The prevalence of dry eye disease is high worldwide and poses a great burden on patients’ daily lives. Accurate diagnosis of the disease is important, and it requires application of various methods. Hyperosmolarity is believed to be the disease marker and thus measuring it provides useful information. In this study we investigated utility of tear osmolarity measured with TearLab osmometer, along with other diagnostic tests  with dry eye disease and 29 healthy controls (58 eyes). Statistical differences between the patient group and the control group were observed for all the tests apart from tear osmolarity, regardless of cut-off value . Moreover, in the receiver operating characteristics curve analyses tear osmolarity measurement could not discriminate dry eye disease pathological scores. Therefore, our study suggests that tear osmolarity measured with TearLab osmometer cannot be used as a key indicator of DED. The symptoms of DED include ocular burning, foreign body sensation, soreness, stinging, irritation, reduced visual acuity, photophobia, and ocular pain1. The burden of DED can vary from mild discomfort to severe complaints that impact daily activities, reduce quality of life, and have significant socioeconomic implications2. The prevalence of DED increases with age and ranges from 5% to 50%2.The International Dry Eye Workshop II 2017 defines dry eye disease (DED) as “… 3, tear film break-up time (TFBUT)4, the Schirmer test5, ocular surface staining6, and meibomian gland functionality7. However, most of the tests lack consistency and reliability for diagnosing DED; therefore, they are subject to clinical interpretation based on experience8. The lack of a strong association between the signs and symptoms of DED is another challenge clinicians face in diagnosing and following-up patients with the disease9. Numerous ancillary diagnostic tests have been developed with the purpose of overcoming these challenges, including several patient-reported DED-specific questionnaires and new tools enabling the quantification of tear film characteristics. One of these tools is the measurement of tear osmolarity. Previous studies highlight tear hyperosmolarity as a significant pathophysiological factor in the development and the clinical course of DED15. Patients with DED present with elevated tear osmolarity compared to healthy controls16 and the hyperosmolarity increases with dry eye severity level (DESL)18.Accurate diagnosis of DED is complex and requires the application of a battery of tests, including questionnaires of patient-reported symptomsIn vivo measurement of tear osmolarity was not widely available for clinical practitioners until the TearLab osmometer  was approved by the US Food and Drug Administration in 2008. Since then, several studies have demonstrated that it is a reliable test with good performance for complementing DED diagnosis20. In situ analyses have also shown the inherent accuracy and precision of the TearLab osmometer21. Some studies have concluded that the TearLab osmometer is the best single marker for diagnosing and classifying DED levels of severity11 and for distinguishing DESL18. On the other hand, several clinical studies have raised questions on the diagnostic ability of the tear osmolarity measured with the TearLab osmometer in DED24. The varying results in the aforementioned studies might be due to the limitations of small sample size, studies conducted at multiple centers, and different diagnostic criteria for DED. Therefore, we conducted a single-center study with a large group of patients with DED (n = 757). Here, we explore whether tear osmolarity measurement with the TearLab system can be used as a diagnostic test along with clinical examination diagnostic methods for distinguishing patients with DED from healthy subjects.This single-center, retrospective study was carried out at the Norwegian Dry Eye Clinic in Oslo, Norway. The study was conducted in accordance with the Declaration of Helsinki. The use of the data for the study from the Norwegian Dry Eye Clinic has been reviewed and approved by The Regional Committee for Medical & Health Research Ethics, Section C, South East Norway (REC). All participants gave a written informed consent for the data to be used for research purposes.1514 eyes of 757 patients  diagnosed with symptomatic DED of different etiologies in at least one eye, with osmolarity measured using the TearLab osmometer in both eyes. The examinations took place from 2014 to 2018. The inclusion criteria were presence of clinically diagnosed DED of different severity levels (I–IV) according to the classification recommended by the 2007 Dry Eye Workshop (DEWS) developed by the Tear Film and Ocular Surface Society (TFOS)25 based on subjective feeling of ocular dryness , negative effect on visual ability , conjunctival injection, conjunctival and corneal staining, signs of DED on cornea and tear film, presence of meibomian gland dysfunction, abnormal tear film break-up time and Schirmer test values. It is important to note that these signs and symptoms vary based on severity levels. For the control group, we recruited 29 healthy participants (58 eyes). These participants were recruited from another ongoing study in collaboration with the Faculty of Dentistry, University of Oslo, Oslo (REK 2015/363). The participants were deemed healthy if the Ocular Surface Disease Index (OSDI)3 score was <12 and TFBUT ≥ 5 seconds. Control participants were excluded if they had any symptoms of DED (controlled with OSDI), seasonal allergy, used contact lenses or lubricating drops, had a history of refractive surgery, used of medications or had a systemic disease affecting tear production. Initially we had access to 65 control subjects who didn’t report symptoms of DED. However, clinical examinations revealed positive dry eye signs in some of them, and they were therefore excluded. We wanted to ascertain that controls had neither symptoms nor signs of DED. This rather strict inclusion criteria resulted in a smaller sample size of the control group.We included a total of 3 , tear osmolarity measurement using the TearLab osmometer11, inter-blink interval (IBI)26, TFBUT4, ocular surface staining (OSS)6, Schirmer I test5, meibum expressibility and meibum quality evaluation7, and meibography27. The examinations were carried out between 9 AM and 4 PM, and the participants were instructed not to use any eye drops within 2 hours prior to examination. Table 28. The patient was seated with the chin tilted upwards and eyes directing towards the ceiling. The tip of the osmometer was positioned just above the lower lid in the lateral canthal area. The osmometer was gently lowered until the bottom of the tip touched the thin line of the tear film. Successful tear collection was confirmed by a beeping sound and disappearance of the green light. The osmometer was docked into the reader immediately (5–10 seconds), the test card code was selected, the OK button was pressed, and the test result was obtained. TFBUT was evaluated after instillation of 5 μl 2% fluorescein sodium, and an average of three readings was recorded. The ocular protection index (OPI) was calculated as the TFBUT divided by the IBI29. Ocular surface staining was graded according to the Oxford grading scheme30. Finally, meibomian gland expressibility and meibum quality were assessed by application of light pressure to the lower eyelids using a cotton swab7. Based on symptoms, ocular surface staining, TFBUT, Schirmer test, and meibomian gland functionality, the DESL in each eye was determined according to the TFOS DEWS dry eye severity grading system25.The participants underwent comprehensive dry eye assessment in the following order: subjective dry eye symptom evaluation using the OSDI questionnaireStatistical analyses were performed with commercial software SPSS for Windows, version 23 . The normal distribution of variables was verified by the Kolmogorov-Smirnov test. Intergroup analyses were carried out with Kruskal Wallis test. The Mann-Whitney U test was used to compare two groups. The ability of tear osmolarity measurement for predicting the pathological scores of other clinical dry eye tests was assessed using receiver operating characteristics (ROC) curve analysis. Pathological TFBUT, Schirmer I test, OSS, meibum expressibility, and meibum quality scores were defined as <5 sec, <10 mm/5 min, <1, >1, and >1, respectively. Values from the right and left eyes were analyzed separately. In addition, we calculated the average values from the right and left eyes. A p-value of <0.05 was considered statistically significant throughout the study. The analyses results are presented as the mean ± standard deviation (SD).Among the patients, 629 (83.1%) were female and 128 (16.9%) (4.9:1) were male; the control group comprised 19 female (65.5%) and 10 male subjects (34.5%) (1.9:1). The mean age of the patient group was 52.2 ± 16.0 years; that of the control group was 44.9 ± 17.7 years (p = 0.201).Avg), , . Osmolarity levels in the right and left eye were 275–398 mOsm/L (95% CI 311.9–314.7) and 272–346 mOsm/L (95% CI 296–315.8), respectively. In the control group, the OsmAvg ranged between 281.5 and 347.5 mOsm/L . Osmolarity levels in the right and left eyes were 281–369 mOsm/L (95% CI 304.7–323.7) and 275–398 mOsm/L (95% CI 306.6–309.3), respectively. Comparison of the parameters from the right and left eyes separately produced results that were consistent with those in Table The patient group presented with worse subjective symptoms, shorter TFBUT, higher OSS, shorter Schirmer I test results and higher values of meibomian gland functionality. Apart from tear osmolarity, none of the mean test values in the control group reached pathological levels. In the patient group, the range of OsmAnalyses of the right eye revealed that 58% of the patients and 50% of the control subjects had osmolarity levels exceeding the suggested cut-off of 308 mOsm/L. The respective levels in the left eye were 42% and 39%. When the cut-off value of 316 mOsm/L was employed, 38% of right eyes and 27% of left eyes in the patient group had osmolarity levels exceeding the cut-off value. Similar results were obtained for the control group: 38% of the right eyes and 24% of the left eyes had osmolarity >316 mOsm/L. Whilst 62% of patients had inter-eye difference of >8 mOsm/L  mOsm/L, the same was true for 74% of the controls .Among the patients, about 10% had DESL 1. The vast majority of the patients had DESL 2 (72.5% of right eyes and 71.3% of left eyes), followed by DESL 3 in 14.4% of right eyes and 16.6% in left eyes. Only 1.3% of patients had DESL 4. We combined the participants based on DESL and created new groups: non-DED = DESL 0; mild DED = DESL 1 + DESL 2; severe DED = DESL 3 + DESL 4. Comparison of the groups based on DESL  and evaporative dry eye (EDE). In our dataset, 90.1% of the patients had a diagnosis of EDE due to MGD with various severity levels, while 9.9% of the patients were diagnosed with ADDE. Comparison of osmolarity levels between ADDE and EDE showed values of 326.8 ± 28.9 mOsm/L and 312 ± 18.2 mOsm/L (p = 0.123) in the right eye, respectively. Results for ADDE and EDE in the left eye were 304.5 ± 15.8 mOsm/L and 306.8 ± 17.5 mOsm/L, respectively, p = 0.809.This retrospective study focused on investigating tear osmolarity measurement as compared to other subjective and clinical parameters of DED, including the scores of the OSDI, TFBUT, OSS, Schirmer I, and meibomian gland functionality tests. Our data show that osmolarity was not significantly different between the healthy controls and the patients Table  and that31. Several measurement techniques have been used in the past33, with the Clifton and vapor pressure osmometers being the most commonly used methods. Despite high accuracy, sensitivity, and specificity33, these methods are not available for use at the point-of-care and require special setups that would need a significant amount of time. Clinicians did not have point-of-care diagnostic tests for measuring tear osmolarity until 2008, when the TearLab osmometer became commercially available. This technology uses a microchip, requiring only a 0.2-μl tear sample obtained without direct contact with the ocular surface. Lemp et al.11 concluded that tear osmolarity is the best single metric for diagnosing and classifying DED and suggested 308 mOsm/L as the most sensitive threshold between normal and mild DED. Similarly, Jacobi et al.19 proposed that a cut-off value of 316 mOsm/L demonstrated superior accuracy to other single tests for diagnosing DED. However, other studies demonstrated high variability of tear osmolarity in DED diagnosis24, and suggested that the TearLab osmolarity results should be interpreted with caution and in the context of other established methods. Accordingly, our data show that tear osmolarity levels measured with the TearLab osmometer were not significantly different between the controls and the patients.Tear film hyperosmolarity is believed to be an etiological factor in DED, hence the measurement of tear osmolarity has been considered important for diagnostic purposeset al.24. Our data differ from that of Lemp et al.11. The inconsistency might be caused by differences in the study population and DED diagnostic criteria. For example, the 314 subjects in their study were recruited from 10 centers, whereas we included 757 participants from a single center. In the present study, all clinical tests were performed by one experienced ophthalmologist with one or two assistants, which could have minimized the inter-observer variation in performing dry eye tests. The larger sample size might also have contributed to the statistical power of the analyses.If, as was done in the present study, osmolarity measured with the TearLab osmometer were used as the only diagnostic criteria for DED, the recommended cut-off value of 308 mOsm/L would exclude a significant proportion of patients (58% of right eyes and 42% of left eyes) with otherwise clinically diagnosed DED in our cohort. On the contrary, around 50% of the controls could have been diagnosed with DED when the same cut-off value was used. Using the cut-off value of 316 mOsm/L yielded similar results. Furthermore, another reference value commonly used for diagnosing DED, i.e., inter-eye difference >8 mOsm/L, failed to distinguish clinical DED from healthy eyes in the present study. Our data show that 62% of the patients and 74% of the controls had inter-eye difference >8 mOsm/L. These findings imply that tear osmolarity measurement with the TearLab osmometer has high variability and that it failed to distinguish patients with clinically diagnosed DED from healthy controls, which is in line with a recently published review by Baenninger 11, indicating a high accuracy of the TearLab osmometer in classifying DESL, the low discriminating ability of the TearLab osmometer and the overlap in osmolarity values have been reported previously16. In the present study, comparison of eyes with different DESL showed that in the right eye, all tests except the tear osmolarity measurement demonstrated inter-group differences. In the left eye, the severe DED group had the highest tear osmolarity level compared to the non-DED group  and mild DED group , but no statistically significant difference was detected between the non-DED and mild DED groups  (Table Despite the finding of Lemp and associates13, where the tears were collected by the TearLab system. The precorneal tear film in DED apparently has higher osmolarity levels, even spiking up to 800–900 mOsm/L in areas of tear film break-up34. Direct measurement of the precorneal tear film might show a stronger correlation with other clinical dry eye tests. Moreover, with the TearLab system, we probably measured the reflex tear, while earlier studies have suggested that pathological changes would be best obtained in basal tears rather than in reflex tears36. Using the TearLab osmometer might have led to reflex tear production, resulting in varying values.There are several possible reasons for the lack of association between TearLab-measured osmolarity and the other clinical parameters. The osmolarity of the precorneal tear film differs significantly from that measured in the tear meniscusThe strength of the present study is the largest sample size in a published investigation of the utility of tear osmolarity measurement for diagnosing DED. Moreover, the data were collected at a single center. Well-trained ophthalmic assistant carried out standardized diagnostic examinations, and a senior ophthalmologist confirmed the examination results. One limitation of the present study is its retrospective design. The patients were not controlled for contact lens or systemic/ocular drug use, which might have affected tear osmolarity. Moreover, tear osmolarity levels were not measured repeatedly, thus it was not possible to detect the reliability or repeatability of the test. However, the patients were instructed not to use any eye drops two hours prior to their visit to the clinic, and the TearLab system was operated daily according to the manufacturer’s instructions before use. High prevalence of DED in age-matched population was another limiting factor in recruiting a large number of healthy controls with no DED signs and symptoms. Nevertheless, the number of control subjects was verified to be sufficient on statistical grounds. The study may appear to be underpowered due to a smaller sample size in the control group, as it is the smallest group is the one that mostly determines the study power. However, whether the study could possibly be under-powered also depends on the effect size. Since many highly significant results were found, it is evident that there are quite large effects in our data. Future prospective studies comparing a large number of DED-free controls are warranted.In conclusion, tear osmolarity measured with the TearLab osmometer shows a large overlap between patients with DED and healthy individuals. Therefore, DED diagnosis relying on tear osmolarity measured with the TearLab osmometer can be inconclusive.


Oral squamous cell carcinoma (OSCC) is an aggressive human malignancy. Because of late diagnosis and recurrence of OSCC, the treatment of patients with OSCC is often ineffective. Thus, finding novel biomarkers of OSCC are essential. Here we derived a methylation marker by utilizing methylation microarray data and testing its capacity in cross-sectional study designed for OSCC detection and screening.NID2) methylation was selected for evaluation. Next, we confirmed the methylation status by bisulfite sequencing from the microdissected OSCC cells in comparison with the microdissected oral epithelia. Subsequently, we developed a simple technique using real-time PCR with the specific probe to examine the ability for the detection of OSCC in the oral epithelial samples, which included 103 oral rinse and 82 oral swab samples.According to bioinformatics analysis of total of 27,578 cg sites, cg22881914 of Nidogen 2 (NID2 was proved to be methylated in most OSCC cells but unmethylated in the normal oral epithelia. Furthermore, the methylated NID2-relied quantitative PCR approach has demonstrated that this marker assists in distinguishing among patients with OSCC from normal oral epithelia, smokers, and patients with oral lichen planus using the non-invasive oral rinse and swab samples.Based on the comparison of microdissected tissue, cg22881914 of NID2 of OSCC could be used as an important potential marker for detecting OSCC. Thus, to certify the utility of this marker, further studies with a larger sample size are needed.Specific methylation at cg22881914 of  Oral cancer is a major health issue, with an incidence rate more than 280,000 patients, of which almost 50% died. Notably, oral cancer is more prevalent in men –3. MoreoThe treatment options for oral cancer depend on the cancer stage at which it is diagnosed. The main approach is surgery, which is usually considered in use for those who have locally advanced and resectable lesions. On the other hand, in patients with unresectable lesions, radiotherapy and chemotherapy would be a recommended treatment option for oral cancer, particularly metastatic OSCC . To dateTRH gene, which was a novel marker for screening. In this study, more interestingly and effectively, we have proposed another screening marker, nidogen 2 (NID2), and developed a method better than using cg01009664 of TRH for OSCC screening [Tissue biopsy is the gold standard method for the early detection of oral cancer, but such procedures are expensive and invasive, which may cause patient discomfort . As suchcreening .The study protocol was approved by the Institutional Review Board (IRB 426/58 and 135/59), and informed consent forms were obtained in patients in the oral rinse and swab groups.NID2 at cg22881914 was selected as they distinctively distinguished the difference between the normal oral epithelial cell and cancerous cell methylation percentage values. The bioinformatics process is illustrated in Fig. The bioinformatic approach was previously reported . In brieA total of 20 formalin-fixed paraffin-embedded (FFPE) tissue samples  were collected from the Department of Pathology, Faculty of Medicine, Chulalongkorn University. The FFPE blocks were recut and stained with hematoxylin and eosin (H&E) for the histopathological review by a certified pathologist (SK). Next, manual microdissection technique was performed according to previously described report . BrieflyIn the oral rinse, the samples from 43 patients with OSCC, 40 smokers, and 50 healthy controls were included. Additionally, in the oral swab collection, there were 22 matched OSCC patients who allowed collecting oral swab, 30 patients with oral lichen planus (OLP), and 50 healthy controls. All volunteers were enrolled in the study were collected from the Department of Otolaryngology, Faculty of Medicine, Chulalongkorn University during January 2016 to December 2017. Sample size of OSCC group was a total of OSCC patients in that period. Detailed data are shown in Table The oral epithelial cells were collected using an oral rinse  and oral swab . In the oral rinse, 0.9% normal saline solution was gargled for 15 s, whereas in the oral swab, a foam-tipped applicator  was applied over the OSCC, OLP lesion, and normal buccal mucosa of healthy controls for 5–10 s. Then, the oral rinse solutions and oral swab foams were kept in a sterile tube and stored at 4 °C until the DNA extraction process.Genomic DNA was extracted from the cell pellet of the oral rinse and swab samples and 10-μm-thick unstained slides (10 slides per sample). The unstained slide samples were prior deparaffinized by xylene. Thereafter, all sample groups were lysed using a lysis buffer  that was mixed with 10% sodium dodecyl sulfate and 20 mg/mL proteinase K for digestion, followed by standard phenol–chloroform extraction . FurtherNID2 by direct sequencing of the PCR products. The forward primer was 5′-GYGYGTAGGTTAGTAGTYGTATT-3′, and the reverse primer was 5′-CCCRAATCATCCTCTCATCCRA-3′.All microdissected samples were checked for the methylation sequence at cg22881914 of NID2, two real-time PCRs were conducted from bisulfite-modification DNA 35 ng in each PCR reaction. The NID2 methylation set was composed of the forward and reverse primers 5′-CGTATTCGTCGTTGCGGG-3′ and 5′-CCGAATCATCCTCTCATCCG-3′, respectively, and the probe 5′-Fam-CGTTGAGTTTATTTTTTGTAACGTC-MGB-3′, with an annealing temperature of 59 °C. The beta-actin set served as the internal control, for which the forward and reverse primers were 5′-GTGTATTTGATTTTTGAGGAGA-3′ and 5′-CCTTAATACCAACCTACCCAA-3′, respectively, and the probe was Cy5–5’AAGGTGAAYGTGGATGAAGTTGGTGGTGAGG3’BHQ, with an annealing temperature of 59 °C [To detect methylation at cg22881914 of of 59 °C . The reaNID2 level in each individual sample was calculated from the standard curve using the following equation: y = 7E + 10e − 0.695x  was prepared in the concentrations of 10 to 1 pg as the standard, which were diluted with unmethylated DNA up to 10 ng/μL in total concentration for the investigation of its sensitivity. The threshold cycle (Ct) value of the methylated 95x Fig. b. The CtP values < 0.05 were considered statistically significant (two-sided).The SPSS software for Windows version 22  was used to analyze all data. Moreover, ANOVA was performed to determine the difference between healthy controls, smokers, and patients with OSCC in the oral rinse samples and between healthy controls and patients with OLP and OSCC in the oral swab samples. Also, the effect of age, gender, histological grade, and disease stage on the methylation status was investigated using the Pearson chi-square (χ2) analysis. NID2 displayed prominent differences in the methylation value among healthy controls, premalignant patients, and patients with OSCC  in all samples of the healthy controls, smokers, and patients with OLP for which individual epithelia oral rinse or swab samples were collected. Consequently, we analyzed the methylation data in qualitative instead. With the rule of the methylation quantitative level, more than 0 ng was defined as methylated status, whereas the quantitative level equal to 0 was determined as unmethylated status. Afterwards, the qualitative data was used to measure the methylated status frequency in individual subgroups.As shown in Fig. NID2 methylation with a higher frequency in the oral swab samples than oral rinse ones  as displayed in Table NID2 methylation in screening OSCC in the oral rinse sample with 79.07% sensitivity and 100% specificity, whereas the screening OSCC capacity in the oral swab has 90.91% sensitivity and 100% specificity.During screening, our results demonstrated OSCC showing 0/22 90.9% vs. 34/Among the total 43 matched OSCC cases between oral rinse and oral swab, only 22 patients were allowed to collect the samples with swabbing because of physical suffering. The results showed that 20 of 22 cases in oral swab were methylated (90.9%), whereas 17 of 22 cases of oral rinse were methylated (77.3%). Notably, both the cases defined that the unmethylation of oral swab samples were in the unmethylated cases of oral rinse samples, suggesting a higher sensitivity of oral swab compared with oral rinse. In addition, no statistically significant methylation level for the histological grade and clinical stage was noted in both samples collected by oral rinse or swab.To date, OSCC is mostly detected at an advanced stage via the conventional gold standard methods, including clinical examination and biopsy . Biopsy NID2 using both oral rinse and lesion swab samples from patients.In this study, using our previous candidate gene analyzed from bioinformatics data, we developed an identification method for OSCC by utilizing a cancer-related methylated gene with noninvasiveness and high sensitivity. Moreover, the technique to score the methylation was improved by counting number that was easier to analyze. This technique can detect specific site methylation of NID2 in the oral tissue types lined up from normal to cancerous tissue and sequencing of bisulfite-conversed DNA from microdissection biopsy samples, demonstrated that hypermethylation occurred when the normal tissue became cancerous. Additionally, real-time PCR results indicated that the methylation was not detected in both epithelia of smokers and patients with OLP, which indicated that this marker was specific to cancerous epithelia. Furthermore, no significant difference was observed between the NID2 methylation and clinical stage or histological grade of OSCC. Collectively, this suggests that at cg22881914 of NID2, the methylation had transition when normal epithelial cell completely transform to cancer cells.The present results of the bioinformatics calculation clearly show the correlation between increased methylation level and OSCC carcinogenesis. A comparison of the methylation level of cg22881914 of Alu and TRH site-specific methylations for OSCC detection. However, the cutoff values of both markers have to be evaluated for cancer [NID2 methylation clearly indicated totally zero value in the normal oral epithelium, and eventually, the samples were used up to a concentration of 10 ng/μl. After we change to detect the quality level (met vs. unmet), this test was easier and more convenient to OSCC screening application.We previously reported the r cancer , 21. In  NID2 hypermethylation and cancer had been reported in terms of downregulation of NID2 expression in several cancers, suggesting tumor-suppressor activity of NID2 [NID2 mRNA and protein level has been determined in cancer tissue samples as well as in nude mice xenograft model. Further investigation using microarray analysis and other techniques revealed that NID2 was hypermethylated, while its demethylation or overexpression could decrease many signs of cancer, such as proliferation, migration, invasion, including apoptosis [NID2 methylation status and its dysfunction, which results in cancer, have been consistently published, increasing the reliability and importance of NID2 [NID2 related to cancer in cases of OSCC. Hence, it may have a tumor-suppressor function and be appropriately used as a biomarker for cancer detection.Nidogen is a component found in the basement membrane. In particular, NID2 is one of nidogen family proteins that play a role in balance of integrity and stability of basement membranes through cooperation with laminin and collagen in the extracellular matrix , 13. Mor of NID2 –11. Recepoptosis . Researc of NID2 –25. Via NID2 methylation had been suggested as being a potential biomarker in the diagnosis of OSCC using noninvasive samples such as saliva. The site of cg22881914 on NID2 has been previously reported by Guerrero-Preston et al. with a very high sensitivity and specificity when a frozen tissue was tested [NID2 level because no control sample had shown that level. This is a significant factor for effective screening and is different from the results of the study by Guerrero-Preston et al. using only quantitative methylation-specific PCR primers, which eventually needed cutoff evaluation [s tested . Howeveraluation .We also improved the sensitivity rate using the swab approach in the sample collection, Matched oral rinse and oral swab OSCC samples show the higher frequency of methylated in oral swab than oral rinse group leading to an increase of up to 90%. The false-negative outcomes in two cases may have resulted from the improper clinical swab technique, in which the swab only covers necrotic tissue over the lesion. Moreover, this swab approach could be used only in patients who presented lesions but not in those without lesions.NID2 methylation is a promising marker for cancer screening, a larger sample size is necessary, and a different cohort should be used for unequivocal results. Because NID2 is a cell-adhesion protein on the basement membrane, NID2 methylation could lead to a loss of NID2 expression, resulting in a false negative detection in epithelial dysplasia or non-invasive cancerous lesion. On the other hand, a false positive result is likely potential in examination applied to the sample of some basement membrane damageable mucosal lesions. In general, epigenetic change is always related to some systemic conditions/diseases [NID2 methylation is measured using real-time PCR based technique. It is validated using a standard universal methylation DNA of the positive control. This validation among laboratories can minimize the technical limitation that might influence an error on methylation level.Although diseases , 27. To NID2 was an altered specific condition that was observed in OSCC but not in normal mucosa, smokers and OLP epithelium. Therefore, the assessment of methylation status in NID2 was a potential OSCC screening method that could be used to detect oral cancer in sample collected from invasive method such as oral rinse and swab approaches.In this study, the methylation at cg22881914 of


Bacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced B. subtilis strain (“midiBacillus”) expressing the immunodominant Staphylococcus aureus antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midiBacillus during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midiBacillus secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell’s array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell. In 1974, Wacław Szybalski provided a contemporary interpretation of synthetic biology and prophesied that biology would eventually evolve from a descriptive discipline to a re(designing) one . The almBacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes and biopharmaceuticals production  IIG-Bs27-47-24 expressing IsaA  of B. subtilis . The free-cell supernatant containing subtilin was collected and subsequently incubated at 80°C for 10 min, prior to freezing at −20°C.For subtilin production, 600] of 0.9) were induced with 1% subtilin (v/v), and samples were taken 120 min after the onset of induction. Bacteria grown without subtilin were collected at the same time point and used as control. For every experiment, three independent biological replicates were analyzed. 30 mL of bacterial cell culture was harvested by centrifugation , and cell pellets were washed three times with TE buffer . The corresponding supernatants were filtered and stored at −80°C for further preparation of the extracellular protein fraction. Cells were mechanically disrupted using the FastPrep24 instrument . Cell debris was removed by centrifugation , and the recovered supernatant was designated as whole cell extract. Protein concentration of these extracts was determined by a Bradford-based assay . The supernatant was designated as cytosolic fraction and the pellet was detached from the bottom by adding 0.75 mL high salt buffer  and incubation in an ultrasonic bath for 5 min at room temperature. This was followed by pipetting the suspension up and down until the pellet was homogenized. The pipette was then rinsed with 0.75 mL high salt buffer and the solution was incubated in a rotator at 8000 r/min and 4°C for 30 min, followed by ultracentrifugation under the same conditions as above. Pellet resuspension and ultracentrifugation were then performed with alkaline carbonate solution , and in a final step with TEAB (50 mM). The pellet containing the final crude membrane extract was resuspended in 70 μL 6 M urea/2 M thiourea.An aliquot of the whole cell extract with a protein content of 5 mg was used as starting material for membrane preparation. This lysate was filled up to 1.5 mL TE buffer and subjected to ultracentrifugation . The cytosolic fraction was also used for quantification of the IsaA protein in this particular sub proteome. UPS2 proteins (Sigma–Aldrich–Merck) were added in a 1:4 ratio (2.5 μg). For LC/MS analysis, 4 μg of peptide mixture per biological replicate was desalted using C18–Zip Tips (Merck Millipore). Peptide concentration was determined using the Pierce Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific). Preparation of whole cell and membrane extracts for targeted-MS followed the same digestion protocol as described above, except for the addition of UPS2 standards. Instead, samples were spiked with heavy peptides of the anchor proteins used in this study–QcrA and YwbN–to a final amount of 5 pmol. A detailed list of used peptides and their optimized transitions is available in Extracellular samples have been prepared according to Bacillus cells were photographed with a size scale using a light microscope (Leica DM2500 LED) coupled to a digital camera (Leica DMC2900). Length and width of the cells were determined with the help of ImageJ software ]. For this purpose, midisoftware  as descrsoftware . In summProtein samples were separated by SDS-PAGE as described previously . Before R = 60,000 with lockmass correction activated. After acquisition of the Full-MS spectra, up to 20 dependent scans (MS/MS) according to precursor intensity were performed in the linear ion trap after collision-induced dissociation (CID) fragmentation.Digested protein mixtures of cytosol (2 μg), membrane (2 μg), and extracellular (1 μg) fraction were separated on an Easy nLC 1200 coupled online to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). In-house self-packed columns  were first loaded with 12 μL of buffer A [0.1% (v/v) acetic acid] at a maximum pressure of 400 bar and subsequently eluted. Elution of peptides took place with a non-linear 166 min gradient from 1 to 99% buffer B [0.1% (v/v) acetic acid in 95% (v/v) acetonitrile] at a constant flow rate of 300 nL/min. Spectra for the eluting peptides were recorded in the Orbitrap at a resolution of r2 were calculated. Based on the calibration curve, 5 pmol of each heavy labeled peptide was added to three biological replicates of the digested whole cell and membrane extract samples for each tested condition–control and 1% subtilin induction–enabling the calculation of absolute amounts of target peptides    analyzed1.6.3.3) , and pro1.6.3.3) . Databasdatabase  with mandatabase  partner A permutation-based FDR approach implemented in the Perseus platform was used to calculate significantly changed membrane protein abundances during overproduction . The folFor targeted MS analysis, we analyzed three independent biological replicates per tested condition for two different fractions: whole cell and membrane extract. Raw files were processed using Skyline 4.2 , and might simply be a reflection of biological variance. The same calculations were performed for YwbN by determining the ratio between membrane and whole cell extract molar amounts, resulting in values of 1.70 and 1.36 for control and 1% subtilin induction conditions, respectively , have unknown function , or are part of two-component sensor kinase complexes, usually related to sporulation   .Bacillus, which is in agreement with the previously published data for Escherichia coli  for the induced and 6.33E+11 molecules/mL (0.3 mg IsaA/mL or 0.21 molecules of IsaA/cell) in the control samples . To validate if the number of molecules/cell dedicated to translational functions increased during induction, we analyzed the cytosolic fraction and found that∼4% more molecules are dedicated to this particular cell function. We were able to accurately quantify ∼50% of proteins predicted to exhibit translational functions and, of these, ∼23% significantly increase their abundance (data not shown). This supports the observation that the translational machinery is boosted upon IsaA induction, resulting in an increase in the total number of molecules/cell. Hence, in order to be able to compare the changes happening within the membrane fraction between production and control conditions, the number of molecules/surface area (μm2) was normalized by the total amount of molecules/μm2 of the corresponding replicate.Our data show a significant increase in the total number of membrane protein molecules/cell during induced secretion of IsaA in comparison to control conditions  (2 during induction conditions) , YfnI , and YvgJ   and LTA, encoded by the dlt operon (2 during induction), a protein described to be under the regulation of cell surface stress sigma factor σM  . This unditions) . A previditions) . Therefoditions) . LTA is ditions) . Our resles/μm2) . Besidest operon . Our datactor σM  , which is in the same range as the cytochrome-c oxidase cluster–CtaC , CtaD , CtaE , and CtaF   .Protein translocation is an essential mechanism that establishes the movement of proteins onto the surface or into the extracellular milieu, thereby ensuring cell survival. The majority of bacterial proteins directed to cross the cell membrane are exported via the highly conserved Sec-dependent secretion pathway . Upon inE. coli YajC) (E. coli YidC)   , and Spoli YidC) . SecA isctively) , it does2)  , which i2) , 5–IsaA,nduction , corroboBacillus suggest that cells cope with the effects of secretion/folding stress caused by IsaA induction by compensatory up- or down-regulation of genes. For instance, our results show that a big part of the proteins annotated as ABC transporters are either OFF during IsaA induction, or they significantly decrease their abundance , suggesting that these proteins might be present in only a small subpopulation of the cells and are only expressed under very specific physiological conditions. This behavior has already been reported in literature as a way for the bacterial community to optimize its resources by differentiating into distinct cell types, having numerous metabolic processes activated at the same time, but not in all the same cells    (Supplemctively) . This mictively) . For insBacillus is successfully secreting 2.41 molecules of IsaA/minute, there is still a considerable amount of IsaA accumulating in the membrane fraction–72 molecules/cell  leads to increased secretion of specific proteins  of E. coli, suggesting a role of these proteins in promoting extra-cytoplasmic protein folding (In addition, modulation of disulfide bond formation might be an additional strategy for higher yields in heterologous protein production, as this reaction is one the most important processes for the activity and stability of many exported proteins. However, little is known about disulfide bond formation in Gram positive bacteria , which t folding .Bacillus cells already show some growth deficiencies and present a bigger surface area. Hence, one might speculate that these phenotypes resultant of vast genome reduction might hamper the correct placement of integral membrane proteins in the cell surface, as the spatial orientation might be somehow disrupted. All these features should be considered when engineering microbial cell factories. Even though midiBacillus is already quite efficient in secreting IsaA to the growth medium (Apparently, induced cells are dealing with severe secretion stress by rearranging their array of transporters in order to maintain a viable cell structure, one that permits a viable growth and survival. Moreover, one should bear in mind that midih medium , the resBiology is, without a doubt, one of the most complex fields of science as the slightest alteration has the potential to become catastrophic for the homeostasis of a biological system.A minimal bacterial strain is highly attractive for the biotechnological sector, as all its remaining biological functions will be uniquely dedicated to the maintenance and production of high yields of heterologous proteins. However, what is the impact on the cell? How does the membrane proteome adapt upon overproduction of a “strange” protein?2. We were able to detect the physiological changes ensuing heterologous protein production, offering a clear visualization of alterations in protein patterns upon the onset of induction. Our results show that, even though IsaA production is being successfully induced, part of this protein is being accumulated in the membrane fraction, leading to a severe secretion stress response, and a reprogramming of the cell’s translocation machinery. Due to the exactness, the data described in this study can be implemented in predictive mathematical models aiming to understand the consequences of secretion stress and define the processes to be modulated. These data will be of the foremost importance to develop new-generation secretion systems for the biotechnological industry.In this study, we answer this question by applying a highly accurate method for absolute membrane protein quantification at the level of molecules/μmThe datasets generated for this study have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015496.MA-V, SM, and DB conceived and designed the experiments. MA-V analyzed the data and wrote the manuscript. RA engineered the strain used in this study. MA-V, JB, MB-C, and TiS performed experiments. ThS performed the sample measurement. JD, SM, and DB supervised the project. SM, JD, and DB provided all necessary corrections. All authors have read and approved 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.


Aedes albopictus, a highly invasive species that is vector of numerous arboviruses, including Dengue, Chikungunya, and Zika viruses. In this study, we investigated the composition of the bacterial community present in the water from a natural larval breeding site in which we separately reared wild-collected larvae and hatched eggs of the Foshan reference laboratory strain. Using sequence analysis of bacterial 16S rRNA gene amplicons, we comparatively analyzed the microbiota of the larvae and that of adult mosquitoes, deriving information about the relative impact of the breeding site water on shaping mosquito microbiota. We observed a higher bacterial diversity in breeding site water than in larvae or adults, irrespective of the origin of the sample. Moreover, larvae displayed a significantly different and most diversified microbial community than newly emerged adults, which appeared to be dominated by Proteobacteria. The microbiota of breeding site water significantly increased its diversity over time, suggesting the presence of a dynamic interaction among bacterial communities, breeding sites and mosquito hosts. The analysis of Wolbachia prevalence in adults from Foshan and five additional strains with different geographic origins confirmed the described pattern of dual wAlbA and wAlbB strain infection. However, differences in Wolbachia prevalence were detected, with one strain from La Reunion Island showing up to 18% uninfected individuals. These findings contribute in further understanding the dynamic interactions between the ecology of larval habitats and the structure of host microbiota, as well as providing additional information relative to the patterns of Wolbachia infection.The mosquito body hosts highly diverse microbes, which influence different physiological traits of both larvae and adults. The composition of adult mosquito microbiota is tightly linked to that of larvae, which are aquatic and feed on organic detritus, algae and prokaryotic microorganisms present in their breeding sites. Unraveling the ecological features of larval habitats that shape the structure of bacterial communities and their interactions with the mosquito host is still a poorly investigated topic in the Asian tiger mosquito  Aedes mosquitoes is known to play a significant role in host physiology, including egg production, blood digestion  is a highly invasive species of growing public health concern due to its ability to transmit at least 22 arboviruses, including Chikungunya, Dengue, and Zika viruses  to what extent is the breeding site microbiota affecting endosymbiont community assemblage in larval and adult Ae. albopictus? (ii) Is the bacterial community of the breeding site water changing over time during mosquito development? (iii) What is the frequency of Wolbachia in adult samples reared as larvae in the wild-collected water?In this exploratory study, we aimed at expanding the current understanding of the dynamic interactions between the ecology of larval habitats and the structure of host microbiota, as well as the patterns of Wolbachia presence in Foshan and five additional laboratory strains with different geographic origins.To answer these questions, we compared bacterial community composition among (i) water from natural larval breeding sites, (ii) mosquito larvae, and (iii) adult individuals. In water from a natural breeding site, we reared both wild-collected larvae and larvae of the Foshan reference strain to derive information about the relative impact of the environment and the genetic background of mosquitoes on shaping mosquito microbiota. Finally, we analyzed Ae. albopictus larval breeding sites was collected in a private garden in Crema, Italy (45°21′51.2′′N 9°40′57.7′′E), which was an accessible site characterized by high population density, at the end of August 2018, when climate conditions are optimal for mosquito development. Domestic collection of water, eggs, and larvae originated from two plastic buckets of a maximum volume of 500 ml that were placed in the same garden next to ornamental plants. Environmental water collected in these buckets derived from dew and rain and was monitored for its level and clarity every day for 4 days. No sedimentary layer was observed. Water and eggs/larvae were then collected in sterile 50-ml Falcon tubes and transferred to the insectary of the University of Pavia, Italy. The collected water was divided into three aliquots and used as follows: (i) an aliquot of 200 ml was brought to the ZooPlant laboratory in Milano, Italy, to characterize its microbial composition ; (ii) 300 ml were aliquoted in two different pans (150 ml/pan) and daily monitored for natural larval hatching in the Pavia insectary (samples named CR); (iii) 600 ml were used to hatch eggs from the Foshan strain  and adults . In the case of adult collections, pupae developed in the larval rearing pans were individually transferred to cups containing 10 ml of the same wild water until adult eclosion. When mosquito development was completed and all adults had eclosed, the remaining water from each rearing pan was transferred into sterile 50 ml Falcon tubes for the analysis of the microbiota .All mosquito life stages were maintained in the Pavia insectary at 28°C and 80% RH, with a photoperiod of 12:12 hrs light:dark.2), nitrates (NO3), ammoniacal nitrogen (NH4-N), and phosphate (P_tot) were measured with a spectrophotometer . Moreover, pH and conductivity were also recorded. Analyses were performed using the following kits, according to the manufacturer’s instructions: Ammonium Test Photometric Method NH4-N, Nitrate Test Photometric Method NO3-N, Nitrite Test Photometric Method NO2-N, Total Nitrogen Test Photometric Method, Phosphate Test Photometric Method PO4-P, Merck Spectroquant®.For each water sample, total nitrogen (N_tot), nitrites (NOGenomic DNA was extracted from (i) water samples at the two time-points described above; (ii) pooled larval (L) samples; (iii) individual adult (A) mosquitoes collected immediately after eclosion.® tubing, sterile reservoirs and filtration modules. The used tangential flow filter was a VivaFlow® 200 cassette (Sartorius) made of polyethersulfone (PES) with a nominal pore rating of 10000 MWCO and a surface area of 200 cm2. The system was scaled up with an additional unit connected in parallel to increase the filtration surface area and the flow speed. The TFF system was run at a transmembrane pressure of 1.5 bar. The initial water samples were concentrated to a final retentate volume of 100 mL, which was further reduced to 1 mL through the Vivaspin® 20 ultrafiltration unit (Sartorius) made of PES with a nominal pore rating of 10000 MWCO. The final volume was allowed to be adsorbed onto a membrane filter before DNA extraction.Water samples were processed according to a modified version of a previously described protocol . First wAll tubing, tubing connections, and containers were sterilized with sodium hypochlorite or autoclaved prior to each experiment and among samples. Every step was conducted in the laminar flow cabinet in a pre-amplification dedicated laboratory.® PowerSoil® Kit (Qiagen) with the QIAcube (Qiagen) automated system, following the manufacturer protocol. DNA was eluted in 75 μL of warmed (40°C) elution buffer, to increase DNA concentration. DNA extraction negative controls were included.Environmental DNA was extracted separately from each of the filters obtained by water filtration using DNeasyAe. albopictus samples were processed, namely 11 larval pools  and 14 individual adults . The finding that sequencing pools of six mosquitoes allowed to capture a level of bacterial diversity comparable to that of single mosquitoes  with Illumina MiSeq 2 × 300 paired-end chemistry (MiSeq Reagent Kit v3).Illumina MiSeq 16S libraries were generated following the standard protocol “16S Metagenomic Sequencing Library Preparation, Part # 15044223 Rev B.” Amplicon PCR was performed using PCR primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) with Illumina library adaptors, for 25 cycles of amplification. DNA extraction negative controls  and amplicon PCR negative controls were included in library preparation. Libraries quantification through TapeStation 4100 (Agilent) showed no amplicon signal for amplicon PCR negative controls, which were excluded from the sequencing. In the case of The raw paired-end FASTQ reads were imported into the Quantitative Insights Into Microbial Ecology 2 program   and demuphyloseq R package (The resulting Amplicon Sequence Variants (ASVs) with less than a 50x coverage were discarded from further analyses. The classification of the obtained ASVs was run using the feature-classifier plugin implemented in QIIME2 against the SILVA SSU non-redundant database (138 release), adopting a consensus confidence threshold of 0.8. The analysis on the bacterial diversity as well as the corresponding figures were done using the  package .Wolbachia were filtered using the filtered taxa plugin of QIIME2.Any reads assigned to Microbiota diversity was described in terms of within  and between (beta) sample diversities. The Shannon index and Observed Features alpha diversity metrics were calculated to estimate the variation of bacterial diversity in the water, larvae and adult samples. Values were compared using the pairwise Kruskal–Wallis test.phyloseq R package.To explore the bacterial diversity of our samples, we used a relative abundance bar plot to show phyla and family distribution between our three sample types . The distribution of the 100 most abundant ASVs was studied with a heatmap plot. Both visualizations were obtained with the Rarefaction is used to subsample and calculate distances among samples. Beta diversity was estimated with quantitative distance metrics using the diversity QIIME2 plugin based on the rarefied dataset with a sampling depth of 10,000 sequences. Samples with lower depth were automatically discarded from beta diversity analysis.diversity plugin. The structure of microbial communities was explored by Non-Multidimensional Scaling (NMDS), an ordination approach  assays were performed targeting the 16S rRNA gene to verify the sensitivity of our approach in detecting the microbiota of water samples and to compare relative bacterial abundances between W Start and W End, according to a protocol previously described . Water s–1 primer solution, 2 μl DNA sample, and 2.8 μl of Milli-Q water. Assays were performed on an AB 7500 thermocycler (Applied Biosystem) and results analyzed as previously described , 0.1 μl each 10 μmol lescribed .Wolbachia infection status in the 25 adult samples used for metagenomic analysis, PCR reactions for Wolbachia detection were performed in 15 μl reaction volumes consisting of 7.5 μl DreamTaq Green Master Mix , 4 μl of autoclaved Milli-Q water, 1 μl primers (10 μM), and 1.5 μl of DNA (∼20 ng). The PCR cycling conditions were as follows: 3 min at 95°C for the initial denaturation step, followed by 35 cycles of 1 min at 95°C, 1 m at 55°C, 1 m at 72°C and 10 min at 72°C for the final extension. The published wsp primers were used  and 691R for wAlbB. PCRs reactions were run on the same samples also using the primer set Aealbo18S_F1 (5′-TGCCATGGATGCTTTCATTA-3′) and Aealbo18S_R1 (5′-GTACAAAGGGCAGGGACGTA-3′) to test for DNA quality. All PCR products were visualized on 1% agarose gels and sequenced.To obtain a qualitative validation of ere used : 328F (5Wolbachia was assessed by PCRs in Foshan (generation 37) and five additional long-established laboratory strains deriving from Canton , Recife , Tapachula , Tampon , and Crema . These strains were selected for several reasons. First, to compare Wolbachia prevalence estimated through metagenomics in CR and FO samples (reared in wild-collected water) with estimates of Wolbachia presence in the Foshan and Crema laboratory strains. The Crema strain was established in 2016 from mosquito eggs collected in the same breeding site used in this study. Second, to determine whether insectary rearing for several generations have led to a 100% Wolbachia frequency in adult individuals.Moreover, the presence of Ae. albopictus samples was examined by sequencing of the bacterial 16S rRNA gene. Sequences of a total of 37 libraries  resulted in 8,191,150 million sequence reads, ranging from 14,277 to 264,318 . No reads were reported in negative controls. Total number of reads per sample before and after Wolbachia-associated reads removal is reported in The microbiota of breeding site water (W), larval (L), and adult (A) post hoc test: p < 0.05).W End samples showed higher variability in the number of ASVs than W Start samples; larval samples showed a much higher variability than adults, irrespective of their origin . These di.e., oxygen and conductivity) were found to be associated with microbiota composition in Ae. aegypti   . Moreove < 0.05) .A total of 28 bacterial phyla and 262 families were identified across all samples. Taxonomic analysis showed that most of the sequences are associated with the phylum Proteobacteria (59.7%), followed by Bacteroidota (16.4%), Firmicutes (3.7%), and Spirochaetota (3.6%).In W Start samples, the main bacterial phyla were Proteobacteria (>95%), followed by Bacteroidota (0.5%) and Verrucomicrobiota (0.3%). In addition to these phyla, in W End samples we also identified Patescibacteria, Dependentiae, Cyanobacteria, and Acidobacteria . The treThe trend of decreasing ASVs variability from water to adult samples is also depicted in a heatmap generated using the 100 most abundant ASVs assigned at the taxonomic level of Family (unweighted UniFrac distance) . The heaHerbiconiux, Leifsonia, and Leucobacter genera.Wild and laboratory larval samples differentiated by the presence of Verrucomicrobiaceae, Chitinophagaceae, Pirellulaceae, Cellvibrionaceae, and Terrimicrobiaceae exclusively in laboratory samples. The only bacterial family uniquely present in wild larval samples was Microbacteriaceae, with the Aedes mosquitoes, such as Sphingomonas and Chryseobacterium  ordination plot . Using pDietzia, Blautia, and Leifsonia; in wild adults, unique genera were Tepidimonas, Cloacibacterium, Haemophilus, and Neisseria. An unidentified uncultured bacterium from the family Rhodobacteraceae was found to be unique of Foshan larvae; Dietzia, Meiothermus, Porphyromonas, Blautia, Micrococcus, and Fructobacillus were the unique genera in Foshan adults.The intersections among the list of bacterial genera found in analyzed samples were calculated to identify the components of the microbiota that are unique of each sample . As expeProsthecobacter, Solimonas, Ancylobacter, Anaerococcus, Schlesneria, and Micrococcus , emphasizing the role of the breeding site in shaping mosquito microbiota. Among the bacteria absent in water samples (both W Start and W end), Vulcaniibacterium was the only present in both larvae and adults, of both CR and FO samples. Escherichia–Shigella was instead found only in CR adult samples. Several unclassified bacteria were found to be conserved in larval samples and/or adults, absent from the W Start samples but detected in the W End samples  than samples of the second group, thus we called these two groups “Wol_low” and “Wol_high,” respectively, and we compared their alpha diversity after excluding Wolbachia reads. Wol_high samples displayed a significantly higher number of ASVs than Wol_low samples   with Shannon diversity index < 1.5; all the other samples had a Shannon diversity index higher or equal to 2 . Interes < 0.05) .Wolbachia in four adult samples prompted us to further investigate Wolbachia prevalence in six long-established laboratory strains. The prevalence of wAlbA and wAlbB strains was also determined. In all mosquito strains, both wAlbA and wAlbB were detected. However, while Wolbachia was present in all tested individuals in Foshan, Canton, and Recife strains, some individuals were shown to not carry Wolbachia in Crema, Tapachula, and Tampon , the comparison between W Start and W End water samples suggests the presence of a dynamic interaction between larvae and their breeding site. Such a difference was also detected by qPCR amplification of the 16S rRNA gene, which confirmed a significantly higher number of bacterial DNA copies in W End in comparison to W Start. Although unculturable candidate phyla are likely present in our water samples, the procedure of relying on 16S rRNA quantification to infer bacterial load in breeding site water and mosquito samples is routinely used  from the larval anal papillae, as shown in Ae. aegypti  but they are absent in the W End sample. This pattern may indicate that members of this family are acquired and digested. In triatomines, Nocardiaceae play the role of nutritional symbionts able to provide the host with essential nutrients  was shown to contribute to mergence . This prAfter such loss of components, adults shape a new microbiota and diet plays a key role. Mosquito hosts, such as plants and animals, are a source of bacteria, as well as viruses and other microorganisms. Indeed, it is known that the midgut of the two sexes harbors different microbial communities: the microbiota of females is typical of blood feeding insects, and it is mostly colonized by Gammaproteobacteria; males, instead, display a midgut mainly colonized by Firmicutes .i.e., Vulcaniibacterium, was found to be shared by both Foshan and Crema samples. This bacterium has been previously found in the gut of both sexes of Bactrocera oleae , the prevalence was particularly low with respect to what detected in the other five strains and previously reported  showed that wAlbA titer was very low in about half of the collected males from both sites  under the BioProject: MB and MCas conceived and designed the experiment. FS, AS, MCar, and AB performed the experiments. FS, AS, and AB analyzed the data. FS, AS, AB, and MB wrote the manuscript. All the authors provided critical comments on the manuscript, read and approved the final version of the manuscript.AS is employed by the company Quantia Consulting srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit , MagMAX™ Cell-Free DNA Isolation Kit , Urine Cell-Free Circulating DNA Purification Midi Kit , and Quick-DNA™ Urine Kit . To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection. Liquid biopsy has emerged as an important non-invasive tool for cancer diagnostics. Liquid biopsy utilizes genetic material from body fluids; this method can overcome the limitations associated with traditional tissue biopsy such as invasiveness and the difficulty of repeated sampling ,2,3. CelAlthough circulating cfDNA in blood is most widely used in liquid biopsy for malignant disease, cfDNA can also be isolated from various body fluids. Urine is an ideal body fluid for liquid biopsy as it can be collected in a truly non-invasive manner with a relatively reduced limit in volume. Specifically, urine could be more useful in liquid biopsy for urologic malignant disease. Several studies have demonstrated that urinary cfDNA can be utilized as an important source for liquid biopsy in urologic malignant disease ,12,13,14In this study, we aimed to compare the urinary cfDNA isolation efficiency of four commercial kits with samples from healthy individuals. The four commercial kits were QIAamp Circulating Nucleic Acid Kit , MagMAX™ Cell-Free DNA Isolation Kit , Urine Cell-Free Circulating DNA Purification Midi Kit , and Quick-DNA™ Urine Kit . Isolation efficiency was assessed based on the level of urinary cfDNA yield and genomic DNA contamination in an electropherogram. We also compared four different sample storage conditions according to temperature and preservative.g for 10 min followed by 3000 g for 20 min to remove cellular material. The supernatants after two centrifugations were mixed with or without 10 mM EDTA and stored at −20 or at −70 °C until used for experiments  and 33.8 (range 26–45), respectively. This study was approved by the Institutional Review Board of Ewha Medical Center (IRB No. 2018-08-027-004) and all participants gave their informed consent in writing. The first morning, urine was collected and the urine samples were centrifuged at 200 eriments . g for 10 min to remove impurities in the samples. The starting volume was determined according to the manufacturer’s recommendation of each kit; 4, 4, 10, and 24 mL of urine were used for QC, MM, NU, and ZQ, respectively. We compared the isolation efficiency of four commonly used commercial kits: QIAamp Circulating Nucleic Acid Kit , MagMAX™ Cell-Free DNA Isolation Kit , Urine Cell-Free Circulating DNA Purification Midi Kit , and Quick-DNA™ Urine Kit . The frozen urine samples were thawed at 4 °C and centrifuged again at 3000 To assess DNA fragment distribution and concentration, cfDNA was electrophoresed on an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips . The cfDNA concentration was measured from the defined range of 50–300 bp on electropherograms . The cfDg for 10 min and was then subjected to cfDNA isolation. The average cfDNA yield of 10 samples was compared according to each storage condition. To compare different sample storage conditions for minimizing cfDNA loss, urine supernatant from 10 healthy individuals was aliquoted into four tubes and kept under four different conditions: (1) storage at −20 °C with the addition of 10 mM EDTA, (2) storage at −20 °C without the addition of EDTA, (3) storage at −70 °C with the addition of 10 mM EDTA, and (4) storage at −70 °C without the addition of EDTA . EDTA waThe cfDNA was isolated immediately after sample collection using the four commercial kits, and cfDNA concentration was analyzed using electropherograms from an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips . The cfDNA yield was calculated as the amount of cfDNA from 1 mL of urine. In the range of 50–300 bp, MM and QC exhibited the highest average cfDNA yield. Of 10 samples, five (50%) and four (40%) showed the highest cfDNA yield by MM and QC, respectively. For more fragmented cfDNA in the range of 50–100 bp, the MM and NU showed the highest average cfDNA yield. MM and NU showed the highest cfDNA yield in four (40%) and six samples (60%), respectively A Supple. DNA fragments in the region of over 1000 bp were considered to be derived from urinary cellular DNA , and theTo compare the effect of storage conditions on urinary cfDNA extraction, urine samples were stored under four different conditions for 3 months . As the In this study, we compared four commonly used commercial kits for urinary cfDNA isolation and our results show that each kit has its own characteristics and advantages. We summarize the characteristics of each kit in Although it is recommended that cfDNA be extracted from urine as soon as possible, it is inevitable that samples would be stored for extended time in a clinical setting. Therefore, it is important to find the best conditions to maintain cfDNA stability during long-term storage. Urine has a stronger activity of DNase I than tissues or other body fluids , thus itFor quantification of cfDNA, real-time PCR has been most widely used targeting specific DNA sequences. Recently, various quantification methods such as spectrophotometry or fluorometry have been used for cfDNA quantification. In this study, we used electrophoresis-based instruments  which enabled both fluorometric quantification and cfDNA sizing. Compared to spectrophotometry, the fluorometric assay is the preferred method for quantification of cfDNA as spectrophotometry has low sensitivity for analyzing low concentrations of cfDNA . HoweverIn summary, we found that the four commercial kits each have advantages. Both MM and QC were efficient in the isolation of urinary cfDNA. For isolation of more fragmented cfDNA, NU could be the choice of method. ZQ permits the largest urine starting volume with the lowest cost. Considering that urinary cfDNA yield is relatively low, ZQ could also be the method of choice if a large volume of sample is available. In terms of storage conditions, −70 °C storage is preferred to −20 °C and the addition of EDTA reduced the loss of urinary cfDNA.


Olive somatic embryos have been successfully cryopreserved using the droplet-vitrification method on aluminum foil strips. Although acceptable recovery rates have been obtained after rewarming, the influence of this cryopreservation protocol on the somatic embryogenesis process is unknown. To evaluate the effect of cryopreservation on olive somatic embryogenesis, the behavior of cultures established from cryopreserved somatic embryos was compared with that of control, non-cryopreserved cultures in the different phases of the somatic embryogenesis process. In order to analyze the influence of the genotype, this investigation was carried out in two independent lines. During the proliferation step, only the line T1 was affected by cryopreservation, with higher fresh weight increases. Although similar total embryos were produced per culture, freezing in liquid nitrogen significantly improved the maturation pattern in the line P5. Better germination results were also found in this embryogenic line. The genotype plays a key role, largely determining the effect of cryopreservation on olive somatic embryogenesis. A specific genotype-dependent response was found depending on the culture step. Variations observed could not be associated to differences in the embryogenic lines’ instability to maintain their morphogenic competence after cryopreservation. Embryogenic cultures established after rewarming retained their regeneration capacity, with no evident negative effects affecting their regeneration capacity. Olea europaea L.) is the second most important oil fruit tree crop worldwide after oil palm. Its production has been traditionally concentrated in the Mediterranean basin, where this crop has a great impact on the economy, history, culture, and environment [The olive  , is a goCryopreservation allows the long-term storage of valuable embryogenic lines used in propagation or breeding programs or utilized for application of biotechnological tools based in somatic embryogenesis systems. Apart from permitting the conservation and management of selected lines, it enables the preservation of transgenic material while field trials are on-going  or of thBoth classical, based on slow cooling protocols, and new, vitrification-based methods have been used to conserve olive embryogenic cultures . In 2017However, during cryopreservation, embryogenic tissues are exposed to different processes, such as conditioning pretreatments, incubation in cryoprotectants solutions, cryostorage, and post-rewarming manipulations, which have a stressing effect and potentially may cause changes in their morphogenic capacity. Consequently, tissue regrowth after rewarming cannot be the only criterion for successful cryopreservation , and befThe objective of the present investigation was to evaluate the effect of cryopreservation on culture regeneration capacity, determining its effect on the efficiency of the different phases of the somatic embryogenesis process. Considering the influence of the genotype on embryogenic cultures behavior , the invControl and cryopreserved cultures of each embryogenic line did not exhibit morphological differences during the proliferation phase . NonetheThe production of somatic embryos in maintenance medium was significantly affected by the genotype and the interaction genotype × cryopreservation . Thus, wThe aspect of control and cryopreserved cultures did not show significant differences after eight weeks in maturation medium . Fresh wAlthough, as revealed by the number of total somatic embryos produced per gram of culture, the ability of embryogenic cultures to form somatic embryos did not change due to cryopreservation, a significant effect of the genotype, cryopreservation, and interaction genotype × cryopreservation was found in TrSE ≥ 5 mm . While iGermination of somatic embryos was significantly affected by the genotype, cryopreservation, and the genotype × cryopreservation interaction . NeverthIn the line T1, cryopreservation did not influence embryo germination . No effeHevea brasiliensis [Castanea dentata [Araucaria angustifolia [Pinus nigra [Abies cephalonica [Picea abies [Persea americana [Vitis vinifera [Five to six months after rewarming, cultures established from somatic embryos cryopreserved following the protocol of Bradaï et al.  presenteiliensis , Castane dentata , Araucarstifolia , Pinus nus nigra , or Abiehalonica . Neverthea abies , Persea mericana , and Vitvinifera , where sMost investigations reported maintenance of morphological appearance and growth pattern after cryopreservation ,21,22 orGentiana spp. [Gentiana cruciata [In relation to the maturation phase, no significant influence of cryopreservation was found in olive embryogenic cultures. Although similar results were reported in ana spp.  and Gentcruciata , investicruciata ,25,26.Abies cephalonica, however, Krajňáková et al. [In relation to the culture developmental pattern, a significant impact of cryopreservation was observed in the line P5, with increased production of TrSE ≥ 5, both proportionally and per gram of culture. Number of TrSE ≥ 5 increased at the expense of TrSE < 5, thus revealing an effect of cryopreservation inducing embryo development to more advanced stages. In á et al.  reportedQuercus suber, Valladares et al. [The effect of cryopreservation on germination capacity of olive somatic embryos varied significantly depending on the genotype. Thus, while in the line P5 a significant positive effect was found; in the line T1, no variation could be observed. In s et al.  obtaineds et al.  observeds et al. . These rDifferent causes may justify the effects of cryopreservation on somatic embryogenesis. Higher morphogenic potential after cryopreservation has been explained by a differential response of embryogenic and non-embryogenic cells to this technique . Hence, According to Barra-Jiménez et al. , alteratThe present investigation reveals a primary role of the genotype on culture response to cryopreservation. Differences among genotypes could be due to differences in the nature of embryogenic tissues related to the proportion of hyperhydric vacuolated cells within them , to the Differences in tissues stability have also been related to differences in their genetic make-up whereby some components of the genome make it more prone to display variation . FurtherIn conclusion, the genotype plays a key role largely determining the effect of cryopreservation on olive somatic embryogenesis. A specific genotype-dependent response was found depending on the culture step. Thus, while in the proliferation phase significant variations were only observed in the line T1; in the maturation and germination steps, only the line P5 was affected by cryopreservation. Variations observed could not be associated to differences in the embryogenic lines’ instability to maintain their morphogenic competence after cryopreservation. Nevertheless, embryogenic cultures established after rewarming retain their morphogenic capacity for subsequent regeneration of plants, with no evident negative effects affecting the somatic embryogenesis process executed following the procedure described by Sánchez-Romero . TherefoOlea europaea L.) embryogenic lines were initiated from radicle of mature zygotic embryos “Picual” following the protocol of Orinos and Mitrakos [Olive (Mitrakos . RadicleMitrakos  supplemeEmbryogenic cultures obtained from single zygotic embryos were maintained as independent lines by repetitive embryogenesis on olive cyclic embryogenesis (ECO) medium ,45. Subc−1, except for ECO medium, which was gelled with 3 g L−1 phytagel. Media sterilization was carried out by autoclaving for 20 min at 121°C and 0.1 MPa.The pH of the media was adjusted to 5.74 before adding the gelling agent, consisting of agar 6 g Lw/v) ascorbic acid. Twenty-four hours later, cultures were transferred to ECO standard proliferation medium [Cryopreservation of olive somatic embryos was carried out following the protocol of Bradaï et al. , using tn medium  and incuIn order to investigate the effect of cryopreservation on somatic embryogenesis, the behavior of cultures established from cryopreserved somatic embryos was compared with that of control, non-cryopreserved cultures in the different phases of the somatic embryogenesis process, five to six months after rewarming. To examine the effect of the genotype, control and cryopreserved cultures of two independent embryogenic lines (T1 and P5) were examined during the proliferation, maturation, and germination steps.For evaluation of culture proliferation, 200 mg of embryogenic tissue were selected from control and cryopreserved stock cultures six weeks after the last subculture and inoculated in 25 × 150 mm test tubes containing 25 mL of ECO medium. After a six-week culture cycle at 25 ± 1 °C in darkness, culture aspect, fresh weight increase, and number of somatic embryos at different developmental stages )  were assSomatic embryo development was carried out by culturing 100 mg of embryogenic tissue in 90 × 25 mm Petri dishes containing 50 mL of basal ECO medium, i.e., ECO medium lacking plant growth regulators and cefotaxime. Eight weeks after incubation at 25 ± 1 °C and darkness, culture appearance, fresh weight increase, and number of TrSE < 5 mm, TrSE ≥ 5 mm, WOSE < 5 mm, and WOSE ≥ 5 mm were recorded.−2 s−1 irradiance provided by Grolux lamps . Germination percentage was recorded after two recultures of six weeks each.Somatic embryos equal or larger than 3 mm derived from maturation conditions were individually cultivated in 85 × 80 mm jars containing 50 mL of Clavero-Ramírez and Pliego-Alfaro  medium fIn the proliferation and maturation phases, 20 cultures were used per treatment and embryogenic line. The number of somatic embryos included at each repetition of the germination experiment ranged from 298 to 547, depending on the performance of the previous maturation phase, uneven for the different treatments and embryogenic lines. All experiments were repeated twice. Somatic embryos were considered germinated when shoot and/or root elongation was equal or larger than 2 mm.Percentage data were analyzed with an R × C test of independence or a three-way log-linear analysis, using the BIOMstat software . The rest of the data were analyzed by one- or two-way ANOVA followed by mean comparison by the LSD test, using the software package SPSS 26.0 . The significance level used was 0.05 in all cases .


Intensive farming of carnivorous fish species relies on the use of feeds, where fish meals have represented, for a long while, ideal sources of protein to ensure optimal growth, health and quality to cultured fish. Due to the gap between the demand for these commodities by the growing aquaculture industry and the unsustainability of further exploitation of the alieutic resources, their levels of inclusion in fish feeds have been mostly replaced by terrestrial plant protein-rich derivatives and conventional animal processed proteins. Recently, novel ingredients  have been proposed to this end. While the impact of different alternative proteins on fish growth and health has been studied, limited information exists on the effects of such dietary changes on quality traits of cultured fish such as skin pigmentation. The present study was aimed at assessing the pattern of yellow pigmentation of the skin in gilthead seabream fed various alternative protein sources  included in different proportions in fishmeal-free diets, in order to evaluate new feed formulations on the basis of their coloring capacity, as intense skin coloration have been associated with high-quality of farmed fish products.Sparus aurata) fed for 12 weeks iso-proteic (45%) and iso-lipidic (20%) diets deprived of fish meal and containing either a blend of vegetable protein-rich ingredients or where graded levels of the vegetable protein blend were replaced by insect  pupae meal, poultry by-product meal , red swamp crayfish meal (10%) and marine microalgae (Tisochrysis lutea and Tetraselmis suecica—10%) dried biomass. Digital images of fish fed diets differing in protein sources were analyzed by means of an automatic and non-invasive image analysis tool, in order to determine the number of yellow pixels and their dispersion on the frontal and lateral sides of the fish. The relationship between the total carotenoid concentration in the diet and the number of yellow pixels was investigated. Test diets differently affected gilthead seabream skin pigmentation both in the forefront and the operculum, due to their carotenoid content. The highest yellow pixels’ number was observed with the diet containing microalgae. Fish fed poultry by-product meal were characterized by the lowest yellow pixels’ number, diets containing insect meal had an intermediate coloring capacity. The vegetable control, the microalgae mix diet and the crayfish diet had significantly higher values of yellow pixels at both inspected skin sites.The pattern of yellowish pigmentation of the skin was assessed in gilthead seabream ( Pagrus pagrus or red sea bream Pagrus major, a natural red skin pigmentation similar of that of wild specimens is pivotal from a commercial point of view, increasing consumer acceptance of aquatic food [Farmed fish external traits, such as skin or muscle color and body shape, are increasingly important for consumers’ acceptance of aquatic food products ,2. Produtic food  and, thutic food . Final ptic food ,9,10,11.tic food . Red andSparus aurata L. is characterized by golden yellow coloration on its forefront and operculum. Farmed fish often exhibited darker gray or fade pigmentation [Wild gilthead seabream entation ,14,15,16entation ,18,19, eThe available knowledge of the effects of diet on skin pigmentation in Sparidae is reported in Previous studies were mainly focused on the effect on skin pigmentation of diet supplemented with synthetic carotenoids ,17,18,22Hermetia illucens pupae meal, poultry by-product meal, red swamp crayfish meal and dried biomass of two marine microalgae (Tisochrysis lutea and Tetraselmis suecica). To this end, a specific automatic, repeatable and non–invasive image analysis tool was developed and used to quantify yellow pixels in a digital image.Modern and sustainable fish feed formulations are developing to minimize the inclusion of fishmeal and optimize the use of conventional vegetable protein-rich ingredients and novel ingredients such as insect meals, microalgae and processed animal proteins . While t−1). The ingredient composition and proximate analysis of the test diets are shown in Hermetia illucens pupae meal (H), poultry by-product meal (P) singly or in combination, red swamp crayfish meal (RC) and of a blend (2:1 w:w) of the dried biomass of two marine microalgae (MA—Tisochrysis lutea and Tetraselmis suecica), while maintaining a same 67:33 vegetable to fish lipid ratio as in the CV diet. All diets were manufactured by SPAROS Lda  by extrusion in two pellet sizes (3 and 5 mm) and stored at room temperature, in a cool and aerated room until used.Nine test diets were formulated to be grossly iso-proteic (45%), iso-lipidic (20%) and isoenergetic . After stocking, fish were adapted over 2 weeks to the experimental conditions. At the end of this period, fish attained an average size of 49 ± 0.4 g and the 27 groups were assigned to the nine dietary treatments according to a random design with triplicate groups (tanks) per treatment. Fish were hand-fed the experimental diets over 12 weeks in two daily meals (8:00 am and 4:00 pm) until the first feed item was refused.The feeding trial was carried out at the aquaculture facilities of the Department of Agrifood, Environmental and Animal Sciences of the University of Udine. It used 486 gilthead seabream juveniles selected to be uniform in size, from a resident stock of 1200 specimens. Fish were randomly allocated to 27 groups, each consisting of 18 individuals kept in an indoor marine re-circulating aquaculture system including twenty-seven 250-L tanks. The system was kept under constant day length and intensity (12 h per day at 400 Lux) by supplemental fluorescent light tubes. It also ensured nearly constant and optimal water quality conditions to sea bream  mounted on a tripod. Fish were illuminated with a photographic lamp (illuminating power 6000 K). The focal distance between the camera and the fish was 40 cm for lateral side and 20 cm for frontal one. Color calibration was carried out with a Color Checker 24 color patch. Background was eliminated from each image using Photoshop CS6. The RGB range of yellow was determined randomly sampling five yellow pixels in three images for each tank (9 for treatment), to define upper and lower values for red, green and blue values. The obtained RGB range was: R = 205–255, G = 130–255 and B = 000–015. These values were perturbed by ± 5%, but the results did not change.https://cran.r-project.org/web/packages/countcolors/vignettes/Introduction.html). Yellow pixels, once identified, were plotted on each image in a different color (magenta), to increase the contrast with the background, and mean images for each treatment were generated.For each processed image (n = 481), the following were determined: (1) the total number of yellow pixels in the defined RGB range, and (2) the Standard Distance Deviation (SDD), computed as the standard deviation of the mean distance among yellow pixels. SDD can be considered as a dispersion index of yellow pixels on the image: the higher is the value, the larger is the yellow pixels scattering along the profile. All data were collected with “countcolors” v. 0.9.1 R package  with poAll analyses were carried out with R 3.9.9 software .All procedures involving fish manipulation were carried out in accordance with the EU legal framework relating to the protection of animals used for scientific purposes (Directive 2010/63/EU). They were approved by the Animal Welfare Committee of the University of Udine (OPBA) and authorized by the Italian Ministry of Health (permission n. 290/2019-PR).p < 0.001) and final weight . Significant differences among dietary treatments are reported in At the end of the trial, the dietary treatments resulted in significant changes in fish total length , but not among replicates  clustered together, but while MA10 and RC10 showed similar pattern of pigmentation of frontal and lateral sides, P20 was poorly colored on the lateral side and showed an intermediate yellowish coloration intensity on the forefront. The vegetable control was intermediate between the MA10, RC10 and P20 groups and the H20, H40 and H10P30 groups, and was pigmented mainly on the lateral side. Seabream fed insects  showed a poor yellow pigmentation on the lateral side, while a gradient was evident on the forefront, with at lower levels of substitution of vegetable ingredients with 2r values: 2rtreat = 0.05, p < 0.01; 2rrep = 0.01, p < 0.05 (ANOSIM). Therefore, no clear indications can be drawn from these data, which show very high intra-treatment variability. On average, lateral SDD was higher than frontal one, the lowest in H10 (145.2 ± 1.2), the highest in P40 (153.6 ± 0.6), while the dispersion of points on frontal profile was on average the highest in H10P30 (87.1 ± 7.9) and the lowest in H20 (73.1 ± 7.2). In this study, the yellowish pigmentation in lateral and frontal areas, expressed as number of yellow pixels, was positively correlated with the total carotenoid concentration in the diet .The SDD was significantly different both among treatments and among replicates, even if with low −1 [−1 [T. lutea and T. suecica dried biomass at 11% in weight (MA10) was definitively the one showing the highest number of yellow pixels as a consequence of a notably high content of carotenoids in the corresponding diet  [Oreochromis spp.) and ornamental carp (Cyprinus carpio) fed diets containing dried biomass of other microalgae such as Arthrospira platensis [H. pluvialis [H. pluvialis was also tested in diets for P. pagrus alevins, giving their skin an acceptable pink color, compared to the grayish one of farmed fish fed with diets not supplemented with carotenoids [Chlorella vulgaris was also added to a basal diet for S. aurata, determining a significant carotenoids deposition in four skin zones , but not in the muscle [Phaeodactylum tricornutum did not impact organoleptic properties of S. aurata fillets [All test diets in this study contained vegetable ingredients, such as cereal glutens, that are good sources of yellow carotenoids  and may −1 ,44, much−1 [−1 ). No stuing diet . Both mi suecica , and the labrax) ,48. Imprlatensis ,50, and luvialis ,51. Astaotenoids . The gree muscle . Diets i fillets  but resuP. clarkii have been already used as a source of pigments in fish diets [−1, consistent with values found by Pérez-Gálvez et al. [Plesionika sp. meal at 12% by weight in a commercial diet did not affect growth and survival of red porgy alevins but resulted in an intense pink color of the skin like that of wild fish [Paramola cuvieri showed significantly elevated skin redness and carotenoids concentration in skin samples [In this study, skin pigmentation pattern in fish fed the diet including a meal obtained from red swamp crayfish was very similar to that attained by those fed diet MA10. Processed products derived from sh diets  and as ash diets . Astaxanz et al.  and may ild fish . Includiild fish . Red por samples .H. illucens pupae or poultry by product meals had minor impact on skin pigmentation possibly due to a marginal contribution of carotenoid supplied by this ingredient. The inclusion of a mix of dried microalgae T. suecica and T. lutea and red swamp crayfish meal had a beneficial effect on S. aurata skin pigmentation, making these ingredients candidates as potentially effective natural sources of carotenoids. Mechanisms by which pigments from these sources are made available for storage in fish tegument should be studied in order to optimize their levels of inclusion in fish diets [Farmed gilthead seabream often exhibit gray pigmentation, different from the golden yellow coloration on forefront and operculum of their wild counterparts; thus, commercial feeds are often supplemented with carotenoids to enhance skin pigmentation and increase consumer acceptance of farmed products. The present study, through an automatic and non-invasive image analysis tool, has shown that the pattern of yellowish pigmentation of the skin in gilthead seabream could be affected by varying nature and levels of major protein sources in diets deprived of fish meal and already including vegetable protein-rich ingredients. Replacing graded levels of vegetable ingredients rich in carotenoids with defatted sh diets .