Results indicate that the OLP tissue microbiome was different from the microbiota of OLP saliva. Compared with the healthy controls, Capnocytophaga and Gemella were higher in OLP saliva, while Escherichia–Shigella and Megasphaera were higher in OLP tissues, whereas seven taxa, including Carnobacteriaceae, Flavobacteriaceae, and Megasphaera, were enriched in both saliva and tissues of OLP patients. Furthermore, FISH found that the average optical density (AOD) of bacteria in the lamina propria of OLP tissues was higher than that of the healthy controls, and the AOD of bacteria in OLP epithelium and lamina propria was positively correlated. These data provide a different perspective for future investigation on the OLP microbiome.The specific etiology and pathogenesis of oral lichen planus (OLP) remain elusive, and microbial dysbiosis may play an important role in OLP. We evaluated the saliva and tissue bacterial community of patients with OLP and identified the colonization of bacteria in OLP tissues. The saliva ( Oral lichen planus (OLP) is a common oral mucosa disease characterized by chronic inflammation, mainly affecting the buccal mucosa, tongue, gingiva, and lower lips . FemalesCandida, and Helicobacter pylori infection . This could provide experimental data for future studies on the potential correlation between saliva and tissue microbiome of OLP patients and healthy people.This study aimed to evaluate the microbial communities of different sample types and whether there are differences between them through bacterial 16S rDNA gene amplification sequencing, to analyze the role of the microorganisms with high abundance co-enrichment in the development of OLP of the two sample types, and to locate the bacteria in OLP tissues by fluorescence This study was approved by the Ethical Committee of the Affiliated Hospital of Stomatology, Nanjing Medical University (permission number PJ2016−034−001) and the Institutional Review Board of Nanjing Medical University (permission number 2014−132). Written informed consent was obtained from each participant and all procedures were conducted following the Declaration of Helsinki.In this study, saliva samples were collected from 40 OLP patients (20 reticular OLP and 20 erosive OLP) and 20 healthy controls. Tissue samples were collected from 24 OLP patients (12 reticular OLP and 12 erosive OLP) and eight healthy controls. All subjects were enrolled from the Department of Oral Medicine of the Affiliated Hospital of Stomatology at Nanjing Medical University from November 2017 to May 2019. Based on the clinical and histopathological diagnostic criteria for OLP proposed by the World Health Organization in 1978  and the The study inclusion criteria were as follows: (i) 22–62 years of age; (ii) no OLP treatment or prescription drug use at least 2 months before sampling; (iii) no history of antibiotic use at least 1 month before sampling; (iv) no immunomodifier use within 3 months before sampling; and (v) no history of serious systemic disorders.Individuals were excluded from the study for fulfilling any of the following conditions: (i) other known oral mucosal diseases; (ii) patients with tumor; (iii) lichenoid reactions caused by drugs or amalgam filling; (iv) pregnancy, lactation, and contraceptives; (v) use of any mouthwash within 7 days; (vi) patients diagnosed as periodontitis, presence of visible caries, and removable or fixed dentures.Due to the diurnal variation of saliva, saliva samples were collected between 8 a.m. and 12 noon, to minimize the variability in salivary flow and compositions. No consumption of any food or beverage at least 1 h prior to sample collection was necessary before conducting a comprehensive oral examination of all subjects. After gargling, about 2.5 ml of spontaneous, unstimulated whole saliva (UWS) samples was collected through the technique suggested by The tissue samples of OLP lesions were all from the representative areas of the buccal mucosa or tongue of OLP patients, while the healthy control tissues were obtained from normal oral mucosa around the tooth extraction wound and sublingual cyst. To reduce sample contamination, tissue samples were fixed in 10% neutral-buffered formalin for 24 h within 1 h after surgical resection and paraffin-embedded using standard histology methods. Tissue samples were taken in duplicate; one was cut into 8−μm−thick serial scrolls and placed into a sterile 2-ml centrifuge tube, which was stored separately at −20°C for 16S rDNA sequencing, and the other was taken from six healthy controls, five reticular OLP, and five erosive OLP for FISH and hematoxylin and eosin (H&E) staining.® Soil DNA Kit . Polymerase chain reaction (PCR) products of all samples were amplified by using the following parameters: denaturation at 95°C for 2 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 30 s, and incubation at 72°C for 5 min. The V3–V4 region of the bacterial 16S rDNA gene was amplified by PCR using primers 341F: 5′-barcode-CCTAYGGGRBGCASCAG-3′ and 806R: 5′-GGACTACNNGGGTATCTAAT-3′, where the barcode was an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20-μl mixture containing 4 μl of 5 × FastPfu Buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions and quantified using QuantiFluorTM-ST .Total genomic DNA was extracted from saliva and tissue samples according to the manufacturer’s instructions using the E.Z.N.A.®3.0 (Life Invitrogen), and every 24 amplicons whose barcodes were different were mixed equally. The pooled DNA product was used to construct the Illumina pair-end library following Illumina’s genomic DNA library preparation procedure. Then, the amplicon library was paired-end sequenced (2 × 300) on an Illumina MiSeq platform  according to the standard protocols.Purified PCR products were quantified by QubitHematoxylin and eosin staining and FISH on tissue sections only included 10 OLP patients and six healthy controls due to insufficient tissue surplus. All formalin-fixed, paraffin-embedded (FFPE) specimens were fixed onto uncoated slides, and the slides were deparaffinized and then hydrated. For each subject, two 4-μm-thick FFPE tissue biopsy sections were prepared, one using standard H&E staining to identify and mark the lesion site of interest, and the other using digoxigenin (DIG)-labeled FISH analysis of 16S rDNA gene universal probe . The spe1) and chimeric sequences were identified and removed using UCHIME. Afterward, the phylogenetic affiliation of each 16S rDNA gene sequence was blasted against the Silva (SSU132) 16S rDNA database and analyzed by Ribosomal Database Project (RDP) Classifier2 using a confidence threshold of 70%  were clustered with 97% similarity cutoff using UPARSE  was the main platform of clustering and statistical analysis. The relative abundance of bacterial taxa in each sample between groups was calculated and analyzed by the Mann–Whitney U-test. Mothur v.1.21.1  and the erosive OLP (n = 4) from 32 FFPE tissue samples of 16S rDNA gene sequencing did not yield enough DNA to be detected or detectable PCR products after 40 cycles of PCR. These samples were excluded from further statistical analysis, which means that tissue samples, including four healthy controls and 20 OLP patients, were eventually analyzed. In the H&E staining and FISH of tissue samples, 10 OLP patients and six healthy controls were finally selected due to insufficient tissue surplus.The healthy controls . However, there were strong variations of the Chao1 index and Shannon index between OLP saliva and tissues  and Shannon index  between the healthy controls, reticular OLP, and erosive OLP in saliva  and tissP = 0.146). However, the OLP group was completely separated from the healthy controls in tissue (P = 0.004), healthy controls and reticular OLP (P = 0.001), and healthy controls and erosive OLP (P = 0.002) were also statistically different. Except for reticular OLP and erosive OLP, which has no statistically significant difference in the weighted measurement (P = 0.150), this indicates that the overall structure of the bacterial community in the population was significantly different, while the structure of reticular and erosive OLP microbial community was not significantly different. There was a partial overlap between saliva and tissue samples of OLP patients (P = 0.001), suggesting that OLP microorganisms between saliva and tissue samples were partially close to each other but still different.The weighted UniFrac phylogenetic distance matrices were used to calculate the beta diversity, indicating the difference in bacterial community structure, which can be seen in Bray–Curtis distance PCA plots . From Fin tissue , and thepatients  and signNeisseria had the highest average relative abundance at the genus level in saliva samples. Compared with healthy controls, the average relative abundance of 11 genera, such as Capnocytophaga, Gemella, and Granulicatella, was significantly higher in OLP saliva (P < 0.05). In the tissue samples, 41 genera such as Escherichia–Shigella, Phyllobacterium, and Megasphaera were significantly higher in OLP than those in the healthy controls (P < 0.05). In particular, compared with healthy controls, Phyllobacterium and Megasphaera were not only significantly higher in both reticular and erosive OLP tissues (P < 0.05), but also higher in reticular and erosive OLP saliva (P > 0.05). In tissue samples, Escherichia–Shigella, Bacteroides, and so on only existed in OLP (P < 0.05). At the species level, in the saliva samples, the average relative abundance of Prevotella saccharolytica JCM 17484, Lactobacillus plantarum, Lactobacillus sp. ARSOA BB, and Neorhizobium huautlense in the OLP was significantly higher than those in the healthy controls (P < 0.05). In tissue samples, the average relative abundance of seven known species including Methylobacterium aquaticum, Achromobacter xylosoxidans subsp. Xylosoxidans, Sphingomonas mali, Pseudomonas poae, Tsukamurella tyrosinosolvens, Rhodopseudomonas pentothenatexigens, and Megasphaera elsdenii DSM 20460 was significantly higher in OLP than that in the healthy controls (P < 0.05).Bacterial communities in OLP and healthy controls were analyzed at different taxonomic levels . RepreseCapnocytophaga, Gemella, Granulicatella, and so on.To further identify the specific bacterial taxa related to OLP, we used the linear discriminant analysis (LEfSe) to compare the microbiota of OLP and healthy controls. Streptococcus, Micrococcus, Sphingobium, and so on; in OLP, Escherichia–Shigella, Megasphaera, Phyllobacterium, and so on.Linear discriminant analysis (LEfSe) was used to determine which taxa could explain the changes observed by UniFrac in the tissue samples . A totalBacteroides and Lactobacillus were the most positively correlated (ρ = 0.87), whereas Fusobacterium and Streptococcus were the most negatively correlated (ρ = −0.48) in saliva samples between healthy controls and the OLP. However, Pelagibacterium and Curvibacter were the most positively correlated (ρ = 0.99), while Stenotrophomonas and Sphingomoas were the most negatively correlated (ρ = −0.77) in tissue samples between healthy controls and the OLP  has been found to promote an inflammatory response by inducing TH1/TH2 imbalance . JanardhMegasphaera was significantly higher in both reticular and erosive OLP tissues than in the healthy controls, and was also higher in reticular and erosive OLP saliva than in the healthy controls. Megasphaera may be associated with viral infection .The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The raw reads for all samples used in this study have been deposited into the NCBI Sequence Read Archive (SRA) database  and the Institutional Review Board of Nanjing Medical University (permission number 2014-132). The patients/participants provided their written informed consent to participate in this study.YF conceived and designed the research. XW, ZZ, and NT performed the experiments. XW, NT, and ZZ processed and analyzed the data with help from JZ. YZ, JX, LQ, and LL recruited volunteers and collected samples. XW drafted the manuscript with inputs from all authors.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.


Visual outcomes in diabetic macular edema (DME) after anti-vascular endothelial growth factor therapy vary across individuals. We retrospectively reviewed the clinical records for 46 treatment-naive eyes of 46 patients with DME who underwent intravitreal ranibizumab (IVR) monotherapy with a pro re nata regimen for 12 months. Overall, mean best-corrected visual acuity (BCVA) improved. Multivariate analyses adjusted for age and baseline BCVA showed that the area ratio, compared with the retinal area, and the number of intraretinal cystoid spaces evaluated on OCT  images at baseline positively correlated with LogMAR BCVA and the extents of ellipsoid zone and external limiting membrane disruption at 12 months, and negatively correlated with central retinal thickness at the time of edema resolution. Therefore, a high area ratio and large number of intraretinal cystoid spaces resulted in a disorganized outer retinal structure at 12 months, a thin and atrophic retina after edema resolution, and a worse visual outcome. The area ratio and number of intraretinal cystoid spaces on initial OCT images were predictors of the visual outcome after IVR therapy in DME irrespective of baseline age and BCVA. The factors were related to retinal neurodegenerative changes in DME and could help in obtaining proper informed consent before treatment. Diabetic retinopathy (DR) is a major complication of diabetes mellitus and a leading cause of visual impairment ,2. In coSpectral domain optical coherence tomography (SD-OCT) is commonly used to evaluate DME before and during treatment. OCT images show the presence of edema and provide varied information regarding the condition of the retina. Previous reports have shown that DME can be divided into three categories characterized by the distribution of the extracellular fluid , i.e., sOther predictive factors of treatment outcome involve initial best-corrected visual acuity (BCVA) ,13, centIn this study, we evaluated initial OCT images to determine biomarkers that would be easily applicable in daily clinical practice to predict visual outcomes and to elucidate the underlying pathogenesis of the DME-affected neural condition of the retina, aiming at helping patients and clinicians to plan the treatment and explore the mechanisms of retinal neural damage due to DME.This was a retrospective study based on a detailed medical chart review. The study included 46 eyes of 46 patients with treatment-naïve DME that were treated with intravitreal ranibizumab (IVR) at the Medical Retina Division Clinic of the Department of Ophthalmology, Keio University Hospital  between April 2014 and October 2018. All patients attended the clinic for at least 12 months, during which the only treatment they received was IVR. All patients underwent BCVA measurement with the refraction test, a slit-lamp examination, and binocular indirect ophthalmoscopy after pupil dilation with 0.5% tropicamide. These examinations were performed at every follow-up visit. Follow-up was carried out every month, but when intra- and/or sub-retinal exudative changes were absent for more than 2 months, the interval was extended to up to every 2 months until recurrent exudative changes were observed. Exudative changes were mainly evaluated by OCT images, referring to the results of fundus examinations. The severity of DR was graded by using fundus photographs and referring to the Fluorescein Angiography (FA) at baseline. Fluorescein Angiography (FA) was performed using a Topcon TRC50DX retinal camera  to determine the grade of the DR. Horizontal sections of Optical Coherence Tomography (OCT) images passing through the fovea were obtained with a Heidelberg Spectralis OCT  instrument in OCT Section mode . Repeat mode was applied at the time of the follow-up visits to obtain OCT images at the exact same location. The images were used to evaluate the CRT, intraretinal cystoid space and subretinal fluid (SRF), and hyperreflective foci. The CRT was defined as the distance between the internal limiting membrane and the presumed retinal pigment epithelium (RPE) at the fovea. The following parameters were evaluated on horizontal OCT images within a 2000 μm diameter around the foveal center. The total area of intraretinal cystoid spaces and the retinal area were calculated using Image J , and the area ratio of the intraretinal cystoid spaces to the retina was defined as the area ratio of the cystoid spaces. SRF was not involved either in the intraretinal cystoid space or retinal areas. The number of intraretinal cystoid spaces with a width larger than 200 μm was counted. The extents of the disorganization of the retinal inner layers (DRIL) ,19,20 anRanibizumab  was injected intravitreally under sterile conditions via the pars plana at first injection. Re-injections were recommended if the OCT image and/or fundus examination showed evidence of any exudative changes in the macula, identified as macular edema or SRF at the time of the follow-up examinations, and performed under informed consent. p < 0.05.All results are expressed as the mean ± standard error. Commercially available software  was used for the statistical analyses. The demographic characteristics of the baseline and outcomes were compared using the repeated measure ANOVA, Pearson correlation coefficients, stepwise multiple linear regression models, and multiple regression analyses. Statistical significance was set at Forty-six eyes of 46 patients with DME  were included in this study. The baseline characteristics of the patients are shown in 2; range, 997–37,290 pixels2) and the retinal  areas. The mean CRT, extents of DRIL, and ELM and EZ disruptions were 418 ± 21 μm, 1056 ± 51 μm, 273 ± 80 μm, and 580 ± 95 μm, respectively. Sixteen eyes (34.8%) had hyperreflective foci, and nine eyes (19.6%) had SRF at baseline. All eyes (100%) exhibited CME, 37 eyes (80.4%) were categorized into the CME type with no SRD type, nine eyes (19.6%) were SRD type, and no eyes were sponge-like retinal swelling type. [The mean area ratio of the intraretinal cystoid space to the retinal area was 19.7% ± 1.9%, and the mean number of cystoid spaces was 3.7 ± 0.3; the area ratio was calculated using the data of absolute amounts of the intraretinal cystoid space   , CRT , and extents of DRIL  and ELM disruption  were also associated with BCVA at 12 months  a and num< 0.001) b of the 2 months .p < 0.001), and number  of the cystoid spaces at baseline. There were no significant correlations between BCVA at 12 months and other baseline findings.We further analyzed the correlations using stepwise multiple linear regression models . After aTherefore, a high area ratio and large number of intraretinal cystoid spaces at baseline predicted worse BCVA at 12 months. Representative cases with or without a high area ratio and large number of cystoid spaces at baseline are shown with their outcome data at 12 months in R = 0.577, p < 0.001, R = 0.589, p < 0.001, R = 0.377, p = 0.009, R = 0. 468, p = 0.001, R = 0.485, p < 0.001, R = 0. 483, p < 0.001, BCVA at 12 months was positively correlated with the extents of ELM disruption  and EZ  disruptions at 12 months after adjusting for age and initial BCVA (Multiple linear regression models showed that the area ratio and number of cystoid spaces at baseline were positively correlated with ELM (ial BCVA . This inR = −0.369; 95% CI, −0.004 to −0.0003; p = 0.024; Next, we analyzed the CRT of 37 eyes whose cystoid spaces and DME had resolved at least once during the 12-month follow up; the other nine eyes did not achieve complete resolutions of DME during these 12 months. The mean CRT at the time of DME resolution was 204 ± 8 μm, and the CRT at that time point was correlated with BCVA at 12 months (R = −0.707; 95% CI, −0.839 to −0.496; p < 0.001; R = −0.561; 95% CI, −0.749 to −0.290; p < 0.001; Finally, we found that CRT at the time of cystoid space resolution was negatively correlated with the area ratio (A representative case with a high area ratio and large number of cystoid spaces, with a thin CRT at the time of DME resolution, is shown in Here, we examined initial OCT images to determine biomarkers that can be easily applied in daily clinical practice to predict the visual outcome after IVR monotherapy. Overall, the patients with DME were successfully treated with IVR monotherapy in terms of both visual function and retinal morphology . The areOne of the main molecular mechanisms underlying DME is BRB disruption by inflammatory cytokines such as VEGF . RanibizOCT images are essential for managing the treatment of DME, and various predictive factors obtained from OCT images have been reported for anti-VEGF therapy. CRT is a major clinical factor for macular diseases, and a previous report showed that the correlation between CRT and BCVA after treatment was modest in DME . In the By contrast, the area ratio and number of intraretinal cystoid spaces were new and significant predictive factors for the visual outcome after IVR treatment for DME irrespective of the patients’ age and initial BCVA. Therefore, when the cystoid spaces are growing and/or increasing, it may be advisable to start treatment soon, even if BCVA is acceptable and photoreceptor damage is not evident in OCT images, although further studies are warranted. It is an advantage that the intraretinal cystoid spaces on OCT images are easily evaluated visually during daily clinical practice.Longer extents of ELM and EZ disruptions at 12 months were related to worse BCVA at 12 months. Given that the ELM and EZ disruptions reflect photoreceptor disorganization, it is natural that they would be related to to BCVA at the same time point. More importantly, a greater area ratio and number of cystoid spaces were correlated with longer extents of ELM and EZ disruptions at 12 months, suggesting that the severity of cystoid spaces may have afterward influenced the ELM and EZ conditions. In addition, eyes with a greater area ratio and number of cystoid spaces at baseline showed a very thin retina at the time of cystoid space and DME remission; a thin CRT, such as that under 200 μm, most likely indicated neural atrophy rather than a healthy retina after DME remission as previously reported.  Karst reCystoid spaces developed as a result of extracellular fluid, i.e., edema, mainly spread to the inner retinal layers ; howeverInner retinal changes may also affect Müller glial cells whose cell bodies are in the inner retinal layer. In particular, the human and macaque foveolas contain specialized Müller glial cells, called foveal Müller cells . They prThe hypothesis that the presence of intraretinal cystoid spaces is related to the progression of photoreceptor degeneration to ultimately inflict permanent ELM and EZ disruptions and a thin retina despite treatment merits further investigation.The limitation of this study was the relatively small sample size and the retrospective design. Moreover, multiple physicians managed the patients, and treatment requirements were determined by each physician and not by the reading center. If the patients refused additional injection although it was recommended, treatment was not performed at that time point; however, there were few such patients. The range of initial BCVA was relatively wide, and eyes with good and bad BCVA at baseline were involved; however, the significant associations were found after adjusting for age and initial BCVA. In addition, because continued treatments would be ideal to attain the best possible outcome , more agIn conclusion, the area ratio and number of intraretinal cystoid spaces were predictors for the functional and morphological prognoses of IVR monotherapy for DME. A greater area ratio and number of cystoid spaces may reflect greater retinal neural vulnerability at baseline, and these may be biomarkers for the progression of retinal degeneration as shown by the ELM and EZ disruptions and/or thin retina on the OCT images despite treatment. Although further studies are needed to validate the present findings, these factors could help to predict treatment efficacy and obtain proper informed consent before treatment.


Zea mays L.) under field conditions is not particularly clear. Mesh-barrier compartments were applied to monitor the distribution of hyphal P uptake throughout the experimental period under different planting densities and soil depths, over two consecutive years. AM symbioses enhanced plant P-acquisition efficiency, especially during the silking stage, and hyphae of AM fungi was assessed to contribution 19.4% at most to total available P content of soil. Moreover, the pattern of AM depletion of soil P generally matched shoot nutrient demand under the high planting density, which resulted in significantly increased yield in 2014. Although the hyphal length density was significantly decreased with soil depth, AM fungi still had high potential for P supply in deeper soil. It demonstrates the great potential of indigenous AM fungi to maize productivity in the high-yield area of China, and it would further provide the possibility of elimination P fertilizer applications to maintain high yields.Arbuscular mycorrhizal (AM) symbioses are an attractive means of improving the efficiency of soil phosphorus (P) that difficult to be used by plants and may provide a sustainable way of maintaining high yields while reducing P applications. However, quantifying the contribution of indigenous AM fungi on phosphorus uptake and yields of maize ( Thus, a sustainable management plan for P applies in these farming systems to meet the food requirements of China’s growing population is urgently needed. On the one hand, P is an essential resource that is necessary for plant growth and development. Crop yields are constrained by available P content and, therefore, the application of P fertilizers is necessary to achieve global food security2. On the other hand, long-term and excessive application of P fertilizer has resulted in substantial P accumulation in the soil, which have led to accelerate the consumption of the finite supply of P from rock and diminish environmental quality through the widespread eutrophication of surface water4. To enhance the efficient use of P, reduce the environmental damage associated with P fertilizer applications, and yet maintain future food security, one option for sustainable P management is to improve the utilization of the large reserve of residual P in soils left behind by the long-term overapplication of P fertilizer in cultivated areas5.High-density planting systems  fungi form symbiotic associations with the roots of more than 80 percent of terrestrial plants, including agricultural plants such as maize. Mycorrhizal symbioses can benefit plants and soils in many ways, such as enhancing plant growth and drought tolerance by improving mineral and water uptake, root architecture11, and check the losses of applied nutrients12. Meta-analysis had confirmed that AM fungi have a positive effect on the increment of crop grain yields13.It may be possible to fully exploit the soil P legacy and reduce dependence on P fertilizer inputs by maximizing root/rhizosphere efficiency, such as mycorrhizal symbioses, which promote the efficient uptake of P from soil15, and the morphological characteristics of crop roots, which adversely impact nutrient absorption via the root pathway16. Consequently, to understand how AM fungi are associated with P nutrition in well-fertilized, high-density planting systems is a first step toward assessing whether P applications could be reduced to improve the management of intensive cropping systems. However, most of these communities were studied in inoculated ecosystems or in pots under experimental conditions19. Furthermore, growth depressions are caused by fungal demands for organic carbon from the host plant that outweighs any benefits; it might be produced by P transfer via the fungus. Moreover, Li et al.20 found that wheat growth depressions caused by AM associations decreased with increasing plant density in calcareous soil, but not just caused by carbon drain. Here we are wondering that how the mycorrhizal pathway responds to adequate carbon sources, and the contribution of indigenous AM fungi to P acquisition in high-density planting systems under in situ field conditions in the black soil region of China remains unclear.High-density planting systems have been reported to negatively effect the diversity and abundance of AM fungal communitiesThus, the primary objective of this work was to measure AM hyphal P uptake in the field by a modified compartment system, estimate the potential contribution of the indigenous AM fungi to P uptake of maize plants to enhance the final product in the high-yield area of China under high planting density mode.The above-ground biomass , AM fungal density, and mycorrhizal colonization generally increased with the growth stage; however, the amount of increase differed between the cropping years Fig. .Figure 1Shoot biomass significantly increased during the vegetative stages, and the average shoot dry weight at the R1 stage was approximately five times higher than that at the V6 stage , representing 19.4% of the total available soil P contributed by the mycorrhizal pathway to maize. This indicates that AM fungi played a vital role in the supply of P to the plant during the V6 to R1 stages even though the depletion of soil P by hyphae was inconspicuous. Furthermore, R1 might be a critical stage for the efficient acquisition of P for a dense planting system.Furthermore, two consecutive years of experimental results showed that Even though P > 0.05) , and the different slope of the line indicated AM fungi might much more efficient in making soil Olsen-P available in 2013 than in 2014  over the whole experimental period, hyphal length density showed a significant relationship to shoot P content in the high-density planting system, P uptake linearly increased with increasing AM hyphal length density, but no correlation was obtained under the low plant density system (05) Fig. a. Corres05) Fig. b. Furthe23. Root infection increased as the growing season progressed and usually peaked in July and August in the maize system24. In the present study, hyphal length density per unit area was found to increase in association with the growth of maize slightly, and AM colonization simultaneously increased until the R1 stage, except for individual samples. This is similar to the previous study; the hyphal length density was very low at the V6 stage, increased significantly between V12 and R1, then declined somewhat by R625.Previous work has demonstrated that AM fungi interacting with plants can shift with time within the whole growing season27. AM fungi may also respond strongly to soil fertilization and planting densities29. Our results confirmed that changes in hyphal P uptake levels during growth coincided with those in the AM colonization rate and the shoot P acquisition by maize, hyphal P which measured by hyphal compartments was increased as the growing period progressed, and peaked at the R1 stage followed by a significant decline at R3 stage under the high planting density. However, it differed under low planting density between two cropping years. This implies that the absorption of P by maize is most dependent on AM fungi in the high-density planting system during the vegetative stages, and R1 is the essential stage at which to measure the contribution of AM fungi to the growth of maize.Maize is a mycorrhizal-dependent plant. The extra-rhizosphere mycelium of AM fungi plays an essential role in balancing nutrient demand and supply by highly branched networks in the rhizosphere and absorbing P and organic matter non-rhizosphere soil that cannot be absorbed by plant roots or root hairs30; mycorrhizal colonization was significantly decreased when soil P supply up to over 10 mg kg−1 in the wheat plant system31. P fertilizers are regularly recommended based on target yields and soil test values to maintain soil fertility and achieve higher crop yields. However, in Northeast of China, low soil temperature during spring inhibits development of maize, due to low root growth and subsequent nutrient acquisition. In practice, some farmers still tend to apply excessive quantities of P to maximize yields and improve soil P fertility. Many studies have shown that the maximum AM colonization rate of maize roots was more than 40% overall growth period24. In our study, the results from both consecutive years showed that the AM colonization was very low and that the highest infection intensity was no more than 16%. This low infection intensity may due to a high level of basal P supply (16.75 mg kg−1 before fertilization) or the usage of chemical fertilizer, pesticide, and tillage practices before this study, such as uniformity of the crop species32. Moreover, the colonization of roots by AM fungi is also controlled by shoot P status33. As we have known, the demand for P nutrient increases gradually with the growth of maize; simultaneously, shoot P concentration decreases with the distribution of the accumulated P to support for the development of various organs, especially at the reproductive stage , the maximum hyphal length density occurred in the topsoil. Even though roots were still readily found in the deep soil layer, the distribution of AM fungi in the soil layers is likely explained by the overall decrease in plant root density with increasing soil depth. The escape of some AM fungi may have caused the abundance of fungi in the deep soil layer into deep soil layers under adverse conditions46, such as the application of large quantities of fertilizer to the topsoil. Fertilizers can adversely affect AM fungi by inhibiting their growth, development, and function47; however, these effects gradually decrease with increasing soil depth. Therefore, there was only a slight (but significant) difference in hyphal P between the surface and deeper soil layers. Furthermore, the hyphal uptake of P and hyphal length density in the different soil profiles showed a strong linear relationship, irrespective of planting density and cropping year, indicating that Olsen-P uptake by maize from the subsoil might also rely on AM fungi because of the limited number of roots and that AM fungi play an immeasurable role in the deep soil.Soil available P is not homogeneously distributed in the vertical soil profile due to the low mobility of P in the soil−1; alkali-hydrolyzed nitrogen content, 120.18 mg kg−1; total phosphorus content, 0.28 g kg−1; and NH4Cl-exchangeable potassium content, 140.37 mg kg−1. Additionally, soil Olsen-P content in different soil profiles was 16.75  mg kg−1, 10.75 mg kg−1, 6.95 mg kg−1, 6.05 mg kg−1, respectively from four soil layers .The experiment was carried out at the Experimental Station of Jilin University, Changchun, China (43Zea mays L. cv. Zhengdan 958) was sown on 1 May 2013 and 30 April 2014 at densities of 50,000 (50 K) and 90,000 plants ha−1 (90 K) in both years, with 70 cm row spacing.The field study was conducted from 2010 to 2014, incorporating four cropping years. In this report, all preliminary experiments were organized in 2013 and 2014 cropping years, referred to as 2013 and 2014 in the context, except for maize yield determination (from 2011 to 2014). Maize  according to the local tillage regimes and plant root distribution range. Then hyphal compartments were buried in the middle of each soil layer in pairs on either side of a maize plant fifteen days after maize emergence Fig. b, three Trial 3: Meanwhile, to eliminate the interaction between AM fungi and bacteria, specific hyphal compartments filled with 500 g of sterilized soil were buried in the field in different depths from 0 to 80 cm in cropping the year 2013. All experimental procedures were the same with trial 2, and soil samples were collected only at milk growth stages (R3) to determine the hyphal length density and hyphal P concentration. , twelfth leaf stage (V12), silking stage (R1), and milk (R3) growth stages in both 2013 and 2014. The plants were cut at the shoot–root junction. Shoots were de-enzymed by heating at 1052 of maize (2 rows−1) was calculated.To determine maize yield, 9.8 m49. Soil available P level was determined as Olsen-P using the molybdo-vanadophosphate method by extracting soil samples with 0.5 M NaHCO50. Extraradical hyphae were extracted from each soil sample and stained with Trypan blue. The hyphal length density (HLD) of AM fungi was determined using the grid line intersect method in which AM fungal hyphae were distinguished from non-AM hyphae by the presence of irregular septa, dichotomous branching, irregular wall thickness, and connection to chlamydospores51, the net increment of HLD in the rhizosphere was calculated as follow. The reduction was the presence of other soil organisms with which AM fungi interacts:Shoot P concentration was measured by the molybdo-vanadophosphate method after samples were digested in concentrated HWhere The soil samples were collected from the hyphal compartments at each growth stage, and analyzed for Where 52 was used to pretreat and stain the root samples. The proportion of the maize root system that was colonized by mycorrhizal fungi was estimated using the method described by Phillips and Hayman53. AM fungal colonization is usually presented as infection intensity (M%) and arbuscular abundance (A%), which are calculated by analyzing 30 randomly selected root segments per growth stage using MYCOCAL software . And then, we transformed the infection intensity (M%) and arbuscular abundance (A%) to the value of angle using the following equation:To evaluate AM fungal colonization, the harvested root samples were cut into fragments approximately 1 cm in length. A modified version of the method described by Koske and GemmaWhere ASIN represent the Arcsine function to normalize distributions of the infection intensity (M%) and arbuscular abundance (A%).PPAll data presented are means of untransformed values (±SD), the significance of differences in shoot dry weight, hyphal length density, infection intensity, arbuscular abundance and P content from different growth stages, and hyphal length density and P content from different soil depths between two planting densities was tested using Fisher’s least significant difference (LSD) at Supplementary information 1Supplementary information 2


Acropora tenuis. Genes/proteins related to oogenesis were investigated: Vasa, a germ cell marker, vitellogenin (VG), a major yolk protein precursor, and its receptor (LDLR). Coral branches were collected monthly from coral reefs around Sesoko Island  for histological observation by in situ hybridisation (ISH) of the Vasa (AtVasa) and Low Density Lipoprotein Receptor (AtLDLR) genes and immunohistochemistry (IHC) of AtVasa and AtVG. AtVasa immunoreactivity was detected in germline cells and ooplasm, whereas AtVG immunoreactivity was detected in ooplasm and putative ovarian tissues. AtVasa was localised in germline cells located in the retractor muscles of the mesentery, whereas AtLDLR was localised in the putative ovarian and mesentery tissues. AtLDLR was detected in coral tissues during the vitellogenic phase, whereas AtVG immunoreactivity was found in primary oocytes. Germline cells expressing AtVasa are present throughout the year. In conclusion, Vasa has physiological and molecular roles throughout the oogenic cycle, as it determines gonadal germline cells and ensures normal oocyte development, whereas the roles of VG and LDLR are limited to the vitellogenic stages because they act in coordination with lipoprotein transport, vitellogenin synthesis, and yolk incorporation into oocytes.This study aimed to elucidate the physiological processes of oogenesis in  They engage in a multispecific spawning event around the designated moon phase once a year4. Approaching the spawning season, corals undergo gametogenesis until their gametes are fully matured; oogonia appear alongside the mesoglea in each gastrodermal mesentery and differentiate into oocytes, which accumulate yolk in their cytoplasm5. Scleractinians like Acropora and Montipora have matured oocytes and sperm packaged as egg-sperm bundles6, which then appear at the mouth of coral polyps and are released into the seawater column a few hours after sunset. Other broadcast spawners  do not form egg-sperm bundles but the eggs and sperms are released directly from the mouth of their polyps. In some cases, certain coral species spawn during the day. For example, Pocillopora meandrina, P. acuta , P. verrucosa and P. eydouxi  spawned early in the morning, while Porites rus  and Pavona sp. (Gulf of Thailand) released sperms in the noon8. Although this process of oogenesis in corals is well documented3, there is limited information on the internal system of oogenesis in corals.Approximately 70% of scleractinian corals are hermaphroditic broadcast spawners and have both male and female gonads developing within the polyp of the same colonyvasa, piwi, nanos, oskar, gurken, aubergine etc.) are discovered10. Among the germline cell specific genes, vasa and piwi were identified in the stony coral, Euphyllia ancora12. Vasa, a member of the DEAD-box RNA helicase family, acts as a translational regulator of germline-specific target mRNAs and piRNAs to determine cell outcome within germline cells16. Vasa was originally identified in Drosophilla melanogaster17 and afterwards, in various vertebrates  and invertebrates . On the other hand, Piwi is a member of the Argonaute (AGO) protein family, characterized by two major motifs, the Piwi/Argonaute/Zwille (PAZ) domain and the PIWI domain. Piwi proteins associate with piwi-interacting RNAs (piRNAs) and regulate epigenetic programming and posttranscriptional regulation, which may be involved in germline specification, germ cell maintenance, transposon silencing, and genome integrity30. In the stony coral, E. ancora, Vasa and Piwi are expressed in early stage germline cells and is proven to be reliable molecular markers to identify germline cells in corals12. It was also reported that Vasa and Piwi in marine invertebrates  acts as a maternal factor in formation and determination of germline and multipotent stem cells during embryogenesis. Furthermore, both Vasa and Piwi plays a crucial role in germ cell formation, maintenance, and development, as well as gamete maturation38. Post-transcriptional studies showed that a null mutation removing the entire Vasa coding region interfered with reproductive capabilities resulting in female sterility with severe defects, including abnormal germline differentiation and oocyte determination38. In D. melanogaster37 and C. elegans35, defects in germline proliferation and abnormal/absent of oocytes in the gonad were observed when Vasa gene was neutralized by RNAi. In addition, delayed spermatogenesis and defective sperm was observed in C. elegans35. While in zebrafish36 and mice38, mutation in Vasa genes hinders the progress of spermatogenesis and defects in germ cell proliferation and differentiation. As germline cells ensure the continuous production of gametes, germline-specific genes  that are expressed in germline cells may have a significant role in gametogenesis in the reproductive cycle of scleractinian corals.Oogenesis is a complex process involving successive mitotic and meiotic phases that are controlled by several reproductive genes and proteins. Gametes, such as egg and sperm are derived from germline cells in the gonadal region. In order to identify germline cells from other somatic cells, some germline cell specific genes  and transported to the gonad to be accumulated in the oocytes45. On the other hand, VG of corals46 and sea anemone48 is synthesised within putative ovarian tissues by mesenterial somatic cells in the vicinity of the oocyte. In the sea anemone, yolk synthesis involves in both autosynthetic and heterosynthetic processes including the biosynthetic activity of the Golgi complex and the uptake of VG via endocytosis48. Ultrastructural studies of the sea anemone48 and jellyfish49 revealed the presence of specialized gastrodermal cells (known as “trophocytes” and microvilli around the oocytes), which play an important role in nutrients transportation during vitellogenesis. There is also evidence that extraoocytic uptake of VG is manifested by the proliferation of endocytotic pits and vesicles over the surface of the oolemma in sea anemone47. The VG uptake process in corals has yet to be elucidated, although two possibilities have been proposed: either via transmesoglea pores or the somatic cell-oocyte contact through transmesoglea pores and would be subsequently taken up by oocytes through receptor-mediated endocytosis50. Due to the large size of the VG protein, the latter possibility is more likely. VGR belongs to the supergene family of low-density lipoprotein receptor (LDLR)-related proteins that have co-evolved in egg-laying and viviparous animals51. Regarding VGR studies in vertebrates, proteins with a high affinity for VG have been identified and characterised on the oocyte membrane from chicken53, frog53, and several fish species, including the rainbow trout55 and the sea bass56. On the other hand, VGR and LDLR have been identified in invertebrates such as insects57, mites58, ticks60, shrimps63 and crabs64. These findings showed that the VGR transcript was up-regulated during the initial phase of VG uptake into the oocyte, indicating that VGR is critical for yolk protein absorption and ovary maturation. Although VGR/LDLR has yet to be identified in corals, it is probable that VG is synthesised in the somatic cells of ovarian tissues and incorporated into oocytes via receptors during vitellogenesis.Vitellogenesis is the process of vitellogenin (VG) synthesis and incorporation into developing oocytes. In most animals, VG is synthesised in the somatic tissues  and the immunoreactivity against AtVasa and vitellogenin (AtVG) using immunohistochemistry (IHC). In addition, we investigated the relationship between these gene and protein profiles and oocyte development during the annual reproductive cycle of this species. This study holds great significant as it helps us to understand the flow of oogenesis in Acroporid corals, as well as the physiological and molecular role of the reproduction related genes (Atvasa and AtLDLR) and proteins (Atvasa and AtVG) throughout the oogenesis process. This study will be the first to show molecular evidence on the involvement of LDLR during vitellogenesis in corals.The aim of this study was to clarify the oocyte development of the scleractinian coral The results of this study will be of great benefit to understand the physiological process underlying oogenesis in scleractinian corals as it would provide as; (1) Benchmark for reproductive studies of other species of scleractinian corals, (2) Molecular indicators to identify the internal factors that promote oogenesis in scleractinian corals, (3) Stepping stone for future coral reproductive studies such as endocrine/neuroendocrine pathways, effect of environmental factors on gametogenesis/spawning and coral culture experiment under artificial conditions, (4) New insights of reproductive mechanisms in other cnidarians.Histological observations revealed that the site of gametogenesis (in the gonad) was located within the mesoglea of the mesentery, which was located between the mesentery filament and the retractor muscles in the endoderm Fig. . One polA. tenuis are shown in Fig. Annual changes in environmental factors (sea surface temperature [SST] and photoperiod) and oocyte diameters of Early vitellogenic oocytes were observed at Stage II of oogenesis in November (114.33 ± 4.51 µm) and December (175.33 ± 5.38 µm), which then continued to develop into Stage III in January (201.27 ± 5.73 µm) and February (247.26 ± 9.38 µm). The mean diameter of vitellogenic oocytes (Stage IV and V) was 278.19 ± 11.50 µm in March, which increased in April (312.44 ± 6.54 µm) and peaked in May (376.19 ± 5.28 µm) before the release of fully matured eggs (Stage VI) in June (605.53 ± 24.04 µm). This value significantly decreased in July (41.27 ± 2.70 µm). The beginning of Stage I of oogenesis was observed from July to September (74.52 ± 1.92 µm) Fig. .AtVasa, AtLDLR and AtVG were partially cloned in the present study. The cDNA of AtVasa had an open reading frame (ORF) composed of 1242 bp (413 amino acid residues). It contained conserved domains that were also found in the ORFs of other cnidarians and invertebrates . Based on the deduced amino acid sequences, the cDNAs were identified as members of the LDLR superfamily. The sequence contained LDLR Domain Class A, the DXSDE motif, the FWTD motif, cysteine-rich repeats, and the following conserved modules: a calcium binding site, a putative binding site, and epidermal growth factors, which were also found in human and other cnidarian ORFs . The sequence contained a domain of unknown function (DUF1943) and cleavage sites of subtilisin family endoproteases, which were also found in other scleractinian corals  and distributed throughout the gonadal regions in the retractor muscles of the mesentery along the mesoglea  and oocytes from A. tenuis were observed, and positive signals were strongly detected at Stages III, IV and V (the vitellogenic phase), but weak/no signal at Stages I and II (the non-vitellogenic phase)  Fig. .Figure 8A. tenuis tissues at different stages. Tissues (putative ovarian tissues in the mesentery and mesentery filament) and oocytes from A. tenuis were observed, and immunoreactivity against AtVG was faintly detected in the cytoplasm of oocytes or not detectable at Stages I , ATPase motif , the motif involved in ATP binding and cleavage  and the RNA unwinding motifs (SAT) were observed. They were also confirmed in E. ancora11, H. magnipapillata26, and H. echinata33. Thus, AtVasa most likely encodes an ATP-dependant RNA helicase. Through partial cloning of AtLDLR, on the other hand, typical functional motifs – calcium binding site, putative binding site, epidermal growth factor (EGF)-precursor domains – of LDLR superfamily and cysteine-rich LDLR Domain Class A repeats were observed and are present in chicken52, teleost55, insects67 and crustaceans63. Thus, it is likely that AtLDLR is a cell-specific lipoprotein receptor. Through partial cloning of AtVG, the deduced amino acid sequence contained specific domains – DUF1943 and cleavage sites of subtilisin family endoproteases – that are conserved in the VG of invertebrates40 and vertebrates31. They were also confirmed in E. ancora46 and G. fascicularis70. Thus, it is likely that AtVG is one of the major precursors of the egg yolk proteins.The cDNAs of A. tenuis. Oocytes and spermatocytes disappeared in coral branches after the full moon in June, indicating that the spawning of A. tenuis in Okinawa occurs before this moon phase in June. The observation of small oocytes in July indicates that oogenesis begins one month after the mass spawning. We found that vitellogenesis begins in December and actively continues until June. Previous studies of other Acropora spp. revealed that the oogenic cycle time varied among different regions. For example, oogenesis in A. millepora in the Philippines (~26–28 °C throughout the year)71, in broadcasting coral species in the Red Sea 72, and in Acropora spp. in the Great Barrier Reef (~29–32 °C in summer)74 occurred over a period of 8, 6 to 7, and, 9 months, respectively. The shorter oogenic cycle in these regions can be associated with higher seawater temperatures and different climates , which may promote oocyte development in corals. This association may be supported by the fact that warmer water temperatures can increase the rate of development of both gametes77 and embryos79 in corals. Furthermore, our results regarding the oogenesis stages correlate with the changes in SST and photoperiod in Okinawa  and Nanos (Nvnos1) in the starlet sea anemone, Nematostella vectensis9. On the other hand, maternal expression of Nvvas1 and Nvnos2 are detected in unfertilized and fertilized eggs of N. vectensis9. In addition, E.ancora also demonstrated that EaVas and Eapiwi gene products (transcripts and proteins) are maternally inherited factors as they were present in unfertilized eggs12. Nevertheless, it should be taken note that the AtVasa mRNA are diluted by other various proteins accumulating within the oocyte cytoplasm, which may cause faint or undetectable ISH signals. During oogenesis, the A. tenuis germline cells in the retractor muscles of the mesentery differentiated into oogonia and developed into oocytes freely in the mesoglea. This results differed from the previous study in the starlet sea anemone, in which the oocytes bulge basally into the mesoglea and undergo vitellogenesis while retaining intimate contact with the gastrodermis via trophonema48.E. ancora11, which found that Vasa protein may play a role in germline cell formation and maintenance, as well as ensuring normal oocyte development. Several reports have demonstrated the involvement of Vasa as a mitotic and meiotic factor in germline cells, as it regulates the localisation of chromosome-associated proteins that mediate chromosome condensation and segregation87. Proteins and mRNAs stored in the oocytes may contribute by maximising translation during embryogenesis, as recent reports have shown that Vasa is essential for protein synthesis in early embryos of the sea urchin, Strongylocentrotus purpuratus88. A decrease in Vasa immunoreactivity in the developing oocyte may be due to an arrest in Vasa protein synthesis and the accumulation of other proteins, leading to the dispersion of Vasa proteins throughout the oocyte cytoplasm91. It is suggested that other essential proteins  are synthesised at high levels as the oocyte volume expands and maturation approaches.Localisation of AtVasa immunoreactivity was observed via IHC in the germline cells and in developing oocytes. The observed signals were strong in the germline cells and small immature oocytes, but became weak in the oocyte cytoplasm as the oocytes developed and yolk accumulated. The results of the present study are in agreement with previous reports in AtLDLR transcripts and AtVG immunoreactivity in the coral branches of A. tenuis. AtVG immunoreactivity was observed in the cytoplasm of the developing oocytes, putative ovarian sites, and mesentery filaments. The results of the present study agree with those of previous study in E. ancora46 that yolk protein synthesis in A. tenuis is heterosynthetic. It is likely that the immunoreactivity detected in the mesentery (putative ovarian site) cells was synthesised vitellogenin, suggesting that these cells are the major sites of vitellogenin synthesis. Conversely, the immunoreactivity in oocytes was likely yolk, which is a cleavage form of vitellogenin and used as a source of nutrition and energy during embryogenesis after fertilisation93. It is suggested that the mesoglea of the putative ovarian site is a nutrient storage organ in cnidarians94. In contrast, it was suggested that a significant amount of yolk synthesis is autosynthetic within the oocyte due to the presence of synthetic organelles such as rough endoplasmic reticulum and Golgi complexes in developing oocytes during vitellogenesis of the strawberry anemone (Actinia fragacea)96. Aside from vitellogenin, novel egg proteins have been discovered in G. fascicularis 70 and E. ancora (EaEp and euphy)97. Among the egg proteins identified, GfEP4 in G. fascicularis and EaEp and euphy in E. ancora did not exhibit sequence similarity to VG or proteins identified in other phyla97, nor did they exhibit immunoreactivity with anti-EaVg/anti-EaEp antibodies97. It is likely that some proteins with functions other than larval nutrition are present in coral oocytes. The major yolk components of coral oocytes may differ from those of the oocytes of other marine invertebrates and vertebrates. In addition, the gonochoric G. fascicularis VG (GfEP-1) were expressed not only in female colonies, but also expressed weakly in male colonies69. It was also reported that G. fascicularis possess pseudo-oocytes in male colonies69, which were not observed in the present study. AtVG immunoreactivity was also not observed in neither testis nor its surrounding tissues  surrounding the oocytes, mesoglea of the mesentery, and mesentery filaments, but not around the membranes of the developing oocytes. This differs from the results of previous studies in other organisms , which found the VGR transcript exclusively localised in oocytes within the ovary57. One possible explanation is that coral oocytes possess a VGR that is different from the VGR of other phyla. This may be supported by the identification of a novel lipoprotein receptor (Lrp13) in white perch (Morone americana)98 and striped bass (Morone saxatilis)100. Lrp13 specifically binds to only one type of VG out of the three VGs in white perch, and its transcript specifically localised in the oolema and zona radiata of the vitellogenic oocytes. Another possibility is that corals possess multiple LDLRs. Our recent investigations confirmed that A. tenuis possess more than one LDLRs (unpublished data), which highlight the possibility of them being VGR. As discussed above, AtLDLR belongs to the LDLR superfamily and is responsible for mediating functions in the somatic cells where VG and other egg proteins are synthesised. The LDLR gene superfamily comprises different genes encoding membrane receptors involved in the endocytosis of a variety of ligands. The members of this family that have been characterised in vertebrates include LDLR, LDLR-related proteins, sortilin receptor, and very low-density lipoprotein (VLDL) receptor. The ability of an LDLR to bind to a specific type of ligand  depends on the ligand binding properties of the receptor98. These receptors typically consist of a unique configuration of epidermal growth factor precursors (EGFP), class B YWxD (LDLB), and O-linked sugar domains, which define their identities, and class A ligand binding (LDLA) repeats, which determine their particular ligand specificities. A molecular study comparing the presence of a number of ligand binding domain repeats for VGR, LDLR, and VLDL receptor revealed a high degree of similarity (around 85%) among the receptors101. Because VGR also belongs to the LDLR superfamily, it shares the same amino acid structure with an eight-repeat ligand binding domain104. In addition to binding to VG, VGRs in the plasma membrane of the oocyte can bind major yolk lipoproteins and VLDL, which are subsequently transported into the cytoplasm of the oocyte105. The mechanisms and components operating in the ovarian tissue of A. tenuis may be similar to the LDLR and VLDL receptor systems in somatic tissues of the ones in vertebrates as shown in mouse102, chicken105, rabbit106 and fish100. We suggest that the AtLDLR protein identified in the present study plays an important role in the synthesis of low-density lipoprotein in extraovarian sites and is directly or indirectly involved in oocyte development. It would be interesting to investigate other LDLRs present, as well as the vitellogenesis-related lipoprotein receptors of other vertebrates (such as Lrp13) in corals.ISH analyses revealed that the A. tenuis. Signals were weak at the early stages of oocytes (Stages I and II), but became stronger towards the later stages of gametogenesis (Stages III–V). These characteristics are consistent with the previous reports in E. ancora46, in which immunoreactivity against VG becomes stronger in oocytes as oogenesis progresses. Additionally, in ISH observations, the expression of AtLDLR mRNA was not detected in A. tenuis tissues in the immature oocytes stage. However, positive mRNA signals were observed in the somatic tissues of the mesenteries in late Stage II of oogenesis. It is suggested that yolk incorporation starts at Stage III (in November and December) and is activated at Stage IV (March and April) to Stage V (April and May). Histological and IHC analyses of the present study demonstrated that vitellogenesis began after the oocytes entered the mesenterial mesoglea, which is supported by previous reports in N. vectensis48, G. fascicularis70, E. ancora46 and Astrangia danae107. It was also reported that vitellogenesis of the sea pen (Pennatula aculeate)108 and strawberry anemone (Actinia fragacea)96 can be initiated before or after the entry of germ cells into mesoglea. Because the present study revealed that the expression of AtLDLR mRNA and immunoreactivity of AtVG changed as oocyte development progressed, AtLDLR may be activated as VG is synthesised in the somatic cells, and transported into the oocyte as it develops.In this study, IHC analyses were performed in oocytes at different stages, revealing that AtVG immunoreactivity detected in oocytes was correlated with the reproductive cycle of AtVasa) and LDLR (AtLDLR) genes, as well as immunoreactivities against AtVasa and AtVG, in A. tenuis. Our results revealed that these proteins are closely related to oogenesis and can be used as markers for oogenesis in corals. Based on the ISH and IHC analyses, it is possible that Vasa plays a role in germ cell determination and development, as well as oogonia-oocyte differentiation. Therefore, the physiological and molecular roles of Vasa last throughout the year. Conversely, the physiological and molecular roles of VG and LDLR are limited to the vitellogenic stages, which last for at least 6 months beginning in December, as these proteins interact with the processes of vitellogenesis, such as lipoprotein transport, vitellogenin synthesis in somatic cells, and incorporation of yolk into oocytes. Because the oogenic cycle of corals was found to correlate with changes in SST and photoperiod, further studies and culture experiments are required to determine the effects of these environmental factors on oocyte development and reproduction-related genes in corals.In conclusion, we investigated the expression of the Vasa (A. tenuis samples were collected from reefs (at depth of 2 m during low tide) around Sesoko Island, Okinawa, Japan  by skin diving. Six colonies of A. tenuis were tagged, and branches were collected from each colony on a monthly basis for one year (November 2016 to October 2017). Coral branches were collected by removing the branches from the base of the colony using pliers. Healthy coral branches 5–10 cm in length were sampled. Immediately after collection, coral branches were transferred to Sesoko Station, Tropical Biosphere Research Centre of the University of the Ryukyus, Okinawa, Japan. Molecular samples were flash-frozen in liquid nitrogen and stored at −80 °C until further processing. Histological samples were preserved in 4% paraformaldehyde (PFA)  for 8 h at 4 °C. The collection of corals was approved by the Okinawa Prefecture government (Nos. 27–76 and 28–84).A. tenuis branch samples were crushed to a fine powder using a liquid nitrogen-cooled mill. Each crushed sample was then mixed with TriPure isolation reagent  on ice. Total RNA was extracted according to the manufacturer’s protocol. RNA concentrations were determined using a NanoDrop spectrophotometer . Reverse transcription (RT) was performed to synthesise cDNA from 1000 ng total RNA using a PrimeScript RT reagent Kit with gDNA Eraser , according to the manufacturer’s protocol.AtVasa and AtLDLR (Supplementary Table Acropora digitifera genes in the whole genome database obtained from OIST Marine Genomics Unit (http://marinegenomics.oist.jp)66. Partial fragments of AtVasa and AtLDLR were amplified by long-range polymerase chain reaction (PCR) using the following conditions: LA Taq DNA Polymerase, 35 cycles of denaturation (10 s at 98 °C), annealing (30 s at 60 °C), and extension (2 min at 68 °C). PCR products were subcloned into the pGEM-T Easy vector  and transformed into JM109 competent cells (Takara Bio). Plasmid samples were sent to Macrogen Japan  to determine DNA sequences using a 3730xl DNA analyser .The primer sets for AtLDLR and AtVasa nucleotide sequences were identified and then translated into amino acids sequences using a Web-based ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The identified ORFs were checked using the Basic Local Alignment Search Tool (BLAST) program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the identity of each sequence. The verified amino acid sequences of AtLDLR and AtVasa were aligned with related sequences from other closely related cnidarians, other invertebrates, vertebrates, and several further taxa as outgroups using ClustalW109. The alignments were then used to construct a phylogenetic tree using the maximum likelihood method with the Jones-Taylor-Thornton (JTT) model110 and 1,000 bootstrap replications. The sequence alignment and phylogenetic construction were performed in MEGA7 software111.The ORFs of the AtLDLR and AtVasa was assessed in samples with matured gametes and those with or without immature gametes by amplification using reverse transcriptase polymerase chain reaction (RT-PCR) with the following conditions: 38 cycles of denaturation (45 s at 94 °C), annealing (45 s at 60 °C), and extension (1 min at 72 °C). PCR products were electrophoresed on 2% agarose gel  containing ethidium bromide at 110 V for 25 min, and visualised under UV. A. tenuis β-actin, a reference gene, was used as a positive control112. The PCR product are then subcloned to T-easy vector (Promega) and we sequenced 8 clones to confirm that PCR successfully amplified the targeted cDNAs.The gene expression of 11 , serially sectioned at 4 μm, and then stained with haematoxylin and eosin Y (H & E staining) for microscopic observations. The development of gametes was scored and classified according to cell size and histological characteristics with brief modifications from the previously reported methodThe stages of oogenesis were classified and characterised as follows:After mass spawning, the coral enters this stage in June. No oocytes are visible in the mesentery. Traces of the spawned oocytes are seen as distinct gaps or spaces in the mesentery, and appear stretched and distorted in shape  and nucleus (pink in colour) can be observed . Yolk polarity in the cytoplasm and peripheral migration of the nucleolus in the nucleus are observed  reaches 600 µm in diameter. The yolk and lipid droplets are visible in histological observation -labelled sense and antisense probes were synthesised using partial AtVasa and AtLDLR cDNA fragments (350–500 bp) via in vitro transcription  and RNA polymerase T7. The probes were then purified with the Agencourt RNAClean XP Kit  and stored at −30 °C until further use.Additional two pairs of PCR-based method RNA probesCoral tissues embedded in paraffin were cut into 4-μm serial sections for anti-sense, sense, and H & E stains. The sections were deparaffinised with xylene, hydrated, and processed for ISH with DIG-labelled RNA probes. The hydrated sections were subjected to digestion with proteinase K (10 μg/mL) at 37 °C for 15 min, fixation with 4% PFA, and then pre-hybridised with 2× SSC/50% formamide. Next, the sections were hybridised with a hybridisation mixture of 500 ng/mL DIG-labelled RNA probes, 10% dextran sulphate, 2× SSC, 0.02% SDS, and 50% formamide. After hybridisation at 60 °C for 16 h, the sections were washed as follows: once in 2× SSC at room temperature for 5 min, 3 times in 1× SSC/50% formamide at 60 °C for 30 min, 3 times in 1× SSC at 60 °C for 30 min, and 2 times in TBS for 5 min.The sections were immersed in blocking solution containing 10% bovine serum albumin, which was mixed with maleic acid for 1 h at room temperature. The DIG-labelled RNA probe was detected using anti-DIG-AP Roche, Basel, Switzerland antibody, which was diluted 1:1000 with the same blocking solution for 1 h at room temperature or overnight at 4 °C. The immunoreactive signals were visualised using the NBT/BCIP liquid substrate system (Sigma-Aldrich). Sections were observed and photographed under a microscope.A. tenuis tissues. Polyclonal antibodies against Vasa and VG were gifted from National Taiwan Ocean University  and were generated against the polypeptides C + AVGTSYGPAGGRFASRD (anti-EaVas) and CGDADGEQWNEYKDPQGR (anti-EaVg) of E. ancora. The polypeptides were conjugated to ovalbumin and immunised to rabbit (anti-EaVas) and guinea pig (anti-EaVg). The antiserum was purified using an affinity column containing 3 mg of the respective peptide antigen 46. The avidin-biotinylated-peroxidase complex (ABC) kit  was used to detect immunoreactivity against antibodies to Vasa and VG. The antibody specificity was confirmed by Western blotting  and incubated for 16 h (or overnight) at 4 °C. After washing with PBS, the sections were incubated with a secondary biotinylated goat anti-rabbit/anti-guinea pig IgG antibody  for 30 min. Then, the sections were incubated in ABC solution and visualised using 3–3′-diaminobenzidine . For control samples, no primary antibodies were added (only serum and PBS were used). The sections were observed and photographed under a microscope.Coral tissues embedded in paraffin were cut into 4-μm sections. The sections were deparaffinised with xylene and hydrated in an ethanol hydration series, followed by incubation in HistoVT One antigen retrieval solution  for antigen retrieval. The sections were then incubated for 10 min in 3% Hhttp://www.rstudio.com). One-way analysis of variance (ANOVA) and Kruskal-Wallis non-parametric analyses were applied according to Bartlett’s homogeneity and the Shapiro-Wilk normality test. Multiple pairwise analyses using Tukey’s honestly significant difference (HSD) test were applied to compare means among analysed groups. The statistical significance level was set to P < 0.05.Data are shown as the mean ± standard error of the mean (SEM). Statistical significance was analysed using RStudio software (Integrated Development for R, RStudio, Inc., Boston, MA, USA; Supplementary Figures.


One of the main goals of dentistry is the natural preservation of the tooth structure following damage. This is particularly implicated in deep dental cavities affecting dentin and pulp, where odontoblast survival is jeopardized. This activates pulp stem cells and differentiation of new odontoblast-like cells, accompanied by increased Wnt signaling. Our group has shown that delivery of small molecule inhibitors of GSK3 stimulates Wnt/β-catenin signaling in the tooth cavity with pulp exposure and results in effective promotion of dentin repair. Small molecules are a good therapeutic option due to their ability to pass across cell membranes and reach target. Here, we investigate a range of non-GSK3 target small molecules that are currently used for treatment of various medical conditions based on other kinase inhibitory properties. We analyzed the ability of these drugs to stimulate Wnt signaling activity by off-target inhibition of GSK3. Our results show that a c-Met inhibitor, has the ability to stimulate Wnt/β-catenin pathway in dental pulp cells in vitro at low concentrations. This work is an example of drug repurposing for dentistry and suggests a candidate drug to be tested in vivo for natural dentin repair. This approach bypasses the high level of economical and time investment that are usually required in novel drug discoveries. Small molecules are organic compounds of low molecular weight  that have the potential to regulate biological process. Owing to their small size, these molecules have the advantage of passing across cell membranes to reach targets, rendering them suitable for many targeted therapies. The attractive characteristics of small molecules makes them ideal for dental applications. However, discovery of novel drugs is typically associated with high risks and high levels of financial and time investment. In recent years, drug repurposing has been successful for identifying novel uses for approved or investigational drugs outside their original medical indication, contributing to substantial financial and time savings in preclinical and phase I and II stages . Drug reIn dentistry, one of the main goals is the maintenance of the natural structure of the tooth and provision of good and natural dental pulp protection when the tooth is damaged. While shallow tooth damage usually results in activation of odontoblast and formation of reactionary dentin that protects the pulp, deep cavities affect the pulp and jeopardize odontoblast survival. This results in activation of resident dental pulp stems, their proliferation and differentiation into new odontoblast-like cells and recruitment into the damage site to form reparative dentin ,16. ThisTo evaluate the potential of selected medications on the promotion of Wnt pathway activity and subsequently dentin/pulp damage repair, we first tested the effect of a range of concentrations of each drug on the viability of murine multipotent molar pulp cells. 17IA4 mouse dental pulp cells were incubated for 24 h with different concentrations of each drug. Cytotoxicity analyzed with the MTT assay determined the highest concentration of each drug that can be used in the subsequent stages . The higTo test whether these drugs stimulate the Wnt signaling pathway, the two highest concentration of each drug that were non-toxic to pulp cells were assessed for their ability to induce Axin2 expression. For each drug, 17IA4 mouse dental pulp cells were incubated with selected concentrations in triplicate for 24 h. For controls, media, DMSO and Bio, a known Wnt agonist, were used. After 24 h, media and drug were removed, and cells were analyzed for expression of Axin2 by qPCR . HighestThe expression of Axin2 in murine dental pulp cells at low concentrations of Tivantinib (50 nM) prompted us to select this drug for further analysis. To demonstrate the potency of Tivantinib, we measured its half maximal inhibitory concentration across a wide range of Tivantinib concentrations with a large scale MTT assay .Inhibition of GSK3 is paramount for targeting many conditions, such as neurodegenerative, psychiatric disorders, diabetes and cancer ,9,39. ThDespite the different kinase inhibitory properties of the investigated drugs, they have all been shown to have the ability to also inhibit GSK3. This was demonstrated in separate invitro studies ,26,27,28Cromolyn is a mast cell stabilizer and a non-steroid antiasthma medication that blocks the release of inflammatory mediators ,36. It bOf the drugs studied here, Famotidine, Olanzapine and Naproxen did not affect viability of murine dental pulp cells at high concentrations. This was 5 µM for Famotidine and Olanzapine and 10 µM for Naproxen. Cromolyn and Tivantinib affected the viability of dental pulp cells at concentrations higher than 100 nM and 200 nM respectively. This suggests that these two drugs could potentially be candidates for in vivo trials if they also demonstrate the potential to promote the Wnt signaling pathway at such low concentrations. Cromolyn did not induce Axin2 expression and Famotidine, Olanzapine and Naproxen induced little Axin2 expression even at high concentrations. Tivantinib did however stimulate Axin2 expression at the low concentration of 50 nM although the level of Axin2 expression was lower than that observed with Bio . Bio is,Tivantinib is a non-ATP–competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Mass-spectrometry-based and chemical proteomics approaches contributed to the identification of GSK3 alpha and beta as novel tivantinib targets ,27. TivaOverall, our results show that a c-Met inhibitor used in a cancer trial had the lowest toxicity for dental pulp cells and the ability to stimulate Wnt/β-catenin pathway at low concentrations in vitro. We are currently investigating the in vivo effects of delivering Tivantinib into exposed pulp cavities via collagen sponges. The results presented here demonstrates an approach where drugs currently used in the medical field can be investigated and repurposed for potential dental application.2 and incubated  for 24 h using standard culture medium. Thereafter, the medium was replaced with conditioned (drugs + media) and control media  for another 24 h. To determine the cell metabolic activity, MTT -2,5-diphenyltetrazolium bromide, Sigma Aldrich, St. Louis, MO, USA) was added to the controls and the conditioned media after 24 h. The resulting formazan product was then dissolved in 200 μL of dimethyl sulfoxide per well . A colorimetric plate reader (Thermo Multiskan Ascent 354 microplate reader) was used to read the absorbance at 540 nm with background subtraction at 630 nm.17IA4 cells were plated in 96-well plates at 20,000 cells/cm2/95% air, 100% humidity) for 24 h using standard culture medium. Selected drugs at the appropriate concentrations were used in triplicates to treat the cells for 24 h. Media only, DMSO and Bio  were used as controls. Bio was used at 50 nM concentration. This is a safe concentration on pulp cell viability as mentioned before [17IA4 cells were plated in 24-well plates and incubated  as recommended by the manufacturer’s instructions. The RNA was reverse transcribed using random primers  according to the manufacturer’s instructions. Gene expression was then assayed by real-time qPCR using Syber Green  on a Rotor-Gene Q cycler  system. Beta-actin was used as housekeeping gene  and Axin2 was the read-out for Wnt pathway activity (Forward-TGACTCTCCTTCCAGATCCCA, Reverse-TGCCCACACTAGGCTGACA. Reactions were performed in triplicate and relative changes to housekeeping gene were calculated by the 2171A4 Cells were seeded in 96-well plates and treated with 1 nM, 10 nM, 250 nM, 500 nM, 1 µM, 50 µM, 250 µM, 500 µM and 1 mM Tivantinib for 24 h in triplicate for each concentration. Following treatment, the percentage of cell survival was determined by MTT assay. The IC50 value of Tivantinib was determined by fitting a sigmoidal dose-response curve to the data, using the GraphPad Prism8. X values for concentration were transformed into scale. The Y values for MTT readouts were normalized to %. A linear regression analysis was performed using “dose response inhibition” and “log inhibitor versus normalized response”. The results were presented as IC50 and R square values.


Acinetobacter, Campylobacter, Escherichia/Shigella, Helicobacter, Listeria, Proteus, Pseudomonas, Salmonella, and Staphylococcus, were shared between sample types. Some OTUs highly represented in the fecal microbiota and associated with Lactobacillus and Streptococcus genera, were absent from eggshells, suggesting a selection during the microbiota transfer and/or the potential role of environmental contamination. To the best of our knowledge, this is the first study using 16S rRNA sequencing to describe the contribution of the transfer from the fecal microbial ecosystem of laying breeder hens to the establishment of the microbiota on the surface of laid eggs, as well as the bacterial communities at both the broiler breeder feces and the eggshell levels.In broiler chicken production, microbial populations on the eggshell surface following oviposition are still poorly characterized, though they may significantly impact both poultry and public health. The aim of this study was to describe the microbiota of both broiler breeder hens’ feces and the surface of their eggs to assess the contribution of the parental fecal microbiota to the eggshell microbiota. A total of twelve breeder flocks in Quebec, Canada, were sampled at two different times, and a total of 940 feces and 16,400 egg surface samples were recovered. Using 16S rRNA gene sequencing, we showed that even if the microbiota of both feces and eggshells were mainly composed of the phyla Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes, the bacterial community compositions and structures differed between both types of samples. Our results also showed that both the sampling time and the flock identity significantly influenced the alpha- and the beta-diversities of the studied microbiomes. Using a Venn diagram, we showed that 1790 operational taxonomic units (OTUs) were shared between feces and eggshell samples. Sequences associated with genera of potentially pathogenic and spoilage bacteria,  Chicken meat is an important source of high-quality proteins, vitamins and minerals. It is the leanest and most affordable meat product available worldwide, which together explains its economic worth . ConsequThe chicken intestinal tract can be colonized by such pathogenic bacteria but also by numerous microorganisms, including commensal and transient bacteria , which tIn broiler chicken production, microbiota studies have been conducted mainly at the broiler chicken level rather than at the breeder hen level, and they have mainly focused on describing the microbiota composition of different segments of their gastrointestinal tract , with onImmediately after oviposition, the egg temperature, which is around 42°C in the hen’s reproductive tract, brutally drops due to its contact with an external colder environment. This creates a negative pressure inside the freshly laid egg which increases the probability for bacteria present on the eggshell surface to penetrate the shell and to contamine the egg content . SeveralBacterial communities present on the eggshell surface are still poorly characterized although they are among the first bacteria encountered by the broiler chickens after hatching and they may impact both poultry and public health. The majority of data available on eggshell bacterial communities comes from research conducted primarily on eggs intended for human consumption and these data come from culture-based studies. The few 16S rRNA gene sequencing studies conducted on wild birds  and layiHere, we aimed to evaluate the transfer of the parental fecal microbial ecosystem and its contribution to the establishment of the eggshell’s microbiota. To do so, a 16S rRNA gene sequencing approach was used to describe both the fecal microbiota of broiler breeder hens and the microbiota found on the surface of the eggs layed by these birds at two different sampling time points spaced by a 4-week interval and among different flocks.n = 24) at a 4-week interval, from October 2016 to June 2017  which was transferred in liquid nitrogen for transportation to the laboratory where it was stored at −80°C until further analyses.During each visit and for each breeder flock, 70 eggs were collected directly from the nests following a stratified sampling plan. Each eggshell was swabbed for 1 min with a sterile wipe  pre-saturated with saline (0.85% NaCl), after which, each egg was returned to its nest. One sterile wipe was used for every 10 eggshells and a new pair of nitrile gloves was used between each wipe. In addition, during each farm visit, an extra sterile wipe pre-saturated with saline serving as a negative control was taken out of the bag and waved in the air in the farm for 30 s, without coming into contact with any surfaces. Immediately after collection, each wipe was transferred to a 50 ml Screw Cap tube  and frozen in liquid nitrogen for transportation to the laboratory where they were stored at −80°C until later processing.g. The supernatant was discarded and the DNA was extracted.Each frozen wipe was aseptically transferred in a 24 oz sterile Whirl-Pak Bag  containing 20 ml of phosphate buffered saline (PBS) and blended for 90 s using a Seward Stomacher 400C Lab Blender . Each wipe was squeezed and twisted to extract as much liquid as possible. The recovered volume was transferred into a 50 ml Screw Cap tube (Sarstedt AG & Co. KG) and centrifuged in a Sorvall Legend XTR Centrifuge TX-1000 (Fisher Scientific) for 25 min at 4,500 × g for 5 min at room temperature (20°C) and the remaining steps of the DNA extraction protocol were conducted according to the manufacturer’s instructions. DNA concentrations were measured using the Qubit 3.0 dsDNA broad-range assay (Fisher Scientific) for feces samples, and the Qubit 3.0 dsDNA high sensitivity assay for eggshell samples, both using a DeNovix QFX Fluorometer (Fisher Scientific). Purified DNA samples were stored at −20°C.The total bacterial DNA was extracted using the DNeasy PowerLyzer PowerSoil DNA Isolation Kit  according to the manufacturer’s instructions with some modifications. A 250 mg (±10 mg) of feces sample or the entire pellet from an eggshell lab wipe, previously resuspended with a bead-beating solution, was transferred to a PowerBead tube with glass beads. Two heat treatments were performed; a first one at 65°C for 10 min and a second one at 95°C for 10 min. Cells were mechanically lysed twice using a Fastprep-24 5G Sample Preparation Instrument  set at a speed of 6,5 m/s for 45 s, with a 10 min waiting period on ice between both bead-beating runs. The PowerBead tubes were centrifuged at 10,000 × 2O, and a positive control, the ZymoBIOMICS Microbial Community DNA Standard  were included. A 5 μl volume of each reaction was run on a 2% agarose gel and visualized following staining to confirm presence of the 292-bp amplicon. Barcoding and DNA sequencing were done on an Illumina Miseq PE250 at The McGill University and Génome Québec Innovation Centre .The 16S rRNA gene amplicon libraries were prepared using the universal primer pair 515FP1-CS1F ACACTGACGACATGG TTCTACAGTGCCAGCMGCCGCGGTAA and 806RP1-CS2R TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTC TAAT  which am1 (accessed April 2018). First, feces and eggshell sequences were analyzed together. Reads from each sample set were combined using the make.contigs command. Sequences containing polymers or ambiguity were rejected using screen.seqs and identical sequences were merged with the unique.seqs command. The remaining sequences were aligned using the Silva reference files, release 1282 and the chimeras were removed. Feces and eggshell sequences were segregated using remove.groups. Sequences originating from the control eggshell sample were removed from the eggshell sequences. The new eggshells dataset and the feces dataset were merged into merge_dataset for the remaining analysis using the merge.count and merge.files commands.Reads were cleaned and analyzed using Mothur v.1.39.5 following the Miseq standard operating procedure3 databases. Only the bacterial and archaeal sequences were kept and clustered into operational taxonomic units (OTUs) at a genetic distance dissimilarity of 3% using the classify.otu command.The sequences were classified at the phylum, class, order, family and genus levels based on homology searches using both Silva version 128 and Ribosomal Database Project (RDP) trainset 16t-test (unpaired and paired) and Kruskal–Wallis test with a significance level of 0.05. These statistical analyses were run on GraphPad Prism 8 . For beta-diversity analysis, a measure of similarity between sample pairs, a distance matrix with the similarity values for all pairwise comparisons  was created using the Jaccard index based on shared or distinct species, and the Yue & Clayton index which includes the species proportions of both the shared and distinct species. The different groups were statistically compared using the Analysis of molecular variance (AMOVA) with a significance level of 0.05 and visualized using 2D non-metric multidimensional scaling (NMDS). Biomarkers associated with feces or eggshells were highlighted using the linear discriminant analysis effect size (LEfSe). Finally, OTUs rarely observed in the final merge_dataset (nseqs = 1) were removed using the remove.rare command and a Venn diagram was generated to reveal the OTUs shared among groups. Command lines used in Mothur are available at https://github.com/CRSV. Raw sequences can be found on CNBI SRA database under accession number PRJNA602334.For alpha-diversity analysis, the species diversity within a sample, the number of observed OTUs, Inverse Simpson’s and Shannon even indices were calculated using a subsample with the size of the smallest library with 1,000 iterations; feces and eggshell samples were treated separately. The results were compared between groups using Student’s The number of DNA sequences for the 96 feces samples ranged from 39,680 to 105,243 with an average of 62,661 sequences per sample, and a total of 5,876,676 sequences. The vast majority of sequences, 99.8%, were of bacterial origin, whereas 0.2% were from Archaea.A total of 22 and 20 different phyla were revealed based on searches against the Silva and RDP databases, respectively . For allClostridium_sensu_stricto_1 (3%) revealed by Silva and Bacteria_unclassified (1%) by RDP.Likewise, a total of 169 and 193 families were found according to Silva and RDP databases, respectively . For allGenera relative abundance by sample at the genus level was also described at both sampling times .The number of DNA sequences from the 168 lab wipes ranged from 546 to 6,216 with an average of 1,233 sequences per lab wipe and a total of 207,225 sequences. Most sequences, 99.6%, were bacteria, and 0.1% were Archaea.A total of 26 and 29 different phyla were identified based on the Silva and RDP databases, respectively . For allMollicutes_RF9_fa, Enterococcaceae, and Bacillaceae according to Silva and the presence of Porphyromonadaceae, Bacteroidales_unclassified, Actinomycetales_unclassified, Firmicutes_unclassified, Peptostreptococcaceae, and Bacillaceae_2 according to RDP. These differences were mostly attributed to the capacity to assign an identity to the analyzed sequences.A total of 344 and 257 families were found according to the Silva and RDP databases, respectively . For allBoth databases appear complementary, but because some sequences could not be assigned an identity using Silva and remained unknown, the RDP database was retained for the remaining analyses.–1 for Staphylococcus to 3.4 × 10–5 for Salmonella in feces samples and from 1.7 × 100 for Acinetobacter to 9.7 × 10–4 for Listeria on eggshells. Interestingly, members of some bacterial genera, namely Acinetobacter, Campylobacter, Proteus, and Salmonella, showed a relative abundance 100-fold higher on eggshells that in feces, and members of Listeria and Pseudomonas showed a relative abundance 10-fold higher on eggshells than in feces.Potential pathogens and spoilage bacteria of interest in chickens were found in feces and on eggshells. A flock was considered positive when at least one feces or eggshell sample contained a related bacterial sequence. Percentages of positive flocks, positive samples and relative abundance are listed in A total of 25,038 OTUs were found in all 96 feces and 168 eggshell samples. The number of OTUs for the 96 feces samples ranged from 506 to 1,608 with an average of 975 OTUs per sample. Likewise, the number of OTUs for the 168 eggshell samples ranged from 190 to 667, with an average of 358 OTUs per sample.Comparisons of alpha-diversity indices, Sobs, Inverse Simpson’s and Shannon even, between both visits, at time = Day 0 and after 4 weeks, for each of the 12 flocks, and for both types of samples, feces and eggshells, are listed in p < 0.05), and were flock-specific (p < 0.001). Moreover, feces and eggshell microbiota were significantly different (p < 0.001). LEfSe was used to highlight biomarkers that were the most strongly associated with each type of sample (LDA Score [log 10] > 4). Six genera with high LDA scores indicative of marked abundances in feces, Lactobacillus, Streptococcus, Romboutsia, Enterococcus, Turicibacter, and Clostridium_sensu_stricto, were revealed  composed of members of the phyla Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Similar results have been reported for wild birds , laying Lactobacillaceae was the most dominant family found in feces, which is in agreement with the works of others who described the intestinal microbiota of broiler chickens  to the shell of their laid eggs (Salmonella, it is known that the bacterium can contaminate the eggshell, and for some serotype, the inside of eggs through an infection of the hen’s reproductive tract or feces (Pseudomonas, it has been reported that the watery content of the fecal material which contaminates the egg surface would increase the capacity of the bacterium to digest the cuticle, giving bacteria better access to the pores, and consequently, increasing the risks of internal contamination of the egg by pathogens (The current study identified several potential pathogens and spoilage bacteria that were shared between the broiler breeder feces and the surface of their laid eggs such as  systems . A recenaid eggs . For Salor feces . For Pseathogens , 2005. GOur study documented for the first time the contribution of the fecal microbiota of commercial broiler breeders to the establishment of the microbiota found on the surface of their laid eggs. This work also provided the first description of feces and eggshell microbiota of broiler breeders using high throughput sequencing methodology. Our results showed that these microbiomes evolved over time and varied among flocks under these conditions. Our results also revealed that the presence of potentially pathogenic bacteria on the eggshell was not always related to their presence in fecal matters. Therefore, it is important that all stakeholders, including producers, breeders, veterinarians, inspectors and researchers, be aware of the microorganisms present upstream in the broiler breeder production chain in order to better manage the risks for poultry and public health.PRJNA602334.The datasets generated for this study can be found in the Raw sequences can be found on CNBI SRA database under accession number The experimentations presented in this manuscript did not require the approval of an ethic committee as they did not involve the manipulation of birds. Strict biosecurity measures were followed at each visit and all sampling were in accordance with and under the supervision of a representative of the establishments visited. Freshly voided feces were collected without the manipulations of birds and eggs were gently sampled.AT, M-LG, and PF designed the experiments. ST performed the experiments. ST, AT, and PF analyzed the results. ST, AT, M-LG, J-CC, and PF discussed the results and wrote the manuscript.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.