Cordyceps, Fusarium, Harpochytrium, Emericellopsis, Volutella, Cladosporium, Stachybotrys, Trichoderma, Cochlonema and two unknown fungal species) was present in emperor Yang’s coffin chamber. The predominant species were Stachybotrys, Fusarium, Trichoderma and Cochlonema. Redundancy analysis (RDA) indicated that humidity, temperature, height and illumination were the most significantly related factors shaping the fungal communities. Humidity showed the highest degree of variance description (19.2%) than all other environmental factors, followed by illumination (18.3%) and height (12.8%). Furthermore, fungal richness and diversity indices showed a positive correlation with humidity (p < 0.05). These results help in understanding the fungal community in tombs, promoting the mitigation of deterioration phenomena of such building heritage for the present and future.Biodeterioration caused by filamentous fungi is often a threat to the architectural heritage (i.e. tombs and historic sites). To specifically understand the deterioration phenomena caused by microorganisms in tombs and how these are shaped due to various environmental factors, the fungal communities in the coffin chamber of the Chinese emperor Yang (BC 569–618) were investigated at different heights using denaturant gradient gel electrophoresis (DGGE) fingerprinting. The associated environmental conditions, such as humidity, temperature, height and illumination, were also assessed. The results showed that a great diversity of fungal species ( Environmental factors such as sunlight, temperature, water content, rain, wind and relative humidity also contribute to that. Physical and biophysical damages in such structures are often related to the loosening of the intergranular bonds caused by porous volume fluctuations (expansion and shrinkage). this weakens the strength of the building materials2 and subsequently its structure. Microbial populations, and their corresponding volume, often develop into the brick or plaster of these structures. This is also a key factor that contributes to deterioration of these structures. Fluctuations in the volume of the microbes at the developed voids in the masonry structure is also a factor that compromises the structural capacity of these buildings. A very common ‘suspect’ of such microbial/fungal fluctuations are fungi or lichen hyphae4.Brick masonry structure is the most widespread building material that accounts for a major fraction of the ancient architectural history worldwide. In China, most of historical artifacts are constructed of black brick masonry. However, the main drawback of such masonry is the fact that is prone to deterioration due to its porous and rough surface6. As a result, deterioration usually causes an aesthetically detrimental effect7, rendering the preservation and maintenance of these valuable cultural heritage extremely challenging and financially unattainable. The porous structure of blue bricks makes such material easily adhered by various microorganism8; then these microorganism utilize the water and nutrition in pores to reproduce to a larger scale. Wang et al.5 isolated the bacterial strains from the surfaces of weathered bricks, and considered that Bacillus, Massillia and Brevibacillus could contribute to brick weathering. Vasanthakumar et al.9 assumed that the brown spots on the walls of King Tutankhamun tomb was related to microbiological activity. Their research showed that Penicillium chrysogenum, which could produce malic acid in vitro, was very likely responsible for the formation of these spots. However, previous research/studies only provide with a rough outline of the relationship between microorganism and building heritage deterioration, while the mechanism is not yet very clear. Therefore, we shout that there is a need to extend the knowledge on the microbial communities or the species developed on the surface of these valuable cultural heritage, so we can further understand the nature of the problem and subsequently protect our heritage for the future.A wide range of studies demonstrated that microbes could promote the biodeterioration of historical artifacts that were constructed by black bricks or stones11. Porosity, surface roughness, moisture content, pH value of substrate, the mineral and nutrient concentrations all influence both microbial colonization and reproduction13, and subseqenty deterioration. Liu et al.14 reported that a biological community grows easier under conditions of water seepage and/or of high humidity. Moreover, several environment-related means are developed and/or improved to limit the biological colonization, i.e. reduction and maintenance of humidity and condensation levels15, effective drainage of rainfall precipitation16, and minimization of the moisture levels on the substrate14. Nevertheless, the control and the mitigation of biodeterioration processes are still far from being implemented, since the impact of the environmental factors on such sites is ignored or not thoroughly assessed. Further understanding will certainly tackle bio-deterioration of our historical heritage.Generally, the degree of biodeterioration is dependent on the environment factors and the characteristics of the substrate (masonry)17, this study investigates the possibility for mitigation of such deterioration phenomena of brick masonry structure heritage, from the perspective of controlling the microorganisms that could be developed or not by manipulating environmental factors.The tomb of Emperor Yang of the Sui Dynasty, an ancient tomb with special historical and cultural value, was accidentally discovered in March 2013 at Yangzhou, China. This ancient tomb is constructed by black brick at a size of 3.45 m in width, 4.08 m in length, and 2.95 m in height  along vertical direction. The temperature of WC30, WC80 and WC150  and positively related to air temperature (p < 0.05). The illumination in all sampling sites on the passage wall were “very low”, while the assessed points on the top sites of the main wall (height 185 cm and 105 cm) were high; illumination at the underlayer (height 30 cm) was found low.The results of the environmental characteristics of the tomb are shown in Table PCR-DGGE fingerprinting was conducted to reveal the shifts in fungal community composition in different positions in WCC and WC Fig. . In geneCluster analysis, based on the band pattern on the DGGE gel, revealed the differences among the samples at different sites. The profiles were mainly divided into three clusters Fig.  at a simp < 0.05) among the samples collected from different sampling sites in the tomb. Among the 6 sampling sites, WC30 showed the highest richness and diversity, followed by WCC30 and WCC185, while WC150 was the lowest.In this study, Richness and Shannon indices were selected to estimate the fungal diversity. As shown in Table Cordyceps, Fusarium, Harpochytrium, Emericellopsis, Volutella, Cladosporium, Stachybotrys, Trichoderma, Cochlonema and two unknown fungal species .Phylogenetic analysis showed that 18 fungal genotypes obtained from the DGGE profiles Table . The seqes Table . Specifip < 0.05).The result of DCA showed that the gradient length of the first axis was computed to be 2.90. According to this, RDA was used to analyze the correlation between fungal community composition and environmental factors. As shown in Fig. 19. Emperor Yang’s coffin chamber is one of the most valuable buildings in China. Our previous study on this ancient remnant has revealed some bacterial-related groups that were associated with the tomb20, and managed to isolate a novel bacterial species, Paenibacillus tumbae21. In this study, we firstly investigated the fungal communities in the walls of Emperor Yang’s coffin chamber, and hypothesized that the existed diversity fungal species. The results, based on DGGE profile, showed that the dominant fungal genera were belonging to Stachybotrys, Fusarium, Trichoderma and Cochlonema  in the Monastery of St Damian in Assisi. Similar to S. chartarum, Fusarium spp. have recently emerged as a significant human pathogen34. So, the presence of Fusarium spp. in coffin chamber or buildings is becoming a potential threat for both humans and environment.Trichoderma (DGGE band 15) was the dominant fungal species on the walls of Emperor Yang’s coffin chamber, observation very well aligned with the findings of Rao et al.35. Species of Trichoderma genus are versatile in nature with varying activities, for example, they are often related to suppress soil-borme diseases and/or produce celluases and xylanases37. They are also commonly found in indoor environments39, and considered as one of the indicators of moisture-damaged building materials40. Trichoderma and Fusarium have been identified in Godoy’s House, a historical building41. Samson et al.42 highlighted that Stachybotrys spp. and Trichoderma are likely to arise when the water activity (aw) is greater that 0.85. In this study, DGGE band 15 showed the highest densities in samples WCC105 and WC30, respectively, where the air humidity was 88%, observation in line with previous studies42. In addition, Trichoderma species are not known to be harmful to humans, but there is accumulating evidence that a few species may be infectious for humans44. Particularly, we also found the dominance of Cochlonema, corresponding to DGGE band 18, in emperor Yang’s coffin chamber; however, to our knowledge, there is no available report that associates this specie with indoor environment, building or human disease. In this study, we also identified the presence of Cordyceps, Harpochytrium, Emericellopsis, Volutella, Cladosporium and two unknown fungal species, most of which were previously found in buildings. In this study, we could not discuss them in detail due to their low relative abundances.One more finding from this study is that 46. In this survey, we highlighted that fungal communities in the walls of Emperor Yang’s tomb were predominantly affected by humidity, illumination, air temperature and height of sampling sites  levels of 0.5, 0.85 and 1.0, the results showed that the metabolic processes of fungal communities at aw level of 0.5 only referred to cell maintenance, but displayed more diverse secondary metabolic processes at 1.0. These results totally highlighted that humidity or moisture have a pronounced impact on controlling fungal growth. Actually, moisture is considered as the only limiting factor for fungal growth in buildings45. As we know, fungi need adequate temperature, nutrients and water to grow. Usually, building materials, even fiberglass fabric coverings, could provide with nutrients such as cellulose and starch for fungal growth50, and some phototrophic microorganism such as cyanobacteria and lichens could also supply with organic nutrients for bacteria and fungi52. As for temperature, in this study, this factor ranged between 24.07–25.47 °C, suitable for fungal growth54. Although the result of RDA revealed the significant influences of temperature, height of sampling site and illumination on fungal communities, here, we did not discuss their effects because they were directly or indirectly related to humidity  related to fungal richness and Shannon indices. This provides with useful information to further understand the fungal species emerge in a tombs of such kind, and reveals the key environmental factors that shape fungal diversity and community composition.The fungal community structure developed at different points in emperor Yang’s coffin chamber was investigated to understand the plausible links between fungal community individuals and biodeterioration, and their potential responses to various environmental factors. 18 dominant 18S rRNA gene sequences were recovered from DGGE gel, the majority of which belonged to The bottom of Emperor Yang’s coffin chamber is 2.95 m below the ground surface, which means the chamber is embedded in the soil Fig. . BecauseMicrobial DNA genomes of 0.5 g dust sample from various positions was extracted using a FastDNA® SPIN Kit for soil  according to the manufacturer’s instructions. The extracted DNA was dissolved in 50 μl TE (Tris-EDTA) buffer, quantified by spectrophotometer and stored at −20 °C until further use. Extraction was carried out within 24 hours after sampling.56 was used to amplify ~390 bp fungal 18 S rRNA gene fragments of different samples in the coffin chamber. A DCode Universal Mutation Detection System  was used for DGGE analysis. Approximate 150–250 ng PCR amplicons from each sample were electrophoresed on a 8% acrylamide- bisacrylamide gel, with 45% to 75% denaturant at 100 V for 10 h in 1×TAE running buffer at 60 °C. The gels were stained for 20 min with SYBR Green I nucleic acid gel stain (1:10000 dilution) . The gels were visualized and digitalized by using a Gel DocTM EQ imager  combined with Quantity one 4.62 . The representative bands were excised, left overnight in 25 μl Milli-Q water, reamplified and run again on the DGGE system to ensure purity and correct mobility of the excised DGGE bands. Correct PCR products were purified using the QIAquick PCR Purification kit (QIAGEN) before cloning.The primer set used was FF390 (5′-CGA TAA CGA ACG AGA CCT-3′) and FR1 (5′-AIC CAT TCA ATC GGT AIT-3′) with a GC-clamp (5′-CCC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GCC G-3′) at 5′ endEscherichia coli DH5α competent cell. Six random clones containing correct gene size for each DGGE band were sequenced by Invitrogen Sequencing Department in Shanghai. DNASTAR software package was used to manually check and compare the clone sequences. One representative clone sequence with high quality after sequence comparison from each band was used for phylogenetic analysis.The purified PCR amplicons of the excised DGGE bands were cloned into a pMD18-T vector  and transformed into 57. The tree topology was further evaluated by different algorithms including Minimum Evolution and Maximum Parsimony. The phylogenetic relationships of fungal 18S rRNA gene sequences to the closest homolog in the GenBank were then inferred.Together with the top three BLAST hits of homologous gene sequences, the gene sequences from cultured and well characterized species in Genbank, the DGGE band sequences were used to build a basic phylogenetic tree by the neighbor-joining method using the software package of MEGA 7.0 version p < 0.05. DNA fingerprints obtained from the fungal 18S rRNA gene banding pattern on the DGGE gels were photographed and digitized using Bio-Rad’s Quantity One software (Version 4.62). Shannon index (H) is calculated based on the mathematical formula: H = −∑(pi)(lnpi), which pi stands for the proportion of species i relative to the total number of species. Richness is calculated according to band number of each sample. Spearman correlation was used to determine the relationships between diversity indices and environmental factors. A detrended correspondence analysis (DCA) was performed for the response variable data to estimate the heterogeneity through the length of the community composition gradients. After confirming the gradient length of axis 1, redundancy analysis (RDA) was performed to arrange fungal communities based on physical properties using Canoco for windows (version 5.0)58.Statistical analysis was carried out with the software package SPSS 13.0 for Windows. Data were expressed as means with standard deviation (SD). Mean separation was conducted based on Tukey’s multiple range test. Statistically significant differences were considered at Supplementary information


Novel human astroviruses (HAstV) were discovered 10 years ago and have been associated with fatal cases of central nervous system infections. Their role in gastroenteritis is controversial, as they have been identified in symptomatic and asymptomatic subjects. The aim of the study was to investigate novel HAstV in a gastroenteritis case-control study including a pediatric population in Spain over a one-year period. We included stool samples from patients with gastroenteritis and negative results for viruses screened by routine diagnostics, and stool samples of control subjects who sought for a routine medical consultation. All samples were screened by real-time RT-PCR assays for novel HAstV. An additional screening for rotavirus, norovirus GI, GII, sapovirus, classic HAstV and adenovirus was also performed for the control group. Overall, 23/363 stool samples from case patients (6.3%) and 8/199 stool samples from control patients (4%) were positive for ≥1 novel HAstV. MLB1 was predominant (64.5% of positives). Seasonality was observed for the case group (p = 0.015), but not the control group (p = 0.95). No difference was observed in the prevalence of novel HAstV between the case and control groups . Nevertheless, MLB genome copy numbers/ml of fecal suspension was significantly higher in the control group than in the case group (p = 0.008). In our study, we identified a lack of association between novel HAstV and gastroenteritis in the studied population, which could indicate a potential role of reservoir for children, especially given the higher viral load observed in the asymptomatic group for some of them. In immunocompromised hosts, they can cause chronic infections6 and disseminated disease with severe complications7. Recently, several cases of central nervous system infections have been associated with HAstV, mainly the novel clades HMO-VA and MLB14, which were identified ten years ago15. The emergence of these novel clades, which are genetically very distant from the classic HAstV16 and are associated with unexpected clinical presentation in humans and also various animal species19, raises the question of their origin and the pathophysiology of central nervous system infections. Interestingly, a recent study reported the retrospective identification of an astrovirus strain belonging to the HMO-VA clade in a brain biopsy of a muskox that had died of suppurative encephalitis in 198220. Another study suggested a common ancestor between the MLB clade and astrovirus identified in rats21. These data suggest a cross-species transmission23, which could have occurred long time ago. Sequences of novel HAstVs have been found in human stools of individuals with diarrhea26, but also in asymptomatic healthy controls28; unfortunately, the two latter reports have contradictory results in their case-control study, especially for MLB1, which so far is the most common. We also recently reported a high rate of co-infection with other enteric viruses during episodes of gastroenteritis where a novel astrovirus was identified29, raising the question of their causal role in gastroenteritis.Human astroviruses (HAstVs) classically cause acute mild to moderate diarrheal illness in young children and the elderlyThe main aim of the study was to compare the prevalence of novel HAstV in a pediatric population with and without symptoms of gastroenteritis in Spain, in order to assess the association of novel HAstVs with acute gastroenteritis in children. In addition, we also aimed at characterizing the prevalence of infection by novel HAstVs as well as other common enteric viruses among asymptomatic children and determining the risk factors for being asymptomatically infected.10 RNA copy numbers/ml of fecal suspension were 2.35 (IQR 2.13–3.76) and 3.23 (IQR 2.87–8.86) for HAstV-MLB and HAstV-VA, respectively Thirty-five out of 363 (9.6%) patients had a positive coproculture. There was no significant difference in the prevalence of novel HAstVs between patients with  and without  a positive coproculture (p = 0.48), but the median log10 RNA copy numbers/ml of fecal suspension was statistically higher in patients with a positive coproculture  than in those with a negative coproculture  (p = 0.02). The prevalence of novel HAstV differed significantly between month (p = 0.015) , including 21 MLB  and 4 VA . Two patients had a double infection (MLB2 + VA1 and MLB1 + VA3). Median log15) Fig. , while t15) Fig. .Figure 110 RNA copy numbers/ml of fecal suspension were 6.52 (IQR 4.52–6.84) and 2.70 (IQR 1.73–9.05) for HAstV-MLB and HAstV-VA, respectively. The prevalence of novel HAstV did not significantly differ between month (p = 0.95) , including 5 MLB  and 3 VA . There were no co-infections observed with several novel HAstV. Median log95) Fig. , nor bet95) Fig. , sex , 22 noroviruses , including 5 GI and 17 GII noroviruses, 17 rotaviruses , 10 sapoviruses , and 7 classic HAstVs  Fig. . Co-infe10 RNA copy numbers/ml of fecal suspension did not significantly differ between case and control subjects (p = 0.07), it was the case for MLB when analyzing both clades separately (p = 0.008) .Globally there was no statistically significant difference in the prevalence of novel HAstV between the case and the control population  Table . By analge Table , the dif15, in order to analyze whether circulating strains were specific to each group. Analysis of a partial capsid-coding sequence (357 bp) revealed that although sequences isolated from controls and cases were not identical, strains infecting controls and cases were genetically closely related . Yet, a statistically significant difference in novel HAstV prevalence according to the month of sample collection could be observed here, with January-April months being the ones with the highest detection rate. This seasonality is in agreement with the study of Jacobsen et al.30, although the latter focused on both novel and classic HAstV.We found that 6.3% of case patients were positive for one or several novel HAstV. This is slightly lower than our previous study, which investigated a similar Spanish pediatric population, with a positivity rate of 10%et al.28 and the Gambian cohort of Meyer et al.27. We could not identify factors predicting the asymptomatic carriage of novel HAstV in children, but we demonstrated that asymptomatic children can harbor a high variety of eukaryotic viruses, with adenovirus and norovirus being the most frequently identified. In a case-control study screening the presence of several enteric pathogens by conventional PCR assays, the global positivity rates for norovirus, rotavirus A and sapovirus in control subjects were 16%, 14%, and 2%, respectively31, which is in accordance with our results. Rotavirus is classically associated with severe gastroenteritis in infants32, but asymptomatic infections can occur33, and may reflect an infection during the period of maternal antibodies protection or a reinfection with a previously mounted immune response allowing to control/prevent the disease33. Additionally, there could be a lower susceptibility to rotavirus infection correlated with the genetic variability of the histo-blood group antigen (HBGA) cell receptor33. Surprisingly, we found no association between rotavirus asymptomatic infection and vaccination status, but this could be due to the small number of rotavirus infections. After logistic regression, only children between 0 and 1 year old were significantly at increased risk for rotavirus infection, which is consistent with the lack of immunity in this population group32. As for norovirus, asymptomatic shedding in children has been reported to be in the range from <1% to >30% in different European and non-European countries38. The higher prevalence of norovirus in patients vaccinated for rotavirus could be explained by a shift in the type of viral infection as has been observed in patients in certain countries where rotavirus vaccination has been implemented43.The novel HAstV positivity rate among control subjects was 4%. This is comparable to findings in the Indian cohort of Holtz et al. did not find any association between MLB1 infection and diarrhea28, Meyer et al. reported an association between MLB1 and diarrhea and between MLB3 and asymptomatic disease, and no significant association of VA viruses with any of the groups27. The results of the present study are also in agreement with the high rate of co-infections observed in our previous study: MLB and VA were identified in co-infection with ≥1 other enteric virus in 78% and 54.8% of cases , respectively, raising the question of their contribution to the digestive tract symptoms29. In this previous study, we could not observe a difference in the novel HAstV mean viral RNA titer during mono- and co-infection. In the present study, the RNA concentration in fecal suspensions significantly differed between both groups, particularly for the MLB clade, and was unexpectedly higher in asymptomatic infections than during episodes of gastroenteritis. This interesting finding points at a potential mechanism of protection towards other pathogenic enteric viral infections: the presence of MLB HAstV at higher viral load in asymptomatic patients could confer a protection due to viral interference40. Our hypothesis is supported by a recent study by Ingle et al., who demonstrated that a specific strain of murine HAstV was able to protect immunodeficient mice from infection by murine norovirus and rotavirus through high level of IFNλ 2/3, without inducing intestinal inflammation41. In addition, novel astroviruses were identified at higher viral load in presence of a pathogenic bacteria, raising the question of whether astrovirus could influence the equilibrium of the gut microbiome42.No difference in the prevalence of novel HAstV between the case and the control groups was observed. While Holtz 43. Therefore, our analysis of seasonal pattern of novel astrovirus has to be confirmed by larger studies extending over several years, and our conclusions are so far limited to the 2017/2018 period. Also, according to the mentioned difficulty to recruit healthy controls, it is still possible that our study was underpowered to demonstrate a difference of prevalence between both groups, but the higher viral load in the asymptomatic group supports the lack of association with diarrhea. Finally, a deep genomic analysis to compare strains infecting asymptomatic and diarrheic children will be performed in future studies to assess whether molecular determinants of symptomatic or asymptomatic infections may exist.Our study has some limitations: we could not perform a full one-year screening of the case patients, which could have influenced our analysis of seasonality. In the control group, sample collection varied from one month to another probably due to the criteria of exclusion , the randomness of the pediatric healthy controls and holiday periods. Yet, few samples could be collected during months of August through October. Our study also focused on a one-year period and we cannot exclude that there is a variability in astrovirus prevalence from one year to another, as previously suggestedIn conclusion, our study provides additional information on novel HAstV epidemiology among asymptomatic children and reinforces the hypothesis that these emerging viruses are not necessarily associated with gastrointestinal syndromes. The unexpected finding that viral load is higher among asymptomatic than symptomatic children and in presence of pathogenic bacteria should raise our attention on the potential role of novel HAstV in the intestinal homeostasis and drive further investigations to understand the mechanisms underlying severe complications.44. A total of 363 stool samples were randomly selected between August 2017 and May 2018. The study was conducted in accordance with the Declaration of Helsinki, and was approved by the Clinical Research Ethic Committee of the Hospital Universitari Vall d’Hebron (PR(AG)32/2016; February 2016).The case study group consisted of children ≤5 years old attending outpatient clinics in the Barcelona Metropolitan Area for symptoms of gastroenteritis. Stool samples were analyzed for routine diagnostics at the Microbiology Laboratory of Hospital de Vall d’Hebron, Barcelona (Spain). No clinical data were available except the patients’ age and the date of stool collection. Stool samples positive for parasites, classic HAstV, rotavirus or adenovirus by routine diagnostics, including immunochromatographic tests for viruses, were excluded. We decided not to exclude bacteria, according to evidence for transkingdom interactions between some enteric viruses, including astroviruses, and intestinal bacteriaThe control study group consisted in children of ≤5 years old visiting the primary health care center El Serral, Sant Vicenç dels Horts, Barcelona (Spain) for their routine control visits. Patients were excluded in the study if they had shown symptoms of gastroenteritis such as diarrhea or vomiting during the preceding 4 weeks. The following clinical and demographic data were collected: age, sex, date of sample collection, any chronic pathology, breastfeeding history, rotavirus vaccination status, antibiotic or probiotic use in the preceding month, fever and/or respiratory symptoms in the preceding 2 weeks, siblings, school or daycare center attendance, and domestic pet exposition. Parents or legal representatives of all patients signed an informed consent before being included in the present study and the study was approved by the Clinical Research Ethic Committee of the Institut Universitari d’Investigació en Atenció Primària Jordi Gol .45, using the Kapa Probe Fast Universal One-Step real-time RT-PCR Master Mix (Kapa Biosystems) and following the manufacturer’s instructions, on a Stratagene Mx3000P (Thermofischer) and a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). The standard curves for quantification were constructed based on 10-fold serial dilutions of plasmids containing a fragment of the corresponding genome strains .All samples were screened for the presence of novel HAstVs. RNA was extracted from 200 µl of a 30% stool suspension in phosphate buffered saline (PBS) using the Viasure RNA-DNA extraction kit (Certest Biotec) following the manufacturer’s instructions. Real-time RT-PCR specific assays for HAstV-MLB and HAstV-VA were performed in duplex as previously described29.The presence of other enteric viruses in stool samples of the control population were also assayed by real-time RT-PCR assays for classical HAstVs, sapovirus, rotavirus, adenovirus, and noroviruses GI and GII, using commercial kits . This additional screening was performed in order to complete the information on the asymptomatic carriage of novel astrovirus in healthy children, providing a comparison with prevalence of mostly encountered enteric viruses. We did not performed such complementary screening among the diarrheic group, as this was already done in a previous study15. Nucleotide sequences were used for the construction of a phylogenetic tree by the neighbor-joining method using MEGA7.046.Sequence information was obtained after amplification by RT-PCR using SF0073-SF0076 and SF0053-SF0061 primers, targeting the conserved RNA polymerase region and the MLB1 capsid coding region, respectivelyThe Mann-Whitney and Kruskal Wallis tests were used to compare continuous variables and Fischer exact test or Chi2 test were used to compare categorical variables. Linear and logistic regression analyses were performed to adjust for confounding variables. P < 0.05 was considered statistically significant. Statistics were performed by Stata/IC 13.1 .


Mercury (Hg) is a naturally occurring element that bonds with organic matter and, when converted to methylmercury, is a potent neurotoxicant. Here we estimate potential future releases of Hg from thawing permafrost for low and high greenhouse gas emissions scenarios using a mechanistic model. By 2200, the high emissions scenario shows annual permafrost Hg emissions to the atmosphere comparable to current global anthropogenic emissions. By 2100, simulated Hg concentrations in the Yukon River increase by 14% for the low emissions scenario, but double for the high emissions scenario. Fish Hg concentrations do not exceed United States Environmental Protection Agency guidelines for the low emissions scenario by 2300, but for the high emissions scenario, fish in the Yukon River exceed EPA guidelines by 2050. Our results indicate minimal impacts to Hg concentrations in water and fish for the low emissions scenario and high impacts for the high emissions scenario. Permafrost locks away the largest reservoir of mercury on the planet, but climate warming threatens to thaw these systems. Here the authors use models to show that unconstrained fossil fuel burning will dramatically increase the amount of mercury released into future ecosystems. Microbial decay eventually consumes the organic matter2, releasing the Hg. Permafrost is soil at or below 0 °C for at least two consecutive years and the active layer is the surface layer of soil above permafrost that thaws in summer and refreezes in winter. Sedimentation in permafrost regions has buried vegetation over thousands of years, freezing organic matter at the bottom of the active layer into permafrost3. Once frozen, microbial decay effectively ceases, locking the accumulated Hg into the permafrost. Based on soil measurements, permafrost regions store an estimated 1656 ± 962 Gg Hg in the top three meters of soil, of which 793 ± 461 Gg Hg are frozen in permafrost1. Observations indicate accelerated permafrost thaw over the past 30–40 years5. Model projections estimate a 30–99% reduction in northern hemisphere permafrost extent by 21007. When permafrost thaws, microbial decay of the stored organic matter will resume and release Hg, but how much, where, and when remain unclear.Naturally occurring and anthropogenic Hg deposits on land from the atmosphere and bonds to receptor sites in plant organic matter9. Because Hg bonds to organic matter, the terrestrial carbon cycle modulates the terrestrial Hg cycle. Hg has three uptake pathways: (1) bonding to soil organic matter11, (2) stomatal leaf uptake13, and (3) root absorption14. Once absorbed by plants, translocation by phloem assimilates Hg into leaves and wood13. The deposition of dead leaves, roots, and stems transfers additional Hg to the soil15.Atmospheric deposition is the dominant source of Hg to the terrestrial biosphere11, (2) leaf stomata transpiration14, (3) fire11, and (4) leaching into groundwater followed by eventual export by rivers into the oceans16. Microbial decay frees Hg from organic matter, but plants and soil organic matter reabsorb11 most of this liberated Hg. Whether leaves represent an Hg source or sink depends on the concentration gradient between the stomata and the atmosphere14. Fire consumes soil organic matter, emitting carbon dioxide and Hg into the atmosphere11. Once leached into water, bound to Dissolved Organic Carbon (DOC) and Particulate Organic Carbon (POC), Hg can methylate, entering the food chain and accumulating in various species, particularly fish8.Hg has four release pathways: (1) evasion into the atmosphere after microbial decay16. We added Hg to the Simple Biosphere/Carnegie-Ames-Stanford Approach (SiBCASA) terrestrial biogeochemistry model17 16. We estimate total Hg concentration in fish by multiplying the simulated MeHg by an empirical relationship of observed fish concentrations as a function of MeHg concentration in water  discharge, but warming temperatures and thawing permafrost will change the magnitude and timing of the seasonal water flow. We assume constant rates of DOC and POC leaching from soil, constant methylation rates, and steady accumulation of Hg in fish. However, large-scale permafrost thaw will change local hydrology, likely changing these rates23.We assume many processes remain constant that will likely change over time and space. For example, we assume constant atmospheric deposition of HgHere, we estimate potential future Hg releases from thawing permafrost for the circumpolar Arctic and impacts to Hg concentrations in water and fish in the YRB. We leverage a full-physics model of the terrestrial carbon cycle to simulate potential future changes in the terrestrial Hg cycle. We predict minimal Hg releases and small increases in Hg concentrations in water and fish for a low emissions scenario and large Hg releases and increases to Hg concentrations in water and fish for the high emissions scenario.0 fluxes to the atmosphere from permafrost regions comparable to the current total global anthropogenic emissions by 2200 under RCP8525  former ambient water quality criterionL−1 Fig. . We see II export can persist throughout winter. For RCP45, we simulate limited thawing in the YRB. HgII concentrations increase by 14% in 2100 compared to 2003, a change well within the 30% uncertainty of simulated values. In contrast, the HgII concentrations for RCP85 increase by 116% in 2100 compared to 2003, more than double 2003 values. For RCP45, the number of days per year that exceed 12 ng Hg L−1 increase from 132 days to 152 days by 2100 , with 208 days exceeding 12 ng Hg L−1.Warming due to climate change will increase Hg concentrations in the Yukon River, particularly for RCP85  from simulated MeHg  the effects of soil organic matter on soil properties, (2) wind compaction of snow, (3) frozen soil biogeochemistry, (4) a dynamic organic layer, and (5) liquid water content in frozen soils36.We estimated Hg releases from permafrost regions using projections of the Simple Biosphere/Carnegie-Ames-Stanford Approach (SiBCASA) model. SiBCASA is a full-physics land surface parameterization with fully coupled carbon, water, and energy cycles2. We divide the carbon and Hg into 13 pools representing different vegetation and soil components, such as leaves or humus17. The flow of carbon from one pool to the next represents the life cycle of organic matter: photosynthesis produces starch, which plants use to grow leaves, roots, and wood. When the plants die, they produce litter and woody debris, which microbes consume to produce humus. Each time carbon and Hg transfers from one pool to another, some is lost as respiration of CO2 and evasion of Hg0 to the atmosphere. Each soil layer contains a complete set of carbon and Hg pools. SiBCASA accounts for the effects of soil temperature and moisture on microbial decay. In frozen soil and permafrost, SiBCASA limits microbial decay to thin water films surrounding soil particles35. Here, we report total Hg losses and gains summed over all pools and all layers within the soil column.SiBCASA assumes vegetation growth, death, and microbial decay control Hg uptake and release from the terrestrial ecosystem0 and 29% HgII based on observations9. SiBCASA has two uptake pathways, bonding to soil organic matter and root absorption. We assume all Hg gets oxidized to HgII during uptake and evenly distribute the HgII between the two pathways. SiBCASA has two release pathways: aqueous export of HgII by rivers and evasion of Hg0 to the atmosphere after microbial decay. We assume HgII export to rivers results primarily from leaching of Dissolved Organic Carbon (DOC) and Particulate Organic Carbon (POC). We assume a DOC leaching fraction of total respiration of 1.5 × 10−3 g C g C−1 based on laboratory measurements and a POC to DOC ratio of 9:1 to get a total aqueous HgII export fraction of 1.5%21 or 1.5 × 10−2 g HgII g Hg−1. After microbial decay, we assumed an atmospheric Hg0 evasion fraction of 16%2 or 0.16 g Hg0 g Hg−1. The remaining 82.5% of released Hg recycles back into the organic matter. We neglect Hg emissions due to fire and other potential Hg sources, such as glacial melt and leaching from bedrock.We assume total atmospheric Hg deposition consists of 71% HgII export for the Yukon River Basin (YRB) by summing HgII export for all pixels in the YRB and compared the result with observed values of HgII export16. We estimated an annual average Hg concentration in the Yukon River by dividing the annual export by the observed average discharge rate at Pilot Station, Alaska. We assumed daily Hg concentration is proportional to the discharge rate16 and used the median seasonal cycle in observed discharge rates measured at the Pilot Station gaging station37. We estimated methyl mercury (MeHg) concentrations using the observed ratio of 1%16  by multiplying simulated MeHg by a regression of observed Hgfish to observed MeHg  simulations. The spread between models increased over time such that climate uncertainty varied from zero in the modern era to a peak of 37% after 2200. We calculated uncertainties for all regressions as the root mean square error (RMSE) of the residuals between the regression model and the original data. The RMSE varied between 5 and 15% for the various regression coefficients and model parameters, but these did not strongly contribute to overall uncertainty. The largest sources of uncertainty dominate overall uncertainty because we combine them in quadrature using Gaussian error propagation. The overall uncertainty varied from 30 to 60% and tended to increase over time, reflecting the increased uncertainty in climate. The largest, single source of uncertainty in these projections is the amount of soil Hg in permafrost regions.We calculated uncertainty in all estimated parameters using Gaussian error propagation. We assumed independent errors and combined them in quadrature. The largest single source of uncertainty comes from the map of soil Hg used to initialize our model1 and spun up the model for 5000 years to steady state initial conditions in 1901 with randomly selected weather years between 1901 and 1910. Steady state initial conditions in 1901 assumes Hg0 and HgII deposition from the atmosphere balances Hg0 evasion and HgII export. The resulting Hg atmospheric deposition rate of 0.75 Gg Hg year−1 or 0.9 ng Hg m−2 h−1 appears consistent with observed values of 1.4 ± 1.0 ng Hg m−2 h−1 on the North Slope of Alaska9. The simulations start at steady state in 1901, but after 1901 the pools and simulated flux drift in response to climate. The magnitude and range of simulated Hg0 flux and HgII export appear consistent with observed values38  4.5 and 8.5 (RCP45 and RCP85 in the main text). We initialized the Hg pools down to three meters depth with a map of permafrost Hg based on observationsSupplementary InformationSupplementary Data


The purpose of this study is to investigate the natural weathering properties of unmodified and acetylated veneer overlaid wood‒plastic composites (vWPCs) manufactured by one-step hot press molding. The results show that the water absorption and thickness swelling of vWPC with acetylated veneer were lower than those of unmodified vWPC. In addition, the surface tensile strength of vWPC increased with increasing weight gain of acetylated veneer, and the flexural properties of vWPC were not significantly different. Furthermore, the results of natural weathering demonstrated that not only the photostability but also the modulus of elasticity (MOE) retention ratio and surface tensile strength of vWPC with acetylated veneer were significantly higher than those of vWPC with unmodified veneer. Thus, better dimensional stability, surface tensile strength, and weathering properties can be achieved when the vWPC is made with acetylated veneer, especially those containing veneers with a higher weight percent gain. In the past few decades, wood‒plastic composites (WPCs) have been used in various fixtures, such as window framing, fencing, roofing, decking, and siding . The glo3L) were higher than those of homogeneous single-layered BPC.It has long been shown that lignin is liable to photodegrade among constituents of wood, and this leads to radical-induced depolymerization of lignin, hemicellulose, and cellulose at the wood surface ,6. FurthFurthermore, Najafi et al.  and AdhiPinus taiwanensis Hayata), a fast-growing wood species, was sampled from the experimental forest of the National Chung Hsing University in Nan-Tou County. Wood particles were prepared by hammer milling and sieving; particles between 16 and 24 mesh were selected and used in this study. Defect-free rotary-cut radiata pine (P. radiata D. Don.) veneer sheets with a thickness of 2 mm were purchased from Wan Tsai Industry Co., Ltd. . Recycled high-density polyethylene  was kindly supplied by Horng Gee Co., Ltd. . All plastic pellets were ground in an attrition mill to reduce their particle size to less than 20 mesh before composite processing. The chemicals and solvents used in this experiment were purchased from Sigma-Aldrich Chemical Co. .Taiwan red pine (M1 − M0)/M0, where M0 and M1 are the oven-dried weights of veneer before and after acetylation, respectively.Veneers were acetylated with acetic anhydride (AA) using the vapor phase reaction method  at a sol3. The formed sandwich panels (300 mm × 200 mm with 12 mm thickness) were hot pressed at 180 °C and 2.5 MPa for 8 min and then cold pressed until the core temperature of the vWPCs decreased to 40 °C.The flat-platen pressing process was applied to the manufacture of adhesive-free vWPCs according to our previous studies ,16. The For the natural weathering test, composite species were exposed facing south, inclined at a 45° angle for a period of 1185 days at the campus of National Chung Hsing University . During exposure periods, the temperature ranged from 7.7 to 36.6 °C, and the average relative humidity and annual precipitation were 77.0% and 6494 mm, respectively. The exposed samples were periodically removed, and their properties were measured regularly.t/MOE0); MOR retention ratio (%) = 100(MORt/MOR0), where the measurements with the subscript indices 0 and t were for the vWPC data before and after weathering for a time t, respectively.To determine the properties of the vWPCs, several determinations, including density, water absorption, thickness swelling, flexural properties, and surface tensile strength, were made according to the Chinese National Standard (CNS) 2215. In brief, specimens with dimensions of 230 mm × 50 mm × 12 mm were used to evaluate flexural properties by the three-point static bending test with a loading speed of 10 mm/min and span of 180 mm. The surface tensile strength of the vWPC was determined on samples with dimensions of 50 mm × 50 mm × 12 mm at a tensile speed of 2 mm/min. All samples were conditioned at 20 °C and 65% relative humidity for 2 weeks prior to testing. The retention ratios of modulus of elasticity (MOE) and modulus of rupture (MOR) of the vWPCs after natural weathering were determined as follows: MOE retention ratio (%) = 100 spectra of all specimens were recorded on a Spectrum 100 FTIR spectrometer  equipped with a deuterated triglycine sulfate (DTGS) detector and a MIRacle ATR accessory . The spectra were collected by coadding 32 scans at a resolution of 4 cm65 light source with a test window diameter of 8 mm. The color parameters L*, a*, and b* of all specimens were obtained directly from the colorimeter. Based on the CIE L*a*b* color system, L* is the value on the white/black axis, a* is the value on the red/green axis, b* is the value on the yellow/blue axis, and the ΔE* value is the color difference (ΔE* = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2).The color of the composite surface was measured by a color and color difference meter  under a Dp values < 0.05 were considered to be significant.All results are expressed as the mean ± standard deviation (SD). The significance of differences was calculated by Scheffe’s test or Student’s t-test, and 3, and there were no significant differences among them. In addition, after 24 h of immersion in water, the water absorption and thickness swelling decreased with increasing WPG of the veneer. Of these, the vWPC with WPG 16 of acetylated veneers exhibited the lowest water absorption (9.9%) and thickness swelling (1.6%). This phenomenon may be affected by the acetylation of hydroxyl groups of the veneer cell wall with AA, which leads to a decrease in the content of hydrophilic hydroxyl groups and results in more hydrophobic surfaces [The various physical and flexural properties of the vWPC with different WPGs of acetylated veneers are shown in surfaces ,18. Anotsurfaces ,20.In addition, The appearance characteristics of all vWPCs changed significantly during 1185 days of natural weathering. As shown in L*a*b* color system. As shown in L*, a*, and b* values of all vWPCs before natural weathering. In addition, the L* value of vWPC with unmodified veneer decreased during natural weathering  veneers during 1185 days of natural weathering was evaluated using the CIE athering a. This rfference c, but th of time b. After a* and b* values of vWPC with unmodified veneer increased with increasing exposure time in the first 8 days. However, vWPCs with acetylated veneers retarded the browning process during natural weathering, and similar results were observed on acetylated veneer [L* value of all vWPCs with acetylated veneers increased with increasing exposure time in the first 8 days. These results suggest that the acetylation of wood can play a major role in controlling the natural weathering process of wood and wood composites.It is well known that among the constituents of wood, lignin is most susceptible to photodegradation . Most ofd veneer . Meanwhid veneer  and Mitsd veneer  reportedThe changes in flexural properties and surface tensile strength of various vWPCs during weathering are shown in Furthermore, as shown in −1) of lignin decreased with increasing exposure time. After 4 days of natural weathering, the absorption bands of unmodified veneers and acetylated veneers with WPG 6 and 11 almost disappeared. However, the acetylated veneer with WPG 16 retained some of the absorption bands of lignin. Similar results have been reported by Evans et al. [−1) group of vWPC with unmodified veneer increased significantly in the first 4 days of weathering. Afterward, the absorption band of the carbonyl group decreased or even disappeared as the exposure time increased. Accordingly, an explanation for this phenomenon is that photodegraded products of lignin located on the surface of veneer are leached during weathering, causing the absorption band of carbonyl groups to decrease. Furthermore, the absorption bands at 1737 , 1371 , and 1237 cm−1  also decreased significantly with increasing weathering time for all acetylated vWPCs. This result indicated that the deacetylation or partial hydrolysis of these groups occurred during weathering. Similar results have also been shown in some esterified wood [In this study, ATR-FTIR spectroscopy was used to monitor the specific reactions of vWPCs during natural weathering. As shown in s et al. , who sugied wood . AccordiE* values of all vWPCs exhibited no significant differences after 1185 days of natural weathering. Of all vWPCs, the vWPCs with a higher degree of acetylation of veneer exhibited higher modulus of elasticity (MOE) and modulus of rupture (MOR) retention ratios after 1185 days of natural weathering, while the vWPCs with unmodified veneer had the lowest retention ratios. In addition, the absorption bands of acetyl groups decreased significantly with increasing weathering time for all acetylated vWPCs. Accordingly, the mechanical property retention of vWPCs can be enhanced through acetylation of veneers, but the effect of acetylation on improving the photostability of vWPCs was not significant. Further research is needed to improve the photostability of vWPCs for outdoor use.Adhesive-free veneer overlaid wood-plastic composites (vWPCs) were successfully prepared by the one-step flat-platen pressing process. The natural weathering properties of vWPCs are greatly influenced by the acetylation of veneers. In this study, vWPCs with acetylated veneers were subjected by photobleaching upon exposure to UV in the first 8 days and retarded the browning process of veneers during natural weathering. However, the Δ


COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism–polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I ( Manihot esculenta Crantz) performs well in poor soils, even in regions with unpredictable rainfall, where the cultivation of cereals and other crops is challenging or impossible. Ecology and climate models suggest that even though cassava should continue to produce well under extreme climatic shifts, it may become increasingly subject to the geographic shift of pests and diseases2. Models predicting pest incidence suggest that, due to climate change, the distribution range of mites will widen to cover almost all areas where cassava has the chance to predominate1.Cassava  cause the most damage, decreasing root yield by 87% and the number of stem cuttings in 82% of plants3. Recently, there have been reports of mites in Asia causing significant yield reductions: 2–10% in the case of slight damage and up to 60% of serious damage5. Moreover, in the last decade, Mononychellus mcgregori has been reported in new cassava cropping areas in Vietnam and Cambodia7, raising concerns with farmers of its potential spread into new fields. The vegetative propagation of crops like cassava tends to cause a build-up of pathogens and pest infestations, which makes disease diagnosis difficult. Symptoms caused by different pests and diseases  are found commonly in the field, making pest surveillance and monitoring cumbersome. In Africa, the 1970s outbreak of cassava green mites confounded the severity of both cassava mosaic disease and the mealybug, making it very difficult to implement timely control and management measures8. Therefore, an integrated approach to monitor and manage these pests must include a reliable and easy species-identification assay.More than 50 species of phytophagous mites are associated with cassava; among those, the spider mites (COI) gene9 is a useful and reliable species-identification marker in insects, due to its lack of introns, limited exposure to recombination, and the availability of robust primer sites. COI has been employed effectively for species-level discrimination in insects12 and arachnid13. In this study, our objectives were to develop an identification kit based on restriction fragment length polymorphism–polymerase chain reaction (RFLP-PCR) using subunit I of COI to rapidly and definitively identify four species of cassava mites, and to democratize its access to field practitioners to ameliorate the current labor-intensive and time-consuming taxonomic identification methodology.Nowadays, molecular biological methods are used widely for species identification in molecular entomology. DNA barcoding using the ∼650-bp region near the 5′-end of the mitochondrial cytochrome c oxidase subunit I ; an experimental field; and two greenhouses at the International Center for Tropical Agriculture, Palmira  Table . At each14. Each specimen was examined, and the most relevant morphological structures for identification were photographed with scales using a Canon Eos 60D camera attached to an Olympus light microscope BX43, and with a Nikon Digital Sight DS-Ri1 camera attached to a Nikon Eclipse Ni-U 90 microscope. The measurements (micrometers μm) were developed in Adobe Illustrator . The body length represented by the idiosoma, was measured with and without gnathosoma, and the setae were measured from the base to the tip; the description of characters follows Lindquist15. Identification to the genus level was performed using the taxonomic key of Bolland et al.16, and the species were identified with different taxonomic keys17, morphological descriptions, illustrations20 and 55 specimens identified by José Maria Guerrero at CIAT, for each genus identified. All specimens examined were deposited in the CIAT Arthropod Reference Collection (CIAT-ARC).Seventy-seven adults from the four colonies indicated in Table 21, with slight modifications . The DNA barcode region was amplified using 10 μM of universal DNA primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) for the amplification of COI22. PCR was performed in a 20-µl reaction volume using 0.25 U of Platinum® Taq , 1X PCR buffer , 2.5 mM of each dNTP , 2.5 mM MgCl2, and 20 ng DNA. The amplification program, conducted in a Mastercycler® Pro , was 5 cycles , followed by 35 cycles  with a final extension period of 72 °C for 10 min.DNA was extracted from four mite species using the cetyltrimethylammonium bromide methodCOI region of each green mite species was cloned into the PGEM®-T Easy vector . Plasmid DNA from Escherichia coli was purified using an SV minipreps preparation . DNA inserts in this vector were sequenced in both directions using the Big Dye™ Terminator Cycle Sequencing kit with an Applied Biosystems 377 DNA fragment analyzer by the Cornell University Life Sciences Core Laboratories Center  using vector primers SP6 and T7. The resulting DNA sequences were edited and analyzed using Sequencher® 4.5 .The COI sequence contigs were assembled from three clones per species to identify unique haplotypes with a discrimination requirement of 100% identity. All mite sequence identities and similarities were calculated using BLAST. Genetic distances were calculated with the alignment sequences for the four species. Additionally, we included COI sequences reported in GenBank from other species belonging to the same tribe (Tetranychini)  with 1000 bootstrap replications.DNA sequences were edited by eliminating vector and universal primer sequences with Sequencher V5.2.4. COI sequences belonging to each of the four mite species morphologically identified in this study, and the 15 Tetranychini and 1 Bryobia COI sequences obtained from GenBank for the species identified as closely related to them . Of the 36 commercially available restriction enzymes, three allowed a clear differentiation of the four mite species in silico. Two units of the restriction enzymes AseI, MboII, and ApoI were used to digest 2 µg of the PCR-amplified COI region from T. urticae, M. caribbeanae, M. mcgregori, and M. tanajoa. The final reaction volume was 50 µl and included 10% of 1X supplied buffer and ultrapure water. Reactions were incubated for 1 h at 37 °C or 50 °C according to the manufacturer’s specifications. Restriction digestion products were run in 2% agarose gel in BS buffer  at 80 V for 3 h. Gel staining was carried out with ready-made SyBR Safe . Restriction pattern images were captured with a GelDoc™ BioRad documentation system, and size estimation of the digestion patterns was performed using PyElph V.1.4 software27.em Table , were diMononychellus caribbeanae28, M. mcgregori29, M.tanajoa30, and Tetranychus urticae31. A description of the diagnostic morphological characteristics used to identify these species is provided in detail.In the Valle del Cauca region of Colombia, cassava is affected by different mite species. A survey conducted in 2012, in which cassava leaves infested by mites were collected and inspected under a stereoscope, identified at least four morphotypes on a single leaf. Four mite colonies, one from each morphotype, were then established on fresh cassava leaves. From the resulting offspring, mating couples were selected and allowed to breed on new cassava leaves to enable a full taxonomic and molecular identification . Empodium split near the middle into three pairs of hairs. Opisthosoma with longitudinal striae between the e1 setae. Dorsal body setae serrate16.18. Dorsal striae of hysterosoma anastomosed except in the posterior part  where there is a reticulate pattern  and without gnathosoma of 422 (507–533), width of 344 (325–364) Fig. A18. DorsManihot esculenta (Euphorbiaceae).Twenty-seven females and eight males, Cesar (n = 3), La Guajira (1) and Valle del Cauca (22), Colombia; two females, Cocle, Panama; two females, Managas and Sucre, Venezuela; one female, Palmar del Rio, Cuba. All specimens were collected on 18. Dorsal striae of hysterosoma transverse  and without gnathosoma of 364 351–377), width of 281 (260–299) .Eleven females and four males, Cauca (1), Huila (1), Quindio (1) and Valle del Cauca (8), Colombia; two females, Junin, Peru; two females, Quan Ngai, Vietnam. All specimens were collected on 18. Dorsal striae of hysterosoma form a slightly reticulate pattern and transverse  and without gnathosoma of 398 (360–481), width of 281 (273–299) .Forty-one females and seventeen males, Atlántico (1), Guajira (1), Magdalena (2), Meta (1) and Valle del Cauca (36), Colombia; seven females, Anzoategui (1), Apure (1), Carabobo (1) and Monagas (3), Venezuela; two females, Idiho and Fonkome, Benin. All specimens were collected on 16.One pair of para-anal setae. Empodium split distally, usually into three pairs of hairs, and spur absent. Duplex setae of tarsus I well separated. Peritreme anastomosed distally38, with elliptical body shape , on 16 and Flechtmann & de Queiroz17.Note: Based and modified of Bolland et al.Tetranychus urticae.1. Female and male: One pair of para-anal setae…Female and male: Two pairs of para-anal setae (h2-3)…2.Mononychellus caribbeanae.2. Female: Dorsal striae of hysterosoma anastomosed except in the posterior part  where there is a reticulate pattern  from the genus Mononychellus . The T. urticae example from this study clusters together with the GenBank example of the same species collected in Korea. Likewise, the three species of Mononychellus collected in the Valle del Cauca clustered together; they are the first to be sequenced and reported to GenBank.This phylogenetic tree clearly separates the genus COI sequences belonging to 20 mite species were digested in silico with 36 restriction enzymes with the aim of identifying a set of enzymes that could be used for easy identification. We found that the digestion patterns obtained with AseI, MboII, and ApoI allowed for the full identification of the four mite species described here. Restriction digestion of the PCR-amplified COI regions of the four mite species confirmed the in silico results. The banding patterns obtained from the agarose gel electrophoresis showed that AseI and MboII resolved eight and five distinctive bands, respectively, across all mite species with sizes above 100 bp, demonstrating the ease and accuracy of this method. ApoI showed only three distinctive bands, and did not distinguish between M. tanajoa and M. caribbeanae, limiting its potential use for quick diagnosis , confirming the reproducibility and accuracy of RFLP-PCR. AseI showed the best profile for differentiating among the four mite species, followed by MboII. Even though ApoI only allowed the definitive identification of two mite species, its inclusion alongside the other two enzymes would make an identification kit for the four species more robust.The banding patterns obtained after restriction enzyme digestion of the In the present study, we undertook a classical morphological identification of four mite species associated with the cassava crop in Valle del Cauca and then used those results to confirm the feasibility of a proposed molecular kit for easier identification.43 and Guerrero et al.37 reported problems in the identification of Mononychellus specimens, whereby apparent differences in morphological characteristics could have been the result of bad mounting or deterioration of the microscope slide. The other limiting factor affecting the morphological characterization of Mononychellus spp. is the limited number of morphological characteristics available for accurate characterization. For instance, in M. tanajoa, the traditional characteristic is the longitude of dorsal setae that presents a continuous variation among the populations, a factor that does not facilitate distinguishing them from nearby species43. Another important characteristic is the shape of the aedeagus; although its descriptions have presented problems, comprehensive research indicates the same form as documented in this paper43, demonstrating its usefulness.The morphological identification of the four mite species assessed here was challenging because it required extensive technical expertise to prepare and mount the samples as well as a deep knowledge of the taxonomy of mites. Rogo et al.16. The organism’s small size and significant phenotypic plasticity also increase the complexity associated with its morphological identification44.Not only is the identification process for mites using morphological traits time consuming and demanding of specialized taxonomic expertise, but the task is also amplified by the fact that the Tetranychidae family is composed of > 1200 species45. The COI gene sequence has been used extensively for insect identification; for instance, it has been used successfully in the identification of whitefly46, rhopalids47, and mirids48. On this basis, we sought a method to reduce the time and increase the accuracy of mite species identification that would be usable by other scientists or agriculture research practitioners responsible for pest surveillance and monitoring, choosing to characterize the genetic information contained in the COI region of the COX-1 gene of four mite species taxonomically identified and associated with the cassava crop in the Valle del Cauca, Colombia.DNA barcoding could circumvent these limitationsCOI region has been used successfully to characterize the mite species complex51; thus, we believed this type of data would aid the species identification of mites associated with cassava. Among the most important species, only a partial COI sequence for M. progressivus is available at NCBI52; thus, the four haplotype COI sequences reported here constitute the first to be available through GenBank for the genera Mononychellus and Tetranychus. Comparing molecular markers with diagnostic morphological traits allows the building of a species-specific sequence library to aid the accurate identification in the absence of a well-trained entomologist. COI sequences from mites associated with other plants, including T. urticae55, were used to conduct a phylogenetic relationship analysis, permitting the construction of a phylogenetic tree from 20 COI sequences, including the outgroup Bryobia sp.44. Two well-defined clades were apparent: one from a monophyletic group that corresponded to the Tetranychus genus (and in which our T. urticae sample was grouped in close relationship with a sample reported by the Korea Research Institute of Bioscience and Biotechnology); and the second from a polyphyletic group composed of four different genera but including a well-defined Mononychellus clade.The COI sequence. We have shown that a standard PCR amplification of the COI region, followed by enzymatic digestion with AseI or MboII allows the unequivocal identification of all four mite species associated with the cassava crop, with no sequencing of the COI region necessary. Our method does not compare sequences to classify one sample into a species; therefore, our method does not take into account the number of intra- or interspecific variations, which have been problematic, given that in some species of mite, intraspecific variation may exceed interspecific variation44. Our kit is based on the target sequences of the restriction enzymes and the patterns they generate when digesting the sequence of the COI region. This approach to a rapid and accurate mite species identification is cost effective and easy to implement in a very basic molecular biology laboratory. Thus, incorporating RFLP-PCR for the COI gene into a routine mite surveillance and monitoring program should quickly and simply identify any potential outbreak of an exotic type not reported previously. The restriction digestion patterns from AseI and MboII clearly showed how the COI-RFLP is as effective a method for mite identification as that proposed for whiteflies and the oriental fruit fly56.Most mite identification studies agree on the challenge of identifying the species by morphology alone, because that is time consuming and defeats any attempt to rapidly provide information on pest distribution. Large-scale surveys involving DNA sequencing may be possible, but are probably too costly. Hence, DNA-based methods not involving sequencing become attractive. Restriction enzyme-mediated genotyping is a more cost-effective approach for identification with large sample sizes. It requires that a restriction digestion of a PCR product (RFLP-PCR) be obtained from a variable well-known gene, such as the COI-based RFLP-PCR kit, developed using a multi-disciplinary approach that included gene data, morphological traits, and bioinformatics pipelines, has been shown to unequivocally identify T. urticae, M. caribbeanae, M. mcgregori, and M. tanajoa. This kit can identify mite species accurately, cheaply, and rapidly, and could become a useful tool in crop protection programs monitoring and surveying mite pests.In conclusion, our


When severe proximal interphalangeal (PIP) joint flexion contracture is induced in the little finger by Dupuytren’s disease, it interferes with activities of daily living. To extend the little finger, open fasciectomy is selected as a general treatment method. However, postoperative complications have been frequently reported. To solve these problems, finger shortening was undertaken. In this study, we treated two cases of Dupuytren’s disease manifesting severe PIP joint flexion contracture of the little finger with finger shortening by proximodistal interphalangeal (PDIP) fusion in which the middle phalanx is resected and the residual distal and proximal phalanges are fused. For flexion contracture of the MP joint, a percutaneous aponeurotomy using an 18G needle was performed to obtain the extended position of the MP joint. Favorable outcomes with high patient satisfaction, including esthetic aspects of retaining the finger with the nail without complication, were achieved. We report this challenging treatment and its discussion. When severe proximal interphalangeal (PIP) joint flexion contracture is induced in the little finger by Dupuytren’s disease, it interferes with activities of daily living, such as face washing, causes skin disorders, such as tinea manus, and has harmful esthetic effects . To exteTo solve these problems, Watson and Lovallo designed finger shortening by bone resection of the proximal phalanx , and HonThe study was carried out in accordance with the Declaration of Helsinki and the appropriate ethical framework. Written informed consent was obtained from the patients for publication of this case report and any accompanying images.The subjects were two patients who underwent finger shortening for severe PIP joint flexion contracture of the little finger caused by Dupuytren’s disease between January 2012 and December 2017 at our hospital. The first case was a 59-year-old male with no notable past medical history. The disease was Dupuytren’s disease manifesting severe PIP joint flexion contracture (flexion 100°) and metacarpophalangeal (MP) joint flexion contracture (flexion 45°) in the left little finger. There was no deformity or osteoarthritis on the distal interphalangeal (DIP) joint. The range of motion of the DIP joint was flexion 45° and extension 0°. The preoperative Tubiana classification was stage 4, grip strength was 32 kg, visual analogue scale (VAS) was 0/10, and quick disabilities of the arm, shoulder, and hand (Q-DASH) score was 25.00/100. The patient requested esthetic acquisition of the finger extension position. The second patient was an 81-year-old male with no notable past medical history. The disease was Dupuytren’s disease manifesting severe PIP joint flexion contracture (flexion 90°) and MP joint flexion contracture (flexion 30°) in the left little finger. There was no deformity or osteoarthritis on the distal interphalangeal (DIP) joint. The range of motion of the DIP joint was flexion 25° and extension 0°. The preoperative Tubiana classification was stage 3, grip strength was 16 kg, VAS was 3/10, and Q-DASH score was 36.36/100. The patient requested surgery because the little finger was an obstacle in washing his face and interfered with activities of daily living.®, Nihon Medical Next, Osaka, Japan)   and 1E. The patients were clinically evaluated two years after surgery. In the first case, the extension position of the little finger was acquired, VAS was 0/10, the Q-DASH score was 0/100, and grip strength was 21 kg (65% of that before surgery)  and 2B. In surgically treated recurrent cases, the skin condition was worse than that after the first surgery, and the incidence of complications, such as skin necrosis and infection, increased , 12. In Of course, there are some disadvantages to finger shortening. First, from a cosmetic perspective, there are concerns about the acceptance of female patients. Raimbeau et al. performed this procedure on seven female patients and all of whom were cosmetically satisfied . FurtherAlthough we performed surgery following the procedure reported by Honecker et al. (PDIP fusion) , the proBased on this report of two cases, finger shortening by PDIP fusion for Dupuytren’s disease manifesting severe PIP joint flexion contracture of the little finger may be a useful surgical method. The indication of this surgical procedure should be carefully considered in each case. However, finger shortening is capable of overcoming the problems of other surgical procedures in cases in which amputation is considered or multiple surgeries were performed.The authors declare that they have no conflict of interest.


Candida fungal cells remains one of the most life-threatening complications among hospitalized patients around the world. Although most of the cases are still due to Candida albicans, the rising incidence of infections caused by other Candida strains that may not respond to traditional anti-fungal treatments merits the development of a method for species-specific isolation of Candida. To this end, here we present the characterization of the dielectrophoresis (DEP) response of Candida albicans, Candida tropicalis and Candida parapsilosis. We complement such characterization with a study of the Candida cells morphology. The Candida strains exhibited subtle differences in their morphology and dimensions. All the Candida strains exhibited positive DEP in the range 10–500 kHz, although the strength of the DEP response was different for each Candida strain at different frequencies. Only Candida tropicalis showed positive DEP at 750 kHz. The current results show potential for manipulation and enrichment of a specific Candida strain at specific DEP conditions towards aiding in the rapid identification of Candida strains to enable the effective and timely treatment of Candida infections.Bloodstream infection with  Candida species are one of the most prevalent fungal pathogens in hospitals around the world. In the United States alone, 5%–10% of hospitalized patients will acquire a nosocomial infection and 80% of such infections are caused by Candida species [Candida species became recognized as the fourth most common cause of nosocomial bloodstream infections in the United States, and most recently reported as the 3rd most common cause of nosocomial bloodstream infections in the intensive care unit (ICU) [Candida have a crude mortality rate of 39% overall, and this figure can be as high as 47% for patients infected in the ICU [Candida species have been identified as responsible for invasive candidiasis (IC), an umbrella term referring to various severe diseases resulting from Candida infection [Candida albicans remains the most frequently isolated Candida strains from infected blood [Candida albicans, whereas incidence rates were 14% for each Candida glabrata and Candida parapsilosis, 7% for Candida tropicalis and 2% for Candida krusei [albicans species was Candida parapsilosis, followed by Candida tropicalis and Candida glabrata [albicans species as pathogens is concerning because many of them do not respond to conventional anti-fungal therapy, which are generally targeted for Candida albicans. For example, Candida tropicalis is less susceptible to fluconazole, a common anti-fungal medication, when compared to Candida albicans [Candida species, there is a need for a method that allows for rapid identification of the Candida species, so that timely measures can be taken towards species-specific treatment of Candida infections.it (ICU) . Concern the ICU . More thglabrata . The emeCandida species in a label-free fashion towards a rapid and affordable assay. DEP is a relatively simple procedure that works by exploiting the specific response of different cells to an electric field gradient [Candida albicans [Candida strains. However, till date, no DEP characterization of Candida strains other than Candida albicans is available. Hence, there is a knowledge gap preventing the wider use of DEP as a method to sort Candida strains. Methodical characterization of the DEP response of Candida strains can enable the use of different DEP platforms towards a more rapid way to identify the type of Candida causing an infection and an informed approach to combat it. For example, specific Candida strains can be isolated and enriched from a dilute sample in a timely manner in a DEP-based sample preparation protocol previous published by the authors [Dielectrophoresis (DEP) is a technique that offers the potential for sorting different gradient ,10,11,12gradient ,18,19,20albicans ,25,26,27albicans ,29,30 en authors .Candida strains: Candida albicans, Candida parapsilosis, and Candida tropicalis; which are three of the most frequently isolated Candida strains from infected samples. We used 3D carbon microelectrode arrays to obtain the results presented here due to their improved performance over more traditional planar electrodes [In this work, we present the morphological characteristics and a first study on the DEP response of three different ectrodes ,32,33.Candida albicans (ATCC 18804), Candida parapsilosis (ATCC22019), and Candida tropicalis (ATCC750) were cultured in dynamic conditions at 37 °C and 215 rpm in yeast malt broth (YMB) and passed regularly to maintain a healthy culture. To prepare the sample for DEP experiments, 100 µL of 4-day old cell culture were mixed with 2.5 mL of an optimized DEP buffer solution composed of 8.6 wt% sucrose, 0.3 wt% dextrose and 0.1 wt% bovine serum albumin to achieve a concentration of around 106 cells/mL. The electrical conductivity of this DEP buffer solution was 20 µS/cm. Cells were then pelleted through centrifugation at 5000 rpm for 5 min and then resuspended into fresh DEP buffer solution. This centrifugation and re-suspension protocol were repeated three times to ensure complete removal of any remaining YMB culture media.2 substrate. The SU-8 microstructures were carbonized at 900 °C in a nitrogen environment using a heating rate of 5 °C/min to obtain carbon microstructures. The resultant carbon electrode array  featured intercalated 3D electrodes as shown in The microfluidic DEP device used in this study featured 3D carbon microelectrode arrays. The fabrication of the carbon microelectrodes has been reported several times in our previous work ,35,36,37Candida strains [Experiments revolved around characterizing: (1) cell morphology and (2) DEP response. Few studies are available regarding the morphological characterization of few  strains ,40 and app. In the second stage, washing, a cell-free DEP buffer solution was flowed with a flow rate of 10 µL/min through the still polarized electrode array for 5 min to wash any non-trapped cells. In the last stage, release, the polarizing signal was turned off while maintaining the same flow rate. This last stage lasted for 110 s. The entire experiment was monitored through a 10× objective lens in a Nikon Eclipse LV100 microscope. However, only the release stage and the last 10 s of the wash stage were recorded using an Andor Zyla CMOS camera running at a frame rate of 5 frames per second. Hence, each of the video recordings used for data analysis was 120 s, or 600 frames, long. The electric field was turned off at frame 50.The experimental set up for characterization of the DEP response of the cells at different frequencies is illustrated in x) and minor axes (y) of at least 30 cells per strain. The major and minor axes were identical for a perfect circle. The average values and standard deviation for all measurement were calculated using built-in mathematical functions in Microsoft Excel.Images of cell cultures were manually analyzed in the NIS Element Basic software native to the microscope to measure the major  to plot the average pixel intensity at a region of interest (ROI), with area 830 µm × 700 µm and established immediately after the electrode array, throughout each experiment done for a particular cell strain and frequency of interest c–e. The x) of Candida albicans is identical to the minor axis diameter (y). Candida albicans featured an average diameter of x = y = 5.12 ± 0.75 µm, as depicted in Candida albicans a displayx = y = 5.98 ± 0.75 µm, which is consistent with previous findings by other authors [Candida tropicalis exhibited multiple morphologies. The first was a spherical morphology similar to that of Candida albicans but with larger diameter of  authors . As in t authors ,41,42 anx = 6.29 ± 0.83 µm and y = 3.8 ± 0.84 µm, and these values are in the range of the dimensions reported elsewhere for this strain [Candida albicans, Candida parapsilosis was also found in its budding stage but with a featuring morphology that resembled two or more attached ellipsoids instead of spheres.Candida parapsilosis shown in s strain . AlthougCandida strains at will. Upon applying the electric field at specific frequencies, the Candida cells experienced positiveDEP force and got trapped in the regions of high field gradient around the carbon microelectrodes  [Candida cells expected to be in the buffy coat [Candida cells from other blood cells until only particles that may resemble Candida are present in the sample. At this stage, a DEP assay for fine separation could be used to perform isolation and purification of specific strains to increase the performance of detection assays such as PCR as detailed above. If necessary, the DEP properties of blood cells with similar sizes than Candida have been characterized [It should be noted that the value of DEP in a practical solution for rapid diagnosis of ls (RBC) , with thffy coat . Buffer cterized ,50,51 ancterized ,35.Candida strains from infected samples: Candida albicans, Candida tropicalis, and Candida parapsilosis was presented here. The studied Candida strains only exhibited subtle differences in morphology that makes direct observation an incomplete method to sort them. 3D carbon electrode DEP was implemented for characterizing the DEP response of the Candida cells. All three Candida strains showed a strong positive DEP response in the frequency range 10–500 kHz. However, positive DEP at 750 kHz was only observed for Candida tropicalis. Furthermore, the Candida strains exhibited the positive DEP at different strength. Candida tropicalis and Candida parapsilosis showed relative high DEP response than Candida albicans in the frequency range 50–250 kHz, whereas at 10 kHz, the DEP response was strongest for Candida albicans. Together, the morphological and DEP differences among the different strains could provide a framework to enable sorting different strains.The characterization of the morphology and DEP response of three frequently isolated Candida strains. The current results show promise towards using DEP as a tool to enable separation of different Candida species. Ongoing work focuses on characterizing the membrane capacitance of the strains presented here and expanding this study to other relevant strains such as Candida glabrata and Candida krusei. The results presented here indicate that one can potentially manipulate and enrich a specific Candida strain at specific DEP conditions and encourages further work towards rapid identification to the enable effective and timely treatment of candidiasis.This is to the best of our knowledge the first study reporting the DEP responses of different