Punica granatum L. showed higher antioxidant capacity, but bacon smoked with Armeniaca vulgaris Lam had better overall eating quality. This study reveals that selection of woods to prepare antioxidant fumigant is a feasible approach to retard oxidative spoilage of meat products. Future study is need for the development of composite smoke flavorings to improve both oxidative stability and sensory quality of foods.Effects of liquid smoke prepared from different woods on physicochemical parameters, sensory quality, and protein and lipid oxidation were determined in bacons during process and storage. The relationship between the antioxidant activity of smoked liquid and the quality of bacon was further explored through chemometric analysis. Results showed that liquid smoke prepared from different woods differed in phenolic and carboxyl compounds and antioxidant capacity. Bacon processed with different liquid smoke had different antioxidant capacity, lipid and protein oxidation during storage, and sensory quality. The concentration of phenols was positively highly correlated with the antioxidant capacity of both liquid smoke and fresh bacon, but negatively correlated with lipid and protein oxidation in bacon. Among the five woods, liquid smoke made from  Bacon is a processed meat product made from a pig belly or back that is subjected to curing, drying, and smoking. Due to its unique smoked flavor, bacon is widely consumed in western countries and even in the world , while tBacon is rich in lipids and proteins, making it prone to oxidation. Oxidation of lipids, especially poly-unsaturated fatty acids, entails the generation of rancid or off flavor, decreases the nutritional value and reduces the storage period of foods . In addiIn addition to flavor and oxidative stability, the safety of bacon products is also a very important issue . CompareTherefore, in the present study, liquid smoke was prepared from different woods  and implemented on bacon. The antioxidant capacity of liquid smoke, oxidative stability and sensory quality of bacon, as well as volatile compounds in both liquid smoke and processed bacon, were comparatively evaluated with the aims for high quality bacon production and further understanding of the factors influencing bacon shelf life and wholesomeness.Cupressus funebris Endl (CE), Armeniaca vulgaris Lam (AL), Diospyros kaki Thunb (DT), Punica granatum L. (PL), and Ziziphus jujuba Mill (ZM). For each type of wood, liquid smoke was made in six replicates. A commercial liquid smoke (CS) was purchased from Red Arrow International LLC  and used as a reference in the study.Liquid smoke was prepared by dry distillation as reported in literature  with sliThe cuts of bacon (pig tenderloin) were derived from a carcass of crossbreed pig (Duroc × Landrace × Yorkshire). The pork samples were cut into pieces (4 cm × 4 cm × 1.5 cm) before being cured/smoked. For bacon antioxidant capacity and oxidative stability analysis, pork cuts were submerged in curing/smoking solution  and cured for 12 h at 4 °C. Afterwards, samples were cooked in a baking oven at 70 °C for 4 h, individually wrapped in plastic water proof bags and stored at 4 °C for 9 days. On day 0, 3, 6, and 9 the bacon samples were collected and stored at −80 °C until analysis. For sensory evaluation and volatile compound analysis, pork cuts were cured in curing/smoking solution  for 12 h at 4 °C and cooked in a baking oven at 70 °C for 4 h before analysis.2CO3 were added and incubated at 25 °C in the dark for 1 h. The absorbance at 765 nm was determined with a Varioskan Flash microplate reader . Distilled water was used for blank. Gallic acid was used to prepare the standard curve and total phenol in samples was calculated as gallic acid equivalents.The total phenol content in liquid smoke was measured by Folin–Ciocalteu method as in literature  with somThe total carbonyl compounds in liquid smoke were determined according to GB1886.127-2016 National Food Safety Standards–Food Additive Smoke Flavor I and II from Haw-pit. Carbonyl compounds reacted with 2,4-Dinitrophenylhydrazine  in acidic condition to generate red hydrazone derivatives, which were determined by measuring the absorbance at 430 nm. Total carbonyl compounds in liquid smoke were calculated as heptaldehyde equivalents.v:v) at a flow rate of 1.0 mL/min. The excitation and emission wavelength of fluorescence detector was 384 and 406 nm, respectively, the column temperature was 35 °C, and the injection volume for each analysis was 20 μL.Benzo (a) pyrene (BaP) in liquid smoke were determined according to GB5009.27–2016 National Food Safety Standards–Determination of Benzo (a) pyrene in Foods. BaP in meat samples was extracted by n-hexane, purified by BaP imprinted column, and determined by a Waters 1525 HPLC coupled with fluorescence detector and Symmetry C18 Column . Isocratic elution mode was selected, with a mobile phase consisting of 88% acetonitrile-water  scavenging capacity, total antioxidant capacity (T-AOC), and oxygen radical absorbance capacity (ORAC). Liquid smoke was diluted before the assay. Then, 0.1 g bacon samples were homogenized in 1 mL of precooled PBS  and centrifuged at 4 °C and 10,000× g for 5 min. The supernatant was used for antioxidant activity assay.The antioxidant activities of liquid smoke and bacon were evaluated by DPPH  free radical scavenging capacity, ABTSDPPH assay was performed as previously described . DPPH wa734 = 0.700 with PBS to make ABTS•+ working solution. Then, 10 µL of diluted liquid smoke or bacon extract were added with 200 µL of ABTS•+ working solution, mixed well and reacted at 30 °C for 10 min. The absorbance at 734 nm was measured. Ultrapure water was used for a blank measurement. The ABTS free radical scavenging rate was calculated to evaluate ABTS free radical scavenging ability of samples [ABTS assay was conducted as described by Roberta et al. . Briefly samples .The T-AOC of liquid smoke and bacon was measured by using a T-AOC Assay Kit . The ORAC values of samples were determined according to literature . BrieflyCarbonyl content, sulfhydryl and disulfide bond content, protein surface hydrophobicity (PSH), and 2-thiobarbituric acid reactive substance (TBARS) values of meat were measured using our previous protocols . Meat flThe sensory evaluation was conducted using a 10-person trained taste panel. The panelists were selected and trained according to literature . Before p < 0.05. PCA and correlation analysis were performed using OriginPro 2021 .The results reported are the means of six replicates (expressed as the mean ± standard error). The data from various treatments were analyzed by one-way analysis of variance (ANOVA) by using SPSS software package version 25.0 . Levels for significant differences were set at p < 0.05) than that of the commercial liquid smoke (CS). The higher concentration of total phenols indicates that the liquid smoke prepared from PL may be superior in fumigation flavor and antioxidant activity.From the point of view of chemical composition, both smoke and smoke flavorings are very complex mixtures, which depend on the type of wood, the pyrolysis conditions and the treatments of the smoke. According to literature, the smoke produced at 650–700 °C is richest in components able to impart desirable organoleptic properties to treated products . Thus, ap < 0.05) carbonyl compounds than other liquid smokes. As most carbonyls are formed from the thermal decomposition of cellulose and hemicelluloses through carbohydrate degradation, The difference in carbonyl content should be related to the variation in chemical composition of different woods [Carbonyl compounds represent the largest number of compounds in wood smoke . This clnt woods . Based oPolycyclic aromatic hydrocarbons (PAHs) are a group of compounds which are harmful to human health. PAHs are formed in the decomposition of wood, especially at limited access of oxygen in the range of 500–900 °C . Benzoa. BaP is Different assays have been introduced to measure antioxidant capacity of foods and biological samples, which describes the ability of redox molecules in foods and biological systems to scavenge free radicals. Antioxidant capacity assay measures the additive and synergistic effects of all antioxidants rather than the effect of a single compound; therefore, it may be useful to evaluate the potential benefits of antioxidants on food nutrition, quality, and safety ,37. In ap < 0.05) DPPH free radical scavenging capacity before reaching the upper limit of the measurement, which is in accordance with the higher concentration of total phenols in them (p < 0.05) DPPH free radical scavenging capacity  ABTS+ free radical scavenging capacity, followed by bacon made with AL liquid smoke  and the commercial liquid smoke had lower (p < 0.05) antioxidant capacity when compared with others (p < 0.05) ORAC value than CE, AL, DT, and the commercial liquid smoke (p < 0.05) total antioxidant capacity  TBARS values than those of the smoked bacon during the whole storage period, showing that liquid smoke was antioxidative and retarded lipid oxidation in bacon during storage. When the six smoked bacon samples were compared, bacon prepared with the commercial liquid smoke showed higher (p < 0.05) TBARS values than the others. In addition, The lowest TBARS values were determined in the samples of the AL and PL group during the first three days of storage. This is generally in agreement with the content of phenolic compounds in liquid smoke  in the unsmoked cured meat throughout the whole storage period when compared to that of the smoked bacon, especially smoking with AL and PL woods reduced carbonyl content ~35.6% compared to the CM treatment. In agreement with carbonyl content in meat, the sulfhydryl content was significantly lower (p < 0.05) and disulfide bond was significantly higher (p < 0.05) in the unsmoked cured meat (p < 0.05) total sulfhydryl content on day 3, 6, and 9, and lower (p < 0.05) disulfide bond on day 6 compared to all the other groups, further demonstrating that the higher content of phenolic compounds  surface hydrophobicity than all the smoked bacon, indicating a higher degree of protein oxidation in CM and liquid smoke inhibited protein oxidation in bacon. Again, the lower protein surface hydrophobicity in AL and PL groups compared to the DT and CS groups confirmed liquid smoke prepared from AL and PL woods had higher antioxidant capacity  on bacon appearance (redness and yellowness) and flavor . The scores of saltiness and texture attributes  were not different between treatments, showing that liquid smoke prepared from different woods had no significant effect on these sensory quality attributes. The significantly higher overall liking score of AL bacon suggests that bacon smoked with AL wood received sensory panelists’ preference over bacon smoked with CE, DT, and ZM woods. To better explore the impact of smoking woods on bacon eating quality, principle component analysis (PCA) was performed. As shown in The sensory panel evaluation of prepared bacon is shown in r ≤ 0.91, p < 0.001), showing that smoke should be a major resource of antioxidant compounds in processed meat products. TBARS values measure lipid oxidation in meat. The TBARS values of bacon determined on day 9 were negatively correlated with the DPPH and ABTS+ free radical scavenging capacity, T-AOC and ORAC values of fresh bacon . This is logic because antioxidants in bacon protect lipid from oxidation during storage. The antioxidant capacity of liquid smoke measured by DPPH, ABST, T-AOC, and ORAC assays was negatively correlated with the TBARS values of bacon on day 9 , especially the DPPH free radical scavenging capacity of liquid smoke was negatively highly correlated with TBARS values of bacon on day 9 , demonstrating that antioxidants from smoke play a role in inhibiting oxidation in bacon. This is confirmed by the negative correlation between the antioxidant capacity  of liquid smoke and carbonyl content in bacon on day 9 , the positive high correlation between the antioxidant capacity of liquid smoke and sulfhydryl content in bacon on day 9 , the negative correlation between the antioxidant capacity of liquid smoke and disulfide bond content in bacon on day 9 , and the negative correlation between the antioxidant capacity  of liquid smoke and surface hydrophobicity of proteins in bacon on day 9 , which shows that smoking inhibited protein oxidation in bacon during storage. In consistence with literature [r = 0.96, p < 0.001) and total antioxidant capacity  of liquid smoke. In addition, the content of phenolic compounds in liquid smoke was positively highly correlated with the antioxidant capacity of bacon , but negatively correlated with protein oxidation in bacon on day 9. These data suggest that phenols should be the major antioxidant compounds in smoke. Smoking significantly inhibited lipid and protein oxidation in meat during storage  may help to overcome the flaws of smoke liquid made from single type of wood.Liquid smoke made from different woods varies in chemical composition and antioxidant capacity. Smoking modulates the antioxidant capacity and the oxidation of lipids and proteins of bacon. This study reveals that the selection of smoking woods and development of liquid smoke with antioxidant activity are a feasible approach to enhance the oxidative stability and shelf life of produced meat products. Phenols are a major class of antioxidant compounds to modulate the antioxidant capacity of both liquid smoke and fresh bacon. Among the five types of woods studied here, liquid smoke made from


This study highlights the difficulty of simultaneously implementing SDGs and elucidates novel research perspectives and policies to decrease the negative impacts of climate change on socio-economic and environmental well-being.The objective of this paper was to gain novel insights into the complex relationships among Sustainable Development Goals (SDGs) in shaping productivity (GDP/capita) growth. Using dynamic panel regressions on data collected in 138 countries between 2000 and 2017, we found that rising temperatures negatively affect growth and mitigate the impact of other SDGs on growth. We also found that CO Following the Millennium Development Goals (MDGs), the Sustainable Development Goals (SDGs) were introduced in 2015 to emphasise global efforts toward reconciling economic and social with ecological aspirations , implemeMitigating the impact of global warming is vital for the future of humanity. In 2019, the earth’s surface temperature was around 0.95 degrees Celsius (°C) warmer than the 20th-century average . As a reRising temperature is likely to have a continued negative effect on societies and economies. Shared socio-economic pathways explore possible paths for climate change projections that could affect a wide range of future trends . Xu et a2 emissions contribute to per capita growth or total factor productivity (TFP) , physical capital [tK], and labour [tL], determined by past accumulation in period t. α and (1 − α) are the elasticities of capital and labour, and constant returns to scale (0 < α < 1) are assumed in this model. The technical efficiency of production is denoted by [tA], as the residual output, which is not explained by expenditures of labour and capital used in production. Both sides of Equation (1) should be divided by [tL] to determine output per worker (productivity) [ty] Equation (2):The sum of total income [k] represents the capital/labour ratio as the amount of capital available per unit of labour input. The dynamics of capital intensity [k*] is equal to the amount of [ks] and the unit of effective labour (n + δ + g) that needs to be invested in preventing [k] from falling [δ] and technological change [g] are assumed to have little effect on the estimates, resulting in a constant (0.05) increase in employment growth [n]. Likewise, the steady-state level of productivity [ty*] corresponds with [k*] Equation (3):The term  is the dependent variable of GDP per capita at constant prices, i,tlnsk is the ratio of gross capital formation per GDP, and [ln(n + δ + g)] is calculated as the sum of the growth rate of employment.  denotes the exogenous rate of TFP , the model remaining after the capital accumulation. TFP can capture the impact of climate change, emissions, life expectancy, and food security on productivity.Transform and arrange Equation (4) into a linear formula at country i, and time t Equation (5):The frequent misunderstandings about the neoclassical model is that it fails to explain the catching-up countries. However, the real explanation for the economic growth needs to be derived from the model, which can be understood as the changes that the economy itself (endogenously) forms . Arellani,ty] is the growth ratio of real GDP per capita of the country [i] in the period [t]. The first independent variable refers to the lagged dependent variable. The second concerns the share of investment in output. [n] is the average growth rate of employment, and [δ] + [g] is assumed to be constant (0.05). [Temp] denotes the average temperature change. [2CO] refers to carbon dioxide emissions. [2sqCO] is included to test the potential quadratic relationship between productivity and emissions. [Life] is life expectancy at birth, and [MDER] is the minimum dietary energy requirement.After taking the first differences of the dependent variable, the above Equation (5) was transformed as follows Equation (6):k) positively impacts productivity growth, and employment growth (n + δ + g) is negatively related to the dependent variable in both models.The coefficients of temperature change (Temp) are relatively small and range from −0.005 to −0.072, negatively affecting productivity growth. If the average surface temperature rises from 0 to 1.5 °C, productivity decreases by 0.008 units (0.022 to 0.014), keeping all other variables constant, which is approximately 64 per cent lower than without a temperature rise.2 emission: a negative relationship between CO2 and growth, but a positive one between CO2 square and growth (Models 3 and 4). The overall t-test  also supports the presence of a curvilinear U-shaped relationship. First, as pollution increases, productivity growth decreases (negative relationship) until a local minimum, and afterwards, growth starts to increase again (positive relationship). Models (5–8) indicate that the life expectancy coefficient is significant; however, its sign is negative in all regression models. If life expectancy increases by one unit, GDP per capita will decrease by 0.119–1.197.Results also show a U-shaped relationship between productivity growth and COModels 7 and 8 show that food security (MDER) positively contributes to GDP per capita growth, while rising temperature negatively affects it. We also found significant two-way interaction effects between temperature change and MDER. Similarly, we found significant interaction effects between temperature change and life expectancy. 2 emissions, life expectancy, and food security on productivity growth and the interaction of increasing temperature with life expectancy and food security. A dynamic panel regression (Arellano and Bond) model estimates multidimensional data with longitudinal properties. This method eliminates the problem of adding deeper lags of the dependent variable, reducing the number of observations available. Contrary to previous approaches, we also considered the moderating effects of global warming, which is necessary for exploring the underestimated relations between socio-economic and environmental challenges.This study examined how critical sustainable development goals (SDGs) interact in shaping economic growth. We tested the effects of global warming, COOur results indicate that global warming negatively affects GDP per capita growth (H1). Carbon dioxide emissions have a U-shaped relationship with productivity growth (H2). In addition to its direct negative impact, global warming also mitigates the effects of other SDGs on growth. While life expectancy negatively influences growth (H3), it is positively moderated by global warming (H5a). Food security positively impacts growth (H4), which is negatively moderated by global warming (H5b). Hence, our data and analysis support H1, H4, and H5b, partly support H2, and reject H3 and H5a.The adverse effect of increasing temperature on living standards urges policymakers to combat climate change and its devastating impacts worldwide. However, Hasegawa et al.  claim thThe Paris Agreement of 2016 aims to strengthen the global response to the threat of increasing temperature by keeping its increase below 1.5 °C compared to pre-industrial levels . Accordi2 emissions and GDP per capita growth is robustly curvilinear at a global scale. Lower CO2 emissions and higher productivity growth are typical of developing (African) countries, while higher pollution couples with higher growth in China, India, and the United States. Several developed countries  tend to implement innovative green technologies to accelerate sustainable growth with decreasing levels of GHGs. Meanwhile, countries rich in fossil fuel  will also show faster per capita growth with higher environmental degradation [Contrary to Rigas and Kounetas , we founradation . Lower-iradation .We found that food security positively influences growth, but life expectancy has a negative impact on it. It appears that most countries have not achieved the demographic transition yet when population growth decreases because of increasing life expectancy. In the long run, increasing life expectancy will contribute to productivity growth due to accelerated human capital accumulation .Results also suggest that besides its direct (negative) effect, rising temperature moderates the economic impact of other SDGs. Global warming can exert influence on other SDGs in many ways as it impacts health, food and water scarcity, weather conditions, heat exposure, and diseases . SDGs sh2 levels; for instance, green investments accelerate the decarburisation of all aspects of the economy [There are several potential policy implications of the findings. Countries have decided to rebuild their economies; they can only become cleaner, greener, healthier, safer, more resilient, and sustainable by adhering to recovery plans . The pos economy . The ava economy . Rapid p economy . ResponsThis study is mainly limited by omitted variable bias, as the variables in the models reflect only a few SDGs  that we consider vital for the future of humanity. We urge researchers to test further interactions between SDGs  to help policymakers implement them simultaneously by minimising trade-offs. The Cobb–Douglas production function can also be replaced by a constant elasticity of substitution function or an alternative green growth approach.Future research also needs to consider theories from various disciplines  to obtain results and develop global indicators that reflect the complexity of SDGs and their potential future trajectories. Finally, growth should only be supported if it ensures that the people and our planet will continue to provide resources and environmental services for the well-being of all.


Phosphorus is one of the essential elements for plant growth and development, but available phosphorus (Pi) content in many soil types is low. As a fast-growing tree species for timber production, Chinese fir is in great demand of Pi, and the lack of Pi in soil restricts the increase of productivity of Chinese fir plantation. Root morphology and the synthesis and secretion of organic acids play an important role in the uptake of phosphorus, but the molecular mechanisms of Chinese fir root responses to Pi deficiency are largely unexplored. In this study, seedlings of Yang 061 clone were grown under three Pi supply levels  and morphological attributes, organic acid content and enzyme activity were measured. The transcriptome data of Chinese fir root system were obtained and the expression levels of phosphorus responsive genes and organic acid synthesis related genes on citric acid and glyoxylate cycle pathway were determined.We annotated 50,808 Unigenes from the transcriptome of Chinese fir roots. Among differentially expressed genes, seven genes of phosphate transporter family and 17 genes of purple acid phosphatase family were up-regulated by Pi deficiency, two proteins of SPX domain were up-regulated and one was down-regulated. The metabolic pathways of the citric acid and glyoxylate cycle pathway were mapped, and the expression characteristics of the related Unigenes under different phosphorus treatments were analyzed. The genes involved in malic acid and citric acid synthesis were up-regulated, and the activities of the related enzymes were significantly enhanced under long-term Pi stress. The contents of citric acid and malic acid in the roots of Chinese fir increased after 30 days of Pi deficiency.Chinese fir roots showed increased expression of genes related with phosphorus starvation, citrate and malate synthesis genes, increased content of organic acids, and enhanced activities of related enzymes under Pi deficiency. The results provide a new insight for revealing the molecular mechanism of adaption to Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots. Phosphorus migrates slowly in the soil and moves over a short distance . The inoCunninghamia lanceolata) is the largest timber tree species widely grown in Southern China [Chinese fir  and morphology (e.g. specific root area and specific root length) for efficiently explore the soil, as well as altering root exudates and mycorrhizal symbiosis to increase P bioavailability in soils , 54, 58.ClPHR1was expressed in all tissues of Chinese fir, but the highest expression level is found in the root. Eight differentially expressed genes related to phosphorus were found in the transcriptome of Chinese fir root under aluminum stress, which indicated that the transportation and metabolism of phosphate in Chinese fir root system are also affected [PHT1;4 and PHO1, ACP, and MDH in Chinese fir, while high Pi decreased PHT1;4 transcription and increased mRNA level of phosphate transporter PHT2;1 gene. These findings lay a foundation to study further the molecular mechanism of phosphorus deficiency in Chinese fir root system and related genes and metabolites. As the molecular mechanism of morphological and physiological responses of Chinese fir to Pi deficiency is not clear, further studies on the expression and regulation of genes induced by Pi deficiency need to be carried out.Advances in molecular biology in recent decades have revealed the central role of phosphate starvation genes (PHR1) in the transcription response associated with Pi deficiency , 36, 38.affected . The relaffected . It was In this study, the transcriptional characteristics of Chinese fir roots under Pi deficiency were studied by measuring the root transcriptome of the seedlings of Yang 061 Chinese fir clone, which were treated with no phosphorus, low phosphorus and normal phosphorus supply for 3 and 30 days. The differentially expressed genes and the related metabolic pathways under different phosphorus supply conditions were analyzed to screen the candidate genes that might play a role in Chinese fir adaptation to Pi deficiency. Identification and analysis of the functional expression of key genes involved in phosphorus transport, acid phosphatase secretion, and organic acid metabolism, in combination with the determination of some metabolites in the metabolic pathway of significantly different gene enrichment were studied. The results of this study are of great significance in revealing the transcriptional characteristics and molecular mechanism of Chinese fir roots in response to Pi deficiency, and providing gene resources and data support for screening and breeding Chinese fir genotypes for high phosphorus utilization.− 1 KH2PO4 and 0.333 mmol·L− 1KCl; 0 mg·L− 1 P), low phosphorus supply  and high phosphorus supply treatments . After 3 and 30 days of growth under treatment conditions, root samples were collected from NP-, LP- and HP-treated plants, organic acids and related enzymes were extracted, and the transcriptome and gene expression of the roots were analyzed. Seedling height, root morphology, biomass and nutrient content were also determined after 30 and 60 days of growth.The experiment was carried out in greenhouse in Forestry College , Fujian Agriculture and Forestry University. The region has an altitude of 10 m above sea level and a subtropical monsoon climate, with an average annual temperature of 25 °C, an average annual sunshine of 1700–1980 h and an average annual precipitation of 900–2100 mm. The experimental material, one-year-old seedlings of Yang 061 clone of Chinese fir provided by the state-owned forest farm of Yangkou, Fujian, China, were cultured with 1/3 Hoagland nutrient solution  in sand For root morphology determination, whole root systems of intact plants were carefully uprooted and washed with deionized water to remove the nutrient solution. Root length and root surface area were measured using WinRHIZO . The dry weight (DW) of shoots and roots were measured respectively after oven-drying for 45 min at 105 °C and 48 h at 65 °C until constant mass. Nutrient element concentrations were subsequently determined by inductively coupled plasma atomic emission spectroscopy.www.genedenovo.com/).The roots of plants treated with NP-, LP- and HP- supply for 3 and 30 days were harvested for RNA-Seq with three independent biological replicates for each treatment. Total RNA was extracted using a Spectrum™ Plant Total RNA Kit  and RNA quantity and quality were assessed by a NanoDrop spectrophotometer  and Agilent 2100 Bioanalyzer . The mRNA was enriched by magnetic beads with Oligo (dT) for RNASeq. The constructed library was sequenced with Illumina HiSeqTM4000  at GENEDENOVO (P < 0.05). The filtered transcripts were selected to be denovo Chinese fir root transcriptome, used as a reference for further analysis. The denovo Chinese fir root transcriptome sequences were a combination of the differentially expressed transcripts and other transcripts with RPKM ≥1.5 regardless of P treatments. These sequences were annotated using Blast2Go [Raw reads were quality checked, trimmed and de novo assembled using Trinity 5. Gene expression was calculated and normalized using reads per kb per million reads (RPKM) . DiffereBlast2Go  and MapMBlast2Go .The first-strand cDNA was synthesized from 1.0 μg RNA using GoScriptTM Reverse Transcription System . The reverse transcription system was based on 2 μg RNA in a final volume of 20 μL reaction volume. Reverse transcription was performed at 42 °C for 60 min with a final denaturation at 70 °C for 5 min. The resulting cDNA was diluted to100 ng/μL with nuclease-free water and stored at − 20 °C.−ΔΔCt method. Three biological replicates of each treatment and three technical replicates of each sample were used in the analysis.The qRT-PCR reactions were carried out on a StepOnePlus™ real-time fluorescence quantitative PCR instrument  using GoTag qPCR Master Mix  with transcript-specific primers. The qPCR was performed on a mixture containing 10 μL 2× SYBR Premix Ex Taq™, 0.5 uL each of specific forward and reverse primers and 2 uL cDNA. The amplification program was 95 °C for 3 min followed by 40 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s. The specificity of the amplification was controlled by melting curve analysis (from 65 to 95 °C). We used β-Actin1 as a reference gene to normalize expression level [2 = 0.9992) and citric acid  were determined. The injected sample was 10 μL, the flow rate was 1.0 mL / min, the column temperature was 25 °C, the retention time was 40 min and the wavelength was 325 nm. The pyruvic acid  content was also determined. The citrate synthase (CS), NAD-malate dehydrogenase (NAD-MDH), pyruvate dehydrogenase (PDH), phosphoenolpyruvate carboxylase (PEPC) Kits  were used to determine the activity of organic acid related enzymes involved in phosphorus metabolism.The roots of plants treated with NP-, LP- and HP- supply for 3,10,30 days were used for organic acids and related enzymes extraction. Root samples (0.2 g) were added into 1 ml extraction solution, ultrasonically extracted for 60 min, and the supernatant was filtered with needle-type filter and analyzed using HPLC. The HPLC conditions were Agilent 1100 High-performance liquid chromatography , Kromasil C18 reversed phase column , 10 μL injection volume, 0.8 mL / min flow rate, 25 °C column temperature, 20 min sampling time and 214 nm UV wavelength. The contents of malic acid , Amborella trichopoda (7.64%), Nelumbo nucifera (5.32%), Picea sitchensis (2.68%) and Elaeis guineensis (2.04%).We sequenced 18 samples by RNA-Seq technology and transcriptome sequencing data were deposited in the NCBI SRA database under the accession number PRJNA681878. We generated 126,413 unigenes with a maximum length of 16,707 bp, a minimum length of 201 bp, and a mean contig length of 702 bp. The assembled Unigene sequences were compared with the Nr, SwissProt, KOG and KEGG databases using blastx  annotation information of Unigenes was obtained by Blast2go, and all Unigenes were classified by WEGO, and the functional distribution characteristics of Unigenes were obtained . Purple acid phosphatase (PAP) is a special class of acid phosphatase, and its members have different responses to Pi deficiency and their functions are regulated by transcription factors and SPX protein. SPX is an important regulator of phosphorus signaling network, which can interact with PHR1 to regulate the expression of phosphate transporter gene PHT1, thus promoting phosphorus uptake by roots under Pi deficiency. A total of 18 ACP genes, 70 PAP genes, 10 SPX structure and 10 protein genes were screened from the transcriptome data based on all Unigenes annotation in GO and KEGG databases. Among them, 17 PAP genes, three ACP genes and three SPX domain proteins were differentially expressed under Pi deficiency. The differentially expressed PAP, ACP, and SPX genes were analyzed by expression thermography Fig. A, and thAccording to the annotation of GO and KEGG database, 20 differentially expressed genes related to organic acid synthesis and metabolism were screened. The gene expression of these 20 genes under different Pi deficiency was analyzed by thermography Fig. B, and thArabidopsis thaliana, rice and poplar, four genes with high homology to PHT4 gene were screened. These genes were highly similar to the known PHT family genes. The Unigene annotation messages contained three probably anion transporters, whose pathway were annotated as perfuse of the major facilitator superfamily and one possibly organic anion transporter (Unigene 0074757). Furthermore, two PHT5 genes were annotated with membrane proteins containing SPX domains. A total of 41 genes involved in inorganic phosphate transport were obtained.Based on all Unigene annotation results, a total of 86 Unigene entries annotated as phosphate transporter family and phosphate protein carrier were retrieved. A total of 35 Unigene entries were confirmed to be involved in inorganic phosphate transport by sequence signature analysis compared to BLAST analysis. The other 51 genes may be involved in phosphorus transport, the function of which is unknown. Among the 35 phosphorus transporter genes, six genes, which had high homology with PHO1, were annotated as PHO1 family genes by Nr and Swissprot and classified as small molecule transporter by KOG. We found 16 genes with high homology with PHT1 family, inorganic phosphorus transporters; one carrier family with high homology to the PHT2 gene, sodium-dependent phosphate transporter; 12 carrier proteins annotated as mitochondrial phosphate, which were highly homologous and predicted to be PHT3 family. Based on the analysis of the characteristics of the PHT family genes, and the prediction of the coding sequence (CDS) and the characteristics of the PHT gene multiple sequence alignment with Thermographic analysis of seven Unigenes with significant differential expression of the PHT gene family Fig. C showed In order to better understand the role of PHT gene in phosphate uptake by Chinese fir root, and clarify the expression pattern of PHT gene in Chinese fir root under different phosphorus supply treatments, seven known up-regulated PHT gene families expressed in phosphate starvation response were selected and verified by qRT-PCR Fig. D and fluCitric acid and glyoxylate cycle play an important role in the synthesis of organic acids in plant tissues, and the transcriptome sequencing of Chinese fir roots under phosphorus starvation revealed that the main metabolic pathway for enrichment of differentially expressed genes was the biosynthetic pathway of organic acids. The annotated transcripts related to citric acid and glyoxylate cycle were mapped, and 28 genes with significant differences in enzyme coding presumably involved citric acid and glyoxylate cycle acid were identified under Pi starvation Fig.A. DifferIn addition, qRT-PCR analysis was used to study the expression of transcripts in citric acid and glyoxylate cycle pathway, including pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase and isocitrate lyase Fig.C. The reThe activities of NAD-malate dehydrogenase, citrate synthase, pyruvate dehydrogenase and phosphoenolpyruvate carboxylase in the root of Chinese fir seedlings after 0, 3 and 30 days of different phosphorus treatments were determined Fig. A. There The contents of malic acid, citric acid and pyruvic acid in roots of Chinese fir seedlings were measured after 0, 3 and 30 days of different phosphorus treatments Fig. B. The coOur study showed that the morphological and physiological responses of plants to Pi deficiency become prominent around 30 days after Pi deficiency. Pi deficiency inhibited seedling height growth but promoted root elongation. The root-shoot ratio of Chinese fir seedlings under Pi deficiency condition was higher than that under normal phosphorus condition, which is similar with the general morphological characteristics of Chinese fir under Pi deficiency condition , 55, 58.The root transcriptional characteristics of Chinese firYang061 clones were studied after 3 and 30 days of NP-, LP- and HP- treatment, and a total of 126,413 Unigenes were obtained, of which 50,808 Unigenes were annotated in the database. The analysis of differential expression of Unigenes showed that the stronger the Pi deficiency stress was, the larger the differentially expressed genes were, particularly after 30 days than 3 days of Pi deficiency. GO annotation revealed that the distribution of cell and cell subclass catalytic activity, protein binding and transport activities were enriched to a much higher proportion of genes than other items in the same category. This result showed that the root system of Chinese fir seedlings responded to P stress by changing its cell components, adjusting its catalytic activity and adapting to the stress environment through various metabolic pathways. The results of KEGG enrichment analysis showed that the response genes could be divided into more than 30 subclasses and distributed in more than 120 pathways, which include redox processes, transmembrane transport, osmotic stress response, pyruvate metabolism, tricarboxylic acid cycle and acid phosphatase.Arabidopsis thaliana WRKY45 regulates the high expression of PHT1 in the roots. In other plants, MYB member PHR1 can positively regulate miR399, SPXs, PHT1, and the downstream genes PHO2 and PAPs [The genes obtained by trend analysis were annotated, and the up-regulated genes included transcription factors involved in the stress response-signaling pathway. It also included the tonoplast dicarboxylate transporter gene, which is involved in the organic acid transport through the tricarboxylic acid cycle pathway, and isocitric acid and isopropyl malate dehydrogenase, which are involved in oxidative decarboxylation , 37. Pi and PAPs , 23. Theand PAPs . Thus, tArabidopsis thaliana [Among the acid phosphatase ACP genes, purple acid phosphatase PAP gene and SPX domain protein screened from the transcriptome data, 17 PAP genes were differentially expressed, three ACP genes and three SPX domain proteins were induced by Pi deficiency. The expression of ACP and PAP genes increased with the intensity of Pi deficiency and the time of stress, indicating that Chinese fir has the physiological response of synthesizing acid phosphatase to activate the organic phosphorus in its rhizosphere under Pi deficiency. Most of the acid phosphatase was involved in the Pi transfer and recovery process . They cathaliana  and tomathaliana  have shoOne of the typical characteristics of plant roots in response to Pi deficiency is to enhance the synthesis and secretion of organic acids in roots to activate insoluble phosphate in soil. Root transcriptome of Chinese fir in response to Pi deficiency showed that most of the 20 differentially expressed genes were up-regulated after 30-day of Pi deficiency compared to 3-day Pi deficiency stress. This might be related to the time lag in phosphorus activation by organic acid synthesis in Chinese fir roots. Furthermore, genes related to citrate-glyoxylate cycle were generally different. Under 3-day of HP-treatment, the expression level of except-enzyme genes was low, but under 30-day of Pi deficiency, 15 of the genes on the 26 TCA metabolic pathway were up-regulated, nine were down-regulated and two were not detected. The expression of eight Unigenes increased with the increase of Pi deficiency and the extension of time. Compared with 3-day of Pi deficiency, the expression of genes related to the tricarboxylic acid cycle pathway was more active under 30-day of Pi deficiency, with the increase of stress degree. Thus, Chinese fir activated the enhanced synthesis and secretion of organic acids in roots to promote its survival in low soil phosphorus environment.Lupinus albus is evidently increased under Pi deficiency, whereas the exudation of malic acid and oxalic acid increased in soybean root system [The contents of malic acid and citric acid in the roots of Chinese fir under 3-day of Pi deficiency increased, while the contents of citric acid in the roots of Chinese fir under 30-day of Pi deficiency significantly increased. The results showed that citric acid was more sensitive to Pi deficiency than malic acid. Citrate synthase was up-regulated under Pi deficiency with increasing stress intensity. Aconitase was up-regulated under Pi deficiency, however, the expression level decreased with the increase of stress intensity. In addition, isocitrate dehydrogenase always showed a relatively high level of expression, which was up-regulated under 3-day of Pi deficiency treatment, but decreased under 30-day of Pi deficiency. Succinyl-coA synthase was highly expressed under 30-day of Pi deficiency, and its expression increased with the increase of stress intensity. Similarly, under 30-day of Pi deficiency, the expression of isocitrate lyase and malate synthase was increased while the expression of malate dehydrogenase-related genes was down-regulated compared with that under 3-day of Pi deficiency. The increase of organic acid synthesis and secretion in roots of plants under Pi deficiency has been widely confirmed, but the selection of organic acid secretion is different in different species. For instance, the amount of citric acid secretion in roots of rape and t system , 34. SinThe activity of pyruvate synthase was up-regulated under Pi deficiency condition. This may be due to the extraction of organic anions below the detection limit, or pyruvic acid itself as a metabolic intermediate, which is also the raw material for the synthesis of amino acids, the starting substrate of tricarboxylic acid cycle, and the final product of glycolysis. Previous studies have shown that biosynthesis and exudation of organic anions are associated with increased expression of genes encoding PEPC, NAD-MDH, CS and PDH. However, it should be noted that functional compensation exists in genes and proteins, and the expression of genes is not consistent with the increase of enzyme level, enzyme activity and organic acid production. In general, the metabolism of pyruvic acid of Chinese fir was enhanced under Pi deficiency in this experiment, so more pyruvic acid was used to synthesize various organic acids in tricarboxylic acid cycle. The gene for pyruvate dehydrogenase was up-regulated, the synthesis of citric acid was promoted, and the expression of isocitrate dehydrogenase related genes was up-regulated to promote α-ketoglutaric acid synthesis. The α-ketoglutaric acid can synthesize more glyoxylic acid, fumaric acid and malic acid in the presence of dehydrogenase. The expression of ATP-citrate lyase in the glyoxylic acid synthesis pathway is enhanced to promote the synthesis of glyoxylic acid, malate synthase was also up-regulated to promote the synthesis of malic acid under Pi deficiency, thus promoting the synthesis of citric acid, isocitrate, malic acid, glyoxylic acid and other organic acids under Pi deficiency. This result is also consistent with the synthesis and secretion of organic acids by Chinese fir roots under Pi deficiency . The resChinese fir roots showed increased expression of phosphorus starvation related genes, citrate and malate synthesis genes, increased content of organic acids and enhanced activities of related enzymes under Pi deficiency. We obtained three SPX domain genes, 17 purple acid phosphatase genes, three acid phosphatase genes and 35 phosphate transporter genes by screening the genes related to phosphorus starvation response, which in turn increased rhizosphere Pi. We also found 20 genes related to the metabolism of organic acid synthesis and 28 genes related to the metabolism of citric acid and glyoxylic acid that were significantly expressed. The up-regulated expression of the phosphorus transporter gene, promotes the absorption and transport of Pi in roots. The contents of citric acid and malic acid in the roots of Chinese fir were also increased and the activities of malic acid and citric acid synthase were increased after 30 days of Pi deficiency, which may be related to the adaptation of Chinese fir to Pi deficiency. Screening phosphorus starvation response genes laid a foundation for further research, verify the functions of the alternative genes and explore the genetic variation of tree morphology after the establishment of genetic transformation system of Chinese fir in the future. It provides genetic resources and data support for exploring the mechanism of phosphorus uptake by Chinese fir and cultivating phosphorus efficient genotypes of Chinese fir. The findings on the relationship between Pi deficiency and organic acid metabolism of Chinese fir provides a new way to reveal the molecular mechanism of response of Chinese fir roots to Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots.


Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption. Enteric viruses represent a major risk to human health, being responsible for numerous outbreaks worldwide. The best characterised foodborne viral agents are human norovirus (NoV) and hepatitis A virus (HAV), which cause the most significant part of foodborne-associated illness globally. According to Centers for Disease Control and Prevention (CDC) and the European Centre for Disease Prevention and Control (ECDC), NoV-associated foodborne infections are one of the most frequently reported in the United States and European Union (EU), being, for instance, associated with 457 outbreaks, and, most importantly with 11,125 cases of illness in 2019  only in the EU . It is eDomestic and restaurants settings are described as the most common places associated with NoV/HAV outbreaks , and theUsually, since fish are mainly consumed cooked, their ingestion is not a concern regarding a possible contamination with the most common foodborne-associated viruses. However, in recent times raw fish consumption has become increasingly common, and the use of gilthead seabream, Atlantic horse mackerel, and seabass in dishes like sushi, sashimi, poke, and carpaccio has increased. Tthese eating habits create concerns regarding the safety of consuming raw fish since some may be contaminated with several types of potentially pathogenic microorganisms, including bacteria, viruses, and parasites ,7,8. AltThe existing literature suggests that in the assessment of a qPCR assay several factors should be considered, namely, the effect of using different templates to generate standard curves for the absolute quantification of RNA viruses, which can be made from either a plasmid, a synthetic oligonucleotide, or an in vitro transcribed RNA . The advSparus aurata) and seabass (Dicentrarchus labrax), two of the most extensively wild-caught and farmed species in aquaculture, as well as sardine (Sardina pilchardus) and Atlantic horse mackerel (Trachurus trachurus), two of the most consumed fish species in Portugal.Therefore, in this study, we developed a tetraplex qPCR assay to detect and quantify NoV GI, NoV GII, and HAV genomes, using mengovirus as an internal control. This method was implemented either using plasmid or an in vitro transcribed RNA for standard curve construction, which allowed for NoV and HAV genomic quantification to be evaluated by comparing the two standards curve types in terms of efficiency, sensitivity, and detection limit. These tetraplex qPCR assays were used to perform a virological screening in fish captured and farmed along the Portuguese coast, focusing on four of the most economically important species from the Atlantic coast. These included gilthead seabream  was used as process control since this virus is not naturally present in food matrices . Moreove in food . Mengovi in food . Total RTo determine the optimal input for the control process virus, different amounts of mengovirus were used to spike one soft and internal tissue (liver) and one hard and external tissue (gills) to assess the limit of viral genome detection.Additionally, before total RNA extraction, all the samples were spiked with 25 µL of mengovirus suspension to indirectly estimate losses of the target viruses, which can occur at several stages during processing. The recovery rate of mengovirus was calculated using the following formula: % recovery control virus =  × 100.A total of 323 fishes were analysed (post-mortem) in this study. These were either (i) wild specimens caught along the coast of Peniche, Figueira da Foz and Algarve, (ii) available at supermarkets, (iii) discarded from fish markets, or (iv) farmed in the Algarve and Setúbal region , Table 1Approximately 2.0 g of fish tissue was chopped using a sterile razor blade and homogenised in 10 mL of TNE buffer  using a Precellys Evolution Homogenizer . The homogenates were centrifuged 10 min at 2000 rpm at 4 °C to remove particulate debris, and the supernatant used for total RNA extraction, carried out from 250 µL of clarified supernatant, using NZYol , as described by the supplier. RNA was dissolved in 30 µL of DEPC-water, and the concentration and purity of the obtained RNA extracts determined using a NanoDrop 1000 spectrophotometer. RNA extracts were stored at −80 °C until further use. For qPCR reactions, the RNA was converted to cDNA as described in For each target, a distinct set of primers/probes  was usedFor the construction of the standard curves based on plasmid DNA reference templates, three recombinant plasmids were artificially synthesised  harbouring partial sequences of the junction of open reading frames 1 and 2 (ORF1/ORF2) of NoV GI and GII, and the 5′ noncoding region (5′NCR) of HAV. These same plasmids served as a starting point to obtain PCR products from which the in vitro transcribed RNA of each targeted genomic region was subsequently synthesised. Amplicons corresponding to sections of recombinant plasmid DNA were amplified by conventional PCR, and the success of the amplification process was confirmed by their visualisation on 2% agarose gels, followed by their purification with NZYGelpure kit . The primers used to obtain these PCR fragments were chosen to include a T7 RNA polymerase-specific promoter upstream of the viral coding sequence. The fragments were then used as a template for in vitro transcription with the NZY T7 High Yield RNA Synthesis kit . The obtained RNA was purified with NZY RNA isolation kit  and treated with DNase I solution to prevent DNA contamination. RNA concentrations and purity were estimated using a NanoDrop 1000 spectrophotometer. Total RNA was converted to cDNA as previously described and used to construct the quantification standard curves.7 to 10 genome equivalents. Each target was either tested individually or as a mixture.Standard curves were constructed (one for each template) using 10-fold serial dilutions ranging from 10Single and multiplex qPCR reactions for detecting NoV GI, NoV GII, HAV, and mengovirus were carried out in a total volume of 20 μL using SensiFAST™ Probe No-ROX amplification mix . The concentrations of Nov GI, NoV GII, HAV, and mengovirus forward/reverse primers and probes , as wellAdenoviridae), infectious pancreatic necrosis virus , infectious hematopoietic necrosis virus , viral haemorrhagic septicaemia virus , viral nervous necrosis virus  and Hepatitis E virus .All single and multiplex assays for the detection of NoV GI, NoV GII, HAV, and mengovirus genomes were tested for cross-reactivity with other viruses available including, adenovirus type 5  for viral quantification, positive wastewater samples previously shown to contain NoV GI, NoV GII, and HAV genomes (our work—not published), were used to validate the qPCR assays developed. The quantification of genome equivalents was carried out by qPCR using either plasmid or in vitro transcribed RNA standard-based curves. RNA was extracted from these samples with QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions, using 140 µL of each sample and an elution volume of 80 µL (double elution 2 × 40 µL). RNA concentration and purity were estimated using a NanoDrop 1000 spectrophotometer. RNA was converted to cDNA as previously described in ® VR-3234SD™, Quantitative Synthetic Norovirus G2 (II) RNA ATCC® VR3235SD™, and Quantitative Synthetic Hepatitis A virus DNA VR-3257SD™. NoV GI and HAV detection was carried out in a total volume of 25 μL using NZYTaq II 2× Green Master mix . For NoV GI, each 25 µL reaction volume included 0.4 µM of each primer (Three nested-PCR (nPCR) protocols targeting NoV GI, NoV GII, and HAV genomes were developed to further characterise by phylogenetic analysis the positive samples previously obtained by the qPCR protocols. All nPCR assays were optimised using synthetic templates purchase from American Type Culture Collection , including Quantitative Synthetic Norovirus G1 (I) RNA ATCCh primer  and 5 µLh primer  and 5 µLFor NoV GII detection, the NZYTaq II 2× Green Master mix was not efficient, being used the Platinum™ SuperFi II Green PCR Master Mix  in a total volume of 20 μL. The thermal cycling conditions for PCR and nPCR included 1 cycle at 98 °C for 30 s, followed by 35 cycles at 98 °C for 10 s, 60 °C for 10 s and 72 °C for 30 s, and a 10 min final extension step at 72 °C.https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 31 March 2021)).All amplification steps were performed on a Doppio VWR thermocycler , and their success was confirmed by amplicon visualisation on 1.5% agarose gels. Additionally, non-template (negative) controls were used in each run. PCR products were sequenced in both directions using Sanger’s method  sequences used for the preparation of the different sequence datasets were selected among those deposited in the GenBank database, on the proviso that they would be representative of (i) each of the previously described species with (ii) a significant sequence overlap with the sequences obtained during this study to maximise the number of unambiguously aligned nt positions in each sequence alignment. For phylogenetic analysis, multiple alignments of nt sequences were constructed with the iterative G-INS-I method as implemented in MAFFT vs. 7 , followeThe generation of a standard curve for qPCR quantification of RNA viruses can be constructed based on the use of serial dilutions of several possible templates, including a viral genome, a structurally equivalent template prepared from in vitro transcribed RNA, or a plasmid harbouring the targeted sequence in the form of dsDNA. In an ideal situation, standard curves for qPCR analysis should be constructed using serial dilutions of cDNA prepared from RNA extracted from a viral suspension. However, since logistic limitations deter virus isolation/propagation in our laboratory, plasmids and in vitro transcribed RNA molecules prepared from a synthetic template were chosen as the starting model for standard-curve construction.3 and 10 genome copies, respectively. For HAV, the detection limit was 102 genome copies in the single reaction, increasing 1-log in the multiplex format  values between samples using the plasmid and the in vitro transcribed RNA standard-based curve . In term9 genome copies/µL, gave similar multiplex qPCR quantification results  of NoV GI genome was disclosed, corresponding to a pool of seabass brain tissue from specimens obtained from Peniche fish market . Similarly, using in vitro transcribed RNA as a standard curve, this sample was also positive with a Cq value of 32.89 corresponding to 4.64 × 104 genome copies of NoV GI. None of the 323 fish specimens analysed revealed the presence of NoV GII or HAV genomes.The analysis of the 323 fish specimens using plasmid as a standard curve for NoV and HAV detection and quantification, revealed one pool where the presence of 1.55 × 10The positive sample for NoV GI obtained by qPCR was further confirmed by conventional nPCR and characterised by Sanger sequencing of the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the norovirus genome . The norNoV and HAV are the leading cause of foodborne viral infections, being responsible for considerable economic and health burdens globally. Moreover, of all the viruses associated with foodborne infections, NoV and HAV are the most important viral pathogens regarding the severity of the associated illnesses and their common occurrence worldwide . They ar9 genome copies/µL, and in all the performed extractions, its recovery rate was consistently above 10%.In the present study, a virologic screening of the two above-mentioned enteric viruses targeted mainly four fish species highly consumed in Europe and caught/farmed along the Portuguese coast. For that purpose, we used a multiplex qPCR assay designed for the detection/quantification of NoV and HAV genomes, considering two different approaches as standard curves: an external in vitro synthesised RNA and a plasmid. Furthermore, this assay also included a control virus since losses of the targeted viral genomes can occur at several stages during sample preparation and nucleic acid extraction ,25. All Multiplex qPCR-based methods have been designed to detect and quantify several targets in a single reaction. However, they tend to display lower detection sensitivity when compared to standard singleplex qPCR . These lThe standard curve in an absolute qPCR assay is generated by amplifying serial dilutions of a standard DNA, which can be a plasmid, a PCR amplicon, a synthesised oligonucleotide, a genomic DNA, or a cDNA. Among the various types of the standard template, using a plasmid is one of the most common options due to its high stability and preparation reproducibility. However, plasmids could adopt several conformational structures, namely a supercoiled form, which can suppress qPCR assay compared to other templates ,28. In tInterestingly, in this study, NoV GI genomic RNA was detected in one out of a total of 400 pools (50 pools of fish × 8 organs). Although seafood contamination by these viruses is well described ,33,34,35As expected, the phylogenetic analysis of the norovirus sequence detected in this study revealed that it clustered with a major group containing norovirus sequences from genogroup I, in particular GI.1 , since ihttps://wwwn.cdc.gov/norsdashboard/ (accessed on 2 April 2021)). In this system regarding the years between 2007 and 2018, norovirus outbreak data showed that fish as a vehicle of transmission of NoV infection, without accounting for person-to-person transmission, were responsible for 15 outbreaks with 180 infected people and four hospitalisations. Moreover, fish contamination could be due to the presence of enteric viruses in the surrounding water. In fact, waterborne infections due to noroviruses were responsible for 55 outbreaks, 3330 illnesses and 33 hospitalisations ) between 2007 and 2018 in the United States, whereas in the EU, in 2019, were responsible for 11 outbreaks and 26 hospitalisations [The detection of NoV genomes in fish samples can indicate that the water where the analysed fish swam was contaminated with human faeces, probably due to sewage pollution. Despite the fact that the most common categories linked to outbreaks of NoV and HAV are fresh vegetables, fruit, and shellfish since they are consumed raw or undercooked, any type of food could be implicated in an outbreak . InformaIn conclusion, we developed a multiplex qPCR method to identify and quantify NoV and HAV genomes based on two different standard-curve construction approaches. Our data suggest that the one based on in vitro transcribed RNA is a better solution for RNA virus quantification in biological samples. Furthermore, detectingthe human pathogenic NoV GI genome in fish brains supports the hypothesis that these viruses circulate in seawater. On the other hand, while foodborne infection control might be very straightforward by simply cooking the food before consumption, raw or undercooked fish consumption as a common trend makes this control very difficult. Thereby, the monitorisation of these pathogens to properly assess human health risks associated with raw or undercooked fish consumption could be of utmost importance since NoV and HAV are characterised by a high rate of transmission, which makes them even more challenging to control.


Hermetia illucens is a widespread species of fly of American origins, which is increasingly used to develop sustainable waste recycling processes as it is able to develop by consuming a wide variety of wastes as food, while both its body and the residuals of its feeding activity can be re-used in a variety of processes. However, many aspects of its larval biology remain unknown. Among these, there is larval movement and its variation in response to external stimuli and environmental conditions. Larvae of the black soldier fly eventually reach the prepupal stage, during which they stop feeding and seek a shelter to pupate. Sorting prepupae from the younger larvae and feeding substratum can be important in rearing processes, since they are used to obtain adults but are also particularly rich in protein and lipids. We focused our study on behavioural differences between prepupae and younger larvae, describing tonic immobility as an anti-predatory response of both, but also their very different ways of locomotion and reactions to stress. Finally, we developed a practical system to exploit these differences, inducing prepupae to move away from the substratum and other larvae to be efficiently collected.The black soldier fly Hermetia illucens has become a cosmopolite species, and it is considered a highly promising insect in circular and sustainable economic processes. Being able to feed on a wide variety of organic substrates, it represents a source of lipids and proteins for many uses and produces recyclable waste. We investigated the characteristics and differences in the poorly-known locomotory behaviour of larvae of different instars, paying particular attention to the unique characteristics of the prepupal stage, key to farming and industrial processes. Moreover, we attempted to develop a “self-harvesting” system relying on the behavioural traits of prepupae to obtain their separation from younger larvae under rearing condition with minimum effort. Prepupae differ from younger larvae in their response to physical disturbance in the form of tonic immobility and significantly differ in their locomotory movements. Both prepupae and younger larvae reacted similarly to heat or light-induced stress, but low light and high moisture induced only prepupae to migrate away, which resulted in the development of a highly efficient separation methodology. The new data on the behaviour of H. illucens not only shed new light on some unexplored aspects of its biology, but also led to develop an inexpensive self-harvesting system that can be implemented in small-scale and industrial farming.The neotropical insect  Hermetia illucens (Diptera: Stratiomyidae) is native to the Americas, but today has a cosmopolite distribution due to human activities [Hermetia illucens is considered one of the key species of great interest developing novel systems to use insects in sustainable practices including agriculture [H. illucens is usable as a protein and lipids-rich feed and food [H. illucens an extremely valuable tool to achieve pressing sustainability goals [The black soldier fly tivities ,2,3,4,5.iculture ,7,8. In iculture ,10,11. Iand food ,13,14,15and food ,17,18. Pty goals ,20. Morety goals  or as a ty goals ,23.H. illucens larvae may take significantly different amounts of time depending on rearing conditions, and larvae are classified into five to seven instars according to different authors [H. illucens as an asset for agricultural and industrial waste-recycle activities, and several studies have focused on defining the optimal rearing conditions for larval development and facilitating the adults’ reproduction [The development of  authors ,25,26,27 authors . Their poduction ,38,39,40oduction ,42,43,44oduction  offered H. illucens larvae remain so far uninvestigated, one of which is the description of their locomotory movement. Indeed, the locomotion and related behavior of fly larvae are only rarely subject to focused descriptive investigations [H. illucens, it is notable how little is known of this widely distributed and heavily studied species apart from what concerns topics of very direct relevance with laboratory farming and possible applications.Despite the great interest in this insect, many basic aspects concerning igations ,47,48. MH. illucens, documenting the behavioral responses of the apod larvae belonging to different instars to both mechanical disturbance, and stress caused by different light or heat conditions. We compared the prepupae’s locomotory behaviour with the younger instars, which is known to differ mainly since prepupae do not feed and are devoted to finding a shelter to pupate (migration). Exploiting these differences, we attempted to develop and quantify the efficiency of a method to induce prepupae to separate from the younger larvae, resulting in a “self-harvesting” strategy useful under rearing conditions.We investigated larval locomotion in Hermetia illucens larvae were bred at the “Azienda Agraria Sperimentale Stuard”  farm from a colony regularly bred in the lab of the Department of Sustainable Crop Production, Section Sustainable Crop and Food Protection, Università Cattolica del Sacro Cuore . They were kept in plastic boxes (60 × 40 × 20 cm) under the following conditions: T: 29 ± 1 °C, RH: 60 ± 0.5%, photoperiod 12:12 (L:D), 4–5 larvae per cm2. To feed the larvae, we used 1 kg of organic material daily (corresponding to about 100 mg per larva every day): this consisted in 500 g of pumpkin (Cucurbita moschata Duchesne ex Poir.) and 500 g of vegetal wastes: according to their availability in the farm, lettuce (Lactuca sp.), summer squash (Cucurbita pepo L.), spinach (Spinacia oleracea L.), radish (Raphanus raphanistrum subsp. sativus (L.) Domin), common chicory (Cichorium intybus L.), beet (Beta vulgaris L.), parsnip (Pastinaca sativa L.), rhubarb (Rheum × hybridum Murray), and tomato (Solanum lycopersicum L.).We followed the latest classification , which All laboratory experiments were conducted from March to June 2021 under the following environmental conditions: T 26 °C, RH 50% ± 0.5%.We analysed larval reaction to mechanical disturbance and its differences according to instars size. We created seven groups of larvae of different sizes, each composed of 100 individuals : A: 4–6; B: 7–9; C: 10–12; D: 13–15; E: 16–18; F: 18–24; G: 13–21). The groups F and G are entirely constituted by 6th instar larvae and prepupae respectively. Each larva was tested in a 5 × 10 cm arena which it was free to explore. An entomological forceps was used to gently poke each crawling larvae on their sixth tergite, after which we recorded the following data: (i) whether the larvae stopped moving or not; (ii) for those larvae who became immobile, how much time they remained still.www.kinovea.org, accessed on 16 December 2021) by keeping track of the movements of the insects’ 2nd segment: the output was a database containing the horizontal and vertical coordinates of the sternite during each frame [We attempted to determine the differences in the locomotory patterns adopted by 6th instar larvae and prepupae. We recorded 4-min HD videos of crawling larvae  filmed from a lateral perspective on a plastic arena using a 6D camera No light + heated mat (32 °C)Ambient light (24 °C)Ambient light + heated mat (32 °C)Cold white light (24 °C)Warm white light (32 °C)Infrared light (33 °C)We attempted to verify which environmental conditions could affect the crawling speed of larvae and the possible differences between 6th instar larvae and prepupae. We considered increased heat (compared to the rearing conditions) and light exposure (considering that larvae are lucifugous) as stressful conditions to test. The experimental apparatus consisted in a plastic arena (17 × 6 cm), divided in an open portion directly exposed to light and heating (13 × 6 cm) and a portion covered by a small plastic roof (4 × 6 cm). Larvae were introduced to the open part of the arena, and we recorded HD videos of their behavior; they were placed next to the center of the 6 cm side, opposite to the plastic roof, with their bodies directed towards the latter so that they were induced to move in that direction; then we evaluated their movement speed by recording the time they spent to travel for 13 cm . To set different combinations of light and heat conditions, we employed a heated mat  as well as lamps positioned 30 cm above the arena. The characteristics and effects of these lamps were measured using a CRI illuminance meter  to characterize the light spectrum, a luxometer  to quantify the lux on the apparatus, and a laser thermometer  to quantify the illuminance and temperature . The following lamps with the following characteristics were tested during the experiments: a warm white LED ; a cold white LED ; an infrared lamp . In addition, tests were also conducted under the ambient light conditions of the laboratory (210 lux) or under conditions of darkness thanks to night vision device, . As a result, the following 7 treatments were created :Each treatment was paired with the presence or absence of a substratum consisting in residual waste produced by the larvae feeding activity . A total of 40 individuals from the 6th instar and 40 prepupae were tested under each possible combination of heat and light treatments and of substratum conditions .2 of substratum, RH: 22–23%) and high moisture . Moreover, five light conditions were obtained through the use of commercially available lamps or ambient light as in the previous experiment (lux measurement with a Delta Ohm Hd 8366 luxometer). Here we only focused on ambient light, as the simplest solution achievable, and infrared light as the better-performing based on the locomotory speed experiment, and therefore we utilized the following treatments: 6 lux, 12 lux, or 60 lux (ambient light reduced by a filter), 210 lux (ambient light), >7000 lux (infrared light obtained with a lamp). A total of 10 replicas was conducted for each of the possible combinations of moisture and light conditions (n = 3 × 5 × 10 = 150).Based on the results of the previous experiments, we attempted to build an apparatus capable of inducing prepupae to separate from younger instars (self-harvesting). This consisted in a plastic box (29 × 35 × 17 cm) in which a covered area was added. The covered area was placed near one of the short sides of the box, characterized by the presence of a plastic barrier obscuring light. The barrier consisted in a roof  and a lateral semi-wall allowing the larvae to enter through a narrow passage at its base (1 cm wide) which was left opened towards the non-covered side of the box . Larvae Statistical analyses were performed by using the softwares SPSS 27  and R 4.0.3  . Mann WhLarvae of group G (prepupae) were the only instar to consistently respond to disturbance by immobilizing their body, 76% of the recorded responses, see , while aPrepupae and 6th instar larvae remarkably differed in their locomotory patterns see . Yet, soPrepupae crawled forward by performing a relatively quick and continuous sinusoidal movement of their body, while 6th instar larvae relied more heavily on pinning with their head to move forward. This fundamental difference was well-described by Kinovea trajectory-tracking feature .p < 0.001), the arching of the anterior part preceding the pinning movement was more evident in 6th instar larvae  and the angle formed during the pinning movement was also much more acute in the latter   .(6) = 130.69, p < 0.001), 6th instar larvae without waste as a substratum (H(6) = 141.68, p < 0.001), prepupae with waste as a substratum (H(6) = 168.19, p < 0.001) and prepupae without waste as a substratum (H(6) = 123.71, p < 0.001). The waste substratum resulted in an averagely slower movement locomotory speed  and were characterized by a large number of prepupae accompanied by just a very small number of younger instars reaching the collecting area. Expressed as mean ± SD on a percentage basis, this was 87 ± 11% prepupae vs. 4 ± 3% younger instars reaching the collecting area for the high moisture treatment, and 81 ± 12% prepupae vs. 20 ± 9% for the medium moisture treatment  insects [Our data suggest that tonic immobility, hitherto undescribed in its prey ,53. It m insects ,56,57,58H. illucens, here described for the first time, strongly differs between younger larvae and prepupae, with the latter lacking a strong role of the head movement that characterizes the former but also the larvae of other Brachycera [The larval locomotion of achycera . This diachycera ,27. To tachycera ,60,61,62achycera ,65,66,67H. illucens larvae: they are thought to spend their life in a lightless environment within rotten material and soil, and excessive heating normally stimulates dipteran larvae to move quicker in search for more favourable conditions . HoweveIn the separation experiments, coherently with the abovementioned results, we found that the highest numbers of larvae left the boxes and reached the collecting arena whenever illumination was stronger (infrared light). However, this proved to be an ineffective tool to elicit separation of prepupae from the younger instars, since all larvae responded similarly independently from their instar–causing a mix of prepupae and younger larvae to occur in the collecting arenas. On the contrary, the most efficient treatment consisted in the lowest illumination: this was enough to stimulate the prepupae to leave but did not have the same effect on the younger instars, so that almost exclusively prepupae were found in the collecting arenas. These data suggest that prepupae, likely driven by the instinct of locating an adequately safe area to pupate, are more sensible to slight light variations than younger instars, which seem to tolerate a low illumination. Moreover, moisture also played a key role and highlighted another major behavioural difference between prepupae and younger instars: while unaffected by low illumination, younger instars would move away from the substratum if the latter was too dry. Their affinity to higher moisture conditions may perhaps be caused by a need for wet decaying food or a higher vulnerability to water loss through evaporation. However, whatever the reason, this characteristic made it necessary to combine at the same time low illumination with high moisture of the substratum in order to obtain an efficient separation of prepupae from both the substratum and the younger instars.H. illucens, which, despite the wide literature on this insect, remained hitherto unexplored. Further assessment on the behavioural responses of black soldier fly larvae will greatly benefit from investigations targeting the life cycle of wild populations, which is still little-known. However, improving the understanding of basic behavioral aspects of these insects proved immediately promising for applicative uses as well. Our results encourage the development and refinement of insect farming systems based on behavioral data, and particularly of self-harvesting methodologies able to exploit insects’ behavioral attitudes to reduce the need for direct human intervention in the rearing processes. The method we developed, which requires very unexpensive tools and no consume of electric energy to be replicated, proved to be highly efficient in its aim to separate H. illucens prepupae from younger instars, and is the first method with such purpose to be tested and described in detail in the publically available scientific literature. As the separation of these different developmental stages appears key to current and future applications of the black soldier fly, it could be easily adopted to optimize small-scale and industrial farming alike with minimal costs.In conclusion, we were able to start defining some key aspects of larval locomotion in


Grassland systems constitute a significant portion of the land area in the United States and as a result harbors significant arthropod biodiversity. During this time of biodiversity loss around the world, bioinventories of ecologically important habitats serve as important indicators for the effectiveness of conservation efforts. We conducted a bioinventory of the foliar, soil, and dung arthropod communities in 10 cattle pastures located in the southeastern United States during the 2018 grazing season. In sum, 126,251 arthropod specimens were collected. From the foliar community, 13 arthropod orders were observed, with the greatest species richness found in Hymenoptera, Diptera, and Hemiptera. The soil‐dwelling arthropod community contained 18 orders. The three orders comprising the highest species richness were Coleoptera, Diptera, and Hymenoptera. Lastly, 12 arthropod orders were collected from cattle dung, with the greatest species richness found in Coleoptera, Diptera, and Hymenoptera. Herbivores were the most abundant functional guild found in the foliar community, and predators were most abundant in the soil and dung communities. Arthropod pests constituted a small portion of the pasture arthropod communities, with 1.01%, 0.34%, and 0.46% pests found in the foliar, soil, and dung communities, respectively. While bioinventories demand considerable time, energy, and resources to accomplish, the information from these inventories has many uses for conservation efforts, land management recommendations, and the direction of climate change science. We conducted a bioinventory of the foliar, soil, and dung arthropod communities in 10 cattle pastures located in the southeastern United States. In sum, 126,251 specimens were collected, representing 20 orders. Herbivores were the most abundant functional guild found in the foliar community, while predators were most abundant in the soil and dung communities. The transects within a sampling area were parallel to one another and spaced 15.2 m apart.Two sampling areas . Vegetation was swept with a 38‐cm‐diameter net, with 25 sweeps occurring perpendicular to each side of a transect line . All arthropods collected from individual sweep samples were stored in plastic bags containing 3 ml of 70% isopropyl alcohol to preserve and prevent specimens from cannibalism. Samples were kept on ice in the field and returned to the laboratory, where they were stored at −18℃ until arthropod specimens could be separated from loose vegetation in the sample. After which, specimens were preserved in 70% isopropyl alcohol for curation.Foliar‐dwelling arthropods were sampled from pasture foliage at the 22.9‐m mark of each of the three transect lines from the first sampling area in each pasture (n = 8 soil cores/ranch). Dung arthropod community cores were taken from the center of randomly selected dung pats found within the pasture borders (n = 5 dung cores/ranch). Age of dung pats ranged from 2 to 5 days old, as this age of pat has peak arthropod abundance and diversity  was used to collect the soil and dung communities. Cores were extracted at 7.6 and 38.1 m on two of the transect lines for soil arthropod community sampling  beyond the class level, Protura beyond the class level, thrips (Insecta: Thysanoptera) beyond the ordinal level, Symphyla beyond the class level, millipedes (Diplopoda: Julida) beyond the ordinal level, Diplura beyond the family level, nor springtails (Hexapoda: Collembola) beyond the family level. All other specimens were separated to genus or species level. Those for which positive species identifications could not be provided were assigned to a numbered morphospecies. Larvae of holometabolous insects were assigned morphotaxa independent from adult species, and treated as different morphotaxa in all analyses, owing to their discrete differences in ecological function. However, immature hemimetabolous insects were excluded from the data because they are frequently not ecologically distinct from adults and could not be reliably associated with particular adult species. Morphospecies were assigned to functional guilds, based on knowledge and current hypotheses regarding the ecology of these organisms. We recognized nine nonexclusive guilds: predator, parasitoid, pollinator, herbivore, granivore, coprophage, carrion, livestock pest, and other/unknown.Voucher specimens are deposited in the Mark F. Longfellow Ecological Collection and housed at Blue Dasher Farm .33.1In total, 52,128 arthropod individuals were collected from pasture foliage, representing 759 morphospecies from four classes  and 13 orders . A complete list of foliar arthropod specimens and their abundance from this study can be found in the Supporting Information accompanying this article. The five orders that contained the highest number of morphospecies and specimen abundance are listed in Table 3.2A total of 224 soil cores were extracted for this study to examine the soil‐dwelling arthropod community. In sum, 53,292 arthropod individuals were extracted from the soil, representing 436 morphospecies from eight classes  and 18 orders . A complete list of soil arthropod specimens and their abundance from this study can be found in the Supporting Information accompanying this article. The five orders containing the highest number of morphospecies and specimen abundance are listed in Table 3.3A total of 100 dung pats were subjected to core sampling. In sum, 20,831 arthropod individuals, representing 234 morphospecies from six classes  and 12 orders . A complete list of dung arthropod specimens and their abundance from this study can be found in the Supporting Information accompanying this article. The five orders with the highest number of morphospecies and specimen abundance are listed in Table 4Our survey shows an abundant and diverse arthropod community in pastures of the southeastern United States. In total, 126,25 specimens were identified to morphospecies for this survey, making it one of the largest assessments of arthropod diversity in pasture systems in the region. Previous surveys of arthropods in grazing lands of the southeastern United States focused on specific groups of arthropods  or on specific habitats within pastures  Hu, . To our Our bioinventory shows both similarities and differences in community composition compared to previous studies conducted in the region. For example, a survey of dung‐dwelling arthropods in north‐central Florida found the orders with the highest species richness to be Coleoptera (109 species), Diptera (35 species), and Hymenoptera (24 species) Hu, , while oThe assemblage of functional guilds was distinct among the three arthropod communities sampled for this study Figure . To begiDescribing arthropod communities of various habitats and agriculture systems requires further attention in order to provide meaningful data for future studies ; Data curation (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Kelton D. Welch: Formal analysis (supporting); Project administration (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Jonathan G. Lundgren: Conceptualization ; Funding acquisition (lead); Writing‐original draft (supporting); Writing‐review & editing (supporting).Supplementary MaterialClick here for additional data file.


The poor adhesion and chemical and thermal stability of plasmonic nanostructures deposited on solid surfaces are a hindrance to the longevity and long-term development of robust localized surface plasmon resonance (LSPR)-based systems. In this paper, we have deposited gold (Au) nanolayers with thicknesses above the percolation limit over glass substrates and have used a thermal annealing treatment at a temperature above the substrate’s glass transition temperature to promote the dewetting, recrystallization, and thermal embedding of Au nanoparticles (NPs). Due to the partial embedding in glass, the NPs were strongly adherent to the surface of the substrate and were able to resist to the commonly used cleaning procedures and mechanical adhesion tests alike. The reflectivity of the embedded nanostructures was studied and shown to be strongly dependent on the NP size/shape distributions and on the degree of NP embedding. Strong optical scattering bands with increasing width and redshifted LSPR peak position were observed with the Au content. Refractive index sensitivity (RIS) values between 150 and 360 nm/RIU (concerning LSPR band edge shift) or between 32 and 72 nm/RIU (concerning LSPR peak position shift) were obtained for the samples having narrower LSPR extinction bands. These robust LSPR sensors can be used following a simple excitation/detection scheme consisting of a reflectance measurement at a fixed angle and wavelength. Conductive materials in the form of nanoparticles (NPs) are shown to exhibit size-, shape-, and material- dependent properties that are not present in bulk, such as increased chemical reactivity and selectivity  and unusNPs can be produced by chemical or physical means . SputterThree main strategies were suggested for stabilizing plasmonic NPs over surfaces: (i) application of ultrathin inert overlayers encapsulating the NPs ,16,17;  and diffusion  pumps to a base pressure below 5 × 10−4 Pa.Three Au nanolayers of different nominal thickness  were sputter deposited using a pure (99.999%) Au cylindrical target (50.8 mm in diameter by 3 mm in thickness) in a pure Ar (99.999%) atmosphere. The Ar flux in the inlet was set to 30 sccm, corresponding to a constant deposition pressure of 0.3 Pa. The target-to-substrate distance was maintained at 12 cm and the rotation speed of the substrate holder was set to 30 rpm. The glass substrates  were kept at floating potential. A d.c. power supply (Advanced Energy Pinnacle Plus) was used with a constant power of 50 W during deposition. The deposition chamber was pumped with a rotary  during 3 min, followed by rinsing with deionized water.Au NPs were dissolved from the glass substrates by dipping the samples into freshly prepared aqua regia solution  was pressed against the samples’ surface and pulled away. In the case of any poorly adhesive nanolayer or NP configuration, detachment is clearly observable with naked eyes.In order to further test the mechanical robustness of the samples, 1 cm viton balls were adapted into a scratch tester stylus to perform scratch tests using a progressive load setup up to 15 N. The vertical load rate was set to 100 N/min, whereas the horizontal velocity was set to 30 mm/min. All tests were performed on a CSEM Revetest automatic scratch tester. The stability of the optical signal to immersion in liquids was evaluated by comparing reflectance spectrum before and after an ultrasonic cleaning bath in ethanol for 20 min and drying.n = 1.333) and glycerol (n = 1.473) at different ratios . The LSPR sensors were hold horizontally on a homemade sample holder and, prior to each test, an o-ring was placed above the samples surface to confine the variable refractive index liquids. Placing three drops of solution was enough to promote the total wetting of the surface being analyzed from the top side by reflectivity measurements. Between measurements, the samples were thoroughly cleaned up using distilled water baths until no change in the reflectance spectrum was observed. The optical reflectance of all samples was analyzed on a Gretagmacbeth X-Rite ColorEye XTH spectrophotometer.Test solutions with variable refractive index (RI) in the range 1.333 to 1.473 were prepared by mixing distilled water  were evaluated after peak fitting assuming a pseudo-Voigt function. The FWHM of the peaks was corrected by deconvoluting the instrumental line broadening [The structure of the Au NPs/nanolayers was analyzed by X-ray diffraction (XRD) on a high-resolution Philips PANalytical diffractometer with Cu Kαoadening . The cryoadening . The suroadening .Nanometric thin Au layers were deposited over glass slides, using different nominal thicknesses of 4 nm, 8 nm and 12 nm, originating continuous Au nanolayers with varying thicknesses see . The samThe structure of each sample was studied by XRD, before and after each thermal annealing treatment, as shown in The surface morphology of each thermally annealed sample was studied by AFM, before and after Au dissolution in aqua regia solution, in order to infer the size/height of the NPs and their indentation size/depth on the glass surface, respectively. After carefully analyzing the AFM images, a set of statistical data concerning the average NP diameter and height was obtained using Gwyddion software . For eacThe microstructure images shown in The representative 3D microstructure configurations of the NPs over the glass slides were recreated using Wolfram Mathematica software  and are From Just like in previous publications on thermally embedded Au NPs on glass surfaces prepared by evaporation methods ,23,24,25The continued reshaping of Au NPs with annealing time observed in this study contrasts with previous observations of thermally evaporated Au thin films where the NP shape was pretty much unchanging with time after 30 min of thermal annealing at 600–650 °C, while the embedding depth in glass increased ,25. TherThe NP indentation depths increased with both Au content and annealing time. The average NP indentation depth values varied from 14–19 nm, 38–56 nm and 59–81 nm for the 4, 8, and 12 nm Au samples, respectively, with increasing annealing time (corresponding to a ~40% increase from 1 h 30 min to 10 h 30 min), whereas the average NP height values above the glass surface stayed approximately constant at ~12 nm in all samples. These observations, translated into NP thermal embedding percentages, are way larger than the ones observed before in literature for similar Au-glass systems: 54–60%, 76–83% and 83–88% for the 4, 8 and 12 nm Au samples, respectively. Au NP heights of around ~ 20–40 nm and depression depths of ~2.5–7.5 nm were observed by Karakouz et al. , resultiAnother observation we can make is that although the measured average NP diameter and that of their indentations agrees well in the 4 nm Au case , for thThe reflectivity spectra of the as-deposited and annealed samples are shown in Concerning the as-deposited Au nanolayers, the overall reflectivity increased with the Au nanolayer thickness. For all samples the reflectivity spectra showed a stable SPR band minima centered at around 500 nm together with a reflectivity maxima at the higher wavelengths that increased in intensity with the Au nanolayer thickness. The former resonance is an intrinsic light absorption feature present in Au NPs and Au nanolayers alike  ,40. The SPR ≈ 557.9 nm to λSPR ≈ 559.3 nm) due to the increased refractive index surrounding the NPs . This resonance is indicative of embedded ellipsoidal NPs of ~40–100 nm [With the annealing at 630 °C and the formation of large ellipsoidal NPs, typical LSPR scattering bands appeared in the reflectance spectra. Light absorption dominates for NP sizes below 30 nm whereas light scattering becomes important only for NP sizes above 30–40 nm, which is the case with our annealed samples. For the 4 nm Au case, a narrow LSPR scattering band appeared centered at around 558 nm, which slightly red-shifted in position with annealing time  as well as optical stability toward dipping in solvents and drying  .All samples containing thermally embedded NPs passed successfully in the adhesive tape test. In order to further test the mechanical robustness of these samples, 1 cm viton balls were adapted into a scratch tester stylus in order to perform scratch tests using a progressive load setup up to 15 N. No plastic deformation or scratch was observed in any of the samples. This contrasted markedly with the as-deposited samples that were easily scratched. As an example, visual pictures of the scratch tested zones of the as-deposited 8 nm Au sample and of the 1 h 30 min thermally annealed 8 nm Au sample can be found in Additionally, the optical signal of the plasmonic sensors have shown excellent stability towards cyclic cleaning procedures consisting of ultrasonic cleaning bath in ethanol for 20 min and drying, which allows the robust utilization of the sensors in real world applications. An example of the optical signal before and after the RIS experiments using different RI liquids and after additional cleaning procedures can be found in 2O molecules that stay adsorbed at the surface of the nanocomposite, even after the drying procedure (in the case of distilled water baths), provoking a small red-shift of the LSPR extinction peak.Interestingly, after ultrasonic cleaning with ethanol and drying the LSPR extinction peak is slightly blue-shifted. If an additional water bath and drying procedure is performed, the optical signal matches almost perfectly with the original signal. Therefore, this small difference in peak positioning after ethanol bath and after water bath should be related with very small amounts of HUltra-thin Au layers, with thicknesses above the percolation limit, were sputter-deposited on glass substrates and subject to thermal annealing treatments at a temperature above the glass Tg. This process promoted dewetting, recrystallization, and thermal embedding of Au NPs of different average sizes (between 40 and 60 nm) and shapes (ellipsoids of different aspect ratios) in glass. The solid-state dewetting process of our magnetron-sputtered Au nanolayers was slower than the previously observed one in evaporated thin films, allowing for a better control over the final microstructure of the plasmonic nanomaterial. With increasing annealing time, the average NP indentation depth values increased by 14–19 nm, 38–56 nm, and 59–81 nm for the 4, 8, and 12 nm Au samples, respectively, whereas the average NP height values above the glass surface stayed approximately constant at ~12 nm. Moreover, with increasing average Au NP volume, it was observed a change in the preferential embedding orientation of the ellipsoidal NPs, from having its major axis parallel to the glass surface to having its major axis perpendicular to the glass surface. Percentages of NP thermal embedding in glass between 54% and 88% were obtained in this study, by far the largest observed in any Au–glass system.After thermal annealing, the Au-glass samples showed strong optical scattering bands that broadened and redshifted with the Au content. Refractive index sensitivities between 150 and 360 nm/RIU (concerning LSPR band edge shift) or between 32 and 72 nm/RIU (concerning LSPR peak position shift) were obtained for the samples showing narrower LSPR extinction bands. Moreover, the thermally embedded Au NPs were highly stable, resisting both liquid immersion procedures and mechanical adhesion tests, allowing for a full-time cyclic operation of the LSPR sensor in real world applications.