Potential Tick Defense Associated with Skin and Hair Characteristics in Korean Water Deer (Hydropotes inermis argyropus).

The Korean water deer (WD), a predominant wildlife species in South Korea, is listed as vulnerable by the IUCN Red List. Despite belonging to the same family, Cervidae, WD show significantly fewer adult ixodid tick infestations compared to roe deer (RD). Ticks, which cannot fly, engage in questing behavior in natural environments to latch onto hosts. They detect signals like body temperature and host skin chemicals to navigate through the hair coat to the preferred epidermis. In light of this, we performed an extensive comparative study of the skin tissue and hair characteristics of both deer species, focusing on elements contributing to the reduced tick bite incidence in WD. Remarkably, WD exhibited more prominent blood vessels, sebaceous glands, and sweat glands, which are crucial for skin barrier functions (p < 0.005). Moreover, WD had irregular scale patterns on their hair cuticles and possessed hair that was significantly stiffer and 2.83 times thicker than that of RD (p < 0.001). These characteristics potentially impede ticks from reaching the epidermis hair in WD and RD in the context of tick bite prevention. Further investigations in this area could enhance our understanding of tick-host dynamics and contribute to developing preventive measures against tick-borne diseases in other deer species.

Habitat utilization distribution of sika deer (Cervus nippon).

Habitat-specific and movement-related behavioral studies are essential for the development of sustainable biodiversity management practices. Although the number of studies on sika deer is increasing, habitat utilization distribution (UD)-related studies remain limited. In this study, we investigated the habitat UD behavior of sika deer (Cervus nippon) using a literature survey and an experimental study on Suncheon Bonghwasan Mountain, South Korea. We reviewed home range-related literature on sika deer published between 1982 and 2019 in order to assess their estimation methods, study region, and research background. We observed that the number of studies on sika deer has increased. The minimum convex polygon (MCP) has been utilized the most to estimate habitat UD, followed by the kernel density (KD), the Brownian bridge model, and a combination of these methods. The average home ranges (95 % utilization distribution) of sika deer from the literature survey were 236.99 ha and 1183.96 ha using the minimum convex polygon and kernel density approaches, respectively. The five female deer in our experimental study on Suncheon Bonghwasan Mountain had a mean home range of 66.831 ± 15.241 ha using the MCP approach and 78.324 ± 20.82 ha using the KD approach. The UD behavior of sika deer explored in this research is expected to benefit future scholars and policymakers when formulating deer management and wildlife conservation strategies.

Predicting Suitable Areas for African Swine Fever Outbreaks in Wild Boars in South Korea and Their Implications for Managing High-Risk Pig Farms.

African swine fever (ASF) is a highly contagious disease affecting domestic pigs and wild boars, with no effective vaccine or treatment available. In South Korea, extensive measures have been implemented to prevent ASF transmission between wild boars and ASF spillover from wild boars to pig farm sectors, including the search for ASF-infected carcasses in mountainous forests and the installation of fences across wide areas of these forests. To determine the priority search range for infected carcasses and establish pig farm-centered quarantine measures, it is necessary to predict the specific path of ASF outbreaks in wild boars and identify pig farms at high risk of ASF spillover from wild boars. Here, we aimed to predict suitable areas and geographical paths for ASF outbreaks in wild boars using the MaxEnt model and shortest-path betweenness centrality analysis. The analysis identified a high frequency of ASF outbreaks in areas with a suitability value ≥0.4 on the suitability map and in areas within a 1.8 km range from the path on the shortest-path map, indicating these areas were high-risk zones for ASF outbreaks. Among the 5063 pig farms analyzed, 37 were in the high-risk zone on the suitability map, 499 were in the high-risk zone on the shortest-path map, and 9 were in both risk zones. Of the 51 pig farm sectors with a dense distribution of pig farms (kernel density ≥ 8), 25 sectors were in contact with or partially overlapped the high risk zone on the suitability map, 18 sectors were located within the high risk zone on the shortest-path map, and 14 sectors were located within both risk zones. These findings aided in determining the priority range for searches for wild boar carcasses and enabled the establishment of preemptive ASF prevention measures around the pig farming sectors that are at risk of ASF spillover from wild boars.

Genome-Wide Searching Single Nucleotide-Polymorphisms (SNPs) and SNPs-Targeting a Multiplex Primer for Identification of Common Salmonella Serotypes.

A rapid and high-quality single-nucleotide polymorphisms (SNPs)-based method was developed to improve detection and reduce salmonellosis burden. In this study, whole-genome sequence (WGS) was used to investigate SNPs, the most common genetic marker for identifying bacteria. SNP-sites encompassing 15 sets of primers (666-863 bp) were selected and used to amplify the target Salmonella serovar strains, and the amplified products were sequenced. The prevalent Salmonella enterica subspecies enterica serovars, including Typhimurium; Enteritidis, Agona, enterica, Typhi, and Abony, were amplified and sequenced. The amplified sequences of six Salmonella serovars with 15 sets of SNP-sites encompassing primers were aligned, explored SNPs, and SNPs-carrying primers (23 sets) were designed to develop a multiplex PCR marker (m-PCR). Each primer exists in at least two SNPs bases at the 3' end of each primer, such as one was wild, and another was a mismatched base by transition or transversion mutation. Thus, twenty-three sets of SNP primers (242-670 bp), including 13 genes (SBG, dedA, yacG, mrcB, mesJ, metN, rihA/B, modA, hutG, yehX, ybiY, moeB, and sopA), were developed for PCR confirmation of target Salmonella serovar strains. Finally, the SNPs in four genes, including fliA gene (S. Enteritidis), modA (S. Agona and S. enterica), sopA (S. Abony), and mrcB (S. Typhimurium and S. Typhi), were used for detection markers of six target Salmonella serotypes. We developed an m-PCR primer set in which Salmonella serovars were detected in a single reaction. Nevertheless, m-PCR was validated with 21 Salmonella isolates (at least one isolate was taken from one positive animal fecal, and n = 6 reference Salmonella strains) and non-Salmonella bacteria isolates. The SNP-based m-PCR method would identify prevalent Salmonella serotypes, minimize the infection, and control outbreaks.

The functional evolution of termite gut microbiota.

BACKGROUND: Termites primarily feed on lignocellulose or soil in association with specific gut microbes. The functioning of the termite gut microbiota is partly understood in a handful of wood-feeding pest species but remains largely unknown in other taxa. We intend to fill this gap and provide a global understanding of the functional evolution of termite gut microbiota. RESULTS: We sequenced the gut metagenomes of 145 samples representative of the termite diversity. We show that the prokaryotic fraction of the gut microbiota of all termites possesses similar genes for carbohydrate and nitrogen metabolisms, in proportions varying with termite phylogenetic position and diet. The presence of a conserved set of gut prokaryotic genes implies that essential nutritional functions were present in the ancestor of modern termites. Furthermore, the abundance of these genes largely correlated with the host phylogeny. Finally, we found that the adaptation to a diet of soil by some termite lineages was accompanied by a change in the stoichiometry of genes involved in important nutritional functions rather than by the acquisition of new genes and pathways. CONCLUSIONS: Our results reveal that the composition and function of termite gut prokaryotic communities have been remarkably conserved since termites first appeared ~ 150 million years ago. Therefore, the "world's smallest bioreactor" has been operating as a multipartite symbiosis composed of termites, archaea, bacteria, and cellulolytic flagellates since its inception. Video Abstract.

Molecular Identification of Bacillus Isolated from Korean Water Deer (Hydropotes inermis argyropus) and Striped Field Mouse (Apodemus agrarius) Feces by Using an SNP-Based 16S Ribosomal Marker.

Ambiguous, heterogeneous, endospore-forming Bacillus species, notably Bacillus cereus, often produce fatal toxins that threaten human health. We identified Bacillus from wild animal fecal samples (n = 80), including the Korean water deer (n = 25) and striped field mouse (n = 55). Using traditional culture-based methods, 25 animal fecal samples (31.25%; 25/80) were found to be positive for Bacillus species, whereas using molecular techniques, 19 samples (23.75%; 19/80) were found to be positive for the same. In addition, we designed a Bacillus species-specific 16S ribosomal RNA (rRNA) gene marker and utilized it to identify 19 samples by means of PCR amplification and sequencing, using at least one colony from the 19 Bacillus positive samples. The recovered sequences were matched to sequences of three Bacillus species (B. cereus, B. amyloliquefaciens, and B. megaterium) from the GenBank database. Moreover, the phylogenetic tree generated in this study established specific clades for the Bacillus group. In addition, to differentiate between B. cereus, B. anthracis, and B. thuringiensis, we designed a single nucleotide polymorphism (SNP)-based primer by identifying SNPs in the alignment of 16S rRNA gene sequences of B. cereus group strains. The SNPs were used to design primer sets for discrimination between highly similar species from the B. cereus group. The study could be used in surveillance of agricultural fresh-produce-associated Bacillus outbreaks, for accurate identification of each Bacillus species, and in the development of control measures.

Development of Single Nucleotide Polymorphism (SNP)-Based Triplex PCR Marker for Serotype-specific Escherichia coli Detection.

Single-nucleotide polymorphisms (SNPs) are one of the most common forms of genetic variation and as such are powerful tools for the identification of bacterial strains, their genetic diversity, phylogenetic analysis, and outbreak surveillance. In this study, we used 15 sets of SNP-containing primers to amplify and sequence the target Escherichia coli. Based on the combination of the 15-sequence primer sets, each SNP site encompassing forward and reverse primer sequences (620-919 bp) were aligned and an SNP-based marker was designed. Each SNP marker exists in at least two SNP sites at the 3' end of each primer; one natural and the other artificially created by transition or transversion mutation. Thus, 12 sets of SNP primers (225-488 bp) were developed for validation by amplifying the target E. coli. Finally, a temperature gradient triplex PCR kit was designed to detect target E. coli strains. The selected primers were amplified in three genes (ileS, thrB, and polB), with fragment sizes of 401, 337, and 232 bp for E. coli O157:H7, E. coli, and E. coli O145:H28, respectively. This allele-specific SNP-based triplex primer assay provides serotype-specific detection of E. coli strains in one reaction tube. The developed marker would be used to diagnose, investigate, and control food-borne E. coli outbreaks.

The complete mitochondrial genome of Eurasian Sparrowhawk Accipiter nisus (Accipitriformes: Accipitridae).

The mitogenome of the Accipiter nisus is a circular module of 18,352 bp, which consists of 39 genes, containing 2 rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes, 22 tRNA genes, and two non-coding regions (control region and pseudo control region). The mitogenome of A. nisus is composed of 31.3% A, 25.5% T, 30.4% C, 12.8% G, and 76.3% AT. The phylogenetic analysis revealed that A. nisus individuals was well grouped in Accipitridae and more closely related to genus Circus than other Accpiter species.

Genetic integrity and individual identification-based population size estimate of the endangered long-tailed goral, Naemorhedus caudatus from Seoraksan National Park in South Korea, based on a non-invasive genetic approach.

The long-tailed goral (also called the Amur goral) Naemorhedus caudatus (subfamily Caprinae), a vulnerable and protected species designated by IUCN and CITES, has sharply been declining in the population size and is now becoming critically endangered in South Korea. This species has been conserved as a natural monument by the Korean Cultural Heritage Administration since 1968. In this study, using 78 fecal DNA samples with a non-invasive genetic approach, we assessed the genetic integrity and individual identification-based population size for the goral population from Seoraksan National Park representing the largest wild population in Korea. Using the successfully isolated 38 fecal DNA, phylogeographic and population genetic analyses were performed with mitochondrial DNA control region (CR) sequences and nine microsatellite loci. We found seven CR haplotypes, of which five were unique to the Seoraksan population, considering previously determined haplotypes in Korean populations. The Seoraksan population showed higher haplotype diversity (0.777 ± 0.062) and mean number of alleles (4.67 ± 1.563) relative to southern populations in Korea reported from previous studies, with no signal of a population bottleneck. Microsatellite-based individual identification estimate based on probability of identity (PID) indicated a population size of ≥30 in this population. Altogether, we suggest that for future management efforts of this species in the Seoraksan National Park, conserving its genetic integrity as an 'endemic' lineage, and curbing a decrease in its number through mitigating habitat destruction might be key to secure the population for the long term.

Prevalence data of diarrheagenic E. coli in the fecal pellets of wild rodents using culture methods and PCR assay.

Wild animals, such as rodents seem to be competent reservoir of bacteria-borne zoonotic diseases which disseminate in human. We investigated the presence of E. coli, Shiga toxin-producing E. coli (STEC), and Salmonella in the feces of six category wild rodent species (Apodemus agrarius, A. peninsulae, A. sylvaticus, Micromys minutus, Myodes regulus, and R. norvegicus) captured from different agricultural regions in South Korea. Among them, A. agrarius, which account for 65% of total (N = 52) individuals, are most widely distributed and abundant in various agroecosystems in South Korea. The bacterial identification was performed by cultural and molecular methods. In cultural method, the fecal cultures from 26 individuals formed colonies on E. coli-selective EMB agar media. Of them, the fecal cultures from 18 individuals also produced colonies on the Shiga toxin-producing E. coli-selective CT-SMAC agar media as well as the EMB agar media. In molecular method, polymerase chain reaction (PCR) was carried out to detect two virulence genes (stx1 and stx2) of isolated E. coli. The amplified dataset of stx1 and stx2 genes of E. coli were sequenced. In this manuscript, E. coli and STEC were detected but there were no Salmonella species. The wild rodents' data would provide important information on reservoirs of those pathogenic bacteria.

Morphological keys for identifying long-tailed gorals (Naemorhedus caudatus) and population composition in the Osaek Region of South Korea.

The objectives of this study were to select morphological keys for the identification of individual endangered long-tailed gorals through analysis of photographic data and to use these morphological keys to determine the number and population composition of gorals living in the Osaek Region of Seoraksan National Park. Amongst 8149 photos taken using 73 cameras in the Osaek Region, 2057 photos of faces and horns were analysed. The presence and absence of horns, shape of the horns, proportion of the ring to the length of the horn and facial colour pattern were selected as morphological keys to identify individual gorals. To verify the accuracy of the morphological keys for identifying gorals, a blind test was performed on gorals residing in the sanctuary of the Yanggu Goral Restoration Center. The test revealed that the population and age of gorals were discerned correctly by the morphological keys, but there was a 12.5% error in discriminating between sexes in gorals aged over 10 years. Fifty-six gorals were identified from 2057 pictures, based on the morphological keys and methods developed in this study. The population of 56 individuals consisted of 43 individuals aged over 2 years (subadult or adult) and 13 offspring aged less than 2 years, with a ratio of 3.3:1. Of the total 56 individuals, 45% were adults aged 10 years or older, 18% were adults aged 3-10 years, 7% were subadults aged 2-3 years, 23% were offspring aged less than 2 years and 7% were individuals aged 2 years or older, whose age and sex could not be confirmed. The sex ratio of males to females was 1.17:1, with a corrected sex ratio of 1:1 considering the 12.5% error rate for gorals aged over 10 years, amongst the 39 gorals aged over 2 years.

Infections of Intestinal Helminth at Two Species of Field Mice, Apodemus agrarius and A. Peninsulae, in Gangwondo and Chungcheongnam-do, Korea.

Rodents are important reservoirs of diseases affecting people and livestock, and are major sources of parasite contamination of agricultural products. We surveyed the infection status of intestinal helminths in 2 species of field mice, Apodemus agrarius and A. peninsulae, captured in the agricultural fields of Gangwon-do and Chungcheongnam-do, Korea. Total 83 mice (57 A. agrarius and 26 A. peninsulae) were collected in 2 surveyed areas, and the intestines of each mouse were opened with scissors, and then intestinal contents were examined with microscope. Total 6 species of intestinal helminth were detected in 61 (73.5%) out of 83 mice examined. Four species of nematode, i.e., Nippostrongylus brasiliensis, Aspiculuris tetraptera, Heterakis spp. and ascarid, were found in 40 (48.2%), 14 (16.9%), 11 (13.3%) and 13 (15.7%) mice respectively. One species of cestode, Hymenolepis diminuta and 1 unidentified egg were also detected in the intestines of 14 (16.9%) and 1 (1.2%) mice, respectively. Conclusively, this study identified 5 helminth species in the gastrointestinal tracts of wild rodents captured in some areas in central and northern Korea, and N. brasiliensis was the most prevalent (dominant) species rather than zoonotic ones.

Parallel and Gradual Genome Erosion in the Blattabacterium Endosymbionts of Mastotermes darwiniensis and Cryptocercus Wood Roaches.

Almost all examined cockroaches harbor an obligate intracellular endosymbiont, Blattabacterium cuenoti. On the basis of genome content, Blattabacterium has been inferred to recycle nitrogen wastes and provide amino acids and cofactors for its hosts. Most Blattabacterium strains sequenced to date harbor a genome of ∼630 kbp, with the exception of the termite Mastotermes darwiniensis (∼590 kbp) and Cryptocercus punctulatus (∼614 kbp), a representative of the sister group of termites. Such genome reduction may have led to the ultimate loss of Blattabacterium in all termites other than Mastotermes. In this study, we sequenced 11 new Blattabacterium genomes from three species of Cryptocercus in order to shed light on the genomic evolution of Blattabacterium in termites and Cryptocercus. All genomes of Cryptocercus-derived Blattabacterium genomes were reduced (∼614 kbp), except for that associated with Cryptocercus kyebangensis, which comprised 637 kbp. Phylogenetic analysis of these genomes and their content indicates that Blattabacterium experienced parallel genome reduction in Mastotermes and Cryptocercus, possibly due to similar selective forces. We found evidence of ongoing genome reduction in Blattabacterium from three lineages of the C. punctulatus species complex, which independently lost one cysteine biosynthetic gene. We also sequenced the genome of the Blattabacterium associated with Salganea taiwanensis, a subsocial xylophagous cockroach that does not vertically transmit gut symbionts via proctodeal trophallaxis. This genome was 632 kbp, typical of that of nonsubsocial cockroaches. Overall, our results show that genome reduction occurred on multiple occasions in Blattabacterium, and is still ongoing, possibly because of new associations with gut symbionts in some lineages.

Streptozotocin-Induced Autophagy Reduces Intracellular Insulin in Insulinoma INS-1E Cells.

Streptozotocin (STZ), a glucose analog, induces diabetes in experimental animals by inducing preferential cytotoxicity in pancreatic beta cells. We investigated whether STZ reduced the production of intracellular insulin through autophagy in insulinoma INS-1E cells. Typically, 2 mM STZ treatment for 24 h significantly decreased cell survival. STZ treatment led to significant decrease in phospho-AMP-activated protein kinase (p-AMPK) level; reduction in levels of phospho-protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α); significant reduction in levels of p85α, p110, phospho-serine and threonine kinase/protein kinase B (p-Akt/PKB) (Ser473), phospho-extracellular-regulated kinase (p-ERK), and phospho-mammalian target of rapamycin (p-mTOR); increase in levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase; decrease in B-cell lymphoma 2 (Bcl-2) expression; increase in Bcl-2-associated X protein (Bax) expression; increase in levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1; and reduction in production of intracellular insulin. These results suggest that insulin synthesis during STZ treatment involves autophagy in INS-1E cells and, subsequently, results in a decrease in intracellular production of insulin.

Phylogenetic position of the Siberian flying squirrel Pteromys volans based on complete mitochondrial genome sequences.

The mitogenome of the Siberian flying squirrel Pteromys volans is a circular molecule of 16,514 bp, consisting of a control region and a conserved set of 37 genes containing 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes (12S rRNA and 16S rRNA). The mitogenome of the Korean P. volans is AT-biased, with a nucleotide composition of 32.2% A, 30.4% T, 12.5% G, and 24.8% C. The phylogenetic analysis revealed that P. volans is well placed within the tribe Pteromyini (Sciuridae: Sciurinae), which forms a sister clade to the flying squirrels of the genus Petaurista.

Anti-inflammatory effect of torilidis fructus ethanol extract through inhibition of Src.

CONTEXT: Torilidis fructus, fruits of Torilis japonica Decadolle (Umbelliferae), is a medicinal herb traditionally used as a pesticide, an astrictive, or a medicine for various inflammatory diseases. OBJECTIVES: Due to the lack of pharmacological studies on this herbal medicine, we explored the inhibitory activity of torilidis fructus on the macrophage-mediated inflammatory response using its ethanol extract (Tf-EE). MATERIAL AND METHODS: The Griess assay and prostaglandin (PGE2) ELISA assay were conducted with Tf-EE (0-75 µg/mL) and LPS (1 µg/mL) treated RAW264.7 cells in cultured media. Tf-EE pretreated RAW264.7 cells were incubated with LPS for 6 h and semi-quantitative PCR was performed. Reporter gene assays, overexpression of target enzymes and immunoblotting were performed on macrophages to determine the molecular targets of Tf-EE. RESULTS: Tf-EE markedly suppressed the inflammatory response of macrophages, such as lipopolysaccharide (LPS)-induced nitric oxide (NO) and PGE2 production with IC50 values of 35.66 and 62.47 µg/mL, respectively. It was also found that Tf-EE reduced the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 by 80%. Nuclear translocation and activation of nuclear factor (NF)-κB (p65 and p50) were declined by 60% and 30% respectively, and their regulatory events including the phosphorylation of AKT, IκBα, Src, and the formation of complexes between Src and p-p85 were also recognized to be diminished. CONCLUSIONS: The signalling events managed by Src and p85 complex seemed to be critically involved in Tf-EE-mediated anti-inflammatory response. This might suggest that Tf-EE exhibited anti-inflammatory effects through Src-targeted inhibition of NF-κB.

Data on MyoD reduction by autophagy in C2C12 cells.

Autophagy is a highly regulated physiologic mechanism in which cells maintain homeostasis by degrading excessive or unnecessary proteins and damaged or aged organelles through the lysosomal machinery (Yorimitsu and Klionsky, 2005) [1]. MyoD is basic helix-loop-helix (bHLH) transcription factors that regulate myoblast proliferation and myogenic differentiation. MyoD is expressed in adult skeletal muscle (Megeney et al., 1996) [2] and adult fibers (Brack et al., 2005) [3]. MyoD is mainly degraded by the ubiquitin-proteasome system (Floyd et al., 2001) [4] and partly by autophagy (Kim et al., 2012) [5]. Data showed that autophagy decreased MyoD protein in C2C12 cells by Western blotting analysis.

Complete mitochondrial genome of the house bat Pipistrellus abramus (Mammalia: Chiroptera) from Korea.

We determined and annotated the complete mitogenome of the house bat Pipistrellus abramus (Chiroptera: Vespertilionidae) from Korea. The complete mitogenome is a circular molecule of 17,236 bp in length, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding regions (L-strand replication origin and control region). The mitogenome is AT-biased, with a nucleotide composition of 33.7% A, 29.9% T, 23.2% C, and 13.2% G. The phylogenetic analysis revealed that the house bat P. abramus from Korea is well grouped with that from Japan and placed within the genus Pipistrellus clade, which has the noctule bat Nyctalus as sister clade.

Complete mitochondrial genome of the Asian badger Meles leucurus (Mustelidae) from Korea.

The mitogenome of the Asian badger Meles leucurus from Korea is a circular molecule of 16,529 bp, consisting of a control region and a conserved set of 37 genes containing 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes (12S rRNA and 16S rRNA). The mitogenome of M. leucurus is AT-biased, with a nucleotide composition of 33.1% A, 27.9% T, 25.4% C, and 13.5% G. The phylogenetic analysis revealed that the badger M. leucurus from Korea is well grouped with that from China, forming a sister clade to M. meles from Japan.

Complete mitogenome of the red squirrel Sciurus vulgaris (Sciuridae) from Korea.

The mitogenome of the Korean S. vulgaris is a circular molecule of 16,511 bp, consisting of a control region and a conserved set of 37 genes containing 13 protein-coding genes (PCGs), 22 tRNA genes and 2 rRNA genes (12S rRNA and 16S rRNA). The mitogenome of the Korean S. vulgaris is AT-biased, with a nucleotide composition of 32.0% A, 30.9% T, 12.6% G and 24.5% C. The phylogenetic analysis revealed that the Korean red squirrel S. vulgaris is well grouped with the European red squirrel S. vulgaris and formed a sister clade to the Old World flying squirrels of the genus Pteromys.