In hippocampal cocultures containing neurons and mixed glia, we observed that rCD300f-Fc, but not control IgGs induced neuronal death. In accordance, in vivo studies performed by injecting rCD300f-Fc or control IgGs into rat or WT or CD300 KO mice neocortex, showed an increased lesioned area after a penetrating brain injury. Interestingly, this neuronal death was dependent on glia, and the neurotoxic mechanism did not involve the increase of proinflammatory cytokines, the participation of NMDA receptors or ATP release. However, exogenous addition of glial cell line-derived neurotrophic factor (GDNF) prevented this process. Taken together, our results suggest that CD300f modulates neuronal survival in vitro and after a penetrating brain injury in vivo and that CD300f inhibition alters microglial phenotype generating a neurotoxic microenvironment.Emerging evidences suggest that immune receptors participate in diverse microglial and macrophage functions by regulating their immunometabolism, inflammatory phenotype and phagocytosis. CD300f, a TREM2-like lipid sensing immune receptor, that integrates activating and inhibitory cell-signalling pathways, modulates inflammation, efferocytosis and microglial metabolic fitness. In particular, CD300f overexpression was described to be neuroprotective after an acute brain injury, suggesting a role for this immune receptor in neurotrophic interactions. Thus, we hypothesised that CD300f modulates neuronal survival through neuron-microglial interactions. In order to study its biological function, we used in vitro and in vivo approaches, CD300f The CD300 family of immune receptors consist of several activating and inhibitory members2. The existence of a CD300-like molecule in ancient vertebrates and the maintenance of its function predicts an essential role of this family in innate immunity4. On the other hand, CD300f has a particular interest given its ability to mediate activating and inhibitory signals in myeloid innate immune cells6 and its modulatory role in microglial immunometabolic phenotype and synaptic pruning7. In line with its role in modulating inflammatory phenotypes, it has been shown that CD300f mediates anti-inflammatory signaling by decreasing mast cell degranulation and allergic responses8 and counteracting macrophage activation by interfering with several TLR signaling cascades9. On the contrary, the antibody-dependent activation of CD300f in microglia cultures potentiates lipopolysaccharide (LPS)/TLR4 mediated responses10. Moreover, CD300f ligands are present in the CNS in vivo and in nervous system primary cultures11, and CD300f overexpression after an acute brain injury is neuroprotective11. However, the role of CD300f signaling in cell–cell neurotrophic interactions is not well understood.Immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). They primarily function by adjusting the threshold and duration of myeloid cell responses1. In accordance, mounting evidence showed that CD300f has been associated with a neuroprotective response against Tau pathology12 further highlighting its pivotal role in nervous system function in both health and disease13. Interestingly, CD300f is among the most up-regulated genes in brain microglia/macrophages after several proinflammatory insults such as intraperitoneal LPS injection14, spinal cord injury15, or with age in a mouse tauopathy model12. Moreover, its upregulation has been associated to promyelinating microglia after a demyelinating stimuli16. The inhibition of CD300f using a soluble rCD300f-Fc fusion protein delayed peripheral nerve regeneration after a sciatic nerve injury17. Genetic variants of CD300f have also been associated to non-classical inflammatory functions of microglial and potentially CNS barrier-associated macrophages (BAM), leading to the modulation of neuropsychiatric conditions such as major depressive disorder and anxiety18. In spite of all these accumulated functional data indicating a role for CD300f and its ligands in neuronal survival, the underlying cellular mechanisms are not well understood.The CD300f immune receptor shares many characteristics with TREM2, a key immune receptor for determining the phenotype of microglia and macrophages, and one of the main risk factor genes for the development of Alzheimer’s Disease (AD) and Nasu Hakola Disease−/−) and the rCD300f-Fc fusion protein which interrupts the endogenous interaction between CD300f receptor and its ligands, both in vitro and in vivo17. By using hippocampal cocultures containing neurons and glia, we observed that CD300f inhibition promotes neuronal death compared to controls. This effect was dependent on glia, because no cell death was detected in enriched hippocampal neuronal cultures treated with rCD300f-Fc. Moreover, we found that conditioned media derived from rCD300f-Fc-treated cocultures induced neuronal death when applied to enriched neuronal cultures. However, conditioned media derived from enriched glial cultures treated with rCD300f-Fc did not affect neuronal survival. In accordance with these results, in vivo studies performed by injecting rCD300f-Fc or control IgGs into wild type (WT) rat or mouse neocortex showed an increased lesioned area. However, no effect on the lesion volume was observed after injecting rCD300f-Fc in CD300f−/− mice, suggesting that the effect of the rCD300f-Fc fusion protein is specific. Taken together, our data suggest that CD300f plays a key role in neuroprotection and that neuron-glia interactions are essential in this process, probably by promoting the release of gliotransmitters. Moreover, CD300f inhibition may alter microglial phenotype generating a neurotoxic microenvironment.To explore the role of CD300f in neuroprotection, we used CD300f deficient mice (CD300f19. Coverslips were coated by adding a 5 mM acetic acid solution containing 5 μg/ml poly-d-lysine (Sigma-Aldrich) and 0.4 mg/ml rat-tail collagen. Mixed glial cultures were plated with BME-based culture media (GibCo) containing 10% fetal calf serum (GibCo), Glutamax (GibCo), 20 mM d-glucose (GibCo), HEPES (1%), pyruvate (1%) and penicillin/streptomycin (GibCo). After 6–7 days in vitro (DIV), dissociated hippocampal cells were plated onto a glial monolayer and maintained in Neurobasal-based culture media (GibCO) containing B-27 (GibCo), Glutamax, 20 mM d-glucose and penicillin/streptomycin. After 24–48 h AraC  was added to minimize proliferation. Cultures were used for experiments at 14–15 DIV20. Conditioned media were generated by treating mixed glial cultures or cocultures at 12 DIV for 72 h with rat CD300f-Fc  or control IgG (1.0 μg/ml). Enriched hippocampal neuronal cultures were prepared by seeding 150,000 neurons in 12 mm coverslips, which were manually counted by using a Neubauer counting chamber with trypan blue to exclude dead cells. After 12 DIV, they were treated for 72 h with rCD300f-Fc or control IgG, or with conditioned media diluted 1:2 alone or with GDNF; 1.0 ng/ml), CPP (3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid, a selective N-methyl-d-aspartate (NMDA)-type receptor antagonist; 100 μM), or CBX .Animal care and protocols were approved by the Committee of Ethics in Animal Research (CHEA-UDELAR), Uruguay. Dissociated hippocampal neuron-glia cocultures were prepared from P0–P1 rat pups as described previously with minor modificationsCells were fixed in 4% paraformaldehyde , 15 min at room temperature (RT), permeabilized with PBS containing 0.1% TritonX-100 , and blocked in PBS (Sigma-Aldrich) containing 0.2 M glycine, 10% FBS (Sigma-Aldrich), and 0.1% TritonX-100 (Sigma-Aldrich), for 1h at RT. For estimating cell numbers, the following primary antibodies were added in PBS containing 5% FBS and incubated for 2h at RT: mouse anti-βIII Tubulin , mouse anti-APC , rabbit anti-IBA1 , or rabbit anti-GFAP . After three washes with PBS-T cells were incubated with secondary fluorescently conjugated antibodies Alexa Fluor 488 or 594 (Invitrogen) and DyLight 649 (Jackson ImmuneResearch). Negative controls, without primary antibody incubation, were made to rule out non-specific staining. DAPI  was used for nuclei detection. As an assay for evaluating the specificity of rCD300f-Fc neurotoxicity, the following primary antibodies were used: mouse anti-mouse myeloid-associated immunoglobulin-like receptor five (MAIRV)/CMRF-35-like molecule1 (CLM1)  and goat anti-mouse CLM1/CD300f .Cell viability was indirectly estimated by the MTT assay, which is based on the cellular reduction of tetrazolium salts. Cells were incubated with MTT  for 2 h for allowing intracellular reduction of the soluble MTT to the insoluble formazan dye. Then, the media was carefully removed without disturbing the formazan crystals formed by viable cells and 100 µL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. Absorbance was measured at 570 nm using a microplate reader .Images were acquired on an epifluorescence microscope (Olympus IX8) at 20X and analyzed using Fiji software. All pictures were obtained following the same pattern by generating a cross and placing its center at the center of the well. One picture was taken per field, and 15 fields in total were used per well. The number of βIII-Tubulin-positive neurons per well was determined by summing neurons from all photos acquired per well. In each experiment, the number of βIII-Tubulin-positive cells were counted and normalized to control conditions (untreated cells).−/− mice , adult (4 to 5 months old) male Wistar rats. Animals were injected at neocortex  using a stereotaxic frame under isoflurane  anesthesia and a nanoinjector  with saline solution (0.9% NaCl), rCD300f-IgG (30 μg/ml) or control IgG (30 μg/ml). The needle was left in place for an additional 5 min to allow diffusion into the brain parenchyma. Treatments were randomly distributed among animals in each cage. Three days after the lesion was performed, animals were anesthetized and perfused intracardially with 4% PFA in 0.1 M phosphate buffer (pH 7.4). Brains were post-fixed with PFA 4% (2 h), cryoprotected in 30% sucrose, and frozen with CO2. Parallel cryostat coronal Sects. (30 μm) of the entire brain were prepared, stained with Nissl and used for quantification of the lesioned and total area of the hemisphere. The brain tissue sections were immersed in toluidine blue solution (15 ml of solution A composed by 0.5% toluidine blue in water and 200 ml of solution B composed by three parts of acetic acid 0.2 M and two parts of sodium acetate 0.2 M) for 20 min, rinsed with distilled water, dehydrated in ethanol solutions with increasing concentrations and cleared in xylene. Then, the sections were mounted with DPX mounting medium (Sigma-Aldrich), covered with cover slips, and observed under a light microscope. Damaged neurons were identified by loss of Nissl staining, whereas healthy neurons exhibited Nissl substance within the cytoplasm, a more relaxed chromatin structure, and well-defined nucleoli.All experimental work was approved by the UDELAR Ethical Commission (Exp. No. 070153-000528-14) and the Ethics Commission for the Use of Animals (CEUA) of the Institut Pasteur de Montevideo (No. 006-20) and conducted according to directives of the Federation of Laboratory Animal Science Associations (FELASA). Data are reported in accordance with ARRIVE guidelines. The animals used in this study were: adult (8–10 weeks) 6.Cg-Tg(Thy1-YFPH)2Jrs male mouse heterozygote for yellow fluorescent protein (YFP-H) (Jackson Laboratories), adult (4 to 5 months old) male C57BL/6 wild type (WT) mice and CD300fQuantification of the lesioned Nissl pale area was performed using Fiji software with parallel microscope observation (using 10X and 20X magnification). The lesion area and the total lesioned hemisphere area were used to calculate the lesion volume and the total  hemisphere volume; data were expressed as “% of lesioned hemisphere” to correct for the possible edema effect. Neurotoxicity in YFP mouse brains was determined by the number of YFP-positive neurons counted in the contralateral hemisphere minus those counted in the injured hemisphere. Values were expressed as “% of neuronal death”.p < 0.05 as statistically significant. The number of independent experiments performed are indicated in the figures. We did not perform any outlier test and did not eliminate any potential outlier.One-way analysis of variance (ANOVA) followed by Holm-Sidak´s test or Tukey’s post-hoc analysis were used for experimental data with normal distribution. Statistical analyses were performed with Prism 8 software and data was shown as mean ± SEM, considering All experimental work was approved by the UDELAR University Ethical Commission (Exp. No. 070153-000528-14) and the CEUA if the Institut Pasteur de Montevideo (No. 006-20) conducted according to directives of the Federation of Laboratory Animal Science Associations (FELASA).17. Interestingly, treating cocultures with rCD300f-IgG2a induced a 52 ± 17% and 43 ± 17% decrease in the number of βIII-Tubulin positive neurons when compared to the untreated or mIgG2a controls, respectively  mimics the effect of CD300f inhibition by rCD300f-IgG2a, showing 60 ± 16% and 55 ± 16% of neuronal cell death when compared to untreated or mIgG2a controls, respectively . Thus, to avoid the putative non-specific effects of the rCD300f-IgG2a fusion protein, we generated and additional fusion protein composed by the extracellular domain of rat CD300f fused to the IgG1 Fc region, which has lower complement activation capacities and decreased ADCC triggeringTo explore the role of CD300f in modulating neuronal survival in vivo, we injected saline, rCD300f-IgG2a (30 μg/ml) or control IgG2a (30 μg/ml) into rat neocortex and evaluated the lesion volume by Nissl staining H and glycolytic enzymes of the endoplasmic reticulum and therefore, cellular MTT reduction represents a measure of the rate of glycolytic NAD(P)H production9. We next analyzed whether inhibiting CD300f signaling would induce a proinflammatory glial phenotype and thus neurotoxicity. Hence, cocultures were incubated with rCD300f-IgG1 (1.0 μg/ml) or control IgG1 (1.0 μg/ml) and the concentration of diverse cytokines was measured in culture media after 72 h by using the Biolegend LEGENDplexTM Mouse Inflammation Panel (13-plex). No significant alterations in IL23, IL1α, IL1β, IFNγ, IFNβ, TNFα, MCP1/CCL2, IL12p70, IL10, IL6, IL17A, IL27 and GM-CSF cytokine concentration was detected under these experimental conditions glial-derived neurotrophic factor ; (ii) NMDA glutamate receptor inhibitor 3-(2-Carboxypiperazin-4-yl) propyl-1-phosphonic acid , or (iii) GAP junction and connexin hemichannel inhibitor carbenoxolone . As expected, conditioned media derived from rCD300f-IgG1-treated cocultures promoted a 70 ± 12% or 69 ± 12% neuronal death when compared to controls  or control IgG (30 μg/ml) and the % of lesioned hemisphere was evaluated 72 h after by Nissl stain   or damage-associated molecular patterns (DAMP’s) receptors, enabling innate immune cells to rapidly recognize tissue damage or extracellular misfolded proteins that triggers interconnected signaling pathways contributing to microglial and barrier macrophage sensing of the surrounding events occurring in the CNS parenchyma (reviewed in32).Important lessons for understanding CD300f function may be learned from TREM2 biology, as both receptors share functions, part of their cell-type specific expression, they are putatively activated by similar ligands, and their signaling pathways are intermingled. In fact, CD300 immune receptors are the closest relatives to TREM receptors11. Taken together, these observations open the possibility that other molecules apart from the phospholipids, such as proteins, may act as co-receptors with phospholipids generating and additional level of complexity and specificity. We show here that, while the in vivo injection of rCD300f-Fc into WT brain induces an increase in lesion volume when compared to control IgGs, there is a similar lesion volume after the injection of rCD300f-Fc or control IgGs into CD300f−/− brains. This denotes that while the rCD300f-Fc protein inhibits the endogenous CD300f-ligand interactions in WT brains, it does not affect the CD300f−/− brain. More importantly, the lack of effects of rCD300f-Fc in CD300f−/− brains evidences that, though CD300f may share lipid ligands with other lipid-binding receptors, it possesses additional ligands that confer specific activation of this receptor. The lack of in vivo effect of the rCD300f-Fc fusion protein in CD300f−/− brain also ensures that the fusion protein is a specific tool for blocking CD300f-ligand interactions. Finally, the injection of control IgGs or rCD300f-Fc into CD300f−/− brains showed an increased lesion volume when compared to the injection of control IgGs into WT brains, confirming that indeed CD300f signaling deficiency induces enhanced neurotoxicity in vivo.Though sharing similar phospholipid ligands, other molecules may act as co-ligands conferring specificity for different lipid-binding immune receptors. Staining cells with TREM2-Fc or CD300f-Fc fusion proteins show that many, but not all live cells, display ligands for these receptors despite abundant phospholipids ligands such as sphingomyelin exposed in the surface of all cells. Moreover, the staining of cells with CD300f-Fc show a patched profile highlighting specific cell surface domains where the ligands are exposed7. CD300f−/− mice showed increased depressive-like behaviors, that were potentiated by systemic and CNS inflammation induced by intraperitoneal LPS injection7. Depression has been shown to be induced under inflammatory conditions, and CD300f has been show to dampen inflammatory reactions in different systemic conditions33. Thus, CD300f−/− mice could display depressive-like behaviors due to low-grade CNS inflammation induced by the release of the negative signaling brake. However, only minor inflammatory profile of the brain or isolated microglial cells were altered in CD300f−/− mice7. Accordingly, the neurotoxicity observed after CD300f inhibition was not mediated by an increased proinflammatory cytokine release. We also showed that the underlying toxic mechanisms do not involve NMDA-dependent excitotoxicity, nor extracellular ATP-dependent toxicity. Regarding the cell-type responsible for neurotoxicity, as CD300f is only expressed in microglia and barrier macrophages in the CNS and mostly by myeloid cells in the periphery5, it may be exerted directly by CD300f-expressing microglia and/or recruited macrophages or neutrophils after the penetrating brain injury. However, in cocultures, the CD300f-dependent neurotoxicity seems to be dependent on microglial cells, as they are the only CD300f-expressing cells in this experimental conditions35. Alternatively, CD300f inhibition may affect microglia-dependent protective mechanisms, as has been shown in vitro and after CNS lesions, where a TREM2-dependent microglial scavenging of toxic lipids such as oxidated phosphatidylcholine occurs36. Based on their ligand similarities, CD300f could putatively also scavenge oxidated phosphatidylcholine after the penetrating brain injury, while the inhibition of this process would potentiate neurotoxicity. Moreover, microglia can induce an astrocyte neurotoxic phenotype, a toxicity that is maintained in astrocyte conditioned media37. This effect was later found to be dependent on toxic long-chain saturated free fatty acids associated to APOE and APOJ lipoproteins38. Astrocytes have also been shown to be capable of inducing neurotoxic responses by producing reactive oxygen and nitrogen species39, however these species are not stable in conditioned media and thus not responsible of the neurotoxic effect. Further studies will be needed for the identification of the toxic components involved in this process and the cell type that produce them.The inhibition of microglial CD300f-induced neurotoxicity, which was dependent on the presence of both neurons and glial cells, was mediated by soluble factors, and could be reversed by GDNF treatment. Thus, inhibiting microglial CD300f signaling could lead to a proinflammatory state toxic for cocultured neurons. In this sense, it has been shown that CD300f regulates microglial immunometabolic phenotype, being necessary for boosting its metabolism in response to inflammatory stimuli10. However, proteolytic cleavage of surface-expressed immune receptors by matrix metalloproteases may also contribute to soluble forms production1. Evidence shows that the cleavage of cell surface immune receptors terminates their signaling, and reflects their activation level in vivo. Moreover, soluble forms may have their own functions, such as scavenging ligands and further restraining immune receptor activation, or even activating yet unknown receptors. For instance, soluble CD300b activates TLR440. In addition, a beneficial effect has been reported after soluble TREM2 brain injection or overexpression in an Aβ pathology mice model41. Human longitudinal studies of Alzheimer’s disease patients show that a higher CSF soluble TREM2 levels predicts slower cognitive decline and attenuated amyloid and tau signal in positron-emission tomography43. This is in contrast with the neurotoxic effects of soluble CD300f injection into normal mouse and rat brain reported here, suggesting that in spite of all the similarities between these two immune receptors they may also have their particularities.Most immune receptors such as TREM1, CD200R, TREM2, and CD300f display soluble forms. A major source of soluble forms is the translation of an alternatively spliced transcript that lacks the transmembrane domain, and this is the case for both human and mouse CD300f35, as well as the extensive work of other colleagues on CD200R1/CD20045, TREM21, and Siglec47 receptors support the notion that immune receptors contribute to restoring nervous system homeostasis after a lesion or under chronic neurodegenerative conditions, and constitute key regulators of microglial phenotype. Importantly, we also show evidences that, though lipid-binding immune receptors share many lipidic ligands, there are additional conditions or co-ligands that confer different specificities.Taken together, the results presented here and previous results related to CD300fSupplementary Figures.


Fermented foods are often erroneously equated with probiotics. Although they might act as delivery vehicles for probiotics, or other ‘biotic’ substances, including prebiotics, synbiotics, and postbiotics, stringent criteria must be met for a fermented food to be considered a ‘biotic’. Those criteria include documented health benefit, sufficient product characterization (for probiotics to the strain level) and testing. Similar to other functional ingredients, the health benefits must go beyond that of the product’s nutritional components and food matrix. Therefore, the ‘fermented food’ and ‘probiotic’ terms may not be used interchangeably. This concept would apply to the other biotics as well. In this context, the capacity of fermented foods to deliver one, several, or all biotics defined so far will depend on the microbiological and chemical level of characterization, the reproducibility of the technological process used to produce the fermented foods, the evidence for health benefits conferred by the biotics, as well as the type and amount of testing carried out to show the probiotic, prebiotic, synbiotic, and postbiotic capacity of that fermented food. This perspective paper reports the outcomes of that discussion, including a brief review of fermented foods and health, the rising interest in fermented foods, the extent to which fermented foods are sources of biotics, and challenges to moving this field forward.The International Scientific Association for Probiotics and Prebiotics (ISAPP) is a non-profit organization created in 2002 for the purpose of advancing the science of probiotics, prebiotics and related substances. In the period of 2014–2021, ISAPP proposed to the scientific, academic, health professional, industrial, and regulator communities a series of definitions, including probiotics , prebiot2.Fermented foods have been part of the human diet from early civilization. Fermentation transforms foods, serving to preserve them from spoilage, enhance safety, improve nutritional value, and to provide unique, widely enjoyed organoleptic properties. These beneficial properties have led to the development of a various fermented products created from animal and vegetable sources. Increasing evidence of the association of fermented foods with health has further increased their popularity in recent years .The evidence that fermented foods can confer health effects has been derived from, among other sources, randomized controlled trials involving defined interventions  or assocThe variety of food matrices, fermentation microbes, and technologies used, as well as the lack of knowledge on the composition and characteristics of some traditional fermented products, present a challenge in assigning a health benefit to a specific component or components of a fermented food. In addition to the macro- and micro-nutrients present, the beneficial effects of fermented foods are often attributed to the presence of different bioactives such as phenolic compounds, prebiotic-like substrates, and the microorganisms involved in the fermentation process. Some of these components may promote health by their ability to modulate the gut microbiota and/or to interact with the immune system of the host . Indeed,A commonality among most fermented foods is the presence of microbes together with other known bioactives, including potentially prebiotic compounds, either already present in the food matrix or, in some cases, produced by the microorganisms during the fermentation process. In addition to the live bacteria responsible for the fermentation process, dead cells will also be present in fermented foods because of losses of viability during the starter/supplementary culture manufacture and/or a reduction in viability during product processing and storage. Such dead microbes could constitute postbiotic-like substances. For example, there is evidence that the administration of increasing amounts of different fermented foods can modulate the gut microbiota and lead to a decrease of biomarkers of inflammation .3.Consumer and scientific interest in fermented foods continues to grow in Western countries, driven in large part by their association with digestive health and the promotion of the gut microbiome. Nearly one in four Americans cites digestive health as the most important aspect of their overall health  and onliWhile fermentation has been used for thousands of years as a preservation  method, 4.Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, should have a minimum of 107 CFU/g until the expiration date. The European Food Safety Authority (EFSA) has approved a health claim for yoghurt containing live bacteria related to the improvement of lactose digestion in individuals with lactose maldigestion, illustrating the importance of the live microbes in yoghurt on a specific health outcome  matrix] are combined with milk and allowed to ferment . Then, t6.For a fermented food to be labeled as able to deliver a ‘biotic’, the product must be sufficiently characterized and tested beyond the requirement for demonstrated health benefits. In this context, the term ‘probiotic’ should be reserved for live microorganims within fermented foods when there is a demonstrated health benefit conferred by the well-defined and characterized live microorganisms . SimilarTo use the biotic term(s), there must be demonstration of health benefits in the target host and adequate characterization of the material(s) serving in that role. Biotic ingredients must be well-defined and characterized, proven to be safe and stable. For probiotics, this relates to the safety, identity, purity, and potency of the live microorganism in question . Genome Based on these principles, sufficient characterization and testing would be required to label and/or market a fermented food or beverage with inclusion of a biotic term. While some manufacturers may be interested in using an individual term , others may choose to combine more than one biotic substance similar to the synbiotic concept.7.Fermented foods and biotics  are gaining attention as nutritional means to support health. Fermented foods is a broad category of foods that may contain live and defined strains of microorganisms , live and undefined consortia of microbes , non-viable defined  or non-viable undefined microbes  or can be even devoid of significant amount of microbes . In this context, the capacity of fermented foods to deliver one, several, or all biotics defined so far will depend on the microbiological and chemical level of characterization, the reproducibility of the technological process used to produce the fermented foods, the evidence for health benefits conferred by the biotics, as well as the type and amount of testing carried out to show the probiotic, prebiotic, synbiotic, and postbiotic capacity of that fermented food.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.GV wrote the introduction and conclusions, compiled contributions, and drafted the paper. PC and HH covered the complexity of fermented foods section. MF covered the market aspects. MG and PR-M addressed the biotics perspective of fermented milk section. SS and KS covered the health benefits section. MS and CC discussed the outline of the paper and made the comprehensive revision and editing. All authors contributed to the article and approved the submitted version.ISAPP provided the venue and travel funding for some participants who met to discuss this topic, and paid for publication charges for this article.GV and SS serves in the board of directors of ISAPP and have been speakers in scientific meetings supported by the fermented food industry. PC has been funded by PrecisionBiotics Group, Friesland Campine, Danone, and PepsiCo. He has also received funding to travel to or present at meetings by H&H, the National Dairy Council U.S., PepsiCo and Yakult. MF is the employee of Health and Scientific Affairs, Danone North America and board member of the International Probiotics Association. KS serves in the board of directors of ISAPP. MS serves as the executive science officer/executive director for the International Scientific Association for Probiotics and Prebiotics (ISAPP), has been a paid consultant for Bayer, Pepsico, Smith Gambrell & Russell LLP, Bill and Melinda Gates Foundation, has been a paid speaker for Associated British Foods, European Federation of the Associations of Dietitians, Fairlife, Omnibiotic/Allergosan, Probi, Sanofi, Xpeer, has served on scientific advisory boards for Danone North America, Cargill, Winclove, has served in an unpaid (except for travel reimbursement) for the Advancement of Food and Nutrition Sciences and United States Pharmacopeia. CC is currently employed by the National Dairy Council. HH has received grant funding from Bio-Cat, Danone Research, General Mills, and Tate & Lyle. She has received speaking honorarium from Beneo, DSM, National Dairy Council, PepsiCo, and Tate & Lyle.The remaining 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.


Reticulitermes chinensis and R. flaviceps, which live close to each other, were used as research subjects in our study to find out how preferring conspecifics affects reproductive behavior between species. We discovered that neither R. chinensis nor R. flaviceps displayed preference behavior for conspecifics. Males of R. chinensis and R. flaviceps chased and groomed not only intraspecific females but also interspecific females. In a brief period of time, 2 mating behaviors, intra- and interspecific mating, were also observed. There were no significant differences in the duration of each behavior  between interspecies and intraspecies partners. Moreover, genetic analysis showed both interspecific mating and intraspecific mating can produce living offspring when the 2 types of mating occur in a colony. Our findings showed that there was no obvious intraspecific preference between the 2 species of termite Reticulitermes when it came to tandem, grooming, and mating, which not only makes it easier for interspecific hybridization to occur but also sheds light on the genetic diversity.The recombinant genotypes that can be produced when closely related species mate improve the genetic diversity of the population. Among closely related species, the link between interspecific reproduction behaviors and genetic diversity has barely been studied.  The division of labor and caste differentiation in social insects such as termites may be prominent features that make colony members work together in search of food, defend the colony, and care for offspring. Cooperation and the division of labor ensure that everything is completed efficiently and conformably within the colony, which helps social insects gain higher competitive advantages over different animals in the territory. This may be a key reason for their ecological success . The oveAs it has been learned, the presence of asexual queen succession (AQS) can avoid inbreeding and maintain genetic diversity in populations of social termites . In termOutbreeding was considered another mechanism for maintaining genetic diversity due to the fewer genetic relationships among colony founders. Interspecific individuals have more distant genetic relations than intraspecies individuals, so mating between interspecific individuals will produce various recombinant genotypes if hybridization can produce offspring, which contributes to the genetic diversity of the colonies . PreviouPrevious studies have reported cases of breaking down conspecific preferences in some species, such as ants , butterfThe reproductive behavior of termites from swarming to mating has been described in previous studies , 2023. IR. flaviceps Oshima and R. chinensis Snyder. Meanwhile, the genotypes of the parents and their offspring in colonies established by a couple of R. flaviceps Oshima and a couple of R. chinensis Snyder were analyzed using microsatellite loci. Our results revealed that there is no preference for same-species partners in the 2 species when intraspecific and interspecific individuals are present in the same arena, which could provide new insights into the genetic diversity and hybridization between species in the termites.In this study, the preferences and behaviors of inconspecifics in the reproductive processes from tandem to mating were measured in the 2 sympatric species: R. flaviceps and R. chinensis, are consistently recognized as the 2 distinct species on the basis of multiple criteria, including morphology, biology  were collected from Shizi Hill, Wuhan City, Hubei Province, China, during the swarming season (March–April) in 2019. The parts of the nest with alates were brought to the laboratory and promoted dispersal flight using methods described in the document  The tandem and grooming behaviors of 2 R. flaviceps  and 2 R. chinensis  were placed in 60-mm Petri dishes with moistened filter paper and allowed to occur unconstrained. Videos (5 min long) were taken with a high definition camera  3 times, at 10, 30, and 50 min after the establishment of the groups because the period from encountering each other to the end of tandem takes about 1 hour  and 2 R. chinensis  were placed into 60-mm Petri dishes with moistened filter paper. One-hour video was taken every hour and observed continuously for 12 h . We could not monitor the mating behavior of too many different colonies at the same time due to uncertainty about the exact timing of mating after the tandem. Therefore, we prepared 8 replicates for this experiment. After the filming was finished, each group (a couple of R. flaviceps and R. chinensis) was placed into a 120-ml transparent cylindrical vial (Φ = 3 cm) and conditions for feed provided moistened filter paper and pine wood at 20–26 °C in constant darkness.Considering the natural sympatry, the individual dealates  from 4 t 1 hour . We prepP-value of less than 0.05 was considered to be statistically significant.The reproductive behaviors, including tandem, grooming, and mating, have already been described in previous studies and that were paired behaviors between the sexes, initiated by one partner and accepted by the other . We viewcf-032806; Hcf-032810; Hcf-032604; Hcf-032601; Hcf-032705) established 2 months ago by a couple of R. flaviceps and a couple of R. chinensis were used for genetic analysis . Male of R. flaviceps has similar tandem behavior to that of R. chinensis. The 2 tandem behavior frequencies of male R. flaviceps, tandem with R. chinensis and tandem with R. flaviceps, respectively, both decrease with time over, and there was a significant difference in the second observation , but there were no significant differences in frequencies during the total observation time . In tandem duration, there were no significant differences in 2 behaviors of male R. chinensis, tandem with female of R. chinensis and tandem female of with R. flaviceps . Similarly, 2 behaviors of male R. flaviceps showed no differences in duration . Tandem behavioral results indicated that the preferences for conspecifics were absent in R. chinensis and R. flaviceps.Males of both gorously . There wR. chinensis provided grooming not only for female of R. chinensis but also for female of R. flaviceps . Male R. flaviceps groomed female R. chinensis more frequently than conspecific females . In grooming duration, we found no significant effect on species of female grooming provided by R. chinensis  and by the male of R. flaviceps .Males of laviceps . Both ofR. chinensis and R. flaviceps were observed mating with conspecific female and interspecific female in a short interval time. The numbers of mating occurred in conspecific were equal to the mating occurred in heterospecific partners per unit of time . There were no significant differences in the duration of each mating between intraspecies and interspecies . These results indicated that the preferences for conspecifics were absent in mating behavior between R. chinensis and R. flaviceps.We found that termites copulate in the opposite position by joining their abdominal ends  and B anR. chinensis couple and a R. flaviceps couple were able to produce living offspring (R. chinensis and R. flaviceps may not be reproductive interference, but interspecies mating to acquire offspring.Genotyping analyses of progeny showed that both heterospecific and conspecific mating in colony established by a ffspring . AlthougConsidering the life history of the entire termite colony, the reproductive pattern and relatedness of colony founders are closely linked to colony breeding structure, and both of them are considered to be major factors affecting the genetic structure of the population . A singlR. chinensis and R. flaviceps have similar reproductive behaviors and lack preferences for conspecifics when individuals from interspecies encounter each other during swarming. These results suggested that the hybridization between R. flaviceps and R. chinensis may be formed in nature. More importantly, hybridization can also produce living offspring in termites. The abundant genotypes produced by recombination in hybrids may facilitate higher genetic diversity than those of the parents.The behavioral interaction among individuals plays a role in gene exchange among populations and may impede or facilitate gene exchange between species . Our resCoptotermes gestroi and C. formosanus in Florida field, the individual preference to tandem with conspecifics or heterospecifics can change depending on the arena (Reticulitermes species.Preference for conspecifics is an assortative mating behavior at the species level that individuals select in the light of corresponding genotypic or phenotypic traits . It is che arena . Howeverhe arena . Thus, rhe arena . On the Interspecific mating will increase genotypes produced by recombination in hybrids and decrease inbreeding retrogression . It can By mating with other species, the termites can acquire higher genetic diversity, increase the success rate of dealate reproduction, gain greater fitness, and retain the transmission rate of their genes to relative species. The inefficient preference for conspecific partners in congeneric species may also benefit from the genetic diversity of offspring because hybridization has higher genetic diversity compared to those produced by conspecific breeding.iead115_suppl_Supplementary_Tables_S1-S2Click here for additional data file.iead115_suppl_Supplementary_Videos_S1Click here for additional data file.


Labeobarbus discovered in riverine environments of the Ethiopian Highlands, East Africa. To test the adaptive value of thickened lips, identify the ecological niche of the thick‐lipped ecomorphs, and test whether these ecomorphs are the products of adaptive divergence, we studied six sympatric pairs of ecomorphs with hypertrophied lips and the normal lip structure from different riverine basins. Trophic morphology, diet, stable isotope (δ15N and δ13C) signatures, as well as mtDNA markers and genome‐wide SNP variation, were analyzed. Our results show that thick‐lipped ecomorphs partition trophic resources with generalized ecomorphs in only one‐half of the examined sympatric pairs despite the pronounced divergence in lip structure. In these thick‐lipped ecomorphs that were trophically diverged, the data on their diet along with the elevated 15N values suggest an invertivorous specialization different from the basal omnivorous–detritivouros feeding mode of the generalized ecomorphs. Genetic data confirmed an independent and parallel origin of all six lipped ecomorphs. Yet, only one of those six thick‐lipped ecomorphs had a notable genetic divergence with sympatric non‐lipped ecomorphs based on nuclear SNPs data (FST = 0.21). Sympatric pairs can be sorted by combinations of phenotypic, ecological, and genetic divergence from an ecologically non‐functional mouth polymorphism via ecologically functional polymorphism to a matured speciation stage via divergent evolution.The evolution of convergent phenotypes is one of the most interesting phenomena of repeated adaptive radiations. Here, we examined the repeated patterns of thick‐lipped or “rubberlip” phenotype of cyprinid fish of the genus  Speciation continuum was revealed within repeated de‐novo origins of lipped mouth in African cyprinids. Diet, stable isotopes and genomics showed step‐by‐step moving from non‐functional polymorphism via ecologically functional polymorphism (trophic resource partitioning) to ecological speciation when genomic divergence occurred. Their results provided weak support for trophic resource partitioning between fish with hypertrophied and normally developed lips . So, weak trophic resource partitioning between sympatrically co‐occurring ecomorphs with strikingly divergent trophic morphology turned out to be an intriguing finding. In general, whether such mouth structure transition is just non‐adaptive phenotypic variation within highly polymorphic Labeobarbus lineage or whether thick lips are an adaptive trait involved in trophic resource partitioning remains unclear so far. To address this question, similar cases in a broader taxonomic and geographical context have been studied by us. We could show that thick‐lipped ecomorphs evolved several times independently in several Ethiopian river basins. The hypertrophied ecomorphs were indistinguishable by mtDNA from sympatric ecomorphs with a generalized mouth phenotype  and L. intermedius 22.1n = 252; gut length: n = 296; diet: n = 81; stable isotopes: n = 237; mtDNA: n = 213; ddRAD: n = 63) were investigated by a set of various methods  in six rivers draining the Ethiopian Highlands and belonging to its four major river basins Figure : (i) theds Table .2.2C), middle lobe length (MLL), lower lip length (LLL), and upper lip width (ULW) were measured in CorelDRAW 2017 (v. 19.1.0.419) using photographs. The scheme of measurements is given in Figure MLL, LLL, and ULW to head length (C) were used for subsequent analyses. The standard length (SL) and gut length (GL) were measured on preserved specimens using a ruler (to the nearest 1 mm). A ratio GL to SL expressed as % was used for analyses. The SL distribution is given in File The head length .Gut content was dried on filter paper and weighed using a Pioneer PX84/E balance with 0.0001 g accuracy. The diet particles were identified using Olympus CX41 microscope (100–1000× magnification) and Motic DMW‐143‐N2GG stereomicroscope (100–400× magnification). The diet components were grouped into: (i) detritus, (ii) invertebrates, (iii) macrophytes and (iv) fish. The group “Invertebrates” included the larvae of amphibiotic insects, and their fragments. The group “Macrophytes” included any fragments of plants – such as leaves, stems or seeds. A composite measure of diet, an index of relative importance (2.4Rsample/Rstandard) − 1] × 1000, where R is the molar ratio of the heavier isotope to the lighter. The samples were analyzed with a reference gas calibrated against the International Atomic Energy Agency (IAEA) reference materials USGS 40 and USGS 41 (glutamic acid). The measurement accuracy was ±0.2‰. Along with the isotopic analysis, the nitrogen and carbon content (as %) and C/N ratios were determined. In total, 237 white‐muscle samples were analyzed.For stable isotope (SI) analyses, white muscle tissue from the dorsal side of the body under the dorsal fin was sampled from freshly collected specimens. White muscle samples were dried at 60°C for subsequent SI analyses. The samples were weighed using a Mettler Toledo MX5 microbalance (Mettler Toledo) with 2 μg accuracy and were wrapped in tin capsules. The weight of the fish tissue samples varied from 250 to 500 μg. SI analysis was conducted at the Joint Usage Center of the A.N. Severtsov Institute of Ecology and Evolution RAS, Moscow. Briefly, a Thermo Delta V Plus continuous‐flow IRMS was coupled to an elemental analyzer (Flash 1112) equipped with a Thermo No‐Blank device. The isotopic composition of N and C was expressed in the δ notation relative to the international standards : δX (‰) = [ were collected from both generalized and lipped ecomorphs of Labeobarbus from the same six localities in Ethiopian Highlands , 1038 bp in length, were amplified (polymerase chain reaction (PCR) conditions were taken from Palumbi, muscle algorithm  or obtained with a salt‐based DNA extraction method  and filtering. We selected optimal parameters for de novo loci assembly using the approach suggested by Paris et al. (m) of 6, distance allowed between stacks (−M) of 1 and maximum distance required to merge catalog loci (−n) of 1 provided the best balance between data quality and quantity for our data set. We also set the ‐‐max_locus_stacks parameter to 7 to improve binning and avoid paralogs  were excluded from the MSA by IQ‐TREE, resulting in 15,820 nucleotide sites. To take into account the absence of constant sites an ascertainment bias correction (+ASC) model  loci genotyped in at least 80% of all samples were kept; (ii) SNPs with a minor allele count (‐‐min‐mac) less than 3 were pruned. Next, we whitelisted loci that are in HWE for all populations and created a structure‐formatted file with populations while retaining a single random SNP per locus to avoid the inclusion of closely linked SNPs. This dataset consisted of 2411 SNPs. We performed 16 independent runs  of STRUCTURE for each K between 1 and 8 using the admixture model with correlated allele frequencies. Structure_threader . For these runs, we created new SNP sets using the same procedure as described above but limiting genotypes to the individuals from identified genetic clusters. In addition, Principal Component Analysis (PCA) was performed using the glPca function of the ADEGENET 2.1.1 R‐package  that were significantly longer compared to the generalized ecomorph in all studied rivers (3.1.2p < .01), Genale (p < .05) and Gojeb .The guts of thick‐lipped ecomorphs were shorter than in generalized ecomorphs from all studied rivers, sometimes significantly Figure . The gut3.23.2.1Labeobarbus spp. were rather diverse and composed of (i) detritus, (ii) invertebrates , (iii) macrophytes , and occasionally (iv) fish . Feeding of sympatric ecomorphs in all four rivers studied was divergent but at various degrees as estimated by the index of relative importance  and thick‐lipped ecomorphs  to 0.9‰ (Sore). Significant differences in δ13C values between sympatric pairs were found in the Didessa, Gojeb, and Sore Rivers. In all these cases, the lipped ecomorph had higher δ13C values (the difference varied from 1.6‰ to 1.2‰).Basic statistics for both δ5 Figure . When siThe total area (TA), standard ellipse area (SEA), and corrected standard ellipse area (SEAc) were analyzed for all sympatric ecomorphs except for thick‐lipped ecomorphs from the Blue Nile and Birbir Rivers because of the small sample size. The thick‐lipped ecomorph had the largest SEAc values in all rivers apart from the Genale River Table . The nic3.3p‐distances between the geographic basins varied from 0.009 ± 0.003 to 0.031 ± 0.005  Eastern  and (ii) Western  parts of the Ethiopian Highlands  clusters that correspond to the (i) Genale, (ii) Didessa, (iii) Gojeb, and (iv) White Nile populations from the Sore and Birbir Rivers  between Didessa generalized ecomorph and Didessa thick‐lipped ecomorph to 0.66 (0.63–0.68) between Sore and Genale . Enrichment of the lipped ecomorph in δ15N (4 pairs: extra 0.9‰–1.9‰) was usually accompanied by enrichment in δ13C (3 of 4 pairs: extra 1.3‰–1.7‰). Nevertheless, SI Bayesian ellipses showed great trophic niche widths and overlaps in half of the comparisons of pairs of sympatric ecomorphs  that confirmed the usage of SI composition as a diet proxy. Lipped ecomorphs had higher δLabeobarbus as a phenotypic trait are not sufficient to predict the diet of fish. The same was found not only for riverine populations but also for lacustrine. For instance, the thick‐lipped ecomorph in Lake Tana had almost the same food spectrum as sympatric generalized ecomorph  appears to be linked to muddy substrates without pebbles, cobbles and boulders but in warmer localities with a relatively low electrical conductivity compared to sympatrically co‐occurring species with a “generalized” mouth. The presence of the thick‐lipped ecomorph on the muddy substrate is reported by F.N. Shkil  for the Lake Tana basin. Due to an unstable hydrological regime in the mountain rivers of the Ethiopian Highlands, the environment varies largely from season to season. Our assessment of diet and trophic position using SI analyses was done based on the material collected during the dry season while the conditions may change during the wet season. One may suggest that thick‐lipped trophic morphologies may serve as a trade‐off between consumption of commonly available food (detritus) and benthic invertebrates in unstable riverine environments, e.g., when seasonally common prey is rare or even absent in some seasons. Additional ecological  studies are needed for the clarification of the mechanisms of ecological functionalization of thick‐lipped phenotype.What conditions can enable a thick‐lipped mouth to function? Both abiotic  or biotic  factors might promote the adaptive value of the thick‐lipped phenotype in some studied localities. Unfortunately, information on the phenotype‐environment correlation is hard to collect for thick‐lipped 4.2Labeobarbus from East Africa  rise of phenotypic divergence (most likely predetermined by ancient genetic polymorphism), (ii) rise of ecological (trophic) divergence that is based on the phenotypic divergence, (iii) genetic divergence caused by divergent selection upon ecological differentiation. At the last stage when all three types of divergence  begin to be detectable, a species status is achieved. Accordingly, the sympatric pair from the Didessa River can be considered two different species. Notable divergence in population genomic structure based on ddRAD SNPs in the lack of sorting in mtDNA suggests a rather recent speciation in the case of the Didessa River radiation.The cases of divergence between sympatric ecomorphs in various rivers are ranked from 1 to 3 points. The results can be treated as a row of situations from ecologically non‐functional polymorphism (1 point) via ecologically functional polymorphism (2 points) to divergent evolution or speciation event (3 points in the presence of genetic divergence). The case of the Sore and Gojeb (2 points) showing the morphological and trophic divergence but no genome‐wide divergence can be considered an initial stage of speciation with only a few loci involved. Studies on genomic differentiation of thick‐lipped phenotype in Amphilophus, which demonstrates a stage‐specific speciation process from incipient to more advanced stages of speciation  came from the lacustrine environment. Generally, ecological speciation is more common among fishes under the lacustrine conditions . This polymorphism is probably ancestral to both African and Middle East Torinae being inherited from Southern and Southeastern tetraploid Torinae lineages . This finding may be interpreted as a result of the generally adaptive value of the hypertrophied lips. In those populations that could not demonstrate an adaptive value of the hypertrophied lips in terms of trophic resource partitioning, the thickened lips might serve as a latent “adaptation in reserve” or “silent adaptation” that can be recruited into invertebrate foraging at appropriate environmental conditions possible due to being maintained in the population in low frequency and due to simple genetic and developmental programs that might also be facilitated by genetic assimilation based on initially phenotypic plasticity  were at various stages of ecological and genetic differentiation. This example of ecological speciation represents a continuum from polymorphism in phenotype via trophic resource partitioning (with various strengths) to genetic divergence. This assumedly starts as an intra‐population strategy to broaden the food spectrum via temporally/seasonally differential niches. However, in some circumstances, polymorphism in lip size may be a major contributor to trophic shift and consequently to divergent selection and speciation, for example, in the Didessa River ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Aleksandra Komarova: Conceptualization ; data curation ; formal analysis ; investigation ; visualization ; writing – original draft ; writing – review and editing . Evgeniy Simonov: Formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft ; writing – review and editing . Alexei Tiunov: Formal analysis ; investigation ; project administration ; writing – original draft ; writing – review and editing . Marina Levina: Data curation ; formal analysis ; investigation ; writing – original draft ; writing – review and editing . Alexander Golubtsov: Conceptualization ; investigation ; writing – original draft ; writing – review and editing . Fyodor Kondrashov: Investigation ; resources ; writing – original draft ; writing – review and editing . Axel Meyer: Conceptualization ; investigation ; resources ; supervision ; writing – original draft ; writing – review and editing .We prepare our data to archive in a publicly accessible repository – in Dryad and GenBank, in particular.Appendix S1:Click here for additional data file.


FST-outliers were examined. Noticiana reported moderate levels of genetic diversity. The high percentage of short and medium length ROH segments (93% under 4 Mb) is indicative of a within breed relatedness dating back to ancient times, despite the absence of management for the mating plans and the reduced population size. In the worldwide context, the Southern Italian, Spanish and Albanian breeds overlapped in a macro cluster which also included the Noticiana sheep. The results highlighted ancestral genetic components of Noticiana shared with Comisana breed, and showed the clear separation from the other Italian sheep. This is likely the consequence of the combined effects of genetic drift, small population size and reproductive isolation. ROH islands and FST-outliers approaches in Noticiana identified genes and QTLs involved in milk and meat production, as well as related to the local adaptation, and therefore are consistent with the phenotypic traits of the studied breed. Although a wider sampling could be useful to deepen the genomic survey on Noticiana, these results represent a crucial starting point for the characterization of an important local genetic resource, with a view of supporting the local economy and preserving the biodiversity of the sheep species.Among livestock species, sheep have played an early major role in the Mediterranean area. Italy has a long history of sheep breeding and, despite a dramatic contraction in numbers, still raise several local populations that may represent a unique source of genetic diversity. The Noticiana is a breed of the south-eastern part of Sicily appreciated both for its dairy products and for its resistance to harsh environment. In this study, the high-density Illumina Ovine SNP600K BeadChip array was used for the first genome-wide characterization of 48 individuals of Noticiana sheep to investigate its diversity, the genome structure and the relationship within the context of worldwide and Italian breeds. Moreover, the runs of homozygosity (ROH) pattern and the pairwise  In 2022Livestock diversity is crucial for food security, productivity and adaptability of production systems, resilience to climate change, and livelihoods . LocallyAmong livestock species, the sheep genetic resources played an early major role in the Mediterranean area. Italy has a long history of sheep breeding and, despite a dramatic contraction in numbers, still raises several local populations that may represent a unique source of genetic diversity –7. In faThe breeding of the local populations is usually based on the use of a restricted number of males and on the mating between close relatives over generations. This process causes cascading changes in the population, starting with an increase in inbreeding due to the reduction of genetic diversity, to the consequent deterioration of reproduction fitness and survival-related traits. Therefore, the genetic diversity is a key measure for the prevention of genetic animal resources erosion. In addition, an investigation of genomic variation is a crucial step to plan appropriate conservation programs in the framework of sustainable strategy models .The availability of sheep genome-wide single nucleotide polymorphism (SNP) panels allowed retrieving background information concerning genome structure in local and cosmopolite breeds , 11, 12.2.2.1.A total of 48 Noticiana sheep blood samples were collected from some custodian farms. Animals were chosen based on their phenotypic profile (such as color face) and the information provided by farmers in order to collect unrelated individuals. DNA was extracted from blood using the commercial Illustra blood genomic Prep Mini Spin kit . The 48 genomic samples were genotyped using the Illumina Ovine SNP600K BeadChip array, which contains 606,006 SNPs spanning the whole ovine genome . The data are deposited and available at.2.2.plink ver. 1.9  and expected (HE) heterozygosity, inbreeding coefficient (FIS) and average minor allele frequency (MAF) using Plink ver. 1.9 (NOTPOP was investigated for observed (ver. 1.9 . Moreovever. 1.9 .r package detectRUNS ver. 0.9.6 (NROH) and chromosome (NCROH) as well as the average length of ROH in Mbp per individual (LROH) and chromosome (LCROH) were calculated. In addition, the total length of the genome covered by ROH was evaluated for each individual and divided by the total autosomal genome length (~2.4 Gb) in order to evaluate the genomic inbreeding coefficient (FROH).We performed runs of homozygosity (ROH) analysis by the sliding windows method implemented in the r. 0.9.6 , and usir. 0.9.6  and Ferer. 0.9.6 : 1–2, 2–p-values: only the SNPs within the top 0.5% were considering to constitute ROH islands. The genomic coordinates of ROH islands were examined through NCBI Genome Data Viewerp < 0.05. GO terms outcomes corresponded to the highest specificity. Corrections for multiple testing were made by applying the Bonferroni test.Markers in rich homozygous regions (ROH islands), were identified by calculating the standard normal z-score from all the SNPs-within-ROH incidence and deriving the 2.4.indep-pairwise (50 10 0.2) function in Plink ver. 1.9  distance matrix containing pairwise combination of all individuals was generated using Pver. 1.9 . Classicrlequin ver. 3.5.2.2  and expected (HE = 0.324 ± 0.152) heterozygosity, a value of MAF = 0.242 ± 0.146, and low genomic inbreeding coefficients . A continuous decline in Ne was found across generations for both populations . The average length of ROH per individual (LROH) was 2.05 ± 0.20 Mbp. Almost all segments of homozygosity were less than 4 Mb in length (92.96%), 6.67% of ROH had length between 4 and 8 Mb, and less than 1% >8 Mb . The average number and length per chromosome were 177.62 ± 123.16 (NCROH) and 2.11 ± 0.21 Mbp (LCROH) respectively, showing OAR2 as the chromosome most affected by ROH. The ROH and ROH length incidence per chromosome are shown in A total of 4,618 ROH segments were identified, ranging from 29 to 220 ROH per individual with a population average of 96.21 ± 42.85 (The top 0.5% of the SNPs-in-run percentile distribution identified the most recurrent ROH’s regions in Noticiana (ROH islands). The descriptive results are reported in 3.2.To explore the genetic relationships of Noticiana in the worldwide and Italian context, we performed MDS analyses based on pairwise IBS distances. FST values calculated in the ITAPOP ranged from 0.066 (Noticiana vs. Comisana) to 0.136  , while higher distances were found toward AltamuranaFG, Barbaresca, and Delle Langhe . The Neighbor-Net  . Noticiahbor-Net  placed NThe MDS plot  of the Sicilian breeds (SICPOP) reported a narrower picture of between breeds relationships of Noticiana sheep . HomogenThe Bayesian clustering analysis reported the lowest CV error for K = 23 in ITAPOP, corresponding to the number of investigated breeds . The anc3.3.q < 0.0002) in the top 0.005% of FST-values percentile distribution. The chromosome position and the genes to which they are linked are reported in ST values is reported in Results from the Bayesian population differentiation approach between Noticiana and Comisana breeds, identified a total of 20 outlier SNPs  were reported in previous studies     and lowe (0.087) . This es (0.087) , with th  and low (0.087) , 16. A Npulation , can givpulation . TherefoLROH and the LCROH around 2 Mb (92.96% under 4 Mb) suggest distant inbreeding events linked to those identical-by-descent genomic regions from ancient ancestors  that highlighted Noticiana and Barbaresca as the most distant breeds from the other Italian sheep, with the exception of Comisana  and Pinzirita . The Noticiana branched within the Sicilian node and showed the strict closeness to Comisana, and at the same time its evolutionary distance. The Admixture analysis generated a clustering pattern that, at high K values, reflects the individuality of most Italian breeds, except for a few that, according to their history of crossbreeding and/or gene flow in a restricted geographic area, clustered together as reported by Ciani et al.  has been linked to heat tolerance in cattle , STAT6 (OAR3), PKD2 and SPP1 (OAR6), and BDNF (OAR15) are involved in the biological process GO:0010604 which is related to a positive regulation of macromolecule metabolic process.The use of the high-density SNP array allowed to thoroughly investigate the ROH pattern and led to the identification in Noticiana of homozygosity hotspots harboring candidate genes and QTLs mostly connected to production-related factors like milk protein content and meat fat content. According to previous studies on sheep species , 55, 56,ed breed . Similared breed  highlighelopment , 61. Theelopment  in threeelopment  in Sardan cattle . Moreovein sheep . Three Rd cattle . Within sorption , 67, whe quality . The OAR quality , and as  quality . The firda breed , and resda breed , 70, 71.perature . Similarn cattle . These f species . Noticia species , and Bar species  the hots species , 76, par species –79. Thisposition , and othnd sheep –83, ABCGse sheep , 84–87,  species , 88, 89.sistance . The OARn cattle . In acco4.4.FST, relies on the comparison between groups, highlighting those markers that show the greatest differences in allele frequencies as putative signal of differentiation , located in the bovine chromosome 8 and close to the dry matter intake QTL (#4425 according to the Cattle QTLdb), is a putative candidate gene that seems to affect dry matter intake and residual feed intake, as reported in a GWAS in Nellore cattle (ATRNL1 that is a candidate gene potentially involved in improving the heat tolerance in pigs (While ROH segments are typically employed to discover potential signature of selection within breeds, outlier analysis, based on pairwise ntiation . In the n cattle . NEGR1 gf cattle  and in Vf cattle , 95. In e cattle . In OAR2 in pigs , and als in pigs .5.This study provided for the first time the genome-wide assessment of the genetic diversity and population structure of Noticiana sheep breed. The results demonstrated its clear distinction from the rest of the breeds, revealing a moderately low level of inbreeding and its shared ancestry components with Comisana. ROH analysis identified several genes and QTLs positively involved in milk and meat production traits. The information generated in this study is of significant importance because it will help to design and implement conservation strategies in order to recover the Noticiana breed and enhance its local products. However, additional analyses and a wider sampling would contribute to refine and validate these results. The implementation of a high-density panel of SNP made possible an in-depth analysis of genomic structure and variability, suggesting this as a valuable tool for uncharacterized local genetic resources. In fact, whereas our study describes an example of the Noticiana breed, the applied analyses are a valid tool for all vulnerable and endangered breeds.https://figshare.com/s/3f5c15b14a103d8d3a1f.The datasets presented in this study can be found in online repositories. A link to the data can be found at: The animal study protocol was approved by the Bioethics Committee of the University of Palermo: protocol code UNPA-CLE–98597.SB, BP, and SM contributed to conception and design of the study. MT, IR, SR, AM, VM, and MTS organized the database. GC, AC, MT, and SM performed the statistical analysis. GC, AC, and SM wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This research was financed by Co.Ri.A.L, Conservazione Risorse Animali Locali. PSR Sicilia 14/20–Operazione 10.2b “Conservazione delle risorse genetiche animali in agricoltura.” Project number IRIS PRJ-0717, CUP G72C21000580009.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.


Crop wild relatives (CWRs) exhibit a close relationship with domesticated crops  and form a part of the crop’s gene pool with potential for gene exchange. A large number of CWRs are potential donors but receive less attention than domesticated crops. CWRs have also suffered from genetic erosion resulting in a severe loss of genetic diversity . The facTrainin et al. documented from an anatomical perspective, the evolutionary forces involved in the selection of the non-foliar photosynthesis trait in the stems of Prunus arabica (Olivier) Meikle  when compared to the commercial almond (P. dulcis (Mill.) D. A. Webb). The stems of P. arabica favor stem photosynthesis for additional carbon gain through multiple strategies. Higher stem photosynthesis in P. arabica than in P. dulcis is attributed to selective anatomical features such as the presence of a high density of sunken stomata in their stems, a chloroplast-rich mesophyll-like parenchymatous cell layer, higher chlorophyll content, better chlorophyll fluorescence and quenching parameters, and its ability to efficiently regulate water loss at elevated temperatures.In this Research Topic, Zhu et al. identified the molecular mechanism of an R2R3-MYB transcription factor gene underlying a drought tolerance trait, VyMYB24, isolated from a drought-tolerant grapevine wild relative– Vitis yanshanesis J. X. Chen. This gene was identified as a nuclear protein that was significantly upregulated during drought to impart drought stress tolerance and functionally validated through the genetic transformation of tobacco. Transgenic plants overexpressing the VyMYB24 gene were phenotypically dwarfed with a lower leaf area, reduced flower size, and seed weight. The role of VyMYB24 in regulating the gibberellin biosynthetic pathway was studied.Du et al. reported the introgression of stripe rust and Fusarium head blight resistance genes from Leymus mollis (Trin.) Pilg.  into wheat cv. 7182 through an alien disomic substitution line, M862 , using an inter-specific octoploid hybrid Tritileymus . Cytogenetic studies indicate that the disomic substitution line (M862) carried 4Ns in place of 4D chromosomes. A few structural variations in chromosomes 1A, 1D, 2B, and 5A were also reported and the transcriptome analysis identified the genes and Ns-specific markers associated with resistance genes that had utility in wheat improvement programs.Gowda et al. used a genome-wide association approach to mine the candidate genes associated with the straw silica content trait in the germplasm of the rice progenitor, Oryza nivara S.D.Sharma & Shastry, representing the variability of 5-16% silica content among a set of 258 accessions. This study led to the identification of the candidate genes, an ATP-binding cassette (ABC) transporter, casparian thickenings, multi-drug and toxin extrusion (MATE) protein, F-box, and MYB-transcription factors.With the availability of genomics tools, whole genome sequence information, and a reference set for the desired trait representing the complete range of trait variation, it is very possible to identify the candidate genes underlying the trait variation. Singh et al. underscore the difficulties in achieving higher genetic gain with special reference to legume crops. Their research provides an overview of the key strategic approaches such as broadening the genetic base through alien introgression from CWRs. In addition, integration of the strategic approaches with modern tools, such as genetic engineering, genome editing, and speed breeding, along with present-day omics tools will potentially bring a paradigm shift in legume breeding programs to achieve higher genetic gain with minimal costs.An understanding of the factors underlying genetic diversity loss will help identify ways to reverse genetic diversity losses and support the introgression of desirable traits into domesticated ones. Though gene introgression from wild to crops is possible, it is mostly untapped due to the deleterious effects of linkage drag reducing genetic gain and selection efficiency and, thus, gene introgression is considered to be the ‘cost of domestication’ . BesidesBarazani et al. discuss the history of olive cultivation using archaeological evidence that documented the presence of plant remains of wild olive, Olea europaea L. subsp. europaea var. sylvestris (Mill.) Lehr during the Middle Pleistocene  in the Southern Levant region. Selection pressure on this wild form gave rise to the present-day cultivated olives, Olea europaea subsp. europaea var. europaea. This warrants further genetic studies among the naturally grown olive populations with traditional landraces and modern cultivars to understand the loci being subjected to selection pressure in the domestication process. With the proven strength of genome editing tools in the creation of de novo domesticated crop plants in the CWRs of model crop plants such as rice and tomato (An understanding of the genomics of linkage drag  would hed tomato , a broadAuthor PR wrote the first draft and all authors have contributed to finalize the manuscript. All authors contributed to the article and approved the submitted version.


Ceratophyllum demersum in hydrologically different aquatic habitats, i.e. rivers and backwaters separated from the main river bed to a different extent. For this purpose, the first microsatellite primer set was developed for this species. Using 10 developed primer pairs, a high level of genetic variation was explored in C. demersum populations. Overall, more than 80% of the loci were found to be polymorphic, a total of 46 different multilocus genotypes and 18 private alleles were detected in the 63 individuals examined. The results demonstrated that microsatellite polymorphism in this species depends on habitat hydrology. The greatest genetic variability was revealed in populations of rivers, where flowing water provides constant longitudinal connections with distant habitats. The populations of the hydrologically isolated backwaters showed the lowest microsatellite polymorphism, while plants from an oxbow occasionally flooded by the main river had medium genetic diversity. The results highlight that in contrast to species that spread independently of water flow or among hydrologically isolated water bodies, macrophytes with exclusive or dominant hydrochory may be most severely affected by habitat fragmentation, for example due to climate change.The adaptability of plant populations to a changing environment depends on their genetic diversity, which in turn is influenced by the degree of sexual reproduction and gene flow from distant areas. Aquatic macrophytes can reproduce both sexually and asexually, and their reproductive fragments are spread in various ways (e.g. by water). Although these plants are obviously exposed to hydrological changes, the degree of vulnerability may depend on the types of their reproduction and distribution, as well as the hydrological differences of habitats. The aim of this study was to investigate the genetic diversity of the cosmopolitan macrophyte  Climate change has been proven to greatly affect the growth and physiology of these plants, as well as the species composition and ecosystem functioning -propanesulfonic acid]/KOH, pH 7.4). The cells were disrupted by homogenizing for three periods of 15 s using a Waring blender. Cell debris was removed by centrifugation at 3,000 g for 5 min. According to the recommendations, the sample DNA was isolated using Thermo Scientific™ Genomic DNA Purification Kit  and sequenced on Illumina Miseq sequencer using v3 kit for 2x300 bp PE protocol. The sequences were aligned using Mira4 , a distance of at least 30 m was kept between two samples. In this way, the 2, 0.2 mM dNTPs, 0.05 units/μl of Taq DNA polymerase  and 0.5 μM of each primer. Forward primers were labelled fluorescently at their 5’ end. The following cycling conditions were used: initial denaturation for 5 min at 94°C, 40 cycles of 45 s at 94°C, 45 s at 55°C, 45 s at 72°C; final elongation of 10 min at 72°C. The success of PCR was checked by running 2 µl of product on 1.4% agarose gels stained with GelRed Nucleic Acid Stain . After amplification, PCR products were multiplexed in a single reaction and fragment analysis was carried out on an ABI 3130 Genetic Analyser. Allele sizes were estimated using Peak Scanner software .DNA was extracted by homogenizing 200 mg frozen leaves in 800 μl CTAB isolation buffer (2%) following the protocol described in 2.3Checking for the presence of null alleles was performed using Micro-Checker 2.2.3  by MonteThe genetic structure of the populations has been analysed using Bayesian-clustering method . The mosTo reveal the genetic differentiation among plant samples from the five selected habitats, the microsatellite allele frequency data were evaluated by standardized principal component analysis (PCA) using SYN-TAX 2000 computer program package .33.1C. demersum populations . The average number of alleles per locus, the number of effective alleles, allelic richness and Shannon’s information index all reached the highest values in the Danube River and the Soroksári side arm, while among the backwaters, most of these values were the highest in the Mocskos oxbow  proved to be three Figure 4C. demersum in river habitats was higher than in backwaters. Comparing the backwater samples, the plants of the Mocskos oxbow showed the highest variability.The ordination of microsatellite data resulted in two large, overlapped groups of samples taken from the main Danube River and the Soroksári side arm, and three smaller and better-separated groups representing the oxbow samples Figure 54C. demersum. Based on the newly developed loci, this study revealed differences in the microsatellite polymorphism of C. demersum populations related to the hydrological types of the habitats. Although the number of samples obtained from the populations was not exactly the same due to differences in size and vegetation cover of the water bodies, the number of effective alleles and allelic richness make it possible to compare the genetic variability of populations with different sample sizes. Both calculated variability parameters showed greater genetic variability in riverine habitats than in backwaters. In addition, the applied multivariate analyses revealed low genetic variability from backwaters based on both the smaller (Zátonyi and Mocskos oxbows) and larger sample sizes (Schisler oxbow). The results also demonstrated that a high number of multilocus genotypes could be detected from both river and backwater populations, however, the observed genotypes were genetically closer to each other in the latter than in the former habitat.Although microsatellites have become the most widely used markers for genotyping plants, and the benefits of their use were demonstrated even in population genetic studies of macrophytes, microsatellite primers have not yet been published for C. demersum (C. demersum stands in the hydrologically isolated backwaters showed the lowest genetic diversity. The population of Mocskos oxbow, occasionally flooded by the Danube, which enables gene flow, had medium genetic diversity. If the majority of C. demersum propagules were not spread by water but by wind or birds, they could easily reach the backwaters that are hydrologically isolated from the main river but geographically very close to it.Genetic variation is highly dependent on the ratio of sexual and asexual reproduction, as well as the short- and long-distance dispersal of sexual and vegetative propagules . Asexualdemersum  suggest C. demersum.Topography and geographic distance affect population genetic structure of plants, especially in aquatic habitats . HoweverC. demersum.Of course, genetic diversity detected at marker loci in clonally spreading populations may be attributed to somatic mutations, which are enhanced by rapid clonal growth and a large amount of vegetative propagules . HoweverC. demersum, whose population genetic diversity is highly dependent on flow conditions and hydrological connectivity. The harmful effects of this kind of habitat fragmentation may appear as a result of any local anthropogenic intervention (cf. For the above reasons, although habitat deterioration affects all macrophytes, the loss of hydrological connections between habitats is expected to be more severe for species, such as tion cf.  or globation cf. . That isThe sequence data has been deposited in the SRA database of NCBI with the code PRJNA1012867.AE: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft. KN: Investigation, Visualization, Writing – original draft. PK: Investigation, Methodology, Writing – original draft. EM: Investigation, Methodology, Writing – original draft. JB: Formal Analysis, Investigation, Methodology, Visualization, Writing – original draft.