (Sus scrofa leucomystax) using fetuses and corpora albicans

Effective population control of Japanese wild boar (Sus scrofa leucomystax) requires reliable information about population dynamics. Fertility rate is the fundamental component of reproduction to evaluate population dynamics. However, little is known regarding the fertility rate of Japanese wild boar. The traditional hunting practices make it difficult to obtain pregnant females and calculate the fertility rate by checking fetuses as is performed in other countries. Therefore, we focused on the corpora albicans (CA) as the CA remains in the ovaries of postpartum females after pregnancy. This study aimed to evaluate the utility of CA and estimate the fertility rate of Japanese wild boars using CA. Histological analysis of ovaries enabled us to discriminate type 1 CA, which remains for 1 year after breeding. Type 1 CA is a superior indicator compared with lactation in the non-pregnancy season because it allows verification of postpartum females over a long period. The fertility rate was calculated by the combination of pregnant and postpartum females using fetuses and type 1 CA from April to November. The fertility rate of the females captured after the second pregnancy season was 90.3 % during the pregnancy period and 100 % during the non-pregnancy period. The high fertility rate of adult females suggests that intensive adult female harvesting is needed. Our new method to determine fertility rates contributes to developing a monitoring system to adequately control Japanese wild boar population.     

Evolution of the melanocortin-1 receptor (MC1R) in Boobies and Gannets (Aves, Suliformes)

The melanocortin-1 receptor (MC1R) has been linked to intraspecific variation of melanin-based plumage color in several unrelated bird species. However, its involvement in interspecific variation has far less evidence. The Sulidae is a family in the Suliformes composed of 10 species of pelagic seabirds, distributed in 3 genera. There is significant variation in the amount and distribution of melanin pigments among species in the family Sulidae, and 2 species, the brown booby (Sula leucogaster) and the red-footed booby (S. sula), present plumage polymorphisms, with the latter being considered one of the most plumage polymorphic birds. We performed a survey of the MC1R evolution in 68 individuals representing all 9 species in the Sulidae, except the Abbott's booby, to determine the role played by this locus in explaining the melanic variation observed in the Sulidae. We found the amino acid substitution R112H to be in full concordance with the plumage color observed in the brown booby, which shows a unique phaeomelanin-dominant coloration. Furthermore, all amino acid residues known to be important for function at the MC1R were completely conserved in the Sulidae, except for the previously described V85M and H207R substitutions among the 2 red-footed booby's color morphs. A total of 14 substitutions were inferred from estimated ancestral nodes throughout the Sulidae phylogeny. Finally, we found evidence that the MC1R is under strong purifying selection in all Sulid species. This study provides additional evidence of the potential involvement of the MC1R in melanin-based plumage variation at the interspecific level.     

Seroprevalence of Toxoplasma gondii in wild boars (Sus scrofa leucomystax) and wild sika deer (Cervus nippon) in Gunma Prefecture, Japan

The ingestion of undercooked meat from wild animals can be a source of Toxoplasma gondii infection in humans and other animals. In this study, we determined the seroprevalence of T. gondii infection in 175 wild boars (Sus scrofa leucomystax) and 107 wild sika deer (Cervus nippon) hunted in 2004–2007 in Gunma Prefecture, Japan, by using a commercial latex agglutination test (LAT). Antibodies (LAT, 1:64 or higher) to T. gondii were found in 6.3% of wild boars and 1.9% of sika deer. This is the first record of T. gondii infection in wild deer in Japan, and deer and wild boar meat should be cooked well before human consumption.     

Presence of pathogenic Leptospira spp. in the reproductive system and fetuses of wild boars (Sus scrofa) in Italy

Leptospirosis is a re-emerging and globally spread zoonosis caused by pathogenic genomospecies of Leptospira. Wild boar (Sus scrofa) are an important Leptospira host and are increasing in population all over Europe. The aim of this investigation was to evaluate Leptospira spp. infection in the reproductive systems of wild boar hunted in two Italian regions: Tuscany and Sardinia. From 231 animals, reproductive system tissue samples (testicles, epididymides, uteri) as well as placentas and fetuses were collected. Bacteriological examination and Real-Time PCR were performed to detect pathogenic Leptospira (lipL32 gene). Leptospires were isolated from the testicles and epididymides of one adult and two subadult wild boar. Four isolates from the two subadult males were identified as Leptospira interrogans serogroup Australis by MLST, whereas Leptospira kirschneri serogroup Grippotyphosa was identified from the adult testicles and epididymis. Using Real-Time PCR, 70 samples were positive: 22 testicles (23.16%) and 22 epididymides (23.16%), 10 uteri (7.35%), 3 placentas (6.66%), and 13 fetuses (28.88%). Amplification of the rrs2 gene identified L. interrogans and L. kirschneri species. The results from this investigation confirmed that wild boar represent a potential source of pathogenic Leptospira spp. Isolation of Leptospira serogroups Australis and Grippotyphosa from the male reproductive system and the positive Real-Time PCR results from both male and female samples could suggest venereal transmission, as already demonstrated in pigs. Furthermore, placentas and fetuses were positive for the lipL32 target, and this finding may be related to a possible vertical transmission of pathogenic Leptospira.     

Homologous regulation of melanocortin-1 receptor (MC1R) expression in melanoma tumor cells in vivo

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist alpha-melanocyte-stimulating hormone (alpha-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B 16F1 cells, a single injection of 50 to 500 microg alpha-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16-24 h. By continuous administration of alpha-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.