Bhutan is committed to eliminating hepatitis B and hepatitis C, though recent baseline estimates of disease burden in the general population are unknown. In 2017, we carried out a biomarker survey in the general population to estimate the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) biomarkers to evaluate the impact of immunization and guide further efforts.In 2017, a cross-sectional, population-based, three-stage cluster survey was undertaken of the general population (1–17 and 20+ years of age). We visited households, collected blood specimens and administered a standard questionnaire. Specimens were collected for hepatitis B surface antigen (HBsAg) and hepatitis C virus antibody (anti-HCV) testing. We calculated prevalence of infection and selected characteristics, along with confidence intervals (CIs).Of 1372 individuals approached, 1358 (99%) participated. Of those, 1321 (97%) had a specimen tested for HBsAg, and among 1173 enrolled individuals 5 years of age or older, 1150 (98%) individuals were tested for anti-HCV. The prevalence of HBsAg was 2.0% in 775 persons 20 years of age or older (95% CI: 1.0–4.0) and 0.5% in 546 persons 1–17 years of age (95% CI: 0.1–1.8). The prevalence of anti-HCV was 0.3% (95% CI: 0.1–0.8) among persons ≥5 years.Universal hepatitis B immunization of infants has resulted in a low prevalence of chronic HBV infection in persons 1–17 years of age and the prevalence of anti-HCV is low among persons aged ≥5 years. Efforts should continue to reach high coverage of the timely birth dose along with completion of the hepatitis B vaccine series. To reduce the chronic liver disease burden among adults, HBV and HCV testing and treatment as indicated might be restricted to pregnant women, blood donors, individuals with chronic liver diseases, and other groups with history of high-risk exposures. To formulate action plans for elimination, burden of disease estimates are needed. Since most chronic HBV and HCV infections are asymptomatic [WHO estimates that in 2015, viral hepatitis led to 1.34 million deaths worldwide [The Kingdom of Bhutan had an estimated population of 807,000 in 2017 (United Nations population estimates). In 1995–96, a serological survey indicated that 5.9% of the general population were HBsAg positive [In 2016, anecdotal reports from clinicians identified a large number of adult patients with chronic liver disease in health care facilities ; a high proportion of these patients were too old to have received hepatitis B vaccination and had chronic HBV infection. However, the prevalence of hepatitis viral infections among adults in the general population was unknown, since no biomarker surveys have been conducted since 1997. Moreover, a population-based biomarker survey among children was needed to document progress towards reaching the regional hepatitis B control goal. Therefore, a cross-sectional biomarker sero-survey was conducted to estimate the prevalence of HBV and HCV infections among those born before 1997 and after 2000, in order to (1) evaluate the impact of hepatitis B vaccination on the burden of chronic HBV infection in children and (2) quantify the burden of chronic hepatitis B and C virus infections in adults. The findings were used to inform development of a national viral hepatitis plan.The design was a cross-sectional, population-based, three-stage cluster survey. We integrated the survey with a measles-rubella serological survey to reduce cost and improve efficiencies. This report describes the hepatitis B and C results.The survey population consisted of household members living in Bhutan at the time of the survey. Three age groups were defined: 1) persons born from 1 April 2012 to 31 March 2016 who were aged 1–4 years (‘young children’) at the time of the survey; 2) persons born from 1 April 2000 to 31 March 2012 who were aged 5–17 years (‘older children’) at the time of the survey. 1 April 2000 was chosen to include children who had had an opportunity to receive three doses of hepatitis B vaccine after the vaccine was introduced in 1997, excluding period from 1997 to 1999 when coverage was low); and 3) persons born before 1997 who were aged ≥20 years (‘adults’) at the time of the survey.We defined chronic HBV infection as the presence of HBsAg, past or present HCV infection as the presence of HCV antibodies, and chronic HCV infection as the presence of HCV RNA.To address the primary objective of the survey , the sample size was estimated on the basis of the expected prevalence of the biomarker in the surveyed population, the absolute precision, and the intraclass cluster correlation. For younger and older children together, we expected a prevalence of 1% , an absolute precision of 1% and a design effect of 1.5. This generated a sample size of 571 children, using the asymptotic formula, increased to 657 children after adjustment for an anticipated 15% non-response. For adults, we expected a HBsAg prevalence of 5% , an absolute precision of 2% and a design effect of 1.5. This generated a sample size of 685 adults, increased to 788 after adjustment for an anticipated 15% non-response. For younger children aged 1–4 years, we also calculated a minimum effective sample size based on the sample size needed for the concomitant measles-rubella serosurvey. Sample size was calculated based on the assumption of an expected 95% seroprevalence of measles immunoglobulin (IgG), a desired precision of a one-sided confidence interval of − 5% and a significance level α = 0.05, which resulted in an effective sample size of 123 (Fleiss formula). Accounting for the cluster design with a target of eight young children per primary sampling unit, an intraclass correlation = 0.1, and an anticipated 15% nonresponse, the sample size was increased to 245 young children. A design of 30 clusters with 29 households per cluster was chosen to achieve the minimum sample size in each age group.The sample was stratified to separate Thimphu, the capital, from the rest of the country, in order not to oversample in the most populous area of the country. In the first stage, four gewogs  were chosen from Thimphu and 26 from the rest of country, by probability proportional to estimated size with replacement from a national census list. In the second stage, an updated household line list was created from which 29 households were randomly selected and visited. At each household, field workers listed those eligible individuals . One participant was selected from each of the three age groups. If there was more than one eligible individual in an age group, one was selected at random using a random number generated by a cell phone application (third sampling stage). In case of absences, field workers re-visited up to three times on two different days. If the selected individual remained absent after 2 days, the individual was counted as absent and not replaced.Trained field workers administered one of three standardized questionnaires (based on the age group) to the enrolled individuals or their parents after obtaining written consent (and assent as appropriate). The questionnaire included demographic characteristics ; hepatitis B vaccination history; each participant’s or caregiver’s knowledge of HBV or HCV infection status; and exposures that could be risk factors for HBV or HCV infection. Vaccination data were verified by reviewing vaccination cards in those 1–17 years of age when available. Questions were asked in a private setting to ensure confidentiality. Pre-test counselling was conducted as per the WHO guidelines on viral hepatitis testing. Written consent was requested for individuals in all three age groups for hepatitis B testing and among older children and adults for hepatitis C testing. Venous blood was collected from consenting selected individuals following standard quality control. Plasma was separated at the nearest Basic Health Unit (BHU), stored at -20 °C, and sent to Royal Centre for Disease Control (RCDC) under temperature control for testing.At RCDC, quality assurance and quality control in specimen processing were carried out using standard operating procedures of the laboratory. The survey specimens were tested together with quality control specimens supplied by the National Reference Laboratory, Melbourne, Australia . HBsAg tAfter laboratory testing was completed, investigators returned the results of the hepatitis B and C tests to the participants, if positive, and provided post-test counselling according to WHO guidelines on viral hepatitis testing . InvestiData were double entered using Epi Info™ v7 . Weights were applied, taking into account the sampling strategy, non-response, and the population distribution in Bhutan. Concerning vaccination, timely hepatitis B birth dose (HepB-BD) was defined as a dose given on the day of birth or on the day after birth. Frequencies, weighted percentages, and 95% logit confidence intervals were calculated for all items. We compared populations with and without selected characteristics in terms of prevalence of biomarkers through the calculation of prevalence ratios and their 95% CIs.The Research & Ethical Board of Health, Bhutan, and the WHO Research Ethics Review Committee approved the survey protocol after the necessary amendments . This activity was reviewed in accordance with CDC human research protections procedures and was determined to be nonresearch, programme evaluation.In March–April 2017, 870 households were visited in 30 clusters in 13 districts (Dzongkhags). Among them, 781 (90%) households agreed to participate in the survey with at least one member of the household, 18 (2%) households refused to participate, and 71 (8%) were ineligible . From the 781 participating households, 1372 individuals were approached for participation. Of those, 1358 (99%) individuals were interviewed ; 1321 (97%) blood specimens were collected and tested for HBsAg; and specimens from 1150 (98%) of 1173 older children and adults were tested for anti-HCV.Of the 775 adults tested , 15  were HBsAg positive  of the routine series than for the birth dose (70%) Table . Among oOf the 1150 older children and adults tested, four were positive for anti-HCV  could be referred to care (others were lost to follow up). Of those, three (25%) were HBV DNA positive. One HBV DNA positive person had an increased alanine amino transferase level, and was eligible for treatment according to 2015 WHO HBV treatment guidelines; this individual began Tenofovir treatment.Of the four persons who were identified to be anti-HCV positive, none were HCV RNA positive. None of the anti-HCV positive persons was eligible for treatment according to the 2018 WHO HCV treatment guidelines.One of the purposes of this biomarker survey was to evaluate the impact of hepatitis B vaccination among children. In 2016, the Immunization Technical Advisory Group of the WHO South East Asia Region set a goal to reduce the HBsAg prevalence among 5 year old children to < 1% by 2020, in agreement with the goal of the GHSS on Viral Hepatitis . In addiThis biomarker survey was also an opportunity to evaluate the HBsAg prevalence among adults in the context of developing a strategy to identify and manage persons with chronic HBV infection. Overall, among persons 20 years of age or older, HBsAg the prevalence was 2%, lower than in the 1995 survey (6.3% among persons 24 years of age or older) . AnotherOverall, the prevalence of anti-HCV was lower (0.3%) than in the 1995 biomarker survey (1.3%). Projections of the HCV infection prevalence to the adult population in Bhutan, and assuming 80% of anti-HCV positive persons are HCV RNA positive (the proportion estimated for India ) suggestOur survey also provided an opportunity to examine prevalence of HCV infection according to histories of possible exposures. Cross sectional surveys that examine serological evidence of past or present HCV infection are not a good way to explore the modes of transmission of viruses like HCV that lead to chronic infection. However, results of our analysis suggest that the prevalence of anti-HCV was higher among persons who had received invasive procedures from traditional healers. This finding is consistent with what is known of the modes of transmission of HCV . Given tOur survey had a number of limitations. First, some questions relied on recall data, such as history of exposure to invasive procedures, some of which might have occurred long ago, and vaccination status of some children. Secondly, while participation was high, some selected households did not participate, and it is unknown if these households were different from participating households. The subsets of individuals with exposures of interest were quite small; therefore, we might have failed to find significant associations with infection, even if they existed. Overall, our small sample size and low prevalence did not allow us to identify population subgroups that should be tested for HBV or HCV infection. Thirdly, expected differences in HBsAg prevalence estimates between age categories may have been diluted, as older children also had received birth dose while some adults could have been vaccinated as young adults. Fourth, the low proportion of HBsAg and anti-HCV positive persons who were positive for HBV DNA (3/1) and HCV RNA (0/4) may point to a need to confirm positive serological tests with a second test. Finally, we did not examine children born when hepatitis B vaccine was first introduced in the routine childhood vaccination schedule , preventing us from inferring the prevalence estimate found in this study directly to the entire population of Bhutan.In conclusion, our survey documented that the prevalence of HBV infection is likely to be lower than 1% in the children 5–17 years of age, probably because of moderate-to-high vaccination coverage. We also found that the prevalence of HBV infection in the total population was under the threshold of 2% (low endemicity) while the past or present HCV infection prevalence was very low (0.3%). Based on these results, efforts should continue to reach high coverage of the timely birth dose along with completion of the hepatitis B vaccine series . For adu


Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes—10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes—0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10 Embryos cultured in the presence or absence of triiodothyronine (T3) till blastocyst stage upregulate genes contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Little is known about the effects of the thyroid hormones in the earliest stages of human development. The results suggest that thyroid hormones affect mitochondrial function in human embryos: stimulate mitochondrial replication and energy production within mitochondria by switching metabolism from glycolytic pathway to more efficient oxidative phosphorylation. The findings shed a light on metabolic switch in early embryo development and might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Data in the present study also suggest that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.1Thyroid hormones, thyroxine (T4), and its biologically active form triiodothyronine (T3) play vital roles in regulating homeostasis and metabolic rate of human cells and tissues. They are essential for physical and mental development; insufficient production of thyroid hormones before birth or during childhood can lead to reduced growth and mental impairment. In adults, hypothyroidism causes reduced metabolism, poor memory, depression, and reduced fertility.In women of fertile age, hypothyroidism frequently results in menstrual irregularities; ovulation and conception are possible, though resulting pregnancies are liable to miscarry. In addition, women with severe hypothyroidism have been reported to have diminished libido and ovulation failure.THRA) and β (THRB) transcripts and protein levels were detected in developing bovine preimplantation embryos up to the blastocyst stage.Thyroid hormone is detected in follicular fluid and has a role in ovarian follicle cycle and egg maturation.Exposure to T3 of human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of the blastocyst stage embryos and induced pluripotent stem cells triggered expression of multiple genes linked to regulation of gene transcription, cell cycle, morphology, apoptosis, cell viability, and cellular and embryonic development.Here, we report investigations into the effect of thyroid hormone on the transcriptome of developing human preimplantation embryos and show that ICM and trophectoderm (TE) react differently to T3 exposure. Whereas T3 had a profound effect on gene expression in the ICM, TE cells remained almost unaffected.22.1The work described here was done under license from the UK Human Fertilisation and Embryology Authority (research license number: R0075) as well as local ethical approvals . Informed consent was obtained from all subjects, and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the NIH Belmont Report. No financial inducements were offered for donation.2.22 and 5% O2 in a humidified atmosphere. Treated embryos were exposed from thawing till harvesting to 10−7 M T3 (Sigma). The medium was refreshed on a daily basis. ICM and TE were separated mechanically as described.The embryos were thawed using Quinn's Advantage thaw kit according to the manufacturer's instructions (Sage). After thawing, the embryos were cultured in 40‐μL microdrops of Quinn's medium supplemented with 10% synthetic protein serum substitute (Sage) under mineral oil (Sage) at 37°C in 5% CO2.3The recovered ICM and TE fractions were stored at −80°C till processing. cDNA was prepared using SMARTer Ultra Low Input RNA for Illumina Sequencing—HV (Clontech) directly from cells. All experiments were carried out in a PCR clean workstation using protocols provided by the manufacturer. The purification of the first‐strand cDNA was carried out using SPRI Ampure Beads (Beckman Coulter). The amplification of the ds cDNA was carried out in a PCR Clean workstation using an Advantage 2 PCR Kit as per the manufacturer's protocol. The PCR‐amplified cDNA was purified using immobilization onto SPRI beads. One microliter of the amplified cDNA was used for the validation using the Agilent 2100 BioAnalyzer and the High Sensitivity DNA Chip (Agilent) per the manufacturer's instructions. The Covaris AFA system was used for the controlled shearing of DNA prior to the generation of the library for Illumina sequencing. The shearing system led to the formation of DNA sequences in the range of 200‐500 bp. Ten microliters of the fragmented ds‐cDNA sample was used for library preparation. The library purification protocol was similar to that used to purify cDNA. The libraries were sequenced on HiSeq 2000 system (Illumina). The libraries were quantified using Qubit (Life Technologies), and the size profile was analyzed on the 2200 TapeStation (Agilent).GenoSplice Technology performed quality control (QC), processing, and further analyses of the data. For each gene present in FAST DB v2014_2, reads aligning on constitutive regions  were counted. Based on these read counts, normalization and differential gene expression were performed using DESeq (v1.16.0 on R v3.1.3).2.4P ≤ .05; GenoSplice, EASANA) were filtered for mitochondrial identity in MitoMiner 4.0v2018 JUN (accessed December 2018) as defined by the integrated mitochondrial protein index as “KNOWN” mitochondrial.Genes identified to have significant differential expression  stage were used for the experiment; 15 embryos were exposed to 10PN stage NANOG, NODAL, SOX2) and TE  demonstrated separation of two subpopulations , whereas the difference between TE of control and TE of T3‐treated embryos was only in 38 genes (0.3% of expressed genes), indicating that the effect of T3 on ICM was profoundly different from its effect on TE  pathways, and those downregulated were genes in Glyoxylate and dicarboxylate metabolism and Citrate (TCA) cycle , and 13 code for proteins that are necessary for cellular energy production: cytochrome b (CYTB), cytochrome oxidase 1, 2, and 3 , ATP synthase 6 and 8 (ATP6 and 8), NADH dehydrogenase 1, 2, 3 4, 4L, 5, and 6  production, and OXPHOS. Studies on metabolism of bovine embryos have shown that ICM and TE have pronounced differences in carbohydrate metabolism, which may reflect their diverging roles in embryological development.Transmission electron microscopic analyses of mitochondria in human preimplantation embryos have shown that dramatic changes in mitochondrial morphology occur during the differentiation of blastomeres in TE and ICM. Rounded to oval “immature” mitochondria are transformed into elongated tubular forms with well‐defined transverse cristae, a sign of increased metabolic activity.THRA1 isoform p43 which may bind directly to the mitochondrial DNA,TFAM)PPARD), nuclear respiratory factor‐1 (NRF1),POLG), or peroxisome‐proliferator‐activated receptor gamma coactivator‐1a (PPARGC1A).Mitochondria have been shown to be the major sites of T3 accumulation in cells, potentially having a direct effect on mitochondrial activity.Although our data suggest that T3 induced mitochondrial biogenesis in ICM, we did not detect an increase in the expression of these genes, which suggests that our observed increase in number of mitochondria was mediated through a different pathway or pathways. For example, recently, a group of small non‐coding RNAs (sncRNA), known as P‐element–induced wimpy testis (piwi)‐interacting RNAs (piRNA), corresponding to mtDNA, have been described.Mitochondrial respiration contributes to energetic requirements during embryonic development. In the cleavage stage only 10% of available glucose is metabolized through aerobic respiration, whereas this is increased at the blastocyst stage to 85%.In contrast, mitochondrial transplantation has recently emerged as a potential treatment of various diseases, including restoration of fertility.The association of reproductive competence with mitochondrial quantity and function at different stages of human embryo development remains unclear. However, the clinical evidence strongly suggests that T3 plays an indispensable role in the earliest stages of development. It is plausible to hypothesize that this is due to its effects on energy production and mitochondrial biogenesis, as found in the data presented here. Currently, the media used for culturing human embryos in vitro do not contain T3. We suggest that supplementing commercial media with physiological concentrations of T3 might have a beneficial effect on the embryo development and improve implantation and pregnancy rate.COX expression. T3 regulates the expression of COX subunits by both transcriptional and posttranscriptional mechanisms in a mitochondrial content‐dependent manner.Mitochondrial metabolism can also be considered as an alternative indicator of developmental identity and indicator of pluripotency stage. hESCs are considered to represent a later, postimplantation, phase of epiblast development, more similar to mouse epiblast stem cells.5Little is known about the effects of the thyroid hormones in the earliest stages of human development. However, women of fertile age with underactive thyroid glands are prone to suffer from menstrual irregularities and, although ovulation and conception are possible, resulting pregnancies are prone to miscarriage.Our results indicate that thyroid hormones affect mitochondrial function in human embryos; although transcription of mitochondrial DNA (mtDNA) genes was not affected by T3, mitochondrial replication was stimulated, as was energy production within mitochondria by switching metabolism from the glycolytic pathway to more efficient OXPHOS. This effect was restricted to the ICM, the part of the embryo that gives rise to the body of the fetus.Our report suggests a new mechanism of metabolic switching in human preimplantation embryos and highlights an important role of thyroid hormones in the early stages of pregnancy. It is plausible to hypothesize that supplementing commercial media with physiological concentrations of thyroid hormone might have a beneficial effect on embryo development and improve implantation and pregnancy rate.Understanding of the basic biochemistry and roles of the ICM and TE in homeostasis of the blastocyst is still limited. One hypothesis is that the TE is a transporting epithelium, sparing nutrients for use by the ICM.The authors indicated no potential conflicts of interest.L.N., S.E.K.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; A.P.: collection and/or assembly of data, data analysis and interpretation; G.G., N.F., A. Capalbo, L.D., P.K., H.R., N.K., K.K.N.: collection and/or assembly of data; V.M.J., A.M., N.H: data analysis and interpretation; A. Cvoro: conception and design, data analysis and interpretation, manuscript writing; R.F.: provision of study material or patients; M.S.A.: financial support, final approval of manuscript; P.F.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, final approval of manuscript; C.O.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript; Y.K., D.I.: conception and design, data analysis and interpretation, manuscript writing, financial support, final approval of manuscript.Figure S1 Expression pattern of lineage specific markers indicate separation of two subpopulations.A) Expression of ICM lineage markers NANOG, NODAL, and SOX2.(B) Expression of TE lineage markers CDX2, GATA2, and GATA3. values. As indicated in Methods, differential analyses were made using Deseq2 from normalized gene counts, not from RPKM. All normalized gene counts used for this analysis are accessible on GEO under accession number GSE131499.Click here for additional data file.


Lontra canadensis) are highly variable with different institutions providing various whole foods, commercial complete prepared diets, or combinations of both. This study investigated the digestible energy intake, gastrointestinal transit time, and digestive efficiency of three different diets being fed at three North Carolina institutions. Otters housed at Institution A (n = 3) were fed strictly fish. Otters housed at Institutions B (n = 3) and C (n = 2) were fed a majority fish based diet , supplemented with fruits, vegetables, and supplemental protein sources as enrichment. There was an apparent trend between increased percentage of fish in the diet and faster transit time and higher digestive efficiency. As less fish was included in the diets, the GI transit time was longer . Median digestive efficiency was high for all three groups  but was higher for the institutions feeding fish. Additionally, the overall median gross energy intake for the eight animals in this study was 163.1 kcal/kgBM0.75/day . While all three institutions had healthy otter populations, it appears that a higher fish diet should be further studied as the model North American river otter diet.Diets currently provided to captive North American river otters ( Lontra canadensis) is a semiaquatic opportunistic predator that feeds primarily on fish but also a wide variety of other prey items  [The North American river otter (insects) –6. Once insects) , 7, 8.The success of human-managed populations of North American river otters depends on proper nutrition , 10. TheThe objective of this study was to determine the gross energy intake, gastrointestinal transit time, and apparent digestibility of gross energy, crude protein, and crude fat of three different diets being fed to three different managed North American river otter populations.n = 3 ; Institution B, n = 3 ; and Institution C, n = 2   , were usOtters housed at Institution A were fed a fish-only diet. Otters housed at Institutions B and C were fed different fish-based diets  supplemented with fruits and vegetables along with supplemental protein sources  . All aniGastrointestinal transit time and total fecal collection digestibility assessments were performed over a 3-day period. Uniquely colored markers  were added to each diet to identify individual otter scat in the group-housing situations. The rate of food transit was measured on Day 1 of the diet trial. The time of ingestion of the marked diet was recorded, and the animal was observed every 2½ minutes until the appearance of the marked feces. Gastrointestinal transit time was calculated as the interval between the first ingestion of the diet and the appearance of marked feces.Prior to each feeding on Days 1 and 2, the wet weight of each diet component was recorded. All of the food left uneaten (ort) was immediately collected and weighed. Total feces were collected throughout the day and the following mornings and weighed. Four separate samples of each fish species fed, each of the supplemental enrichment portions of the diets, and feces from individual animals were individually homogenized in a kitchen blender , and the homogenates were placed in an ultralow freezer  set at −70°C and stored up to 4 months until analyzed.To determine percent dry matter, aliquots of frozen samples were thawed and then dried under forced air at 60°C for 72 hrs. To determine adequate drying, repetitive weights were acquired after each drying, and when weight change between weighings was less than 0.1 g, a sample was considered dry matter. Percent dry matter was calculated by dividing dry weight by wet weight multiplied by 100 (AOAC method 950.46). Dried samples were ground using a handheld coffee grinder , and gross energy was determined for each fish species fed, the supplemental diet enrichment component, and each fecal sample in duplicate using an IKA C5000 bomb calorimeter . Duplicate subsamples were averaged to yield a single data point. Variation in duplicate subsamples was less than 4.0% for all diet items and feces samples analyzed. Fish, supplemental diet enrichment, and feces were analyzed for crude fat by Soxhlet ether extraction (AOAC method 2003.05) and crude protein using a Leco nitrogen/protein determinator  in duplicate by a commercial laboratory service . All nutritional compositions were calculated and presented on a dry matter basis (DMB). Percent apparent nutrient digestibility was then calculated using the following formula :(1)apparDescriptive statistics were calculated using JMP Pro, version 11.0 software . The data for each variable were tested for normality of distribution by the Shapiro–Wilk test. Summary statistics are presented as both median and ranges.Tilapia and salmon fillets used in some diets had the lowest crude fat  and the highest crude protein  of all fish species tested.The nutrient composition of the commercially available fish used in the diets varied . Gross eGross energy for each of the actual overall diets fed to the animals was within 10% of each other . Median gross energy was the highest for otters held at Institution B , followed by Institution A  and Institution C  , Hawaiian monk seal (Monachus schauinslandi) , and Steller sea lion (Eumetopias jubatus)  as well as several commercially prepared diets   fed North American river otters [The nutritional needs of the North American river otters are often extrapolated from the requirements of domestic cats and diets that have been successfully used to maintain captive otters , 14. Ther otters , 21–23. r otters , 25.Protein, carbohydrate, and fat all provide energy in a diet, with fat providing energy on average twice that of protein and carbohydrates . The fis0.75/day) and Eurasian river otters (Lutra lutra)  and fishers (Martes pennanti)  fed several different diets comprising various whole food items and slightly higher than those reported for other comparable sized terrestrial mammals (domestic cat (Felis silvestris catus), 42.0–134.7 kcal/kgBM0.75/day; bobcat (Lynx rufus), 46.3 kcal/kgBM0.75/day; African wildcats (Felis silvestris lybica), 91.5 to 120.5 kcal/kgBM0.75/day) [The median gross energy intake was numerically higher for the otters housed at Institution B when evaluated against the animals at either Institution A or C. This was expected because the otters at Institution B were fed a greater amount of diet by wet weight when compared to the animals in the other institutions. The median gross energy intake established from the otters in this study was consistent with reports on gross energy intake for both North American otters  –35.The gastrointestinal transit time of the North American river otter is much faster than that reported for many other terrestrial carnivores . This woOdobenus rosmarus) (92.7%) and Steller sea lions fed a diet of herring (95.5%) [Mustela vison), blue fox (Alopex lagopus), and both the domestic cat and dog decreased the apparent digestibility of crude protein, which corresponded with the increase in concentration of plant protein, which is less likely digestible than the animal proteins it replaced [The median apparent digestibility of gross energy for all three groups in this study was consistent with reports measuring apparent digestibility of gross energy in North American river otters fed Nebraska Feline Diet (90.2%) , Mazuri Polar Bear Diet (83.1%) , and Hill's Science Diet cat food (86.6%) , as well as fishers fed several different diets comprising various whole food items (80.9 to 92.6%) and both walrus ( (95.5%) , 27, 39.replaced –42. It wreplaced , 44. Thareplaced .Digestibility trials can be difficult to perform on animals housed in more natural habitats in zoological collections due to the difficulty of conducting total feces collection. This difficulty can be overcome by comparing the changes in concentrations of an inert dietary marker . The aniThis study indicated an apparent trend between an increase in the percentage of fish offered in the diet and a faster transit time and higher digestive efficiency. Knowledge on energy content, gastrointestinal transit time, and apparent digestibility of nutrients associated with a more fish-based diet is important if zoological institutions are to provide a nutrient dense diet sufficient for maintenance, growth, and reproduction in this highly active species. This study did not examine the digestibility of any of the diets in regard to vitamins and minerals, and further studies should be performed to evaluate these nutrient parameters and to ensure that these animals are being provided a completely balanced diet.


Even though ladybirds are well known as economically important biological control agents, an integrative framework of DNA barcoding research was not available for the family so far. We designed and present a set of efficient mini-barcoding primers to recover full DNA barcoding sequences for Coccinellidae, even for specimens collected 40 years ago. Based on these mini-barcoding primers, we obtained 104 full DNA barcode sequences for 104 species of Coccinellidae, in which 101 barcodes were newly reported for the first time. We also downloaded 870 COI barcode sequences (658 bp) from GenBank and BOLD database, belonging to 108 species within 46 genera, to assess the optimum genetic distance threshold and compare four methods of species delimitation  to determine the most accurate approach for the family. The results suggested the existence of a ‘barcode gap’ and that 3% is likely an appropriate genetic distance threshold to delimit species of Coccinellidae using DNA barcodes. Species delimitation analyses confirm ABGD as an accurate and efficient approach, more suitable than the other three methods. Our research provides an integrative framework for DNA barcoding and descriptions of new taxa in Coccinellidae. Our results enrich DNA barcoding public reference libraries, including data for Chinese coccinellids. This will facilitate taxonomic identification and biodiversity monitoring of ladybirds using metabarcoding. Subsequently, the 5’ end of the mitochondrial cytochrome c oxidase subunit I gene (COI) has been suggested as a standardized DNA “barcode” for identifying species of all groups of animals, launching the DNA barcoding technology2. The success of barcode identification based upon genetic distances ultimately depended on differences between intra- and interspecific divergences4. DNA barcoding using the 658 bp 5’ region of mtCOI DNA sequence as a tool, has turned out as very efficient and reliable for identifying specimens of unknown origin and taxonomic status, and also for the identification of different developmental stages. The approximately 1600 base-pairs comprise a range of different functional domains showing heterogenous substitution patterns6. In addition to its utility for distinguishing known species, the COI region has also been found suitable for revealing cryptic species11, and biogeographic and phylogeographic patterns, and also species level phylogenetic relationships14.As species are a fundamental biological category, accurately identifying them is an essential premise of biological studies. Tautz 16. Coccinellids are well known as economically important biological control agents, but this family is ecologically and morphologically very diverse. It comprises about 490 genera and nearly 6000 described species worldwide17. Due to relatively small body size and similar elytral shapes and color patterns , most of the species are very difficult to identify18. Therefore, using DNA barcode identification appears highly desirable. Moreover, the usefulness of DNA barcoding for distinguishing and diagnosing coccinellid species has been proved in previous studies20. Until now, there are 7,845 published records of coccinellid DNA barcodes from 46 countries and 37 institutions, comprising 529 BINs (Barcoding Index Numbers) in BOLD (Barcoding of Library Dataset) database system (http://www.barcodinglife.org). However, there is still a lack of reference data for such a large group of beetles. It is apparent that a comprehensive database is urgently required, and further barcoding is critical to improve the taxonomic resolution.The family Coccinellidae is placed in the superfamily Coccinelloidea within the Coleopteran suborder Polyphaga21. The soft tissue soon dries out and decomposes, resulting in the fragmentation of DNA and negatively affecting the amplification success. The damaged DNA is broken down into small fragments of a few hundred base pairs22. Consequently, amplification with standard DNA barcoding primers for the 658 bp COI region is not possible. To solve this problem, Hajibabaei et al.23 and Meusnier et al.24 developed mini-barcode, which means using a region of about 100–150 bp to replaces the standard DNA barcoding procedure for identifying species. The mini-barcode approach proved successful for this purpose, even using museum specimens. In addition, Van Houdt et al.25 recovered full DNA barcodes by continuously amplifying mini-barcodes. As PCR amplification success rate is related to the length of the target band, the amplification of a sequence of about 200 bp is easier than amplifying standard DNA barcodes with 658 bp.Collecting fresh specimens is expensive, time-consuming and often insufficient to ensure a wide coverage of species. Therefore, it is often necessary to use museum specimens for DNA extraction and target band amplification. However, insect material of natural history collections is often pinned without further preservation treatment25 approach to use universal mini-barcoding primer sets that allowed to reconstruct full DNA barcodes for Diptera, and also a new design with a set of universal primers for recovering the standard DNA barcodes for museum specimens of ladybird beetles. Our specific objectives are (1) develop a set of universal primers to increase the amplification efficiency for museum specimens of Coccinellidae; (2) contribute DNA barcodes for morphologically identified coccinellid species without previous DNA barcoding records. Moreover, we combined GenBank with BOLD published data to (3) screen out the optimum genetic distance threshold to the identification of ladybird or other beetles. As the genetic distance threshold is taxon specific26, the threshold for Coccinellidae was hitherto unknown. The choice of the analytical methods for DNA barcoding data and species delimitation methods is obviously important. Therefore we compared and analyzed Generalized Mixed Yule Coalescent (GMYC)12, Bayesian Poisson tree processes (bPTP)27, Automatic Barcode Gap Discovery (ABGD)28, Barcode Index Number (BIN)29 to (4) find out which approach is more accurate for delimiting species of Coccinellidae and suitable for describing new taxa.In the present study, we used Van Houdt’sThe universal LCO1490/HCO2198 primer pair used to amplify the DNA barcoding region resulted in an extremely low success rate. Only the sequence of 3 specimens collected in 2013 could be amplified successfully. Similar results were obtained with our new design primer pair of WDF/XSR Fig. . The conWe separately amplified four mini-barcoding fragments using both standard PCR and nested PCR methods. The PCR success rate was 93% and 83% for Cocc301 and Cocc413 markers using standard PCR, respectively, while PCR success rate was 97% and 93% for Cocc301 and Cocc413 markers, respectively, using nested PCR. The Amplification success rate of Cocc301 and Cocc413 in the nested PCR was slightly higher than in the standard PCR. However, the Cocc286 and Cocc214 markers resulted in a relatively low success rate of 43% and 57%, respectively, using standard PCR method, especially for samples collected before 1987 Fig. . SurprisHippodamia notata has the largest number of sequences, up to 20 barcode sequences, while Mulasntina picata has only one barcode sequence. The average barcode sequence is 8 for each species   were the only case of two morphology-based species assigned to a single BIN.The ABDG analysis with a 1.3%–3.6% maximum intra-specific divergence yielded 110–121 putative species, which was close to the numbers of the species based on morphology Fig. . In BOLDUsing GMYC with single-threshold calculations yielded 142 lineages, and very similar results with BIN. Surprisingly, the bPTP method yielded 223 putative species and the results twice the number of identified morphospecies. The bPTP species delimitation results were significantly more than GMYC, ABGD and BIN.25. The observed pattern is consistent with DNA fragmentation models performed by Zimmermann et al.22, which predict a fast initial drop in average DNA fragment size in the first 5 years followed by a more gradual change. This also illustrates that DNA increasingly breaks down into smaller fragments with time30, and that small PCR fragments more easily amplify than relatively long PCR fragments25.Apparently, the amplification success rate and efficiency of mini-barcoding were much higher compared to the full DNA barcode31. In this study, we also employed this PCR strategy to amplify four mini-barcoding markers. Our results show that the amplification efficiency of nested PCR is obviously higher than that of standard PCR, especially for the Cocc286 and Cocc214 markers  genetic distance analysis showed that intraspecific divergences range from 0 to 3.1%, and the average intraspecific genetic distance was 0.6%. The ABGD analysis we conducted showed that the initial partition is more stable, with 870 sequences divided into 110–121 putative species based on different prior P. In contrast to this, the recursive partition showed a large undulation, and yielded an overestimation of species numbers. However, both approaches reached the same partition when the prior maximum divergence of P was 0.0359, dividing 870 sequences into 110 putative species. The reliability of the ABGD method was confirmed by Puillandre et al.37 and Ratnasingham and Hebert29, who pointed out that recursive partition is unstable and prone to excessive partitioning. Our results confirm that initial partition results are more stable and more consistent with morphospecies. Taking into consideration what we discussed above, we propose that 3% is a useful genetic distance threshold to delimitate ladybird beetles using DNA barcodes.Even though several attempts have been made to establish a standard limit between intra- and interspecific divergence, none of them can be used for a wide range of groups. Due to variable population sizes and time of divergence in different species, defining such a threshold is a problematic issue and somewhat arbitrary29. Given this situation, it is evident that new methods are needed for efficient biodiversity assessments and for in developing a sound species-level taxonomy38. Analysing DNA barcode sequences with varying techniques for cluster recognition provides an efficient approach for identifying putative species39. In the present study, GMYC and BIN methods yielded similar results , using a superdiverse insect genus Polypedilum Kieffer (Diptera: Chironomidae). Their results indicated a conservative number of species with ABGD, whereas bPTP yielded a much higher operational taxonomic unit (OTU) count than the other methods.The rapidly increasing rate of extinction coupled with the magnitude of unknown biodiversity requires accurate species delimitation methodslts Fig. . BIN was44. Although anchored in a solid phylogenetic framework, this method heavily depends on the correctness of the ultrametric gene tree. Errors in this framework underpinning the analysis affect the final results38. GMYC yielded more OTUs than species based on morphology in our analysis, which may be in fact a result of erroneous ultrametric tree reconstruction. Evaluations of the accuracy and efficiency of ABGD conducted by different researchers reached a consensus that ABGD is more conservative and faster than other methods45. This is confirmed by our results, where ABGD yielded 110 OTUs, a number very close to that of species defined based on morphology. Consequently, we recommend ABGD as the best method to delimit species of Coccinellidae. BINs in BOLD have been yielded results largely conform with traditional taxonomy in many groups of animals46. However, it apparently overestimated species numbers in our study, arguably due to the low intra-specific genetic distance threshold obtained for Coccinellidae with the Refined Single Linkage (RESL) method. Both BIN and ABGD use clustering algorithms to distinguish partitions in the genetic distance among a group of individuals, resulting in a final array of OTUs38. In contrast to BIN, ABGD infers an appropriate intracluster threshold with an automatic statistical approach, resulting in a partition close to the pattern of morphology-based species. bPTP neither requires an ultrametric input tree nor a sequence similarity threshold as input27. Instead it adds Bayesian support values to delimit species on the input tree. Higher Bayesian support value on a node indicates that all descendants of this node are likely species. In our analyses, bPTP yielded an astonishing overestimation of 223 OTUs, more than twice the number of morphology-based species. Similar results were also obtained by other authors47. Therefore, we conclude that bPTP is not an appropriate method to delimit species of Coccinellidae.GMYC has been developed to delimit species based on single-locus data, which has a strong theoretical basis. However, it typically generates more OTUs than other methods48. Furthermore, the comparison of different methods helps to understand their tendency to either split or merge clusters. The disadvantage of using several methods is the increased complexity of the investigation and resulting from this an increased amount of time and effort required for OTU delimitation38. We also suggest to apply different species delimitation methods, preferring delimitation patterns consistent across different approaches, even though the process is complicated and time-consuming. Sauer and Hausdorf49, who investigated the performance of different methods using the land snail genus Xerocrassa (Gastropoda: Hygromiidae) on Crete, suggested that all methods based on single-locus sequences are insufficient for delimiting species. They showed that single-gene species delimitation can be affected by several forms of bias, such as the presence of pseudogenes, incomplete lineage sorting, or introgression51. Consequently, the inclusion of at least one additional independent gene is required. Additionally, the use of morphological, geographical and ecological data is highly recommendable, resulting in an integrative framework to delimit species53. Apparently, decisions based on analyses of single-genes contain an interpretational risk. Nevertheless, they are indifferent if the outcome is viewed as a scaffold for taxonomy, rather than as the sole criterion for the description of new taxa48.As different analytical approaches have different theoretical foundations, it is recommendable to test a wide variety of methods of species delimitation, and to prefer patterns that are congruent across the resultsAll fresh specimens were collected by the authors and deposited in the Insect Collection, Department of Entomology, South China Agricultural University (SCAU). Museum specimens used in this study were also obtained from the Insect Collection of SCAU. All of them were used with permission from the Insect Collection of SCAU.61. In addition to our own data, standard 658 bp DNA barcode sequences without stop codons were searched and downloaded from GenBank and BOLD. In total, 870 COI barcodes were included. The GenBank accession number and BOLD BINs are presented in Supplementary Table A total of 104 species of Coccinellidae , following the protocol provided by the manufacturer. We pretreated the museum specimens with 0.9% NaCl buffer before DNA as outlined in a previous studyely Fig. . With th64. For nested PCR, as the first round of primers we used our own WDF/XSR. In the second round the PCR conditions were the same as in the first round. PCR products were electrophoresed in 1% agarose gel. DNA fragments were sequenced in both directions with sufficient overlap to ensure the accuracy of sequence data. All sequences were confirmed as the correct target fragments using BLAST (GenBank). Raw sequences were assembled and edited in Geneious 7.1.465. The sequences were aligned using the MUSLE algorithm66 and checked for stop codons in MEGA 767.Polymerase chain reaction (PCR) amplifications were conducted in a 25 μL volumes containing 12 μL 2 × EasyTaq PCR SuperMix , 10 μL ultrapure water, 1 μL of each primer and 1 μL DNA template. PCR was performed with an initial denaturation step of 95 °C for 3 min followed by 40 cycles at 96 °C for 15 s, 50 °C for 30 s, 60 °C for 3 min and a final extension at 72 °C for 10 min. We also used the nested PCR to ensure a high success rate of PCR amplification. Nested PCR is a modification of standard PCR that uses two sets of primers in two separate PCR rounds to amplify the target band, where the product of the first round serves as the DNA template for the second round of PCR. The advantage is that nested PCR is more sensitive and specific than the standard PCR68. Then, ABGD analysis was implemented on the website (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html), using relative gap width (X = 1.5) and intraspecific divergence (P) values between 0.005 and 0.100 with the K2P model. All other settings were default.We selected 870 standard COI barcode sequences (658 bp) from GenBank and BOLD. These sequences of 108 morphologically identified species were used for screening the optimum genetic distance threshold. The intraspecific and interspecific genetic distance matrix was calculated using MEGA 7 based on K2P model12. The analysis was conducted using BEAST 1.8.069 under a strict clock model and speciation with Yule process Tree model. The runs consisted of 70 million generations sampled every 5000 cycles. Convergence was assessed by ESS values. A burn-in with 25% was set to obtain an optimal consensus tree. We then used the obtained tree to analyse the data under the GMYC species delimitation approach in the software R with the package ‘splits’70 using the single-threshold method.Initially, we used the GMYC method to delimit species. GMYC delimits distinct genetic clusters by optimizing the set of nodes defining the transitions between inter- and intraspecific processes29. The bPTP analyses were conducted on the web server (http://species.h-its.org/ptp/) using rooted phylogenetic input tree constructed with RAxML 8.071 using 500 rapid bootstrapping replicates and the GTR + I + G nucleotide substitution model. The following setting was employed: 500 000 MCMC generations, thinning interval of 100, and first 25% were discarded as burn-in.In a second step, we used the bPTP model to infer molecular delimitation clades. The bPTP model treats the number of substitutions between branching and speciation as independent events28. The ABGD analyses were performed at the web server (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html). The following setting was used: relative gap width X = 1.5, K2P distance and intraspecific divergence (P) values range from 0.001 to 0.100, other parameter values employed defaults. BIN, which is widely used in the BOLD system, employs varied distance metrics to generate a neighbor-joining (NJ) tree and is established as a persistent registry for life OTUs29.The other two methods are ABGD and BIN. The ABGD detects a gap in the distribution of divergence that corresponds to differences between intra-specific and inter-specific distances firstly. When a gap exists, the method will work well for species delimitationSupplementary information.


Daily exposure of the skin to UVA radiation causes oxidative modifications to cellular components and biomolecules. These include proteins involved in the metabolism and cytoprotection of fibroblasts, and their modification can contribute to the disruption of cell function and the development of skin disorders. Therefore, there remains a need for highly active cytoprotective compounds with antioxidant properties. The purpose of this study was to investigate the effect of ascorbic acid on the activity of rutin against UVA-induced changes in the proteome of human fibroblasts. All analyses were carried out on fibroblasts cultured in a three-dimensional system exposed to UVA radiation and incubated with rutin and ascorbic acid. Their proteomic profile was analyzed using nano-HPLC, which revealed 150 proteins whose expression was significantly altered between treatment conditions. UVA radiation led to changes in the expression of 82 proteins. However, some of these changes were mitigated by rutin and ascorbic acid separately  and rutin and ascorbic acid together (23 proteins). UVA radiation has led to the upregulation of proteins involved in gene expression, catalytic processes and antioxidant pathways, and downregulation of proteins with binding activity. Nevertheless, rutin and ascorbic acid used separately or together have countered these changes to varying degrees. Moreover, rutin and ascorbic acid stimulated fibroblasts irradiated by UVA to increase the expression of the signalling molecules responsible for the opening of the transmembrane channels. In the context of the results obtained, the observed cytoprotective effect of the cooperation of rutin and ascorbic acid results not only from the overlapping properties of the compounds. The effect of rutin alone is probably inhibited by its limited bioavailability. Therefore, its interaction with ascorbic acid increases membrane penetration and improves the cytoprotective effect on skin fibroblasts. Rutin is one of the phenolic compounds found in various plant species, where it contributes to the antibacterial properties of the plant . As a glα-tocopheroxyl radicals in the membranes [The cytoprotective properties of rutin contribute to the protection of skin cells exposed to various types of radiation, considerably increasing cell viability , 9. Howeembranes . Ascorbiembranes –15. The embranes . Also, aembranes  and has embranes –20. CompUV radiation can disturb various metabolic pathways in skin cells, a feature that has been widely documented in published reports , 22. TheWhile the surface layers of the skin disperse most of the long UV wavelengths, UVA radiation 320–400 nm) shows strong penetration deep into the skin, reaching the dermis, which is mainly composed of fibroblasts . As a re–400 nm s2 at 37°C in a medium composed of Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) according to the standard protocol for fibroblasts. To avoid bacterial contamination, medium was supplemented with penicillin (50 U/ml) and streptomycin (50 μg/ml). Following two passages, cells were seeded in 24-well plates  with AlgiMatrix gel  to create a three-dimensional (3D) model. After four days of culturing, the cells were exposed to UVA radiation  at a total dose of 20 J/cm2 (λ = 365 nm). Before irradiation, cells were washed with PBS (4°C). In this buffer, cells in plates put on ice were exposed to the irradiation from 6 lamps with an assembly of 6 W each, which corresponds to 4.2 mW/cm2. The distance between cells and lamps was 15 cm. To analyze the effect of rutin and ascorbic acid on UVA-radiated cells, we followed the corresponding methods of Gęgotek et al. [μM rutin or/and 100 μM ascorbic acid in 0.1% DMSO. Parallelly, control cells were cultured in medium containing 0.1% DMSO. The diagram showing the course of the experiment is shown in The human skin fibroblasts (CRL-1474) were obtained from the American Type Culture Collection (ATCC) and cultured in a humidified atmosphere of 5% COFollowing incubation, using AlgiMatrix™ dissolving buffer , fibroblasts were collected from the 3D gel. Cells were washed with PBS and lysed by sonification. The total protein content in the lysate was measured using a Bradford assay .v/v) and heated 10 min at 95°C. Preliminary protein separation was prepared on 10% Tris-glycine SDS-PAGE gels. All bands were detected by overnight staining with Coomassie Brilliant Blue R-250. Proteins were sliced and in-gel digested overnight with trypsin . Obtained peptide mixture was dissolved in 5% acetonitrile (ACN) + 0.1% formic acid (FA) and separated on 150 mm × 75 mm PepMap RSLC capillary analytical C18 column  using Ultimate 3000 HPLC . Eluted peptides were analyzed using a QExactive HF mass spectrometer  operated in a positive mode. Details of the analysis have been described previously [Before proteomic analysis, proteins were denatured by mixing with Laemmli buffer that contained 5% 2-mercaptoethanol  and searched against the UniProtKB-SwissProt database . Peptide mass tolerance was set to 10 ppm, MS/MS mass tolerance was set to 0.02 Da, and up to two allowed missed cleavages were used for protein identification. Details of the protein identification have been described previously , 20. OnlZ-score transformed. Statistical analysis of data was performed using free available MetaboAnalyst 4.0 software , RStudio software (R version 3.6.2 (2019-12-12)), and Perseus 1.6.10.43. Principal component analysis (PCA) was conducted for exploratory data analysis, with the R package MetaboAnalyst [Analyses of each sample were performed in three independent experiments. Missing values were estimated using half of the minimum value from the original data imputation. Results from individual protein label-free quantifications were log and oAnalyst . ANOVA toAnalyst  using “EThe proteomic data obtained in this study allowed us to create a list of proteins whose expression was significantly altered in fibroblasts cultured in 3D after irradiation with UVA followed by treatment with rutin and ascorbic acid . In this case, the chemical treatment induced the expression of 18 proteins which were not found among the 128 proteins expressed in the cells treated only with UVA irradiation .Using principal component analysis (PCA), we found that the changes in the proteomic profiles of skin fibroblast cells led to the clustering of the experimental groups (PC1 40.9% and PC2 28.6% for nonirradiated fibroblasts and PC1 37.5% and PC2 22.7% for UVA-irradiated fibroblasts) . In the The volcano plots comparing the effect of ascorbic acid and rutin on the cells irradiated by UVA also highlight the number of proteins which changed expression under experimental conditions . UVA radSkin cells are exposed to sunlight, which often disrupts cell metabolism due to harmful UVA and UVB radiation . ProteomFibroblasts are one of the main types of cells that build human skin, but their location in the inner layer of the skin, the dermis, gives them partial protection against external factors by epidermal cells. As a result, fibroblasts are very sensitive to penetrating UVA radiation . To suppThe results obtained in this study corroborate the previously published data, clearly showing that UVA radiation induces large changes in the proteome of skin fibroblasts , 43. TheThe main proteins whose expression was sensitive to UVA radiation were those involved in protein biosynthesis; our data show that UVA radiation significantly upregulates the expression of proteins involved in mRNA transcription processes, including elongation factors , the translation initiation factor 2 (P41091), ribosomal proteins (A0A024R2P0), and ribonucleoproteins . This cellular response is aimed at defending against radio-induced stress caused by UVA rays .The cytoprotective action of rutin after UVA irradiation significantly prevents increased expression of translation-inducing proteins, which, in previously published data, has been suggested as a method of inhibiting carcinogenesis . The actIn our study, we have found that ascorbic acid also modestly reduced the expression of several proteins involved in degradation following UVA radiation. However, some of these proteins were more strongly upregulated than in fibroblasts without treatment with ascorbic acid. A similar complex and ambiguous effect of ascorbic acid on proteasomal activity has also been observed, mainly in cells with disturbed metabolism, such as cancer cells , 50. TheOther groups of proteins highly upregulated by UVA radiation in 3D cultured skin fibroblasts were proteins with catalytic activity, in particular in glycolytic processes. Similar changes have been previously described also for UVB-irradiated fibroblasts . ReducinThe antioxidant action of rutin and ascorbic acid, which according to the literature is mainly based on their free radical scavenging activity , leads tA slightly different situation has been observed in keratinocytes cultured in a 3D model. In these cells, treatment with antioxidant compounds additionally supports the natural cellular antioxidant system . MoreoveThe additional cytoprotective mechanism of skin fibroblasts against UVA radiation is their ability to provide multilayer protection for skin cells . In suchRutin, in combination with ascorbic acid, also causes UV-irradiated fibroblasts to upregulate the expression of Ras-related protein Rab-5C (P51148). This protein activity is also closely linked to the membrane-bound proteins and GTP hydrolysis, which participates in the regulation of the secretion of extracellular signalling molecules . Other pIn conclusion, the strong antioxidant properties of rutin, as well as its effects on intracellular and intercellular signalling, are probably inhibited by its limited bioavailability. In the context of the results obtained in this study, this natural polyphenol requires to be administered with ascorbic acid not only to increase its membrane penetration but also to complement its cytoprotective effect on skin cells irradiated with UV. Analysis of the mechanisms of action of rutin and ascorbic acid in different types of skin cells, such as fibroblasts and keratinocytes , draws p


The data further suggests that stress tolerant cultivars suppress cell death and maintain growth and development via fine tuning of hormone signaling, and primary and secondary metabolism. This study highlights potential targets for the development of stress tolerant potato cultivars.Potato production is often constrained by abiotic stresses such as drought and high temperatures which are often present in combination. In the present work, we aimed to identify key mechanisms and processes underlying single and combined abiotic stress tolerance by comparative analysis of tolerant and susceptible cultivars. Physiological data indicated that the cultivars Desiree and Unica were stress tolerant while Agria and Russett Burbank were stress susceptible. Abiotic stress caused a greater reduction of photosynthetic carbon assimilation in the susceptible cultivars which was associated with a lower leaf transpiration rate. Oxidative stress, as estimated by the accumulation of malondialdehyde was not induced by stress treatments in any of the genotypes with the exception of drought stress in Russett Burbank. Stress treatment resulted in increases in ascorbate peroxidase activity in all cultivars except Agria which increased catalase activity in response to stress. Transcript profiling highlighted a decrease in the abundance of transcripts encoding proteins associated with PSII light harvesting complex in stress tolerant cultivars. Furthermore, stress tolerant cultivars accumulated fewer transcripts encoding a type-1 metacaspase implicated in programmed cell death. Stress tolerant cultivars exhibited stronger expression of genes associated with plant growth and development, hormone metabolism and primary and secondary metabolism than stress susceptible cultivars. Metabolite profiling revealed accumulation of proline in all genotypes following drought stress that was partially suppressed in combined heat and drought. On the contrary, the sugar alcohols inositol and mannitol were strongly accumulated under heat and combined heat and drought stress while galactinol was most strongly accumulated under drought. Combined heat and drought also resulted in the accumulation of Valine, isoleucine, and lysine in all genotypes. These data indicate that single and multiple abiotic stress tolerance in potato is associated with a maintenance of CO Solanum tuberosum L.) is one of the most important food crops in the world where it ranks only behind rice and wheat in terms of global production (2 fixation and chlorophyll loss in sensitive species (Potato (oduction . The crooduction . For exaoduction . Such yioduction . Moreove species . Recent  species . However species .2O2), superoxide anions (O2•−), hydroxyl radical (OH•), and singlet oxygen (1O2), can trigger a multitude of detrimental responses which include lipid peroxidation of cellular membranes, breakdown of photosynthetic pigments, denaturation of proteins, carbohydrate oxidation, DNA damage, and decreased enzyme activities . Temperature and water stresses often occur together leading to decreased yield and quality losses. Many studies have investigated the response of plants to a combination of different abiotic stresses , and plants were grown in environmentally controlled walk-in chambers with a 14 h photoperiod, 24°C day/18°C night temperature, and 60–70% relative humidity until stress treatment. All plants were irrigated regularly until the beginning of stress application, and fertilizer N-P-K (18%-18%-18%) was applied twice to all plants after emergence. Stress treatments were initiated 27 days after emergence by dividing plants randomly into four groups as control, drought, heat, and combined drought-heat treatments. The experiment was carried out with three replications, and each replication consisted of two pots each containing two potato plants. Two identical growth chambers were used for stress treatments, one for control and drought, the another for heat and combined drought and heat. Control plants were grown under optimum conditions as described above until samples were harvested. Drought was applied to plants by withdrawing irrigation for 12 days at 24/18°C (day/night). For heat treatment, plants were exposed to a gradual temperature increase in both day and night for 9 days, until the temperature reached to 39/27°C, then, a constant high temperature of 39/27°C was applied for 3 days . Chlorophyll measurements were performed on five leaves of two individual plants and the average value was considered as one biological replicate. Three biological replicates were used to estimate mean and standard error. Chlorophyll measurement was performed on the 12th day of stress treatment.Leaf temperature was measured on fully expanded upper leaves using an infrared thermometer . Leaf temperature measurements were on three leaves of two individual plants and the average value was considered as one biological replicate. Three biological replicates were used to estimate mean and standard error. The leaf temperature measurement was performed on the 12th day of stress treatment.–2s–1 and CO2 concentration was maintained at 400 μmol mol−1. Measurements of Pn, E, and Gs for each genotype were performed on one plant from each of three biological replicates. The Pn, E, and Gs measurements were taken periodically up until the 12th day of stress treatment.Photosynthetic rate (Pn), transpiration rate (E), and stomatal conductance (Gs) were measured on fully expanded upper third or fourth leaves of genotypes with a LICOR LI-6400XT portable photosynthesis system using a built-in light source. Photosynthetically active radiation (PAR) was maintained at 1,500 μmol m−1 cm−1 was used. The values for MDA content are expressed as µmol/g fresh weight (FW).On the 12th day of stress treatment, the level of lipid peroxidation was quantified by measuring the amount of malondialdehyde (MDA) as determined by the thiobarbituric acid (TBA) reaction described by On 12th day of stress treatment, proline content was determined according to the method of For enzyme extraction from the plants, 200 mg of frozen leaf samples were ground in 4 ml of cold 50 mM K-phosphate buffer (pH 7.0) containing 2 mM Na–ethylenediaminetetraacetic acid (EDTA) and 1% (w/v) polyvinyl–polyvinylpyrrolidone (PVPP). The homogenates were centrifuged at 10,000 × g (4°C) for 10 min and the supernatants were transferred into new tubes. The enzyme extracts were stored at −80°C prior to activity measurements as described below. All enzyme assays were performed on extracts from three independent biological replicates.Superoxide dismutase (SOD) activity was determined by measuring the inhibition of photochemical reduction of nitro-blue tetrazolium (NBT) in the presence of riboflavin under light according to 2O2 according to 2O2 and 30 μl enzyme extract to 2.67 ml 50 mM phosphate buffer (pH 7.0). CAT activity was calculated by quantifying the rate of change in absorbance at 240 nm over 2 min using an extinction coefficient of 39.4 mM−1 cm−1.Catalase (CAT) activity was determined by decomposition of H2O2 (40 mM). POD activity was calculated by using an extinction coefficient of 26.6 mM−1 cm−1.For peroxidase (POD) activity, the oxidation of guaiacol in the reaction mixture was estimated by quantification of the rate of change of absorbance measured at 470 nm for 2 min according to . The rea2, 0.5 mM ascorbic acid, 0.1 mM H2O2, and 25 μl enzyme extract. The activity of ascorbate peroxidase was calculated using an extinction coefficient of 2.8 mM−1 cm−1.Ascorbate peroxidase (APX) activity was measured by following the decrease in the amount of ascorbate as estimated from the rate of change in absorbance at 290 nm for 2 min . One micFully expanded leaves were harvested from the fourth node of three independent replicate plants and immediately frozen in liquid nitrogen. Samples were stored at −80°C prior to lyophilization, extraction, and analysis. Samples were extracted as polar and non-polar phases, derivatized, and analyzed by gas chromatography mass spectrometry (GC-MS) as previously described . Metabolhttp://www.ebi.ac.uk/arrayexpress/; accession number E-MTAB-8298). Briefly, a single-channel microarray design was utilized with leaf RNA samples all labeled with Cy3 dye. RNA labeling and downstream microarray processing was performed as recommended using the Low Input Quick Amp Labeling Kit . Following microarray scanning using an Agilent G2505B Scanner, data were extracted from images using Feature Extraction (FE (v. 10.7.3.1) software and aligned with the appropriate array grid template file (033033_D_F_20110315). Intensity data and QC metrics were extracted using the recommended FE protocol (GE1_107_Sep09). Entire FE datasets for each array were loaded into GeneSpring  software for further analysis. Data were normalized using default single-channel settings: intensity values were set to a minimum of 0.01, data from each array were normalized to the 50th percentile of all measurements on the array, and the signal from each probe was subsequently normalized to the median of its value across all samples. Unreliable data flagged as absent in all replicate samples by the FE software were discarded. Statistical filtering of data was performed using two-way analysis of variance  for the factors “cultivar” and “treatment,” with Bonferroni multiple testing correction.Fully expanded leaves were harvested from the fourth node of three independent replicate plants and immediately frozen in liquid nitrogen. Samples were stored at −80°C prior to extraction and analysis. RNA was extracted from potato leaves as described previously . MicroarStatistical analysis was carried out on the metabolite data sets collated from GC/MS polar and non-polar fractions. Firstly, principal components analysis (PCA), using the sample correlation matrix which gives equal weight to all metabolites, was used to summarize broad scale variation among the samples. A second approach involved an ANOVA of each individual metabolite using the main factors variety, treatment , or an interaction of the two factors. Gene expression data were also analyzed by ANOVA, in this case the data used compared the varieties, the control samples and the heat and drought treatments, and the interaction between the two. Genes were selected according to their p-values from the ANOVA F-test, using a significance threshold based on the estimated false discovery rate (FDR) of less than 1%  . StatistThe key objective of the present work was to identify the physiological, biochemical, and molecular responses to single and combined abiotic stresses that underpinned the tolerance phenotype in potato. We therefore chose to compare the responses of stress tolerant and stress susceptible genotypes. Desiree and Unica were chosen as tolerant to heat and drought, and Agria and Russet Burbank were selected as heat and drought susceptible genotypes, based on previous literature . HoweverA key determinant of potato yield under abiotic stress is the capacity to maintain photoassimilation and carbohydrate transport to developing tubers . TherefoIn order to define an appropriate time point for subsequent analysis of physiological, metabolic, and transcriptomic responses to single and combined stresses, stress severity was monitored by measuring gas exchange parameters in the four different cultivars at different time points following the imposition of stress treatments. The intention was to identify a time point at which all cultivars were experiencing stress so that differences in stress-induced responses between stress tolerant and stress sensitive cultivars could be identified in subsequent analyses.2 assimilation between plants subjected to combined stress and control plants while sensitive Russet Burbank , leaf relative water content was significantly reduced compared with control plants  activity following heat stress  or stress sensitivity (inability to acclimate).Arabidopsis metacaspases and seven transcripts encoding subtilases. Both metacaspases and subtilases play a role in programmed cell death in plants (Arabidopsis transcript (At2g46240) encoding a Bcl-2-associated athanogene protein known to be responsive to heat stress (Arabidopsis transcript (At2g20880) encoding an AP2 domain transcription factor that has been shown to be induced by drought or heat stress , a type I metacaspase. Type I metacaspases have been implicated in the control of programmed cell death in Arabidopsis  with a role in response to water deprivation was also present among these transcripts. A further group of transcripts were highly expressed in response to heat or heat and drought in stress tolerant genotypes but less so in stress susceptible genotypes  that acts as a negative regulator of leaf senescence  and a mitochondrially localized leucine rich repeat receptor kinase  shown to demethylate jasmonate and auxin in vitro and a transcript encoding a 2-oxoglutarate dependent dioxygenase (DMT400041989) homologous to an Arabidopsis transcript (AtAt1g06620) involved in drought and ethylene responses   and that catalase is primarily localized to the peroxisomes while APX is found in most cellular compartments  both exhibited a significant reduction in the abundance of transcripts associated with PSII, particularly transcripts encoding light harvesting chlorophyll-protein complex components, when exposed to drought and heat and no such reduction was observed in Agria or Russett Burbank. Similarly, a strong reduction in these transcripts was observed under conditions of high temperature alone in Desiree Figure 6Arabidopsis transcript encoding ABI5 binding protein 2 . The transcription factor ABI5 is a key regulator of ABA signaling and stress responses and its function is modulated by AFP2  with homology to an  by AFP2 . This pr by AFP2 . GA-medi by AFP2 . Also in by AFP2  and so tInterestingly, a series of transcripts associated with metabolic processes were more highly abundant in stress tolerant compared with stress sensitive genotypes Table 1.Aside from the mechanistic insights gained from our transcriptomic dataset analysis, this information is also of value in genetic studies to identify sources of abiotic stress tolerance in potato germplasm, as it will facilitate the identification of candidate genes within QTL limits or associated with other genetic markers.Developmental stage and severity of stress are important considerations when deciding on abiotic stress treatments and is therefore a limiting factor of any study. We chose to impart stress treatments at the tuber initiation stage as this has been shown to limit foliage, stolon and tuber development and yield . To miniIn summary, we identified unique changes in transcripts and metabolites that were specific to individual and combined heat and drought stresses. We define conditions that highlight differences in sensitivity between different potato genotypes. These experiments indicate that stress tolerant cultivars respond to stress by i) reducing light harvesting capacity and increasing non-photochemical quenching and ii) maintaining capacity for growth and development in part by iii) rerouting metabolism to compensate for reduced photosynthesis. This is achieved by a fine tuning of hormonal signaling. The responses of cultivars with contrasting abiotic stress tolerance provided information on genes/classes of compounds that may be used as targets for future studies aimed at enhancing multi-stress tolerance in potato.http://www.ebi.ac.uk/arrayexpress/accession number E-MTAB-8298.The datasets generated for this study can be found in the UD, WM, MT and RH conceived and designed the study. UD, CY, AA, IT, ZG, SC, EA, MC conducted physiological and biochemical experiments. UD, CY, IT, WM and LD prepared material for transcriptome and metabolome analysis. JM conducted microarray experiments that were analysed by PH. WM and RC conducted and analysed metabolite profiling experiments. SV conducted statistical analysis. UD, WM, MT and RH drafted the manuscript with contributions from all authors.This research was funded by a British Council Newton Fund Institutional Links Grant No. 216394957 and the Scottish Government Rural and Environment Science and Analytical Services Division as part of the Strategic Research Programme 2016-2021.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.