P < 0.05). MDA levels in the serum of birds fed CSFBP-fermented feed were lower than in the control group (P < 0.05). The CAT activity in the serum of broilers increased after supplementation with 3% CSFBP-Fermented feed (P < 0.05). Supplementing broilers with CSFBP-fermented feed enhanced VH in the ileum, jejunum, and duodenum (P < 0.05). The addition of 3% CSFBP-Fermented feed decreased CD in the jejunum (P < 0.05). The addition of 3% and 5% CSFBP-Fermented feed increased the mRNA expression of ZO-1 and Occludin in the jejunum of broiler chickens and reduced the mRNA expression of IL-6 (P < 0.05). The addition of 3% CSFBP-Fermented feed increased the mRNA expression of Claudin in the jejunum of broiler chickens and reduced IL-1β mRNA expression (P < 0.05). Compared to the control group, all experimental groups exhibited decreased mRNA expression of TNF-α and INF-γ in the jejunal mucosa of the birds (P < 0.05). According to research using high-throughput sequencing of microorganisms' 16S rDNA, and an analysis of α-diversity found that supplementing broilers with 3% CSFBP-Fermented feed decreased the number of bacteria in their cecum (P < 0.05). Bacteroidota was higher in all groups after supplementation with CSFBP-Fermented feed. At the genus level, after addition with 3% CSFBP-Fermented feed, the abundance of Bacteroide and Prevotellaceae_Ga6A1_group were higher than the control group . The abundance of Rikenellaceae_RC9_gut_group and Fusobacterium were lower than the control group . In summary, supplementing the diet with CSFBP-Fermented feed can promote the growth of performance by enhancing intestinal morphology, and barrier function, as well as modulating intestinal inflammatory factors and microbial composition in broilers. This research aimed to investigate the effects of supplements containing fermented feed made from Citri Sarcodactylis Fructus by-products (CSFBP-Fermented feed) on the growth performance, immunological function, and gut health of broilers. 1080 cyan-shank partridge birds aged 47 days were chosen and casually distributed to four groups, each with 6 replicates and 45 birds per replicate. The experimental groups were provided with 1% (group T2), 3% (group T3) and 5% (group T4) of CSFBP-fermented feed in the basic diet, while the control group (group T1) received the basic diet. The findings revealed that supplementation with CSFBP-Fermented feed reduced ADFI and FCR and improved ADG in birds ( Yet, the over-reliance on antibiotics in farming has resulted in large amounts of antibiotics in poultry products, which have become a severe threat to humans and the environment, and most countries have restricted or even prohibited the use of antibiotics as supplements in feed3. Unfortunately, bans on antibiotics have led to reduced growth performance and increased rates of gut disease4. A healthy gut is essential for the function of barriers, microbial community, and nutrient uptake and contributes to enhanced growth and economic efficiency6. Thus, there is a need to work on new green feed supplements to replace antibiotics for poultry health.Antibiotics were frequently utilized to preserve health and enhance growth in the chicken industry8. Fermented feeds combine fermentation engineering and enzyme engineering to break down some large molecules into small molecules, which are easily digested and absorbed and produce more active probiotics, various enzymes, metabolites, multiple vitamins, small active peptides, and growth-promoting factors10. Nowadays, fermented feed is widely used in animal production. Feeding fermented feed enhance broilers' growth capacity, resistance to oxidation and immunity12. Moreover, the studies revealed that feeding fermented feed can benefit the health of poultry by enhancing the intestinal micro-ecological balance and increasing the digestive capacity and intestinal morphology15.Citri Sarcodactylis Fructus by-products (CSFBP) are by-products of further processing of Citri Sarcodactylis Fructus, such as the production of functional beverages, CSFBP extract, fudge, cosmetics, dried fruit, and jam. However, CSFBP contains a certain amount of nutrients and bioactive components, and these bioactive components can enhance the antioxidant capacity and slow down or inhibit the peroxidation reaction; thus, to some extent, CSFBP has some value for feedingTherefore, we supplemented the broiler basal diet with different proportions of CSFBP-Fermented feed for feeding. To study the influence of supplemented CSFBP-fermented feed on growth capacity and biochemical parameters of serum in birds, gut barrier function and intestinal microbiota, to provide data support for the rational use of CSFBP-Fermented feed in poultry breeding, and to offer a scientific basis for broiler healthy farming in the era of antibacterial reduction and replacement.Experimental protocols were approved by the Animal Ethics Committee of the Leshan Academy of Agriculture Sciences (LSNK. No20220701). All the procedures were performed following the Declaration of Basel and relevant policies in China. The study was carried out in compliance with the Animal Research: Reporting of in Vivo Experiments (ARRIVE) guidelines.Our trial started on September 24, 2022. For a 42-day feeding study, 1080 47-day-old male cyan-shank partridge birds had separated into four groups (T1–T4). Each group had six replicates (cages) of 45 chickens. Each replicate of 45 broilers was housed in a single cage (120 cm–60 cm–50 cm). Broilers are free to feed and drink throughout the feeding period. Broilers were housed together in a contained way with natural light (about 12.2 h), a temperature range of 18 to 25 °C, and humidity levels of 45% to 55%. Feeding was done twice a day. Group T1 was fed the basal diet, and T2–T4 were supplemented with 1, 3, and 5% CSFBP-Fermented feed in the basal diet, respectively. All chickens in each replicate were weighed at days 21 and 42, after 12 h of starvation, and ADFI, ADG and FCR were measured according to feed consumption.Lactobacillus Plantarum, Saccharomyces cerevisiae and Bacillus subtilis. Enzyme preparations containing xylanase, β-mannanase, β-glucanase and cellulase. They have all been qualified for marketing and the product standard is No. Q/12JX 4450- 2019. Our fermentation process is to inoculate probiotics and enzymes in a glucose solution 24 h before strain activation. Then all raw materials are crushed and passed through 60 mesh sieves, mixed according to the corresponding ratio, put into the fermentation bag containing a one-way breathing valve, inoculated with the activated bacterium solution in advance, and sealed. The fermentation temperature is 36℃, and the fermentation time is 72 h.The basal feed formulation and nutrient composition of this trial are present in Table 2 and promptly dissected. An intermediate 2 cm slice of the ileum, jejunum and duodenum were acquired and preserved in 4% paraformaldehyde liquid for paraffin sections after the intestinal contents were removed using pre-cooled saline. Finally, the jejunum of each chicken was collected, and the cecum microorganisms were stored in a −80 refrigerator for further assessment.After a 12-h fasting period, one chicken was randomly selected from each repetition, for a total of 24 chickens. First, 5 ml of plasma was drawn from the airfoil sense in a vacuum blood collection tube, left at room temperature for 30 min, and then centrifuged for 10 min at 4 °C at 3000 rpm to extract the serum. The serum was then transferred to a 1.5 ml centrifuge tube and kept at −80 °C in an ultra-low temperature refrigerator with further assessment. The chickens were euthanized using CO16. Briefly, adding 50 ml of standard solution or serum sample to a 96-well plate . The second antigen was added to the wells and labeled with horseradish peroxidase. Then the panels were hatched at 37 °C for 60 min. Chromogen solutions were applied and then maintained in the dark for 15 min at 37 °C after the wells had been cleaned five times with wash water. After adding the stop solution, the absorbance was measured at 450 nm using a multipurpose microplate reader.A kit was used to measure the serum's T-AOC, GSH-Px, SOD, CAT and MDA, and the specific methods and processes were carried out by referring to the manual. The enzyme-linked immunoassay (ELISA) analysis was used to evaluate the levels of serum of IgA, IgM and IgGThe paraformaldehyde-fixed jejunal tissue specimens were cut into longitudinal sections, encased with petrolatum, and sliced (5 μm), and then observed morphologically with hematoxylin and eosin staining. The marked segments were initially viewed under low magnification, and the suitable locations were picked for picture capturing under high magnification, all done using a light microscope. With the aid of the picture analysis program Image ProPlus 6.0, villi length and crypt depth were calculated.Extraction of 50 to 100 mg of total RNA from jejunal mucosa according to manufacturer's instructions . By using micro-UV spectroscopy and 1% gel polymeric electrophoresis, RNA quality and concentrations were determined. Following the instructions, the kit  was used to create the first thread of the cDNA. GADPH was utilized as the internal reference gene, and the used primers are displayed in Table Microbial DNA was extracted from 200 mg cecal content samples taken from all groups using the MagPure Soil DNA LQ kit. The content and integrity of the DNA were determined using a Thermo Fisher Scientific NanoDrop 2000 spectrophotometer and electrophoresis on agarose gels. The universal primer pairs 343F (5′-TACGGRAGGCAGCAG-3′) and 798R (5′-AGGGTATCTAATCCT-3′) were utilized, as well as TksGflflex DNA polymerase . Following that, 30 ml of the reaction mixture was used for PCR amplification of the high variation region V3-V4 in the bacteria's 16S rRNA gene. An Illumina NovaSeq6000 was read using two paired-end read cycles of 250 bases each. OE accomplished the sequencing and profiling of 16S rDNA amplicons .P < 0.05 was chosen as the cutoff for statistical significance. Data are presented in the table as mean and combined SEM. GraphPad Prism6 was used to create images .With IBM SPSS Statistics 23, a one-way analysis of variance (ANOVA) was used to assess significance levels. The Tukey multiple range test was used to compare numerous things. p < 0.05). Throughout the trial period, the 3% addition was highest for ADG and lowest for ADFI and FCR compared to all test groups.Table p > 0.05). Supplementation with 3% and 5% CSFBP-Fermented feed significantly reduced GSH-Px in broiler serum (p < 0.05). The addition of 1% and 3% CSFBP-Fermented feed significantly increased CAT in broiler serum (p < 0.05). The MDA content in broilers supplemented with CSFBP-Fermented feed were significantly smaller than the control group (p < 0.05).As shown in Table P < 0.05). Supplementation with 3% CSFBP-Fermented feed increased CD in the duodenum and decreased CD in the jejunum of broilers (P < 0.05). VH/CD in broiler duodenum, jejunum and ileum was elevated by supplementation with CSFBP-Fermented feed.The microscopic photographs of the small intestine morphology are shown in Fig. ZO-1 and Occludin mRNA expression in the broiler chicken jejunal mucosa (P < 0.05). Supplementation with 3% CSFBP-Fermented feed significantly increased the mRNA expression of Claudin in broiler jejunal mucosa (P < 0.05). Supplementing feed with 3% CSFBP-Fermented feed decreased IL-1β mRNA expression in the jejunum, and supplementation with 3 and 5% CSFBP-Fermented feed significantly reduced the mRNA expression of IL-6 in the jejunum of broilers (P < 0.05). Compared with the control group, all experimental groups showed downregulation of TNF-α and INF-γ mRNA expression in the jejunal mucosa of birds (P < 0.05).The expression of mRNA of immune regulatory and genes tight junction proteins in birds jejunal mucosa were presented in Fig. P < 0.01). The addition of 5% CSFBP-Fermented feed significantly decreased the Shannon and Simpson indices in broilers (P < 0.05). The total microbial profiles of the four groups were compared using the beta diversity analysis, as shown in Fig. P > 0.05).The cecal samples taken from broilers contained several 2900 OTUs. 570 OTUs were shared by the four groups, whereas 445, 347, 378, and 510, respectively, OTUs were unique to the T1, T2, T3, and T4 groups . The Firmicutes were lower in the cecum of broilers supplemented with 3% and 5% CSFBP-Fermented feed than in the control group . There was a decrease in Desulfobacterota in each group after feeding CSFBP-Fermented feed compared with the control group . Campilobacterota and Deferribacterota were highest in the cecum of broiler chickens supplemented with 3% CSFBP-Fermented feed (2.11% and 1.52%).To evaluate the differences caused by the CSFBP-Fermented feed in the cecum microbiota, we analyzed the classification and composition of the cecum microbiota at both the phylum and genus levels Fig. . At the tes Fig. A. BacterBacteroides, Rikenellaceae RC9_gut_group, Prevotellaceae_UCG-001, Prevotellaceae_Ga6A1_group, Clostridia_vadinBB60_group, and Faecalibacterium species dominated the four groups. Supplementation of CSFBP-Fermented feed in broiler diets enhanced the abundance of Muribaculaceae and Mucispirillum and decreased the abundance of Clostridia_UCG-014 (P > 0.05). Supplementation with CSFBP-Fermented feed decreased the abundance of Desulfovibrio (P < 0.05). Supplementation with 3% CSFBP-Fermented feed improved the abundance of Bacteroides and Prevotellaceae_Ga6A1_group  and decreased the abundance of Rikenellaceae_ RC9_gut_group and Fusobacterium .We discovered the top 15 most prevalent genera at the genus level . The 18. In the present study, our data suggest that feeding CSFBP-Fermented feed can increase ADG and decrease ADFI and FCR in broilers. It was demonstrated that providing fermented feed can promote growth performance by facilitating the digestion and absorption of feed in poultry. However, it is interesting to note that our results showed that feeding CSFBP-Fermented feed reduced ADFI in broilers, which is contrary to some studies. The reason may be that the palatability of CSFBP-Fermented feed affects the intake of broilers. Still, the increase in ADG of broilers is another good result, which is that CSFBP-Fermented feed increases the digestion and absorption in broilers, thus enhancing the utilization of feed and promoting growth performance. In conclusion, the specific reasons need further verification.The fermented feed is easier to be digested and absorbed by animals. The anti-nutritional factors in the spread can be removed to enhance the nutritional quality palatability and digestibility of the feed, thus improving the growth performance of poultry21. Our study found a slight increasing trend in the levels of IgA, IgG, and IgM in broiler chickens when adding 3% CSFBP-Fermented feed, but there were no significant differences compared to the control group. This may be explained by the combined effect of probiotics in the fermented feed and active substances in the Citri Sarcodactylis Fructus, which promote the synthesis of vitamins, amino acids, and other substances in the intestine of animals and serve as antigens and nutrients to stimulate and encourage the development of immune organs24. In animals, the production of free radicals is in balance with the antioxidant system in the body, and this balance is crucial in sustaining the health of the animals25. Antioxidant enzymes play a vital role in the body's natural defense against oxidative degradation. Antioxidant enzymes mainly include SOD, GSH-Px and CAT, which can cooperate and scavenge free radicals and reactive oxygen species by preventing peroxide production, avoiding lipid peroxidation, and eliminating metal ions to achieve the purpose of antioxidation28. In addition, MDA is also an important indicator to evaluate the body's antioxidant function and cellular oxidative stress. It is a lipid peroxidation product, and its concentration reflects the extent of internal oxidative damage29. It has been found that feeding fermented pine needles (Pinus ponderosa) improved the activity of serum and liver SOD and GSH-Px and decreased the concentration of MDA in broiler chickens30. Feeding fermented ginkgo improved broiler intestinal health and boosted antioxidant capacity by raising SOD and GSH-Px activity and reducing MDA contents in the jejunum and ileum31. In line with our findings, we discovered that providing CSFBP-Fermented feed boosted SOD and CAT activity while lowering MDA levels in broiler serum. These findings imply that adding CSFBP-Fermented feed to broiler diets is useful for preventing lipid peroxidation. This could result from bacteria and functional ingredients , which could also be an effective mechanism to lessen the load on the enzymatic and non-enzymatic antioxidant systems to lower cellular antioxidant consumption.IgA, IgG, and IgM are important immune molecules in the body and are involved in various immune responses. According to reports, adding supplements to diets with Bio-Fermented Malic Acid, fermented feed, and Fermented Fava Bean By-Products can increase serum IgA, IgG, and IgM levels in poultry32. VH and CD are significant measures of digestive and absorption function in the intestine. An increase in VH can increase the contact area of nutrients with the intestine and promote nutrient absorption. At the same time, a decrease in CD indicates a shorter maturation cycle of intestinal epithelial cells, increased secretion and enhanced digestive capacity. The VH/CD of both can also reflect the development of the intestinal tract and digestive and absorption ability to a certain extent34. Fermented feed was reported to raise chicks' VH and VH/CD of the gut 35. Similarly, many studies have shown that feeding fermented feeds can facilitate the digestion and absorption in poultry by improving intestinal morphology38. Our study found that supplementation of CSFBP-Fermented feed reduced CD in broiler chickens. We speculate that this may be due to the ability of CSFBP-Fermented feed to regulate the balance of intestinal microbiota, provide beneficial metabolites, and improve intestinal barrier function, thereby lowering the depth of intestinal crypts and maintaining intestinal health. Our study found that supplementation with CSFBP-Fermented feed can increase the gut morphology of broiler chickens by increasing intestinal VH and reducing CD, thus promoting the body's ability to digest and absorb nutrients and improving growth performance.Animals primarily digest and absorb nutrients through the gut, and the shape of the intestine can provide insight into the animal's intestinal health and capacity for nutrient absorptionZO-1, and tight junctions are cellular gaps between neighboring epithelial cells39. The function of Claudin is mainly to regulate the permeability of the barrier structure; Ocludin can maintain tight junction stability and small intestinal permeability; ZO-1 can maintain epithelial cell polarity and gut barrier permeability41. Our study found that supplementation with CSFBP-Fermented feed increased the relative mRNA expression of Claudin, Occludin and ZO-1 in broiler jejunum. Consistent with this study, supplementation of the basal diet with Fava Bean By-Products, Laetiporus sulphureu, and Fermented Soybean Meal increased intestinal tight junction protein in poultry42. Supplementation with CSFBP-Fermented feed to the diet can enhance the tight junctions of gut epithelial cells by promoting the expression of intestinal tight junction protein-related genes, which has a specific effect on the maintenance of the mechanical barrier function of the broiler intestine. The IL family has a key function in maintaining the integrity of the gut mucosa, controlling gut immunology, and modulating several pathways involved in apoptosis or inflammation. IL-1 and IL-6 are among the most significant regulators of inflammation44. TNF-α, a crucial pro-inflammatory factor, stimulates macrophages and controls inflammation and death of cells. IFN-γ linked to infection, and having excessive amounts of it can cause autoimmune diseases46. Our results show that CSFBP-Fermented feed can exert anti-inflammatory effects by inhibiting the overexpression of IL-1β, IL-6, TNF-α and IFN-γ. This is consistent with previous studies that feeding fermented feed can regulate the mRNA expression levels of inflammatory factors in poultry47.The important protein components in tight junctions are claudin, occludin, and 48. Many factors affect the microbiology of the cecum, including feed composition, breeding environment, age, genetics and other factors50. The gut microbiota controls a variety of host metabolic processes, influencing immune response, protecting gut wall integrity, and secreting helpful enzymes and metabolites52. The OTU data show the number of bacterial communities. The OTU data represent the number of bacterial communities; the alpha diversity index is frequently used to describe the variety and abundance of bacterial diversity. The Shannon and Simpson indices represent the microbial diversity in the samples54. Surprisingly, the addition of CSFBP-Fermented feed lowered the Simpson ACE, Shannon and Chao1 microorganism indices in the broiler cecum, indicating a drop in microbiota diversity and richness in the broiler cecum. This is related to our classification in the phylum and genus of intestinal microorganisms. We discovered that Bacteroidota and Firmicutes make up most of the microbes in the cecum microbiota of birds. These organisms contribute to further than 80% of the abundances of the gut microbiome and are crucial for sustaining gut balance55. The abundance of Bacteroidota in the broiler cecum was higher in the CSFBP-Fermented feed than in the control group, and Bacteroides species can use complex polysaccharides as a source of carbon and energy for fermentation and metabolism, releasing fermentation end-products that also provide nutrients and other beneficial properties for the host56. Therefore, we hypothesise that the higher abundance of Bacteroidota after supplementation with CSFBP-Fermented feed may help broilers adapt to the ration and improve the digestibility of nutrients. It was found that the bacterial community associated with body weight, the body weight of geese was highly correlated with members of the Bacteroidaceae, that is, members of the Bacteroidaceae were significantly enriched in geese with high body weight53. Similarly, this research found that broilers fed CSFBP-Fermented feed had higher body weight and ADG, suggesting that Bacteroidetes bacteria may be positively associated with broiler growth performance. Supplementation with CSFBP-Fermented feed reduced the abundance of harmful bacteria Desulfobacterota in the broiler cecum57 and increased the abundance of some beneficial bacteria such as Campilobacterota and Deferribacterota in the broiler cecum59.The cecum of broiler chickens is an essential region for fermenting and digestion, which is critical to poultry health and immunity, and is the subject of a gut microbial studyBacteroides and Rikenellaceae_RC9_gut_group, similar to Zhu's study54, but inconsistent with Wang's results60. The differences in results may be related to chicken breed, diet, age and rearing environment54. Supplementation of 3% CSFBP-Fermented feed increased the abundance of Muribaculaceae and Prevotellaceae_Ga6A1_group in broiler chickens. A study finds potential benefits of Muribaculaceae in reducing inflammation, inhibiting harmful bacteria and promoting anti-cancer immunity61. Our data showed that feeding CSFBP-Fermented feed significantly decreased the abundance of Desulfovibrio and Fusobacterium. Desulfovibrio genus causes the host to mitigate dietary sulfites and sulfates, aand sulfated mucopolysaccharides, leading to the production of a cytotoxic compound hydrogen sulfide62. According to studies, those with ulcerative colitis had a higher prevalence of Desulfovibrio and a more pronounced inflammatory response63. In broilers treated with CSFBP-Fermented feed, we discovered that the species richness of Desulfovibrio was decreased, indicating that the CSFBP-Fermented feed may exercise its anti-inflammatory action by lowering the quantity of sulfate-reducing Desulfovibrio64. These findings demonstrate that adding CSFBP-Fermented feed to a diet can enhance the number of some helpful bacteria while reducing the prevalence of some harmful bacteria, benefiting the intestinal health of broilers. The reason for the decrease in microbial diversity and abundance after supplementation with CSFBP-Fermented feed may be that supplementation with CSFBP-Fermented feed promoted the colonization of thcolonisation by some probiotic bacteria and inhibiting the growth of some pathogenic bacteria, thus decreasing microbial abundance. However, these causal relationships need to be further investigated.Each group's predominant flora at the genus level is In conclusion, our research indicates that supplementation of CSFBP-fermented feed is beneficial for broiler chickens. In the current context of advocating for the prohibition or reduction of antibiotic use in animal production, we incorporated Citri Sarcodactylis Fructus by-products as part of the fermented feed formulation, which differs from conventional fermented feed. Specifically, CSFBP-fermented feed includes certain advantageous functions derived from Citri Sarcodactylis Fructus, such as antibacterial, anti-inflammatory, antiviral, and immunomodulatory properties. This demonstrates that CSFBP-fermented feed exhibits certain advantages over regular fermented feed. However, due to the complexity of CSFBP-fermented feed itself, which includes components like Citri Sarcodactylis Fructus, probiotics, enzyme preparations, and metabolic by-products, it is difficult to determine which specific component promotes the growth of broiler chickens. In future studies, researchers should conduct more in-depth research to unveil whether it is a single component or the synergistic effect of multiple components that promotes the growth of broiler chickens.The current findings suggest that supplementing CSFBP-fermented feed can enhance growth performance by improving gut morphology, and barrier function, modulating the intestinal inflammatory response and intestinal microbial composition in broilers. Combining all the data analysed, a 3% supplementation level is more advantageous.


Sarcoptes scabiei, and sarcoptic mange is reported in several felid species in the scientific literature. However, the historic classification of Sarcoptes mites into host-specific varieties does not include S. scabiei var. felis. It is unclear whether sarcoptic mange transmission in felids involves canids, other sympatric species, or exclusively felids. This study aimed to characterize the genetic structure of S. scabiei mites from domestic cats (Felis catus) and Eurasian lynx (Lynx lynx carpathicus), comparing them with Sarcoptes mites from sympatric domestic and wild carnivores. Ten Sarcoptes microsatellite markers were used to genotype 81 mites obtained from skin scrapings of 36 carnivores: 4 domestic cats, one dog (Canis lupus familiaris), 4 Eurasian lynx, 23 red foxes (Vulpes vulpes), and 4 grey wolves (Canis lupus lupus) from either Italy, Switzerland or France. Two genetic clusters of S. scabiei with a geographical distribution pattern were detected: mites from cats originating from Central Italy clustered with those from sympatric wolves. In contrast, all the other mites from Switzerland, France and Northern Italy clustered together. These results strengthen the previously advanced hypothesis that genetic variants of S. scabiei have a predominant geographic-related distribution with cryptic transmission patterns. These patterns may rely on the interactions between different hosts living in the same ecological niche rather than a simple infection among hosts belonging to the same taxon, reinforcing the idea that the S. scabiei historic classification into “var” might have little ongoing relevance.Domestic and wild felids are considered suitable hosts for the parasitic mite  Sarcoptes scabiei, affecting 200 million people each year  [Lynx pardinus) [Felis silvestris silvestris) [Lynx lynx isabellinus) [Uncia uncia)  [Panthera pardus), lion (Panthera leo) and cheetah (Acinonyx jubatus) [Sarcoptic mange, also referred to as scabies, is a highly contagious skin disease caused by the burrowing mite HO, 2020 , 17 and HO, 2020 . Concernathicus) , Iberianardinus) , and Eurvestris)  in Europellinus)  and snowspected)  in Asia;jubatus)  in EasteFelis catus), more frequently affected by “feline mange”, a similar condition caused by the related burrowing mite Notoedres cati [Sarcoptic mange is considered a rare disease in domestic cats  in continental Europe [Eudorcas thomsonii and cheetahs in Eastern Africa [Similarly, in wild felids, the source of l Europe ) or in an Africa ). Molecun Africa .Sarcoptes scabiei has been traditionally classified into host-specific varieties. Still, growing molecular evidence shows that the mere taxon-oriented approach is insufficient to embrace the issue’s complexity [Sarcoptes strains affecting different host species and to track transmission pathways more efficiently and objectively. Amongst these tools, microsatellite markers have been shown to be more informative than other markers in characterizing the populations’ “strains” of S. scabiei affecting wildlife in Oceania [mplexity . In this Oceania , Europe  Oceania , 36, 41, Oceania , Asia [2 Oceania , and Sou Oceania , 42, somSarcoptes isolates from feline hosts have never been characterized at a molecular level. This leaves open speculation on the possible transmission pathways and reservoir hosts and possible “strain”-specific diversity regarding pathogenicity. This study aims to investigate the molecular profile of Sarcoptes mites obtained from domestic and wild felids from different European countries and to compare them with Sarcoptes mites from sympatric and allopatric wild carnivores (Felidae and Canidae families), using microsatellite markers.In line with the infrequent occurrence of sarcoptic mange in domestic cats, Skin scrapings from four domestic cats and a dog were collected in Italy, France and Switzerland during a dermatological examination due to severe itch and crusted skin lesions. In contrast, skin samples were also obtained from wildlife with skin lesions compatible with sarcoptic mange and submitted for post-mortem examination .All samples were stored at −20 °C in 70% ethanol tubes until mite isolation and later shipped to the Department of Veterinary Sciences of Turin, Italy. Morphological criteria were applied for the preliminary identification of collected mites . For eacS. scabiei mites  following the PCR protocol of Soglia et al. [TM. The software GeneMapper 4.0  allowed the allele calls and microsatellite visualization. After molecular analysis, mites that did not fulfil the required criteria  were excluded from the genetic analysis. Two population genetics analyses were applied to the 81 mite microsatellite outputs: i) Bayesian clustering and ii) principal component analysis (PCA). The first one requires Hardy-Weinberg equilibrium (HWE), while no assumptions are required for the PCA. Descriptive statistics, such as observed and expected heterozygosis , allelic richness (R) and HWE analysis, were carried out with software R 4.0 using the packages Adegenet 2.1.3 [P-values for the HWE test were based on Monte Carlo permutations of alleles. The Bayesian assignment test was computed with the software STRUCTURE 2.3.4. Burn-in and run lengths of Markov chains were 10,000 and 100,000, respectively, and five independent runs for each K (for K = 1–20) were run. The ancestry model selected was the admixture type. Clusters were estimated as suggested by Evanno [DNA was extracted from individual mites following the HotSHOT Plus ThermalSHOCK technique . Then, aa et al. . Capillaet 2.1.3 . P-valuesupplementary material, Table S1). Observed heterozygosity ranged between 0.07 (Sarms 37) and 0.88 (Sarms 41)  to 11 (Sarms 45). Eleven private alleles were found across the ten microsatellite loci, distributed among six loci . Deviation from HWE was detected only in Sarms 34 (arms 41) .The Bayesian assignment test revealed the presence of two geographically separated clusters . One cluThe results of the PCA are displayed in S. scabiei with a geographical distribution pattern. Cat-derived mites from Central Italy clustered with those obtained from sympatric wolves. By contrast, all the other mites from Switzerland, France and Northern Italy clustered together.The main finding of this study is the identification, using microsatellite markers, of two genetic clusters of European S. scabiei have a geographic-related distribution adding to the expected host-related distribution [These results align with previous evidence, though referring to different animal models, that genetic lineages of ribution . Interspribution , 23, 31.S. scabiei exclusively embraces host-specific varieties. It instead supports the view that various mammal species sharing the same environment have opportunities for direct or indirect contact , which may allow the sharing of pathogens [This finding differs from the classical idea that athogens , 32.S. scabiei have been traditionally associated with various hosts, a felid-specific variety  has never been described. The explanation is possibly the infrequent occurrence of sarcoptic mange in domestic cats, in which notoedric mange, caused by Notoedres cati, is predominant. Nonetheless, in the last few decades, an increasing number of sarcoptic mange cases in both domestic and wild felids have been reported [While numerous species-related varieties of reported , 20, 35.S. scabiei [felis of S. scabiei may not exist. This conclusion needs confirmation by further molecular analyses based, in parallel, on microsatellite markers and innovative genomic techniques [Recently, nuclear molecular markers such as microsatellites have been proven valuable for investigating population genetic differences and putative transmission pathways within  scabiei , 31, 33.chniques , 22, 46.et al., 2004 performed a genetic characterization  of Sarcoptes mites from six out of 25 infected cats involved in a single outbreak in Sweden. These authors noted that “the mites had DNA sequences identical to S. scabiei from naturally infected dogs and Swedish wildlife”. These results agree with our findings , nor in the Northern chamois (Rupicapra rupicapra). In these areas, the disease has been detected only in smaller and rarely preyed animals, such as the red fox [Unlike what was observed in two large wild felids in Eastern Africa , no preyern Alps , where s red fox , 30 and  red fox .Sarcoptes epidemiology in felid hosts. Our results, far from suggesting that domestic cats and wolves from Central Italy infected each other by direct contact , show that the same S. scabiei strain circulates in different carnivore hosts living in the same geographical area. It seems reasonable to assume that infected dogs or red foxes represent the missing link between wolves and cats in Central Italy  class of genetic markers, this study may be considered baseline data in the unexplored field of felid taly see and 2. InSarcoptes mites circulating in European carnivore communities can put additional pressure on species or populations of conservation concern, such as the Iberian lynx and the European wild cat (not included in this study). In these wild felids and other European wild felids not included in this study, the recognition of canids (and most likely the red fox) as the expected source of infection may have practical consequences in the planning of delicate conservation and management interventions, such as restocking and reintroductions.While sarcoptic mange may not always represent a threat to domestic animals, numerous cases resulted in zoonotic transmission to the owners or members of the family in contact with the infected cat . MoreoveSarcoptes strains as those involving sympatric canids. A specific geographical pattern may reveal the transmission pathways of Sarcoptes mites in the investigated carnivore hosts rather than a “host species-related only” pattern. Thus, the continued use of the term “var” in the past scientific literature referring to Sarcoptes host-specific subspecies or “strains” may be outdated or even misleading, appearing of little relevance. Accordingly, we believe that the contemporary understanding of the broad and sometimes unexpected host associations of S. scabiei should cautiously lead to the dismission of the historic nomenclature based on questionable mite morphological differences and the exclusive view of strict host association [In conclusion, our results suggest that domestic and wild felids in Italy and neighbouring countries are affected by the same ociation .https://www.parasite-journal.org/10.1051/parasite/2023012/olm.The supplementary material of this article is available at Results of the Hardy–Weinberg equilibrium test at locus (rows) and populations (columns) showing P values from the Monte Carlo test. P values less than 0.05 (*) and 0.01 (**) are considered significant.


FDOPA uptake rate (Ki), fractional volume of free-water (FW), and iron-sensitive R2* relaxometry were quantified within nigrostriatal regions. Inter-group differences (PD vs HS) were studied using non-parametric statistics and complemented with Cohen’s d effect sizes and Bayesian statistics. Correlation analyses were performed exploring biomarker dependencies and their association with bradykinesia scores. PD patients exhibited a significant decline in nigrostriatal dopaminergic activity, being post-commissural putamen (−67%) and posterolateral SNc (−11.7%) the most affected subregions within striatum and SNc respectively. Microstructural alterations (FW) were restricted to the hemisphere corresponding to the most affected side and followed similar spatial gradients as FDOPA Ki . R2* revealed no relevant significant changes. FDOPA and FW were correlated within the posterolateral SNc, and clinical severity was associated with FDOPA Ki loss. The asymmetry between striatal and SNc changes for both dopaminergic depletion and microstructural degeneration biomarkers is consistent with a neurodegenerative process that begins in the striatal terminals before progressing toward the cell bodies in the SNc.Loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and a profound reduction of striatal dopamine are two hallmarks of Parkinson’s disease (PD). However, it’s unclear whether degeneration starts at the neuronal soma or the striatal presynaptic terminals, and how microstructural degeneration is linked to dopaminergic loss is also uncertain. In this study, thirty de novo PD patients and twenty healthy subjects (HS) underwent 6-[ Striatal dopaminergic loss typically follows a posterior-to-anterior gradient, with the greatest deficit in the posterior part of the putamen contralateral to the clinically affected hemibody7. Moreover, the highly focal clinical presentation of parkinsonism, typically manifested first in the hands, suggests a focal somatotopic origin, probably within the sensorimotor putamen7, although no somatotopical organization has been recognized in the SNc9. However, despite growing evidence supporting a distal origin of the pathological process at the terminals16, a pattern of structural and metabolic changes within the nigrostriatal system supporting this hypothesis is still lacking.The onset of cardinal features  of Parkinson’s disease (PD) is classically associated with a 60-80% dopaminergic denervation17, which quantifies the contribution of non-constrained water molecules to the MRI signal. This MRI metric has become a promising biomarker for the assessment of nigral pathology in PD and other neurodegenerative disorders21. In addition to FW imaging, iron mapping is another MRI-derived biomarker that may be a key to understanding PD nigrostriatal pathology and neuronal vulnerability23. Iron radicals play an important role in the synthesis and metabolism of dopamine24, but on the other hand, they also participate in multiple biochemical reactions associated with neurodegeneration26. Several studies have reported iron overloads in the SN of PD patients, suggesting that iron accumulation within the nigrostriatal system might constitute a potential vulnerability factor in the pathogenesis29. Iron contents can be assessed semi-quantitatively using MRI by exploiting the paramagnetic properties of this metal, which induces magnetic field inhomogeneities that reduce local transverse relaxation time (T2*). As a result, the intensity of the T2* MRI signal, or its inverse R2* (1/T2*), will be proportional to the iron load30.Magnetic resonance imaging (MRI) allows the assessment of brain microstructural changes in vivo, particularly by diffusion-weighted imaging (DWI). Pasternak and colleagues used a diffusion bi-tensor model to introduce free-water (FW) imaging18F]-fluoro-L-dopa, a.k.a. FDOPA), microstructural integrity (FW), and iron accumulation (R2*) within several regions of the nigrostriatal system, including spatial divisions of the SNc and striatum. Further, we tested for a correlation between clinical impairment and the degree and topography of nigrostriatal alterations.Here we aimed to study structural and metabolic alterations within the nigrostriatal system at the clinical onset of motor features in a group of de novo PD patients. We assessed the degree and spatial pattern of dopaminergic denervation  respectively, d > 6.5, P < 0.01, K > 2). In the caudate both divisions displayed a significant FDOPA Ki decay in the MAS , whereas only the anterior division reached significance in the LAS . In the SNc, FDOPA Ki decline was close to 10%  in the posterolateral division in both hemispheres and in the anteromedial division in the MAS . See Supplementary Table Striatal dopaminergic impairment followed a gradient of highest to lowest percentage FDOPA d = 0.96, P < 0.01, K > 0.5) but was also noticeable in the anterior putamen , both caudate divisions , and posterolateral SNc . No significant changes were found in any region of the LAS. Interestingly, the pattern of FW increments paralleled the topography of FDOPA Ki reduction in the striatum and SNc  Table . This waSNc Fig. .Fig. 2Nid = 0.98, P < 0.05, K > 1). Further, we found R2* alterations that approached significance in opposite directions for the anterior caudate  and the anteromedial SNc , with decreased and increased R2* values in PD, respectively. See Supplementary Table R2* was significantly decreased in the posterior caudate of the MAS, showing a modest but significant decrease in PD compared to HS  in the PD group, indicating that higher values of FW were associated with lower dopaminergic activity  and with the caudate for the anteromedial SNc division (P < 0.05). This is indicative of a higher FDOPA deficit in the posterolateral SNc co-occurring with a greater deficit in the putamen.Interestingly, FDOPA activity for the PD group demonstrated a nigrostriatal segregation favoring correlations with the putamen for the posterolateral SNc division  and in the posterolateral SNc (P < 0.05) and this motor quantitative assessment. FW and R2* showed no significant association with clinical motor impairment.Bradykinesia quantitative scores showed a significant negative correlation with FDOPA MAS Fig. . Thus, aKi and FW in several regions of the nigrostriatal system to show that microstructural alterations mirror the spatial pattern of dopaminergic loss within the striatum and SNc early in the evolution of PD. These changes are restricted to the brain hemisphere contralateral to the clinically symptomatic side. Thus, the contralateral post-commissural putamen and posterolateral SNc were the most altered regions in both imaging techniques. Further, we found a direct correlation between microstructural integrity and dopaminergic activity in the posterolateral SNc.We have assessed FDOPA Ki decline in the putamen followed a marked posterior-to-anterior gradient of degeneration31. FDOPA Ki loss was also significant in the caudate nucleus, but to a lesser magnitude, with a relatively homogeneous loss across the structure33. Decreased FDOPA Ki values were also shown bilaterally in the SNc. A mild dopaminergic decline has been described previously in the SNc using dopamine active transporter36, vesicular monoamine transporter type 238, or FDOPA radioligands40. Here we provide further details by splitting the SNc into anteromedial and posterolateral tiers. There is a greater loss in the posterolateral SNc, which agrees with common understanding of SNc neuronal vulnerability in PD. The posterolateral SNc includes the nigrosome-1, a substructure placed in the ventrolateral tier of this nucleus that is known to be affected in the earliest stage of the disease41. Since neurons of the posterolateral SNc project to the posterior putamen42, this fits well with the striatal pattern of depletion. These findings confirm in vivo the concept of selective vulnerability of the posterolateral nigroputaminal projection at the onset of PD43.In line with previous PET studies, the FDOPA 45.Dopaminergic activity was significantly associated between nigrostriatal regions. These associations were enhanced in PD as compared to HS, a result that might be explained by the global impact of disease onset in the nigrostriatal circuit. Interestingly, in PD there was a nigrostriatal segregation favoring dopaminergic connectivity between the anteromedial SNc and caudate on one side and the posterolateral SNc and the putamen on the other, further indicating a pattern of selective vulnerability for nigrostriatal dopaminergic degeneration48, a feature that was correlated with FDOPA Ki reduction in the same region. Accordingly, this increment in nigral FW is congruent with the observed dopaminergic decline49, presumably reflecting neuronal loss. In contrast, no correlation was found between nigral FW increment and striatal dopaminergic denervation, which may be a consequence of the very short time since diagnosis (<12 months), large redundancy of striatal dopaminergic innervation, and compensatory capacity, particularly evident in mild PD patients.FW analysis revealed significant nigrostriatal alterations in the most affected hemisphere. In line with previous studies, our PD cohort exhibited an FW increment in the posterolateral SNc47. Nevertheless, there is clear evidence regarding this occurrence using either classical DTI52 or other multicompartmental models54. We show here that FW is sensitive to microstructural alterations within the striatum, but restricted to the symptomatic side, so it could be considered as a potential biomarker for clinical onset and/or progression in PD. It is noteworthy, that striatal cell loss is not a pathological feature of PD55, but a marked reduction in axonal arborization and dendritic pruning are directly associated with dopaminergic denervation. Accordingly, the diffusion of water molecules restricted by tissue architecture is facilitated, perhaps explaining our finding of increased FW in early PD57. On the other hand, the absence of significant microstructural alterations on the LAS stands in sharp contrast to the extensive dopaminergic hypofunction seen there, indicating a possible early disturbance in the dopaminergic activity prior to axonal degeneration or neuronal loss. Indeed, it has already been suggested that SNc neurons and dopaminergic fibers may survive for some time after losing their dopaminergic function, perhaps being able to recover this capacity if treated early enough16. Further investigation needs to be done, but if confirmed, this feature could potentially unlock new opportunities for novel restorative therapies, further emphasizing the relevance of an early detection of the disease.There also was a widespread increment in striatal FW in our PD cohort, which exhibited a similar posterior-to-anterior gradient as for FDOPA loss, i.e., the post-commissural putamen was the most affected region. Few studies have previously examined striatal alterations in PD with the bi-compartmental diffusion model, and none of them reported significant findingsKi, the highest increment in FW matched the most denervated region, i.e., the posterior putamen, which suggests a relationship between these two processes. Previous evidence points to two principal structural mechanisms that may be linked to striatal denervation to explain this relationship: (1) a loss of synaptic terminals and dendritic arborization58; and (2) a significant reduction of dendritic spines59. Both alterations might well facilitate water diffusion and therefore lead to higher FW values. This is a potentially remarkable aspect that should be validated first experimentally and, secondly, throughout disease progression in PD patients.Although there was no direct correlation between striatal FW and FDOPA 61. In this cohort, we failed to demonstrate significant iron alterations within the SNc, but we did encounter a tendency towards increased iron in the anteromedial SNc. We don´t think the discrepancy with previous results stems from technical factors. We believe it is probably related to the limited clinical severity of our patients who, in addition, were drug naive62. Thus, reduced cell loss and lack of dopaminergic metabolic activation (by levodopa treatment) might explain the insufficient statistical evidence of iron abnormalities in the SNc.Previously, several studies have detected iron accumulation using iron-sensitive MRI techniques6. Importantly, this allowed us to test the validity of our biomarkers in a minimally symptomatic hemisphere. Based on disease characteristics, we should also consider the relatively younger age of our PD cohort. Several studies have described disease-specific traits as a function of the age at PD onset64. Accordingly, we cannot exclude that some of the differences relative to controls might be specific to young PD onset. These differences might explain the lack of findings in the literature when using FW to assess microstructural integrity in the striatum47 or our inability to detect significant alterations in the SNc when assessing iron content. In addition, the possibility that some of the patients included here would actually suffer from a different neurodegenerative condition, principally MSA, is acknowledged. This is always a possibility in studies of recently diagnosed patients. We limited largely this false positive inclusion by studying only patients with very focal motor onset including resting tremor in a high proportion, and a correlatively focal dopaminergic loss in the posterior putamen characteristic of early PD7. The median age of onset was also lower than is typical for MSA, PSP, etc., and they exhibited no atypical symptomatology . Finally, the now available evolution (after 4-5 years of follow-up) has not revealed any atypical manifestation in any patient. Another limitation might be the feasibility of FDOPA as a precise marker of nigrostriatal dopaminergic denervation in early PD. FDOPA activity might be upregulated in early-stage PD3. At the time of the study, other radiotracers such as vesicular monoamine transporter type 2, which might provide better sensitivity in early stages of PD and could lead to more robust intermodal correlations, were not available. Nevertheless, FDOPA has previously demonstrated a significant capacity to define precise topography of striatal dopaminergic integrity loss7. This, in addition to its direct relationship to clinical impairment, seems sufficient to accept its use as an adequate marker of dopaminergic integrity in early-stage PD. Finally, SNc delineation with MRI is challenging. Defining SNc boundaries with respect to the SNr or the subthalamic nucleus is extremely difficult if not impossible with the currently available neuroimaging methodology. Multiple approaches have been proposed based on either manual or semiautomatic methods using different MRI sequences, but no gold-standard exists so far65. Here, we applied a fully automated atlas-based division using anatomical landmarks as reference (red nucleus), which provided a robust inter-subject allocation and minimized observer bias. Further studies should aim to demonstrate the impact that specific parcellation strategies have on these biomarkers.The present study had several limitations. The number of patients in our cohort is moderate in size. This group of de novo PD patients had predominant motor manifestations, with very asymmetric and focal presentationKi vs +10.9% in FW), they differed significantly in the putamen (-67% vs +20%). This nigrostriatal asymmetry for dopaminergic denervation and microstructural abnormalities might indicate greater pathological alteration of the striatum (the posterior putamen mainly) versus the SNc. In addition, the lack of significant changes in the microstructure of the minimally symptomatic side along with a net decrease in FDOPA levels suggests a predominant and initial loss of striatal dopaminergic innervation preceding major SNc neuronal loss. Furthermore, although non-statistically significant (P = 0.11), there were some hints pointing to minor microstructural alterations in the putamen (~11.5% in FW) of the LAS, whereas no sign of change was observed in the SNc (~0). Although we cannot rule out the possibility that neurodegeneration may begin simultaneously in both the striatum and the SNc, these results altogether support the contention of a relatively focal axo-synaptic onset of neurodegeneration in PD16. Further multimodal studies are needed to conclusively verify the focal pathological onset of PD in the putamen, but as a corollary, our findings would suggest the convenience of considering the posterior putamen as the primary target for putative neurorestorative therapies68.In conclusion, we show here that early PD is associated with a specific anatomical pattern of dopaminergic and microstructural changes in the nigrostriatal system as evaluated by FDOPA PET uptake and MRI free-water imaging. In comparison to the SNc, the striatal putamen was significantly more affected in both FDOPA and FW imaging. Thus, even though the percentage change in the posterolateral SNc was comparable in magnitude between dopaminergic and microstructural biomarkers 6. The study protocol was approved by the Ethics Committee of HM Hospitales (protocol number: 16.10.0993-GHM). All participants provided written informed consent in accordance with local regulations. Demographic features are described in Table Thirty de novo PD patients and twenty HS matched for age, sex, and handedness were included in this study. Participants were enrolled between June 2016 and October 2020 at the University Hospital HM Puerta del Sur . The inclusion criteria for the PD group were: de novo patients with less than 12 months of disease progression after diagnosis and with unilateral motor impairment, i.e., signs were predominantly confined to one hemibody. The diagnosis of PD was made according to the UK Brain Bank Clinical Criteria by three neurologists specialized in movement disorders . Neurological examinations included the Movement Disorders Society Unified Parkinson’s Disease Rating Scale Part-III (MDS-UPDRS-III). Bradykinesia tests were complemented with quantitative assessments using inertial measurement units (IMUs) 70. Twenty-two activity timeframes were reconstructed  with an effective resolution of 2.09 × 2.09 × 2.03 mm3. Simultaneously to the PET acquisition, the MRI protocol included: (a) A 3D T1-weighted (T1w) image acquired using a magnetization-prepared rapid acquisition gradient echo (M-PRAGE) sequence ; (b) Single-shell DWI based on a single-shot 2D spin-echo sequence , with 4 b value = 0 s/mm2 scans at the beginning of the sequence. Four additional b value = 0 s/mm2 images but with reverse-phase encoding (posterior-to-anterior) direction were also acquired in order to correct EPI distortions; (c) A 3D multi-echo gradient echo (multi-GRE) sequence recorded with the following specifications: TR: 60 ms; 10 echoes; TEs: [4.36:46.12] ms; inter-echo spacing: 4.64 ms; flip angle: 20°; FoV: 230 mm; in-plane matrix: 256 × 256; in-plane resolution: 0.9 × 0.9 mm2; slice thickness: 2 mm; 60 slices.Whole-brain MRI and FDOPA PET scans were collected using a hybrid 3 T mMR-Biograph system . PET sessions were carried out with subjects at rest and after at least 6-hours of fasting. Following an intravenous injection of ~5 mCi of FDOPA, PET data were acquired in list-mode for 90 min. Images were reconstructed with an ordered subset-expectation maximization algorithm and were smoothed by applying a 3D isotropic Gaussian kernel with 4 mm full width at half maximum. Scans were then corrected for attenuation using a 4-compartment MR-based map derived from a dual-echo Dixon-based sequence , that incorporates bone information using a model-based segmentation algorithm71. T1w scans were corrected for intensity bias72 and skull-stripped with BET-FSL (FMRIB Software Library) v6.074. Then, anatomical images were registered nonlinearly to the ICBM-152 2009c Nonlinear Symmetric (ICBM-152) template computing an affine transformation followed by a diffeomorphic symmetric normalization75. Magnitude images from multi-GRE acquisitions were used to generate iron-sensitive maps. Relaxometry R2* maps were reconstructed by applying nonlinear fitting of the complex monoexponential equation with the autoregressive algorithm76 implemented in the MEDI toolbox77. R2* maps were rigidly registered to T1w space using as reference the magnitude image of the first echo. DWI pre-processing included data denoising78, motion, susceptibility-induced, and eddy current-induced distortion corrections (topup and eddy in FSL)81, and bias field correction72. Non-brain tissue was removed with BET-FSL before DWI pre-processing. Corrected DWI scans were rigidly registered to T1w space using the first b = 0 s/mm2 image as reference. FW maps were estimated with the Dipy library82 by fitting the bi-tensor model with the regularized gradient descent method and applying the hybrid initialization strategy described by Parker et al. 83. To reduce motion effects during PET exams, activity frames were realigned within subjects applying a rigid-body transformation with the Mc-FLIRT tool in FSL84. Voxel-based FDOPA uptake rate maps (described as constant Ki [min−1]) were estimated using the Patlak graphical method85 and taking the average time-activity curve from an occipital lobe mask as a reference. Intensity bias correction and coregistration steps were conducted with ANTs  v2.3.186.DICOM data were converted to NIFTI format using the dcm2niix package87 (Prob. ≥ 0.001). The segmentation was partitioned into anteromedial and posterolateral divisions setting a vertical cutting plane across the centroid of the red nucleus   Fig. . SNc ROI88. Pre- and post-commissural divisions were defined by applying a cutting plane set across the anterior commissure  applying a nearest-neighbor interpolation strategy.Whole-putamen and whole-caudate masks were segmented from structural T1w with the FMRIB integrated registration and segmentation tool (FIRST)ure Fig. . In ordeU tests separately for MAS (DS in HS) and LAS (nDS in HS). Statistical significance was set at P < 0.05. The Cohen d was used to assess effect size. Complementary to P values, Bayes factors (BF) were computed and reported on a logarithmic scale (K = log10(BF)89) to improve text readability. Logarithmic BFs were interpreted as follows 0 < K ≤ 0.5 (barely worth mentioning); 0.5 < K ≤ 1 ; 1 < K ≤ 2 (strong); K > 2 (decisive)89.Mean values were computed within ROIs, that were sorted according to the side of the predominance of motor signs (PD) or hand dominance (HS). Consequently, ROI data were divided into MAS and LAS and dominant and non-dominant sides (DS and nDS). Statistical comparisons between groups were carried out by applying the Mann–Whitney 6. Classical and Bayesian statistics were respectively computed with MATLAB  and JASP v.0.16.1 .Non-parametric Spearman correlation analyses were performed to study the inter-regional interactions and the associations between imaging metrics. All correlations were tested independently for HS and PD groups. Results were adjusted for multiple comparisons using the Storey FDR method. Finally, imaging metrics were compared contralaterally with unilateral bradykinesia aggregated scoresFurther information on research design is available in the Supplementary materialReporting Summary


The understanding of structural defects in basal-faceted sapphire ribbons was improved through X-ray imaging at a synchrotron source. The combination of phase contrast and X-ray diffraction makes it possible to visualize and characterize both gas voids and dislocations in the bulk of the ribbons grown by the Stepanov–LaBelle technology. Dislocations were directly related to gas voids. X-ray diffraction topography was employed to investigate the distribution, configurations, and character of the dislocations. The formation of voids of irregular shapes was detected by large-area mapping with spatial resolution in the μm range. Computer simulations of the experimental phase contrast images of microvoids were performed. The sizes of the spherical microvoids were determined. The results are discussed with reference to the available data on the emission of dislocations from the voids. The evolution of the shape, size, and arrangement of the voids during growth provides clues on the formation of block structure in basal-faceted sapphire ribbons. X-ray topography (XRT) and phase contrast imaging (PCI) methods are optimally combined with each other at the third-generation synchrotron radiation (SR) sources. Natural narrow angular collimation of SR is essential for high angular resolution of X-ray topographs. At the same time, the high spatial coherence makes it possible to observe (through the interference phenomenon) an additional phase shift variable over the cross-section of the beam that has passed through an inhomogeneous object ,2.PCI in combination with XRT exploits instrumental setups on special imaging beamlines, which allow the registration of both types of images using monochromatic or white SR beams. In addition, various X-ray detectors are available for recording such images. Unique facilities provide real-time experiments that shed some light on the effects of growth, solidification, or melting of crystalline materials ,4,5.Moreover, XRT and PCI can be applied sequentially with strict reference to the same area of a specimen. In this case, the spectrum and detectors change depending on the purpose of the experiment. For Bragg adjustment using the white SR beam, so that diffraction conditions are satisfied for a set of Bragg planes, a flat detector is the ideal choice. A large-area panel made with CMOS technology registers several reflections from different crystal planes at once. However, the best results are obtained with high-resolution X-ray films. As for the intensity variation caused by a variable phase shift, it is recorded using a charge-coupled device (CCD) with a micrometer pixel size. First, the X-ray intensity is converted into visible light from a crystal scintillator. Then the lens magnifies the transmitted light onto the detector. A correspondence between crystal lattice defects and inhomogeneities  is established by comparing topographs with phase contrast images ,7,8,9,10Real crystals are not uniform across their area. To make good products, it is important to establish not only the types but also the distribution of defects. Only the PCI technique ,2 with aThe present paper describes the investigation of gas voids and dislocations in sapphire crystals grown by the edge-defined film-fed growth (EFG) method invented by H. LaBelle and even earlier by A.V. Stepanov ,12. Our In earlier works, it was observed that gas voids played an important role in the generation of dislocations in sapphire ribbons ,17. A moThe growth of basal-faceted sapphire ribbons by the Stepanov/EFG method took place in a thermal zone where heat shields were used. The growth rate was 1 mm/min. Laser adjustment of the seed relative to the pulling device and the shaper allowed the achievement of a high orientation accuracy. The orientation of ribbon surfaces deviated only slightly (1.0 ′–3.0′) from the (0001) plane. Optical microscopy showed no blocks. The as-grown surfaces were smooth and mirror-like. Specimens were cut perpendicular to the growth direction . For typical values ν = 0.21 and ρ = 1, this resulted in crσ ≈ G/10. This characteristic value was extremely high and not available under the crystal growth conditions.Lubarda et al.  considerL is the length of the void edge. Since the largest edge of a faceted void is roughly equal to the void radius, L ≈ R, for R = 10 μm and b = 0.5 nm, (4) results in crτ = 5 × 10−5 G.Yan et al.  suggesteG ≈ 150 GPa, which would provide us the value of crτ ≈ 7.5 MPa. This value is approximately 10 times higher than the magnitude of the thermal stresses in growing sapphire crystals in the pre-melting state [At relatively low temperatures, the shear modulus of sapphire is about ng state . Howeverng state .Thus, one can conclude that dislocations can be generated by the edges of faceted voids under the action of the thermal stresses in growing sapphire crystals. The inhomogeneous distribution of these stresses across the growing crystals  is respoHigh technical standards for basal-faceted sapphire ribbons intended for windows, mirrors, and substrates make it necessary to improve single crystallinity and perfection of these products. XRT and PCI is a powerful combination to understand the relationships between the defects and inhomogeneities in crystals. The topographs and phase contrast images obtained with monochromatic SR beam allowed us to directly relate dislocations with gas voids.By using large-area mapping of voids, we found that once nucleated, they tended to group and to spread across the width of the ribbon. When the amount of voids in the growing ribbon increased, they spread along the growth direction as well. The resulting distribution pattern was similar to a “grid” structure. Single spherical microvoids did not possess a dislocation generating activity. Moreover, the determination of void sizes using computer simulations revealed a low activity of larger spherical voids as well. In contrast, dislocations were associated with the strain fields of nonspherical cavities. The analysis of the data suggested that the voids united to form larger aggregates, which eventually developed into cavities of heterogeneous shape and slightly irregular surface. Particularly, the XRT data showed that the dislocation density increased and that the density of the dislocation sources became much higher in association with the “grid” pattern, especially in the vicinity of the dense groups of voids. At the initial stage of plastic deformation, no other mechanisms were observed.g·b analysis revealed basal dislocations with 〈Dislocation semiloops located near the centers of their generation were shown by XRT. A topographic


Linum genetic resources in global genebanks, evaluation for agro-morphological traits, stress tolerance, and nutritional profiling to promote their effective use for sustainable production and nutrition enhancement in our modern diets.Linseed or flaxseed is a well-recognized nutritional food with nutraceutical properties owing to high omega-3 fatty acid (α-Linolenic acid), dietary fiber, quality protein, and lignan content. Currently, linseed enjoys the status of a ‘superfood’ and its integration in the food chain as a functional food is evolving continuously as seed constituents are associated with lowering the risk of chronic ailments, such as heart diseases, cancer, diabetes, and rheumatoid arthritis. This crop also receives much attention in the handloom and textile sectors as the world’s coolest fabric linen is made up of its stem fibers which are endowed with unique qualities such as luster, tensile strength, density, bio-degradability, and non-hazardous nature. Worldwide, major linseed growing areas are facing erratic rainfall and temperature patterns affecting flax yield, quality, and response to biotic stresses. Amid such changing climatic regimes and associated future threats, diverse linseed genetic resources would be crucial for developing cultivars with a broad genetic base for sustainable production. Furthermore, linseed is grown across the world in varied agro-climatic conditions; therefore it is vital to develop niche-specific cultivars to cater to diverse needs and keep pace with rising demands globally. Linseed genetic diversity conserved in global genebanks in the form of germplasm collection from natural diversity rich areas is expected to harbor genetic variants and thus form crucial resources for breeding tailored crops to specific culinary and industrial uses. Global genebank collections thus potentially play an important role in supporting sustainable agriculture and food security. Currently, approximately 61,000 germplasm accessions of linseed including 1,127 wild accessions are conserved in genebanks/institutes worldwide. This review analyzes the current status of  Linum usitatissimum L.) has been cultivated for seed oil and stem fiber since ancient times across the world. Globally, six countries  are the major producers  an overview of the origin, domestication, taxonomy, and gene pool; (2) the status of global ex-situ collections of Linum sp., (3) trait-specific accessions identified through germplasm evaluation for agro-morphological traits, nutrition profiling, and biotic and abiotic stress tolerance in target environments to promote the effective use of these materials; (4) enhancement of the use of trait specific germplasm through the application of recent advances in genomic resources, molecular and biotechnological tools; and (5) documentation and access to germplasm/information through a global exchange system to facilitate transboundary flow.Linseed has emerged as a well-recognized nutrition-rich food because of its high omega-3 fatty acid  content, dietary fiber, high quality protein, and lignans. With respect to omega-3 fatty acids, linseed is considered one of the richest plant-based sources and has around 55% ALA of the total fatty acids , 8. It at cancer . Daily ct cancer , 18. A st cancer  since pet cancer . Hence, Vavilov  describeL. usitatissimum, originated from the wild ancestor L. bienne in geographic isolation. An independent domestication event in Central Asia (Indo-Afghan region) resulted in the development and production of fiber varieties as reported by Duk et al. ; drought and cold hardiness (L. perenne); resistance to linseed bud fly and Alternaria blight ; oil, fiber quality, and yield improvement (L. bienne), number of tillers (L. strictum); resistance to rust ; and medicinal use as purgative (L. cartharticum L.)  for ornamental use in floral arrangements  , 60–62. ex situ collections were presented by Maggioni et al. . The Plorldwide .Fusarium wilt) in multilocation-year environments and reported significant phenotypic variation in both fiber and oil accessions. Similarly, Worku et al. , plant height (17–130 cm), and thousand seed weight (2.8 to 11.5 g), were reported . You et u et al.  evaluateu et al.  evaluateu et al. . In paleu et al.  in Canadu et al.  in TurkiBiochemical characterization and evaluation of linseed for nutritional traits have been performed around the world. Green and Marshall  studied Linseed also has high protein content which generally varies from 10.5 to 31% , 11. Fla−1) than North American cultivars (90.6–246.1 cSt mL g−1). The scarcity of information on the chemical composition and functional properties of flax mucilage has limited its use in industries  was carried out by Diederichsen et al. , who scrdustries . HoweverFusarium oxysporum f. sp. lini has been recognized as the most devastating disease worldwide resulting in 80 to 100% loss in yield , Lucern caterpillar (Spodoptera exigua Hubn.), tobacco caterpillar (Spodoptera litura Fabr.), and Bihar hairy caterpillar , appear as sporadic and region-specific pests with high incidence in some cropping seasons. Bud fly (Dasyneura lini Barnes) is a key pest of this crop in Asia, particularly India, Pakistan, and Bangladesh, while it appears as a pest of less economic significance for flaxseed/fiber flax in Europe , followed by landraces , breeding lines , and cultivars .Different marker systems, such as AFLP, RAPD, ISSR, IRAP, SSR, and SNPs, have been employed in linseed specifically for studying the genetic diversity , 220–222p < 0.01) between genetic and geographic distances in the whole collection. Overall, the moderate to low genetic diversity reported in different studies on linseed using varied marker systems are as anticipated and could be accounted for the self-pollinated nature of the crop.Genetic diversity analysis of 168 linseed accessions predominantly of Indian origin was conducted using 50 SSR loci, which unraveled a total of 337 alleles . The meaFusarium wilt resistance in flax using a doubled-haploid population, two AFLP markers (afB13 and afXR6) were found tightly linked to flax rust resistance  markers on 15 linkage groups with a total length of 1483.25 cM and a mean distance of 0.63 cM between two adjacent markers  have been extensively employed in flax for the genetic dissection of complex traits. In the early years of last decades, SSR markers have been used for association mapping of seed weight trait in linseed, which identified five SSR markers associated with the trait . Later, ay genes , 214. GWay genes . ML-GWASay genes . GWAS waay genes . In a siay genes . Detaileay genes . The idede novo assembly of the flax genome sequence of the cultivar CDC Bethune was made available, having an estimated 81% genome coverage from 302 Mb non-redundant sequences . The evaluated germplasm and trait-specific accessions have been published through catalogs, workshop proceedings, and notification of germplasm registration  maintained by the Plant Genetic Resources of Food and Agriculture Organization, Rome, Italy hosts information on major flax collections.stration . The Invstration  and fibestration  were pubstration . FIBexDBstration .Linum genebank collections through comprehensive phenomics and genomics for the identification of germplasm traits relevant to climate resilience and sustainability is presented in http://eurisco.ecpgr.org), the CGIAR system Wide Information Network for Genetic Resources , and the Germplasm Resources Information Network  of United states Department of Agriculture (USDA), has helped a lot. On a global scale, GENESYS (https://www.genesys-pgr.org/) portal integrates data from the website as a single data entry point for users to mine genetic diversity and order germplasm of interest. Currently, it contains information on around 50% of the global accessions; of which, around 24,555 accessions belong to Linum sp., and this information is continuously evolving over time  from Department of Biotechnology (DBT), Government of India.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ancient DNA studies have begun to explore the possibility of identifying identical DNA segments shared between historical and living people. This research requires access to large genetic datasets to maximize the likelihood of identifying previously unknown, close genetic connections. Direct-to-consumer genetic testing companies, such as 23andMe, Inc., manage by far the largest and most diverse genetic databases that can be used for this purpose. It is therefore important to think carefully about guidelines for carrying out collaborations between researchers and such companies. Such collaborations require consideration of ethical issues, including policies for sharing ancient DNA datasets, and ensuring reproducibility of research findings when access to privately controlled genetic datasets is limited. At the same time, they introduce unique possibilities for returning results to the research participants whose data are analyzed, including those who are identified as close genetic relatives of historical individuals, thereby enabling ancient DNA research to contribute to the restoration of information about ancestral connections that were lost over time, which can be particularly meaningful for families and groups where such history has not been well documented. We explore these issues by describing our experience designing and carrying out a study searching for genetic connections between 18th- and 19th-century enslaved and free African Americans who labored at Catoctin Furnace, Maryland, and 23andMe research participants. We share our experience in the hope of helping future researchers navigate similar ethical considerations, recognizing that our perspective is part of a larger conversation about best ethical practices. The search for genetic connections between ancient and living individuals benefits from access to large genetic databases such as those maintained by genetic testing companies. We discuss the ethical issues we considered during our recent study of African Americans from Catoctin Furnace, Maryland, who were compared with 23andMe, Inc’s genetic database. The remains of the Catoctin individuals have been under the stewardship of the Smithsonian Institution since 1980 following their excavation from the African American Cemetery at Catoctin Furnace in advance of planned highway construction. Preliminary anthropological analyses of the historical Catoctin individuals were performed following their excavation to determine the identity of those buried in the undocumented cemetery,,In 2016, CFHS received a Maryland Heritage Areas Authority grant to undertake a forensic re-analysis of the Catoctin individuals,,,,,In the next phase of the project, this research team sought to investigate the ancestral origins, familial relationships, and legacy of the Catoctin individuals through genetic analysis. Members of the Reich Laboratory at Harvard University were invited to join the study team based on their expertise in aDNA recovery and population genetic analyses. Sampling for aDNA was carried out with the approval of the Anthropology Collections Advisory Committee at the Smithsonian Institution, and the sequenced aDNA were reported in a technical noteWe addressed these ethical considerations in a robust way that respects core academic principles through a formal written agreement between Harvard University and the Howard Hughes Medical Institute (HHMI), which were responsible for generating and sharing the Catoctin dataset, and 23andMe, which was responsible for comparing the Catoctin data with the 23andMe database. During the course of the study design and contract negotiations, we identified three key areas that warranted special consideration: data sharing, study reproducibility, and return of results.,,,,To date, the vast majority of aDNA data have been made fully publicly available in accordance with the principles of open science.While the best data-sharing policies for aDNA data may be context specific, all authors agree that a scenario in which a for-profit company—or any research or commercial entity that may benefit financially, either directly  or indirectly , by restricting access to data—retains exclusive, unrestricted access to an aDNA dataset following study completion would be inappropriate. Instead, clear access guidelines should be created that apply to all parties who would like to access the data. In some for-profit companies, like 23andMe, there is a distinction between that organization’s research and their commercial activity. Thus, even if data are available for non-commercial purposes only, this need not prevent research-focused collaborations with for-profit organizations. Regardless of the data-sharing policy that is implemented, it is critical to ensure that the details and implications of this policy are thoroughly communicated to all stakeholders involved in the study, as was done as a part of Harney et al.The collaboration agreement between Harvard, HHMI, and 23andMe ensures that any non-public aDNA dataset shared with 23andMe can only be used for research and publication purposes. The data can only be used by 23andMe for other purposes after it is made fully publicly available. In the case of the Catoctin project, the terms of the sampling agreement set forth by the Smithsonian Institution required the Catoctin aDNA dataset to be made fully publicly available upon publication, or within three years of sampling. To meet the terms of this agreement, we released a technical report describing the Catoctin data in June 2022,The 23andMe genetic database, like many other private genetic databases, is subject to restrictions on individual-level data sharing that make publishing fully reproducible studies challenging.Through a series of discussions between researchers and legal advisors at Harvard, HHMI, 23andMe, and later editors at the journal where Harney et al. was published, we developed a two-phase approach to maximize the ability of researchers to independently replicate the results of our study: (1) we included genetic data drawn from several publicly available datasets, including the 1000 Genomes ProjectIn recognition of the importance of involving community stakeholders in the research process, the formal collaboration agreement enabled sharing of preliminary 23andMe research results not only with the external research partners at Harvard and HHMI who signed the agreement but also with other research partners, including those at the Smithsonian, and members of relevant stakeholder communities, including CFHS, AARCH, and previously identified descendants of the Catoctin individuals.We also recognize that 23andMe’s re-contactable research cohort presents an opportunity to explore options to return results not only to stakeholder communities with known ties to the Catoctin individuals but also to the research participants whose genetic information was used in the study—an option that is not typically available for studies performed using publicly available datasets. Although the research consent process makes it clear to 23andMe research participants that they should not expect to have individual-level results returned to them as part of the research process, it leaves open the possibility that research could aid in the development of future 23andMe features through which results could be returned.Although the aims of Harney et al. were purely research focused, over the course of the study, we considered how a feature or report in the 23andMe user interface could be used to help realize one of the original goals of CFHS and AARCH: to identify and foster a Catoctin-descendant community. In what follows, we discuss some issues that we believe would be critical for genetic ancestry companies to address when designing a product feature that reports IBD connections shared between customers and ancient or historical individuals.Although use of re-contactable research cohorts in studies like Harney et al. provide a unique opportunity to return results to research participants, participants may not wish to learn about these connections for a variety of reasons. For instance, in the case of Catoctin, learning about genetic connections to a direct ancestor or distant relative who was enslaved could be a painful experience that evokes known family trauma or reveals an unknown personal connection to slavery in the United States. In the case of Harney et al., while community stakeholders expressed an interest in learning about any potential genetic connections they might share with the Catoctin individuals, they were also clear that they would like to be given the opportunity to choose to receive this information rather than have it shared with them without warning. When returning results to research participants or other customers in their database, genetic ancestry companies should give customers the opportunity to actively opt in to receive this information and provide adequate information to help customers anticipate what they might learn by doing so. In making this recommendation, we acknowledge that users who opt not to receive this information from genetic ancestry companies directly may still learn about their likely genetic connections to historical individuals via relatives who do choose to opt in or explore their connections using other means. Our recommendation to include an opt-in step before returning results to customers mirrors the consent that customers at 23andMe and other genetic ancestry companies give before receiving information about living genetic relatives.While identifying a Catoctin-descendant community using a genetic approach was one of the goals of Harney et al., genetic connections are only one of a variety of ways through which stakeholders can be connected to historical communities and may not even be the most important form of connection. Other forms of connection include non-genetic familial relationships through shared culture or heritage or through a collective kinship bond that does not depend on recent shared ancestry. For instance, key community stakeholders associated with the Catoctin individuals self-identify as members of a “collective” descendant community that was intentionally created by the CFHS and AARCH to honor and protect the Catoctin individuals.When returning results to research participants who share a genetic connection to a historical population, it could be tempting for genetic ancestry companies to emphasize these genetic connections over the many other ways through which stakeholders can be connected to a historical group. However, when returning results to research participants or other customers, genetic ancestry companies should be explicit that genetic connections are not necessarily more important than other types of connections. Research on genetic connections to historical individuals has the potential to contribute to the biologization of identity, whether intentionally or not. Thus, when returning results, genetic ancestry companies should take care to identify and describe the presence of existing stakeholder communities. In the case of Catoctin, existing stakeholders have been explicit that they are eager to engage with genetic relatives of the Catoctin individuals. In other cases, genetic relatives who lack any other connection to a historical group may not be welcomed by the existing stakeholder community on the basis of their genetic results alone.When designing product features that reveal genetic connections to historical people, it is critical to use approaches that are optimized to work on aDNA data. Although many population genetic tools are capable of generating output when applied to aDNA data, it should not be assumed that these results are meaningful unless the performance of those tools on aDNA data has been rigorously assessed—paying particular attention to the impact of missing data, C-to-T misincorporations, low-confidence genotype calls, and, when applicable, imputation errors.,,,,In many cases, the identification of a genetic connection to a historical individual may impact customers in profound ways—particularly in cases where customers may have sought out genetic ancestry testing in order to fill gaps in their family and community history. For instance, many descendants of enslaved Africans in the United States have turned to genetic ancestry testing to learn about their family history.Genetic ancestry companies should also recognize that returning inaccurate results or returning results in a confusing way may actively harm community stakeholders, people or organizations who serve as stewards for the human remains, those who serve in other roles that involve educating the public about specific historical individuals or sites, or broader public understanding. In the case of Catoctin, while groups like CFHS, AARCH, and researchers at the Smithsonian Institution are committed to helping potential descendants and other stakeholders learn about Catoctin, they should not have an added responsibility of helping customers of genetic ancestry companies interpret their genetic results. Reports generated by genetic ancestry companies should be created with enough educational content and detail so that customers with shared genetic connections can interpret their results without the need for guidance from outside organizations. In fact, genetic ancestry companies may wish to consult with members of these organizations to ensure that any product features that involve these historical populations are represented accurately and clearly. But in doing so, they should again be careful not to place the burden of responsibility for providing comprehensible results on these organizations.,,,,,,,,,,,We believe that joint analysis of historical genomes and data in genetic databases controlled by for-profit genetic ancestry companies represents an exciting new direction for aDNA research. As aDNA research continues to leverage the power of ever-growing datasets, all parties involved in this work must make a concerted effort to predict ethical issues that may arise and proactively design an ethically sound strategy to ensure that sensitive genetic data remains protected. The continued application of aDNA technology to new contexts is inevitable, and we are encouraged by the robust conversation about best ethical practices that provided a foundation for this work.