Historic Rebel (HR) cheese is an Italian heritage cheese, produced from raw milk during the summer grazing period in the Alps. The aim of this work was (i) to characterize the cheese microbiota, by 16S rRNA gene amplicons sequencing, and the volatile and non-volatile lipophilic fraction, by Gas Chromatography and Dynamic Headspace Extraction-Gas Chromatography-Mass Spectrometry, and (ii) to evaluate their respective associations. HR cheese was dominated by Firmicutes phylum (99% of the entire abundance). The core microbiota was formed by Streptococcus, Lactobacillus, Lactococcus, Leuconostoc and Pediococcus genera together representing 87.2–99.6% of the total abundance. The polyunsaturated fatty acids composition showed a high PUFA n-3, PUFA n-6 and CLA content, two fold higher than typical plain cheeses, positively correlated with pasture altitude. A complex volatilome was detected, dominated in terms of abundance by ketones, fatty acids and alcohols. Total terpene levels increased at higher altitudes, being the main terpenes compounds α-pinene, camphene and β-pinene. The HR cheese showed a great diversity of bacterial taxa and lipophilic fractions among producers, despite belonging to a small alpine area, revealing a scarce cheese standardization and a chemical fingerprint of a typical mountain cheese produced during the grazing period. A deeper knowledge of the variability of HR cheese due to its composition in microbial community and volatile compounds will be appreciated, in particular, by elite consumers looking for niche products, adding economic value to farming in these alpine areas. This approach has been used in several investigations of the microbial diversity of raw milk cheeses7, influenced by different factors, including animal feeding systems9, food processing environments10, and manufacturing techniques11.Cheese is a complex matrix that reflects the complexity of the environment in which is produced, especially in mountain areas. Microbial communities, as well as pasture grazing, contribute to its quality and physiochemical properties. The number of investigations on dairy product microbiota has increased in the last decade12.Autochthonous bacteria, starter and non-starter lactic acid bacteria (NSLAB), originating from milk autochthonous bacteria and the environment, can modify the physical and chemical properties of cheese, throughout the whole production chain, during manufacture and ripeningHR) is produced in the Orobic Alps , during summer pastures (Jun 1st–Sep 30th) at an altitude from 1500 to 2000 m a.s.l. and above, using raw cow milk  with a supplement of 10–20% of fresh raw goat milk (Orobica breed). HR is a semi-cooked, semi-hard cheese, cylindrically shaped (40–50 cm diameter × 9–12 cm height) with concave sides and sharp edges, weighting 9–20 kg. The paste has a compact structure with sparse partridge-eye holes; the color varies from white to straw yellow according to the degree of ripening, from a minimum of 70 days up to 10 years or more. This cheese was initially called Bitto Storico to differentiate the cheese obtained strictly following the traditional recipe from Bitto DOP cheese, whose disciplinary allows to supplement cow diet with a maximum of 3 kg of dry matter per day, and to transform the milk with the use of indigenous starter cultures. A Consortium for the HR (https://www.formaggiobitto.com/en/) and a Slowfood presidium (https://www.fondazioneslowfood.com/en/slow-food-presidia/heritage-bitto/) were created to support HR production as gastronomic heritage considering the cultural, social, landscape, and eco-system aspects. The current production (as of 2020) is of about 2000 wheels per year.The “Storico Ribelle” cheese .The cheese-making process is linked to ancient local traditions: raw milk is processed in the HR product specification, the high variability of organoleptic characteristics depends only on pasture composition and naturally occurring bacteria.Since silage and diet supplementation, as well as the usage of starter cultures in cheese-making are prohibited by 5. These NSLAB are a significant portion of bacteria in most cheese varieties during ripening and can also be used as microbial fingerprint13. In order to fulfill the nutritional needs of lactating cows, farmers use grazing techniques with herds in rotations from the lowest to the highest points of the alpine pasture following the phenological stage of herbs along the summer, helping fast pastures regeneration and guarantying high quality of grass.The absence of commercial starter cultures promotes the development of a natural cheese microbiota composed of bacteria, yeasts and moulds14. Mountain pastures are also rich in terpenoids that possess an important olfactory impact that contribute to the “typicity” of mountain cheeses, and can also be used as markers of mountain production17. The resulting biodiversity of pastures, together with the microbiota of raw milk and of the environment, can lead to unique—complex—cheeses in terms of flavor and paste structure18. Variation of cheeses produced with the milk from different pastures and flowering periods is highly appreciated in particular by elite consumers.During the summer season, in different areas, pastures are characterized by different fatty acid (FA) composition, as well as by a different terpenoid content. Mountain herbs contain a high level of polyunsaturated FA that are recognized as beneficial for human healthHR cheese production has never been thoroughly assessed. This study aims to: (i) assess the characterization of the microbial variability of HR cheese by Next Generation Sequencing, and (ii) analyze the lipophilic fraction of the HR cheese, produced in different alpine pasture areas, to better characterize this heritage cheese in particular in those aspects linked to its production environment.However, the microbiota involved in 19. Scant information is available using HTS approach in traditional mountain cheese production linked to summer pastures biodiversity, including the Italian context19. For these reasons in this study, HTS and GC techniques were used for the first time as systemic approaches to characterize the biodiversity and richness of bacterial communities, the volatilome, terpenes and fatty acid profiles in HR cheese.With the introduction of HTS techniques, several studies addressed the microbiota characterization of the major PDO cheeses, with the aim to improve their quality, safety and commercial valuesEnterococcus spp. formed the least abundant group among LAB bacteria, ranging from 106 to 107 CFU/g. Leuconostoc spp. and Pediococcus spp. levels ranged from 106 to 108 CFU/g. Lactobacillus spp. counts were from 107 to 108 CFU/g, while Lactococcus spp. levels ranged from 107 to 109 CFU/g, among producers. Finally, the Streptococcus spp. counts were equal to 108 CFU/g, the prevalent group among LAB bacteria.LAB quantification was performed in cheeses samples collected at 101 ± 4 days of ripening. For all the producers, the 20 and by Colombo et al.21, in term of genera identification and quantitation, in Bitto PDO Italian cheese, a cheese produced with similar technology, in the same geographic area and in the same period of production of the Historic Rebel.Our results were in line with the LAB levels reported both by Morandi et al.The % of moisture ranged between 34.30 and 37.36 g/100 g, the % of protein ranged between 23.94 and 25.29 g/100 g and the % of fat between 31.11 and 34.33 g/100 g. No significant differences were detected among producers Table .Table 1PHTS produced a total of 41,427,924 reads , which, after filtering, resulted in 24,388,097 reads . For computational reasons, a random subset of 100,000 reads was extracted from each sample. After de-noising and clustering, a high-confidence dataset of 2375 zOTUs (avg. of 1164 ± 192 zOTUs per sample) were obtained.Historic Rebel cheese-associated microbiota was dominated by members of the Firmicutes phylum which accounted for about 99% of the entire abundance, with a minor presence of Proteobacteria. Carafa et al.12 and Dalmasso et al.6 noticed the same pattern in traditional mountain cheese produced with no starter strains in other alpine areas.HR cheese showed a low diversity, with only 5 genera constituting the vast majority of the community: Streptococcus (mean relative abundance: 76.6%), Lactobacillus (mean rel. ab.: 10.5%), Lactococcus (mean rel. ab.: 5.9%), Leuconostoc (mean rel. ab.: 2.3%) and Pediococcus (mean rel. ab.: 2.1%) together represented an average of 97.2% (range among samples: 87.4–99.8%) of the total abundance. Only a minority of samples showed presence of Escherichia  and Enterococcus    and Leuconostoc (5.4%); PP4 was characterized by a high rel. ab. of Lactococcus (15.4%), as well as the lowest presence of Streptococcus (60.9%); PP5 microbiota, instead, showed the highest rel. ab. of Lactobacillus (19.3%) and a subdominant presence of Lactococcus (2.9%). On the other hand, PP6, PP3 and PP1 samples were all characterized by higher percentages of Streptococcus , associated to Pediococcus in PP6 (5.3%), Leuconostoc in PP3 (3.3%) and Lactobacillus in PP1 (9.9%) , revealed how the cheese from each producer had a peculiar bacterial composition. They differed both on biodiversity , with producer PP4 having the most biodiverse community and PP1 the least, and composition . In particular, similar bacterial profiles were observed for PP2 and PP4, and for PP6 and PP3 , whereas PP5 and PP1 appeared to have a peculiar composition . Taking into account also minor contributors of the microbiota, all GP-PPs were statistically different  .Since med Fig. . SpeciesStreptococcus, S. thermophilus represented most of the total, accounting for > 98% of total rel. ab. of this genus, on average. PP2 and PP4 samples only showed about 0.5% of other (unidentified) Streptococcus. These data confirm the findings of Morandi et al20, who reported the prevalence of S. thermophilus in Bitto DOP along with the presence in low numbers of S. uberis, S. salivarius and S. macedonicus. The presence of S. thermophilus in high numbers is a common trait in cooked cheeses where high temperatures are applied during cheese-making, as in HR cheese, and for this reason, S. thermophilus is generally used as starter. In addition, Cremonesi et al.22 found in milk from Rendena dairy cattle, a local Italian breed, at same farming conditions, a greater quantity of S. thermophilus in the local breed milk than in the mainstream breed Italian Holstein, indicating that the animal genetics may have an influence on the composition of the milk microbiota. The abundance of S. thermophilus in HR cheese could be also due to the use of typical wooden equipment during the cheesemaking process, from milk collection to ripening, with wooden poles, bands and shelves, acting as a possible natural reservoir of S. thermophilus strains of wooden vat origin, as demonstrated in other studies24, providing microbial diversity in dairy traditional products. The acidification of curd during cheese-making process, is probably mainly due to the presence of indigenous S. thermophilus, and is of paramount importance for the HR cheese production, where commercial starter cultures are not allowed. The other LAB species found in the Historic Rebel are almost the same as those found in previous studies regarding Bitto PDO21, confirming the close association between these two products. On the other hand, Pediococcus pentosaceus summed up to 98% rel. ab. of this genus, apart for PP6 and PP5 samples, for which 1.6% and 8.6%, respectively, was due to P. acidilactici. Among Lactobacillus, the majority of species were identified as Lacticaseibacillus casei/paracasei group. , with some notable exceptions: PP5 showed 49.8% of Lactobacillus delbrueckii and 13.7% of Limosilactobacillus fermentum, whereas PP2, PP3 and PP4 were characterized by a somehow higher presence of L. coryniformis . Lactococcus population was dominated by L. lactis with PP5, PP4 and PP2 showing the presence of other unidentifiable species . Finally, among Leuconostoc, PP3 was composed for about 87% by Ln. mesenteroides, whereas PP1, PP5 and PP6 showed a consistent presence of Ln. paramesenteroides ; PP2 and PP4, on the other hand, displayed a majority of reads belonging to Ln. citreum/lactis group.Evaluating species composition within each genus allowed the identification of some peculiar traits of prevalence of certain species in determined PPs. Among S. thermophilus was the dominant species, as expected, with rel. ab. ranging from 60.6 to 85.7%. PP1 was composed quite entirely of S. thermophilus and members of Lact. casei/paracasei group. ; PP2 and PP4 were characterized by a higher presence of L. lactis ; PP5 showed a consistent presence (13.4%) of L. delbrueckii; finally, PP6 was composed for 5.2% of P. pentosaceus (compared to an average of 1.4% on other PP).With regard to species-level composition for the six Pastures areas-Producers (PP) Fig. , on an aCheese volatilome represents the final step of catabolism of sugars, proteins and lipids by endogenous and microbial enzymes. Along with the terpenoid content, volatiles characterize the richness of flavor of mountain cheeses, highly appreciated by consumers.HR cheese were: 8 alcohols, 7 free fatty acids, 4 ketones, 3 esters, and 1 aldehyde , and Phenylethan-1-ol , derived from the catabolism of amino acids leucine and phenylalanine, respectivelyRegarding the volatile metabolome as a whole, the comparison among the volatilomes of the six PPs considered, showed some similarities Fig. . Some prHR volatilome, like in other mountain cheeses, derives, mainly, by the usage of raw milk, and by the complexity of the indigenous microbiota of that particular environment and of the whole cheese-making process, starting from feeding (grazing) till ripening in cellars.The differences in volatilome seem unrelated to the valley  is strongly and positively associated with Lactococcus genus, confirming the results of Pogačić et al.35 and of Gallegos et al.36 where 3-Hydroxybutan-2-one and 3-methylbutan-1-ol were positively correlated to L. rhamnosus, L. paracasei, L. sakei and L. lactis subsp lactis, L. lactis subsp. cremoris, L. paracasei subsp. paracasei. In our study 2-Heptanol, derived by the reduction of the corresponding ketone, showed a strong positive correlation with Lactobacillus as found by Dan et al.37, where this secondary alcohol was detected in samples fermented by different strains of Lactobacillus. Hexanal, a result of lipid oxidation, and its corresponding alcohol 1-hexanol, showed a strong positive correlation with Streptococcus, confirming the findings of Dan et al.38 where this compound was present in milk fermented by S. thermophilus. The only (fatty) acid that showed a significant positive correlation with Leuconostoc genus was propionic acid, derived by carbohydrate metabolism, as demonstrated by Porcellato et al.39 and Østlie et al.40, where Leuconostoc have been shown to dominate the cheese microbiota in the later stages of ripening with added propionic acid bacteria.The volatiles that showed high and significant  Spearman’s correlations with bacterial microbiome composition are reported in Fig. versus summer productions or mountain extensive production versus intensive indoor production46.Among the three principal milk macro-components, protein, sugar and fat, the latter is the most susceptible to changes in composition. The most part of fatty acids, in fact, is not produced de novo in the mammary gland, but derives from the bloodstream, after the ruminal activity on feeding matter. When feeding changes, like along the summer grazing period, a change in fatty acid composition takes place. Several studies were carried out on this aspect, in particular with the aim to compare winter 46, when no maize silage is given to cattle. Mean value for CLA (% FA) obtained in our study is more than twice  among the PPs  of terpenoid moiety found in all the HR cheese samples, is typical of a mountain production during summer grazing period, where these volatile lipophilic compounds are transferred to the milk and finally to the cheese49. α-Pinene, camphene and β-pinene were the most abundant compounds in HR cheeses. Their presence is confirmed in many plants from alpine pasture49.A clear distinctive character between mountain and plain vegetation is the content of terpenoids, an important class of compounds derived by isoprene units emitted by mountain herbs, mainly dicotyledons, in defense to predators. Dicotyledons are the main constituents of summer pasture and, among these, Apiaceae, Lamiaceae, and Asteraceae families contain high levels of terpenoids, while Poaceae along with Fabaceae, mainly constituents of the indoor feeding from plain, do notPs Table . A simil15. Panseri et al.28 in a study comparing cheeses from different producers of PDO Bitto, the neighborly produced cheese, didn’t found such difference. The producers PP3, PP5 and PP6, settled at the highest grazing areas , produce cheeses characterized by the higher levels of total terpene contents. Among PPs, PP5 showed the highest abundance of terpenoids. The terpenoids that best differentiated the PPs were allo-ocimene, α-terpinolene, α-pinene, and δ-3-carene. The latter, especially, was directly related to the altitude of grazing within the same producer PP5  in three different valleys, Gerola valley (VG), Brembana valley (VB), and Bitto di Albaredo valleys (VA), were analyzed. The six producers, two from each valley, corresponded to six different alpine pastures at straight-line distances among them of ≥ 12 km, , identified as PP1 to PP6 (Pasture area-Producer). Three grazing periods (GP), at the beginning , in the middle , and at the end  of the summer production season, from Jun 1st to Sep 30th 2015, were analyzed. For additional information on the experimental design, please, refer to Supplementary Files (Table es Table .HR cheese ripened for 101 ± 4 days (range 93–106) were collected, equally distributed among the six Pasture area—Producers (PP1–PP6) and the three grazing periods (GPI–GPIII). Three cheese replicates from independent edges were collected in each week of sampling. A slice of cheese (approximately 250 g) representative of the whole wheel (from the rid to the center) was obtained by means of a sterile knife. Cheese samples were immediately vacuum-packed in polyethylene bags, stored at − 18 °C, and thawed at 20 °C before analysis. Each cheese slice was grated to obtain a homogeneous sample and the total amount was divided into different portions for the different analyses.A total of 54 samples of Lc. lactis subsp. lactis MF 118, L. delbrueckii subsp. bulgaricus MF117, St. thermophilus MF69A, Ln. mesenteroides MF20, Pd. pentosaceus BT154, E. faecium RAF81 strains were provided by the ISPA-CNR  bacterial collection and they were used for the standard curves.The Real Time PCR approach was used to quantify the LAB in the samples. 50 was used, without the pre-treatment step, to extract the DNA starting from 1 mL of overnight reference cultures (approximately 1–3 × 109 CFU/mL). Then, the DNA was ten-fold diluted and used for the set-up of the standard curve (3 points in triplicates); in each assay a negative control was also included. Each sample was tested in triplicates.The protocol described in Cremonesi et al.https://www.ncbi.nlm.nih.gov/genbank/), the primer pairs specific for Lactobacillus spp. , Lactococcus spp. , Streptococcus spp., , Leuconostoc spp. , Pediococcus spp.  and Enterococcus spp. , and were designed using Primer Express software (version 3.0) running standard settings. The primers were synthesised by Eurofins Genomics GmbH  and were resuspended to a final concentration of 100 pmol/μl in sterile double-distilled water.Based on published 16S DNA sequences available in GenBank database  and 0.5 μM of each primer. The analysis was carried out on QuantStudio6 Flex Real-Time PCR System  with a standard programme (50 °C for 2 min/95 °C* for 10′/40 cycles 95 °C* for 15″ and 60 °C* for 1′).Cheese samples were analyzed for moisture, protein, and fat content, at ARAL  laboratories. Moisture was determined by gravimetric method (ISO 5534:2004), protein was determined as total nitrogen × 6.38 (ISO 8968-1:2014), fat content by Rose-Gottlieb Method (ISO 1735:2005).2HPO4 buffer solution  were added to 5 g of each sample; the sample was, then, mixed for 1 min and 30 s in a Stomacher machine . The DNA was extracted starting from 800 µL of the homogenized sample following the protocol described in Cremonesi et al.50 with some modifications. Briefly, 400 μL of lysis buffer  and 300 μL of binding solution  were added to the sample and vortexed for 30 s to obtain an emulsified solution. Then, the sample was incubated for 5 min at room temperature. After centrifugation for 30 s at 550×g, the supernatant was discarded and the silica-DNA pellet obtained was subsequently washed twice with 500 μL of lysis buffer, twice with 500 μL of washing solution  and once with 500 μL of absolute ethanol. After every washing and vortexing, the silica-DNA pellet was centrifuged for 30 s at 550×g and the supernatant was discarded. The pellet was then vacuum-dried for 10 min. After addition of 100 μL of elution buffer , the silica-DNA pellet was gently vortexed and incubated for 10 min at 65 °C. After a 5 min centrifugation at 550×g, the supernatant containing the DNA was recovered and stored at − 20 °C. DNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer .For the metagenomics analysis, 45 mL of 2% (w/v) K51, targeting the V3–V4 hypervariable region of the 16S rRNA gene. All PCR amplifications were performed by using 1 × HotStarTaq Master Mix  following manufacture instructions, with 0.2 µL of each primer (100 µM) and 2 µL of genomic DNA (5 ng/µL). Amplicon cycling conditions were 15 min at 94 °C, followed by 25 cycles of 1 min at 94 °C, 1 min at 58 °C and 1 min at 72 °C, and finally 7 min at 72 °C. Then amplicons were cleaned-up with Agencourt AMPure XP  following 16S Metagenomic Sequencing Library Preparation protocol  and checked for amplicons size with 2100 Bioanalyzer Instruments . Libraries were prepared by a second PCR amplification step using Nextera XT Index 1 Primers FC-131-1002, following 16S Metagenomic Sequencing Library Preparation protocol. The libraries obtained were quantified by Real Time PCR with KAPA library Quantification Kits  pooled in equimolar proportion and sequenced on a paired 2 × 250 bp run on a Miseq platform (Illumina).Genomic DNA extracted from cheese samples was amplified using primers described by Caporaso et al.52. Results are expressed log10 of the peak area (arbitrary units) of the corresponding selected ion.The volatilome produced by bacteria during cheese ripening was determined by Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (SPME-GC-MS) on 2 g of finely grinded cheese by means of a Combi-Pal automated sampler  coupled to an Agilent 6890 gas chromatograph with an Agilent 5975 mass spectrometric detector (Agilent Technologies) and a polar column . Extraction and separation conditions are described elsewhereg, per 1 h at 40 °C, as described in De Noni and Battelli16. Briefly, terpenoid compounds were extracted from the head space of the melted fat by means of a DHS Master  with 500 mL of high grade purity nitrogen during 18 min, trapped on a Tenax-TA trap (270 mg) at 40 °C, and directly desorbed at 260 °C for 3 min in the injection port of the same gas chromatograph, equipped with the same column, used for volatilome determination17. Results are expressed log10 of the peak area (arbitrary units) of the corresponding selected ion.The terpene fraction was quantified by Dynamic Headspace-Gas Chromatography-Mass Spectrometry (DHS-GC-MS) on 2 mL of melted cheese fat obtained without the use of solvents, by centrifugation at 49,000×45.Fatty acid (FA) profile was determined on the same melted cheese fat obtained without the use of solvent for terpene determination. The FA methyl esters in hexane were injected into a gas chromatograph (GC) Agilent 7890 GC system (Agilent Technologies) equipped with on-column injector, and an FID detector. The separation was performed on a 100% dimethylpolysiloxane column . Derivatization and chromatographic condition were previously describedStatistical analyses for chemical and chromatographic data were performed by using the SAS package v 9.4 . The two-way analysis of variance procedure (PROC GLM) was used to analyze the effects of alpine pasture area, grazing periods and the percentage of goat milk added on the variables of the proximate analysis, volatile organic compound evaluation, terpene and cheese fatty-acid profile. The model included as fixed effects the interaction between alpine area and grazing period and the percentage of goat milk nested in the Pasture area/Producer (PP1–PP6). Altitude was considered as covariate. Results are given as adjusted least squares means ± standard error means (LSM ± SEM).53 , and discarding those of length outside 250–900 bases, as well as non-overlapping sequences. Sequences having more than 25% nucleotides with a Phred score of 3 or less were filtered out using the “split_libraries_fastq.py” utility of the QIIME suite54 (release 1.8.0). Filtered reads were de-duplicated and de-noised creating zero-radius Operational Taxonomic Units (zOTUs) by unoise3 algorithm55 in usearch (v. 11.0.667), discarding those with less than 5 supporting reads, and taxonomically classified against the 13.8 release of the Greengenes bacterial 16S rRNA database (ftp://greengenes.microbio.me/greengenes_release/gg_13_8_otus) by RDP classifier56 at 50% confidence. Taxonomic characterization at species-level was performed by BLAST-aligning all reads belonging to five selected genera  to a custom reference database made up collecting all available reference sequences in NIH-NCBI database (ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/) for a total of 7009 strains belonging to 232 species. Potential matches were filtered to retrieve an unequivocal classification for each read. Additional details on species-level classification can be found in Lactobacillus underwent major re-classification into 25 different genera, including 23 novel57. Taxonomy classification in this paper was performed according to the previous classification due to how reference databases were built. In species-level characterization, however, species have been named, where needed according to the new nomenclature. Sample biodiversity  was estimated according to different microbial diversity metrics . Inter-sample diversity (i.e.: β-diversity) analysis was conducted using both weighted and unweighted Unifrac metrics58 and Principal Coordinates Analysis (PCoA). Data separation was assessed with a permutation test with pseudo F-ratios using “adonis” function from R package59 “vegan” (version 2.0-10). For relative abundance analysis, a Mann–Whitney U test was used. Volatile compounds (VOC) quantities were correlated to bacterial composition by calculating Spearman’s correlation coefficient between normalized VOC quantities (both on sample and on single VOC basis) and bacterial genera present ≥ 1% rel. ab. in at least 1 sample; only correlations with a statistically non-zero coefficient were considered. For each statistical analysis, unless otherwise stated, P-values < 0.05 were considered as significant. All statistical evaluations were performed in Matlab .For microbiota analyses, raw sequencing reads were processed performing a local assembly between two overlapping pairs, generating a single fragment covering the whole V3–V4 amplicon, using PandaSeq softwareSupplementary Figures.Supplementary Tables.Supplementary Information.


Spodoptera frugiperda is a serious pest that causes devastating losses to many major crops, including corn, rice, sugarcane, and peanut. Camptothecin (CPT) is a bioactive secondary metabolite of the woody plant Camptotheca acuminata, which has shown high toxicity to various pests. However, the effect of CPT against S. frugiperda remains unknown.S. frugiperda larvae. Histological and cytological changes were examined in the midgut of larvae fed on an artificial diet supplemented with 1.0 and 5.0 µg/g CPT. The potential molecular mechanism was explored by comparative transcriptomic analyses among midgut samples obtained from larvae under different treatments. A total of 915 and 3560 differentially expressed genes (DEGs) were identified from samples treated with 1.0 and 5.0 µg/g CPT, respectively. Among the identified genes were those encoding detoxification-related proteins and components of peritrophic membrane such as mucins and cuticle proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that part of DEGs were involved in DNA replication, digestion, immunity, endocrine system, and metabolism.In this study, bioassays have been conducted on the growth inhibition of CPT on S. frugiperda and for future studies on potential practical application.Our results provide useful information on the molecular basis for the impact of CPT on The online version contains supplementary material available at 10.1186/s12864-021-07726-8. Spodoptera frugiperda (Lepidoptera: Noctuidae), is an important insect pest worldwide. The insect can feed on at least 353 plant species including major crops such as corn, rice, soybeans, sugar cane, and cotton [S. frugiperda is native to tropical and subtropical regions of the Americas, but has been spread to Africa and Asia in recent years [S. frugiperda include pyrethroids, diacyl hydrazides, diamides, and benzoylureas [The fall armyworm, d cotton –3. S. frnt years . The voroylureas . ExtensiS. frugiperda has been investigated. For example, the botanical insecticide azadirachtin is very toxic to S. frugiperda, with LC50 values 0.59 and 0.46 mg/L for 2nd and 3rd instar larvae, respectively, under 0.3 % azadirachtin emulsifiable concentrate (EC) [Toona ciliata, is toxic to S. frugiperda larvae as well [S. frugiperda larvae, causing reduced larval and pupal viability [Actinostemon concolor, Piper aduncum, and Ruta graveolens has also been tested against S. frugiperda caterpillars [Plants are considered the most abundant natural resource in the world for the identification of chemicals with insecticidal activity , 8. Planate (EC) . Cedrelo as well . The flaiability . The toxrpillars –16.Camptotheca acuminata Decne, is a potent pharmaceutical secondary metabolite with antitumor activities in mammalian cells by targeting intracellular DNA topoisomerase I, resulting in inhibition of nucleic acid synthesis and induction of DNA strand breakage [Drosophila melanogaster, Musca domestica, Mythimna separata, and Spodoptera exigua [Nilaparvata lugens (Ståhl) Brevicoryne brassicae (L.), and Chilo suppressalis  [Tetranychus urticae, Acaphylla theae and Brevipalpus obovatus were sensitive to the aqueous CPT-Na+ solution under laboratory and field conditions [Spodoptera litura, which could be involved in apoptosis induction [S. frugiperda and relevant molecular mechanisms remain to be revealed.Camptothecin (CPT), a pentacyclic quinoline alkaloid isolated from the plant breakage , 18. CPTa exigua , 19, 20. . Due to  . Phytophnditions . The toxnditions . Besidesnduction . While tS. frugiperda. Changes in the weight of S. frugiperda larvae were examined after treatments with different CPT concentrations. Histopathological and ultrastructural changes in the midgut of larvae fed on diets containing 1.0 and 5.0 µg/g CPT, respectively, were examined. In addition, comparative transcriptomic analyses were carried out with different midgut samples from larvae under different treatments. Our results indicated that CPT is a growth inhibitor of S. frugiperda larvae and has the potential as an insecticide for controlling this important insect pest in the field.The objective of this study is to investigate the adverse effect of CPT against S. frugiperda, third-instar larvae were fed on artificial diets containing 0, 1.0, 2.5, 5.0, 10, 20, and 30 µg/g CPT, respectively. The weight of larvae for each sample was recorded on 1, 3, 5, and 7 days after treatments. The average weight of larvae fed on CPT-diets for one day showed no significant difference compared with that of controls. Weight loss was observed in larvae fed on CPT diets for 3, 5, and 7 days  staining. As shown in Fig. de novo assembling of all combined reads. The length of the unigenes ranged from 201 to 28,483 bp, with an average of 765.28 bp. N50 and GC content of the unigenes were 1358 bp and 40.55 %, respectively. Original data were deposited to the SRA database with the accession number of SRP242660. Transcripts assembled by Trinity were submitted to the TSA database with the accession number SUB8976341.Midguts dissected from larvae treated with CPT (1.0 and 5.0 µg/g) for 7 days and control larvae were used for transcriptomic analyses. The number of raw reads from nine libraries ranged from 43,911,148 to 51,395,872. High quality reads ranged from 43,587,740 to 51,021,846 , Swiss-Prot, Protein family (Pfam), Cluster of Orthologous Groups of proteins (COG), Gene Ontology (GO), and KEGG. A total of 26,461 (45.53 %) unigenes were functionally annotated. The number of unigenes matched to NCBI NR, COG, GO, Pfam, Swiss-Prot, and KEGG databases were 24,937 (42.90 %), 22,844 (39.30 %), 18,265 (31.43 %), 16,888 (29.06 %), 16,335 (28.10 %), and 14,110 (24.28 %), respectively  to sequences from S. litura, followed by Helicoverpa armigera , Trichoplusia ni , Heliothis virescens , Eumeta japonica , and C. suppressalis   of annotated The 16,888 annotated unigenes were divided into three categories: biological process, cellular component, and molecular function. The GO terms of binding, catalytic activity, and cellular process were with the most numbers of unigenes, with 9310, 8554, and 6308 in each category, respectively , glutathione S-transferases (GSTs), carboxylesterases (COEs), UDP glucosyltransferases (UGTs), and ATP-binding cassette transporters (ABCs). As shown in Table In addition to DEGs with functions in detoxification, several genes encoding mucins were also expressed differentially among control and treated samples. Mucins are high molecular weight glycoproteins covering the surface of epithelial cells that respond to external environmental stimuli such as infection, dehydration, and physical and chemical injury . Two andThe third major group of DEGs included genes encoding cuticle proteins (CPs), which are indispensable structural components for insect tissues such as cuticle and midgut peritrophic membrane. Specifically, Four genes encoding larval cuticle protein LCP-17 DN2495_c0_g1), cuticle protein 6.4-like (DN2113_c0_g1), cuticle protein CP14.6-like (DN38621_c0_g1) and cuticular protein RR-2 (DN1220_c0_g1) were down-regulated in samples treated with 1.0 µg/g CPT. Interestingly, 26 unigenes encoding cuticle proteins were up-regulated in samples treated with 5.0 µg/g CPT, whereas there were only two cuticle protein-encoding genes that were down-regulated . The enriched GO terms for biological process included “carbohydrate derivative metabolic process”, “aminoglycan metabolic process”, “chitin metabolic process”, “glucosamine-containing compound metabolic process”, and “amino sugar metabolic process”. The enriched GO terms for cell component included “membrance part”, “integral component of membrane” and “intrinsic component of membrane”. The enriched GO terms for molecular function included “oxidoreductase activity”, “transporter activity”, and “transmembrane transporter activity” . The most significantly enriched GO term for biological process was “chitin metabolic process” . The most significantly enriched GO term for cellular component was “extracellular region” . The most significantly enriched GO term for molecular function was “structural constituent of cuticle”  , including “DNA replication” (15 DEGs), “Purine metabolism” (15 DEGs), and “Ribosome biogenesis in eukaryotes” (14 DEGs). KEGG analysis assigned 1401 DEGs between controls and samples treated with 5.0 µg/g CPT to 232 pathways. The most significantly enriched pathways included “Purine metabolism” (42 DEGs), “Ribosome biogenesis in eukaryotes” (41 DEGs), and “Peroxisome” (36 DEGs)  or nucleopolyhedroviruses exists against Trichoplusia ni and S. exigua [S. frugiperda larvae. Therefore, CPT might be used as an independent insecticide for controlling S. frugiperda. Alternatively, CPT may be used with other insecticides for enhanced efficiency. One limitation of CPT as an insecticide is its insolubility in water. More efficient derivatives with improved solubility and hydrophobicity may be developed in the future for pest control.of years . Variousof years –29. Somee insect , 30. CPTe insect . The insus pests . In addi. exigua . Our resTrichoplusia ni and S. exigua larvae, including the loss of the single layer of epithelial cells and the disruption of the peritrophic membrane [S. frugiperda after CPT treatments. These observations are consistent with previous findings in other insects, suggesting that CPT holds the potential as an insecticide for controlling S. frugiperda and other insect pests.The insect midgut is an important organ responsible for food digestion and nutrient absorption , 35. CPTmembrane . In thisS. frugiperda in response to 23 pesticides [S. frugiperda larvae treated with azadirachtin were also initially analyzed [S. frugiperda larvae treated with CPT were analyzed for the first time. Our transcriptomic analyses of midguts from S. frugiperda larvae revealed that the expression levels of a large number of genes were affected by CPT treatments.Recently, transcriptomic analysis has become a routine method to identify the differentially expressed genes in insects in response to toxic compounds . For exasticides , 38. DEGanalyzed . In thisB. mori [S. exigua larval midgut [S. litura larval midguts after being treated with tomatine [S. frugiperda, a GST in Ostrinia furnacalis, Pxae18 and Pxae28 in Plutella xylostella [S. frugiperda larvae after CPT treatments. We speculated that up-regulation of these detoxification genes such as the genes encoding P450s, GSTs, and CarEs could accelerate the removal of CPT, thus reducing the toxicity of this chemical to S. frugiperda. Our results indicated that high doses of CPT induced more detoxification-related genes. The dose-dependent upregulation of these genes further indicates their roles in the detoxification of CPTs in S. frugiperda larvae. Assuming the up-regulated detoxification genes are indeed critical to detoxify CPT, application of CPT together with detoxification enzyme inhibitor(s) might increase the efficacy of CPT as a controlling agent for the S. frugiperda pest.Among the up-regulated genes by CPT are genes involved in detoxification. Metabolic detoxification through the overexpression of metabolic genes is considered one of the main ways to handle toxic insecticides by pests . The insB. mori . Sublethl midgut . Detoxiftomatine . The rollostella –45. HereH. armigera after being treated with abamectin, indoxacarb, and lambda-cyhalothrin [B. mori treated with NaF [S. frugiperda larvae treated with 5.0 µg/g CPT. These up-regulated genes belong to either ABCC or ABCG subfamilies. The ABCC transporters in most insects are composed of full transporters (FTs) and half transporters (HTs), while ABCG transporters are typical HTs, which need to form homo- or heterodimers to perform transport functions [S. frugiperda.ABC transporters are membrane proteins that are divided into eight subfamilies (A to H) based on the conserved nucleotide-binding domain (NBD) and their functions . Three Aalothrin . Five ABwith NaF . In the unctions . There iunctions , 50. TheN. lugens exposed to β-asarone [S. frugiperda larvae treated with 1.0 and 5.0 µg/g CPT, respectively. Two UGT genes were previously found involved in the detoxification of benzoxazinoids from maize in S. frugiperda larvae [UGTs perform diverse functions including substrate detoxification, ecdysteroid metabolism, cuticle formation, pigmentation, and olfaction in insects. Lipophilic compounds could conjugate with UDP-glucose of UGTs and be glycosylated as water-soluble products that can be easily excreted . Multipl-asarone . Ten UGT-asarone . In thisa larvae . These fS. frugiperda, mucins are located in the peritrophic membrane and thus may protect epithelial cells and immobilize proteolytic enzymes [S. frugiperda larvae may have gained increased resistance in this insect to CPT. If that is the case, application of CPT with other compounds that can prevent induction of mucin or cuticle protein genes or both may result in enhanced efficiency of CPT in controlling this insect pest. Furthermore, the expressions of DNA topoisomerases were not changed after CPT treatments. The likely reason is that CPT could affect the activities of topoisomerases, but does not affect the transcription level of topoisomerases.Mucins are highly O-glycosylated proteins that are abundant in salivary glands, the midgut, and malpighian tubules of insects . Mucins  enzymes . Multipl enzymes , 59. In  enzymes . SeveralP-values < 0.05). Innate immune response and immune system processes were enriched  as well. KEGG analyses demonstrated that some pathways associated with Carbohydrate metabolism, Amino acid metabolism, Lipid metabolism, Nucleotide metabolism, Xenobiotics biodegradation, and Metabolism of cofactors and vitamins were enriched significantly, as well as the pathways of Digestive system, Endocrine system, Cell growth and death . These results suggest that CPT inhibited the growth of S. frugiperda larvae by disrupting many digestive and metabolic processes.Carbohydrates, lipids, proteins, nucleotides, chitin, and vitamins are important resources of energy for insect growth and development . Our GO S. frugiperda larvae treated with different concentrations of CPT. Damage in the midgut was found after CPT treatments based on HE staining and TEM observation. Comparative transcriptomic analyses identified a large number of DEGs caused by CPT treatments. Genes involved in detoxification, DNA replication, and structural components of the peritrophic membrane were up-regulated significantly. Our results suggest that CPT exerted its impact on the growth of S. frugiperda larvae potentially by disrupting DNA replication, the digestive system, and other metabolic processes. Our studies provided a foundation for future research on the basic mechanism and potential practical application of CPT on S. frugiperda control.In conclusion, growth inhibition was observed in S. frugiperda was generated from larvae collected from a cornfield in Conghua District, Guangzhou City, Guangdong Province, China. The colony has been maintained on an artificial diet since then. The artificial diet of 1 kg was prepared with 100 g cornflour, 80 g soy flour, 26 g yeast powder, 26 g agar, 8.0 g vitamin C, 2.0 g sorbic acid, 1.0 g choline chloride, 0.2 g inositol, 0.2 g cholesterol, and 900 mL distilled water. Adults were fed on 10 % honey water. Insect cultures were kept in an incubator set at 25 ± 1 °C, 60–70 % relative humidity, and a 16:8 h light: dark cycle.A laboratory colony of CPT was purchased from Selleck Chemicals (USA) and was dissolved in dimethyl sulfoxide (DMSO). Third-instar larvae were selected and fed on artificial diets containing CPT at 1.0, 2.5, 5.0, 10, 20, and 30 µg/g, respectively, for 7 days. Twenty larvae were selected as one sample for the experiment and three biological replicates of each concentration were performed. Weights of insects for each sample were recorded after feeding for 1, 3, 5, and 7 days, respectively.S. frugiperda larvae treated with 1.0 and 5.0 µg/g CPT was dissected and washed with cold phosphate-buffered saline (PBS). Midgut tissues were fixed with 4 % paraformaldehyde  at 4 °C for more than 24 h. Midgut samples were then embedded into paraffin wax and sliced into sections of 4 μm thickness. The sections were fixed onto a glass slide and stained with hematoxylin and eosin solution. Histopathological changes in the midgut of larvae fed on CPT were visualized on a microscope .After seven days of feeding, the midgut of Larval midguts exposed to 1.0 and 5.0 µg/g CPT for 7 days were dissected, washed with cold PBS, and then fixed in glutaraldehyde solution at 4 °C for 4 h. After washing with PBS three times, the tissues were postfixed in 1 % osmic acid solution at room temperature for 2 h and then washed with PBS three times again. The tissues were dehydrated in a graded series of ethanol solutions (50–100 %) and then kept in 100 % acetone. The midguts were then embedded in epoxy resin and sliced into sections of 60–80 nm thickness. After staining with 2 % uranium acetate saturated alcohol solution and 2 % lead citrate solution for 15 min each time, the sections were dried at room temperature overnight. The ultrastructures of larval midgut were observed on an HT7800 TEM .Ten to fifteen larval midgut samples exposed to 1.0 and 5.0 µg/g CPT for 7 days were collected, Total RNA was extracted using TRIzol® Reagent . Potential genomic DNA contaminants were removed using DNase I . The quality and concentration of RNA samples were analyzed on a 2100 Bioanalyser  and quantified on an ND-2000 . cDNA library construction and RNA-seq were conducted at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd.  via a commercial contract. Briefly, mRNA was purified from 5 µg total RNA of each sample using oligo-dT-attached magnetic beads. Purified mRNA was fragmented using a fragmentation buffer and the resulting products were used as templates for double-stranded cDNA synthesis with a SuperScript double-stranded cDNA synthesis kit . The double-stranded cDNA was subjected to end-repair, phosphorylation, and ‘A’ base addition. cDNA fragments of 200–300 bp were selected by 2 % Low Range Ultra Agarose and amplified in PCR reaction with Phusion DNA polymerase  for 15 cycles. Then the products were quantified on TBS380 and sequenced on the Illumina HiSeq 4000 platform .http://hannonlab.cshl.edu/fastx_toolkit/). The reads that contained adaptor contamination, low-quality bases, and undetermined bases in raw reads were removed with SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) under the default parameters. The clean reads were assembled using Trinity (https://github.com/trinityrnaseq/trinityrnaseq) with the parameters of k-mer = 25 and min_kmer_cov = 5. Then the false assembled transcripts include chimera, structural errors, incomplete assembly, base errors, etc. were filtered with TransRate (http://hibberdlab.com/transrate/). Besides, the redundant sequences were identified and removed by CD-HIT (http://weizhongli-lab.org/cd-hit/). Subsequently, the assembly integrity of these transcriptomes was assessed by BUSCO . The transcripts that shared sequence content were clustered and the longest transcript in the cluster was selected as the unigene. Assembled transcripts and unigenes were annotated by searching the NCBI protein non-redundant (NR), Swiss-Port, and Clusters of Orthologous Groups of proteins (COG) databases with DIAMOND v0.8.37.99 software using BLASTX with a threshold E-value of 1 × 10− 5. Besides, the Pfam database was also used for unigenes and transcripts annotation with the software of HMMER3 3.1b2. The function of each transcript was assigned according to the first hit. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using BLAST2GO 2.5.0 (http://www.blast2go.com/b2ghome) and KOBAS 2.1.1  with default parameters, respectively.After sequencing, the base quality, base error rate, and A/T/G/C base content distribution statistics of raw paired-end reads from each sample were evaluated with the software of fastx_toolkit_0.0.14 (http://www.bioconductor.org/packages/2.12/bioc/html/edgeR.html). The criteria for DEGs were |log2Fold change| ≥1 and P-value ≤ 0.05. GO functional enrichment analysis of DEGs was performed via Goatools (https://github.com/tanghaibao/Goatools) with Fisher’s exact test. KEGG pathway enrichment analysis was conducted by KOBAS software (http://kobas.cbi.pku.edu.cn/home.do) with Fisher’s exact test and the pathway enriched with DEGs significantly was identified with the judging indicator of corrected P-value < 0.05.The method of fragments per kilobase of exon per million mapped reads (FRKM) was used to analyze the expression level of each transcript in different samples. Differentially expressed genes (DEGs) were identified using R statistical package software named EdgeR  was used for qRT-PCR in a 10 µL reaction solution on a LighCycler480 II machine . qRT-PCR has proceeded as follows: one cycle of denaturation at 94 °C for 3 min, followed with 45 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 20 s, and elongation at 72 °C for 20 s, followed by a melting curve analysis. Two reference genes RPL3 (AY072287.1) and RPL13 (AF400183.1) were selected for normalization of qRT-PCR results. mRNA levels were analyzed by the 2−△△CT method. Each assay was repeated three times.Two µg total RNA of each sample was used for qRT-PCR cDNA synthesis using a FastKing RT Kit (With gDNase)  following the manufacturer’s instruction. Primers were designed using the Primer 5.0 software and synthesized by Thermo Scientific. Primer sequences were listed in Supplement Table F test and different capital letters in figures indicate significant differences between different doses as determined using ANOVA followed by Duncan’s new multiple range method (DMRT) (p < 0.01) in SPSS 17.0 .Each experiment was conducted with three biological replicates and all data were expressed as the mean ± standard deviation (SD). One-way ANOVA was applied for statistical analysis of single larval weight. Homogeneity of variance test was determined by Additional file 1:


Galdieria sulphuraria (Cyanidiophyceae) is able to grow autotrophically, or mixo- and heterotrophically with 1% glycerol as a carbon source. The alga divides by multiple fission into more than two cells within one cell cycle. The optimal conditions of light, temperature and pH  for the strain Galdieria sulphuraria  Merola 002 were determined as a basis for synchronization experiments. For synchronization, the specific light/dark cycle, 16/8 h was identified as the precondition for investigating the cell cycle. The alga was successfully synchronized and the cell cycle was evaluated. G. sulphuraria attained two commitment points with midpoints at 10 and 13 h of the cell cycle, leading to two nuclear divisions, followed subsequently by division into four daughter cells. The daughter cells stayed in the mother cell wall until the beginning of the next light phase, when they were released. Accumulation of glycogen throughout the cell cycle was also described. The findings presented here bring a new contribution to our general understanding of the cell cycle in cyanidialean red algae, and specifically of the biotechnologically important species G. sulphuraria.The extremophilic unicellular red microalga  Galdieria sulphuraria, belongs to Cyanidiophyceae, the class of primitive rhodophytes, which includes the genera Cyanidioschyzon, Cyanidium and Galdieria [Cyanidioschyzon merolae, G. sulphuraria and Galdieria phlegrea [Galdieria is not only resistant to high levels of metals and rare earths, but it can also accumulate them inside the cells [G. sulphuraria is usually cultivated for several biotechnological applications such as recycling of valuable components and nutrients from low pH wastewaters; production of phycocyanin as a main pigment even under heterotrophic conditions; cultivation for biofuels, due to its high resistance to contamination, even under heterotrophic conditions; production of glycogen; nutritional applications . Unlike sources ,21,22. Oglycerol , which iglycerol ,25. Under plants . Glycoger weight .2 sequestration, and the chemical composition of the algae [G. sulphuraria is 40–42 °C. Although, it can grow over a wide range of temperatures from 20 °C to 56 °C [G. sulphuraria, pH is another important abiotic factor affecting its growth. Being acidophilic, it thrives over a pH range from 0.05–5.0. Changes in pH significantly affect growth and chemical composition, particularly the lipidomic profile, of G. sulphuraria [In autotrophically grown algae, two abiotic factors set the growth rate: light intensity (and duration) ,29,30 anhe algae . Furtherhe algae ,37,38. Ahe algae ,37, althhe algae ,37,39 orhe algae . The optto 56 °C ,18. For phuraria ,23,41.G. sulphuraria divides by multiple fission into four, or up to 32 daughter cells [n daughter cells from a single mother cell within one cell cycle. The cell cycle includes processes of cell growth (growth sequence—G1 phase) and reproduction . The two sequences are connected by cell division. Multiple growth and reproductive sequences overlap in the multiple fission cell cycle. Each growth phase leads to an approximate doubling of cell size, cell mass, bulk RNA, and bulk protein per cell . Douiew, see  and the iew, see . There aiew, see ,45). In iew, see ,45,48). luvialis  or Parackessleri .Cultures of algae are routinely synchronized by alternating light/dark (L/D) regimes, which mimic situation in nature. When grown autotrophically, the cells cannot grow in dark but are able to divide. Therefore, they all start growing exactly at the same life stage and time resulting in a synchronous population .G. sulphuraria, only one species was studied extensively, C. merolae dividing by binary fission with the S and M phases occurring in the dark. It was synchronized successfully by an alternating light/dark (L/D) regime [Porphyridium sp. was studied [Porphyridium purpureum [Porphyridium cruentum [G. sulphuraria remains limited. Recently, G. sulphuraria strain 074W was partially synchronized by a L/D regime of 12/12 h under low light conditions (100 μmol photons m−2 s−1). This kind of treatment has led to the enrichment of dividing cells in the dark period, but the percentage of dividing cells within the population was quite low, 10–15% [While the methodology for synchronization of green algae is well-developed , synchro) regime  and its ) regime ,56,57,58 studied ,60 and surpureum ,62 and Pcruentum . Informa, 10–15% . This trG. sulphuraria. For the mixotrophic regime, glycerol was used as a cheap organic carbon source. We synchronized the culture of G. sulphuraria under optimal growth conditions by L/D cycle and characterized the timing of CP attainment, DNA replication, nuclear and cellular division(s). Furthermore, we analyzed the glycogen accumulation profile of synchronously growing and dividing cultures.In the present study, we followed the effect of different parameters—pH, light intensity, temperature, and trophic regimes—on the growth of Galdieria sulphuraria  Merola, 002 was obtained from the Algal Collection of Dipartimento delle Scienze Biologiche, Section of Plant Biology, University “Federico II” of Naples, Italy. The algae were grown in modified Galdieria-nutrient medium [3, to the following final composition of macroelements (g L−1): 1.31 (NH4)2SO4, 0.27 KH2PO4, 0.25 MgSO4·7H2O, 0.02 C10H12O8N2NaFe, 0.14 CaCl2·2H2O, and microelements diluted 500x from the stock solution (mg L−1): 31 H3BO3, 1.25 CuSO4·5H2O, 22.3 Mn SO4·4H2O, 0.88 (NH4)6Mo7O24·4H2O, 2.87 ZnSO4·7H2O, 1.46 Co(NO3)2·6H2O, 0.014 V2O4(SO4)3·16H2O, 0.3 Na2NO4·7H2O, 1.19 KBr, 0.83 KI, 0.91 CdCl2, 0.78 NiSO4, 0.12 CrO3, 4.74 Al2(SO4)3K2SO4·24H2O  in distilled water, autoclaved for 20 min. Glycerol )  was added as a source of energy and carbon for the mixotrophic cultivations [The unicellular red alga t medium , pH 1.5–−2 s−1. The spectral composition of the light with the main peaks at 540 and 610 nm and smaller peaks at 435 and 490 nm was measured by a BTS256-PAR BiTec Sensor Agriculture Light Meter  and is shown in 2 (2% v/v), at a flow rate of 15 L h−1. The experiments were carried out in a batch culture regime, over a temperature range of 25–40 °C and mean irradiance of 110–750 µmol photons m−2 s−1. All experiments were conducted in triplicate.The cultures were grown in glass cylinders (300 mL) or flat (2.5 L) photobioreactors. The cylinders or photobioreactors were placed in a thermostatic water bath and illuminated from one side by a panel of dimmable fluorescent lamps  allowing adjustment of the incident light intensity from 16 to 780 µmol photons mG. sulphuraria from a 3-week-old agar plate were used to inoculate the batch culture that was to be synchronized. The synchronization itself was carried out by alternating light/dark periods, the lengths of which were chosen according to the growth parameters of the cells. For the first two or three cycles, the culture was observed by light microscopy to set the correct length for both the light and dark periods. The optimal time for darkening the cells was when they started their first protoplast fission. The length of the dark period was chosen to allow all cells of the population to release their daughter cells. Once the culture was synchronous, the length of light and dark periods were kept constant. For the cell cycle experiment, a synchronized culture was diluted at the beginning of the light period to the initial dry matter (DM) concentration of 100 µg mL−1. The light/dark (L/D) cycle under optimal conditions was set as 16/8 h L/D.The cells of For light measurement in the cultivation cylinders, a ULM-500 universal light meter  equipped with a microsphere sensor  was used. During the measurement, the sensor was placed directly inside the suspension in the middle of the cultivation cylinders. For light measurement of the flat photobioreactors, a quantum/radiometer-photometer  equipped with flat quantum sensor was used. The mean irradiance was calculated from the incident and transmitted light intensity according to the Lambert Beer law. The adjustment of irradiance was achieved by dimmable fluorescent tubes.g for 3 min in dried and pre-weighed 5-mL test tubes. The pellet was dried at 105 °C for 12 h and weighed on a Sartorius TE214S-0CE analytical balance  [Dry matter was determined from 5 mL of algal suspension centrifuged at 2100× Germany) . Data in2 (v/v) in air. Under these conditions every cell eventually divided if it had passed the commitment point [−1 of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) in S buffer (0.25% (w/v) sucrose, 1 mM EDTA, 0.6 mM spermidine, 0.05% (v/v) mercaptoethanol, 10 mM Tris.HCl, pH 7.6) according to Zachleder and Cepák [2O) at a final concentration 10 μL of Lugol solution per 1 mL of cell suspension. The sigmoidal commitment curves, nuclear division and cell division curves were obtained by plotting the cumulative percentages as a function of sampling time [To determine when the cells were committed to divide, 10 mL samples were taken every 2 h, incubated in the dark at 40 °C and aerated with 2% COnt point . About 3nt point . For round Cepák  and the ing time ,51.For visualization of nuclear and cell division by confocal microscopy, the cells were stained with SYBR Green according to Vítová, et al. , for det2 slush (CryoAlto 2500). Frozen samples were transferred into a preparative chamber (CryoAlto 2500) where they were fractured with a scalpel at −135 °C. Specimens were either without sublimation or sublimed for a short time . The samples were sputter-coated with gold for 30 s. Samples were examined by JEOL 7401F SEM  at 1.5 kV and at −135 °C using the Everhart—Thornley detector of secondary electrons.For cryo-SEM, the cells were transferred to holes within the SEM holder and the holder was immediately frozen in liquid N260).Total nucleic acids were extracted according to Wanka  as modifw/v) was mixed with the samples of total nucleic acid extracts in the ratio 1:1 and the mixtures in the test tubes were illuminated from two sides with fluorescent lamps . The incident radiation from each side was 150 μmol photons m−2 s−1. After 6 h of illumination at 40 °C, the difference between the A600 and A700 nm was estimated. The RNA content was calculated from the difference between total nucleic acids and DNA content.Light activated reaction of diphenylamine with hydrolyzed DNA, as described by Decallonne and Weyns  was usedThe sediment remaining after nucleic acid extraction was used for protein determination. It was hydrolyzed with 1M NaOH for 1 h at 70 °C. The protein concentration was quantified using Folin and Ciocalteu’s phenol reagent  according to the procedure described by Lowry et al. . The samv/v) H2SO4) were added and stirred. The mixture was incubated in a water bath at 100 °C for 8 min. It was then cooled to 20 °C, and the A625 was measured spectrophotometrically . Calibration was carried out simultaneously using glucose as the standard. The values measured for glucose were multiplied by 0.9 to obtain a calibration curve for glycogen determination.Glycogen was determined according to Brányiková et al. . SamplesG. sulphuraria before synchronization experiments, the strain was cultivated autotrophically under continuous light. In the case of a mixotrophic regime, it was cultivated with the addition of 1% v/v glycerol to evaluate and optimize growth conditions of asynchronous cultures for potential biotechnological applications. Different light intensities, temperatures and pH were tested.To establish the optimal growth conditions for −2 s−1) and four different temperatures  were followed  (3 µg mL−1) at 40 °C and 500 µmol photons m−2 s−1  . A similµg mL−1) E,F, with m−2 s−1 .−2 s−1 were chosen as the optimal conditions for further autotrophic cultivation and synchronization. An irradiance of 500 µmol photons m−2 s−1 was chosen as optimal because it is high enough for good synchronization of algae and concurrently, DM was the second highest in the autotrophic cultures; the highest DM in mixotrophic cultures was at 500 µmol photons m−2 s−1.The temperature of 40 °C and the starting irradiance of 500 µmol photons m−2 s−1 at 40 °C under different trophic regimes for 48 h  was reached at pH 3  and cultivated at a mean irradiance of 500 μmol photons m−2 s−1, 40 °C, pH 3 and L/D cycle 16/8 h for one cell cycle. Once the first cell cycle was completed, the culture was diluted to approximately the same initial concentration of 100 µg mL−1 and followed for another cell cycle in continuous light to let it reach a maximal DM.The established optimal conditions were used as the start point for the synchronization experiments. For the first two or three cycles, the culture was regularly observed by light microscopy to set the correct length for both the light and dark periods. Based on the culture behavior, the L/D cycle was set at 16/8 h, which was then used throughout the experiments. For the experiments, a synchronized culture was diluted to an initial DM of 100 µg mLGrowth of the synchronized culture was followed by analyzing changes in several growth parameters: total RNA, total protein, and DM. The total RNA content per cell doubled twice from the starting value in two steps at approximately the 6th and 12th hs A.−1) at the 14th h. The glycogen content increased by about 6 fold and was consumed during the dark phase to a value of 7.1 pg cell−1 (−1) at the end of the cell cycle   . Single ryo-SEM) A,B.Galdieria is well adapted to high temperatures, acidic environment and low light conditions [Galdieria corresponds to the geographic origin of representatives of the genus [Galdieria sulphuraria  Merola 002, originating from Italy, were optimized. Different irradiances of 110- to 750 μmol photons m−2 s−1 were tested under the auto- and mixotrophic regimes . The L/D cycle was established as 16/8 h (G. sulphuraria strain 074W into mostly 4 or 8 and rarely 16 daughter cells [Galdieria species divide into 32 autospores [G. sulphuraria stayed in the mother cell wall through the dark phase (G. sulphuraria strain 074W [To study cell cycle progression, along with determining the optimal growth conditions, it is necessary to synchronize the experimental culture. In this study, the protocol for synchronization of s 16/8 h . Applyiner cells . Other Gtospores  or 16 autospores . The divrk phase F, and weain 074W .G. sulphuraria cultivated under optimal conditions increased total RNA, total protein and DM four-fold in two waves of doublings (C. merolae. Unlike other microalgae, the red algae, including Galdieria, produce glycogen instead of starch as energy and carbon reserves [G. sulphuraria is well-characterized [Galdieria through the cell cycle has not been studied. It can only be examined in parallel with the accumulation of starch through the cell cycle of the model green algae Chlamydomonas reinhardtii, Desmodesmus quadricauda or Parachlorella kessleri grown under optimal conditions. The periodicity of starch content followed the L/D cycling or, in continuous light, starch was consumed during cell division [The synchronous culture of oublings . Such a reserves . The patcterized ,82, and cterized ,83, the division ,36,37,57G. sulphuraria had midpoints at about the 10th and 13th hs of the first cell cycle (C. merolae and C. caldarium were determined with a peak in the 10th h of the cell cycle, even at a lower light intensity [G. sulphuraria was determined to be about 4–7-fold larger than the average daughter cell volume and 4-fold larger for C. caldarium [Cell cycle entry occurs at CP, when a reproductive sequence composed of DNA replication, nuclear and cellular divisions commences. The CPs in the synchronized culture of ntensity , suggestntensity ,29. Jongntensity  also coraldarium .G. sulphuraria affected by different trophic regimes and growth conditions, especially in the context of photo-inhibition under high light intensities, would be a relevant future research direction. For example, photosynthesis of mixotrophic G. sulphuraria was boosted by enhancing carboxylation activity of Rubisco and decreasing photorespiration [G. phlegrea [Evaluation of photosynthetic parameters of piration . Similarphlegrea .G. sulphuraria. All these factors affect its growth rate, production of metabolites or capacity for bioaccumulation [G. sulphuraria reflects progress in our understanding of the cell cycle in red microalgae and the successful achievement of synchronization is an essential precondition for future cell cycle studies.Obtaining an understanding of the optimum temperature, light intensity, pH and cultivation conditions for growth under different trophic regimes, and glycogen accumulation, are important for any further exploitation of the biotechnologically significant alga mulation ,23,85. T


Loss of physical and emotional health due to spinal cord injury (SCI) has been rapidly increasing worldwide. Effective evaluation of the severity of SCI is crucial to its prognosis. Herein, we constructed rat models of SCI with four different degrees of injury , using the surgical approach. Cerebrospinal fluid (CSF), plasma, and spinal cord were sampled at the sub-acute spinal cord (72 h post-injury) from each rat. The LC–MS-based metabolic profiling of these samples was performed according to a universal metabolome standard (UMS). The results demonstrated that 130, 104, and 128 metabolites were significantly altered within the CSF, plasma, and spinal cord samples, respectively. Among them, there were four differential metabolites, including uric acid, phosphorycholine, pyridoxine, and guanidoacetic acid, which were commonly identified within the CSF, plasma, and spinal cord samples. Further pathway analysis of these differential metabolites demonstrated a disturbance in the metabolism of glyoxylate and dicarboxylate and glycine, serine, and threonine which were associated with pathophysiologic consequence of spinal cord injury. In particular, phosphorycholine, pyridoxine, and guanidoacetic acid demonstrated a relationship with SCI severity. Thus, they could be utilized as potential metabolite biomarkers for SCI severity assessment. There is a significant rise in the occurrence of spinal cord injury (SCI) each year due to the fast development of transport, mining, sports, and construction sectors , plasma, and spinal cord from subacute SCI. Changes in metabolites of the CSF, plasma, and spinal cord in respect to different degrees of injury were identified in order to assess potential biomarkers for SCI. With LC–MS, the dataset demonstrated that metabolism of glyoxylate and dicarboxylate, glycine, serine, and threonine metabolism were related to pathophysiologic consequences of SCI. In addition, levels of uric acid, phosphorycholine, pyridoxine, and guanidoacetic acid were significantly altered in the spinal cord, CSF, and plasma samples, which can be prognostic biomarkers for prediction of SCI.Samples from rat spinal cord, CSF, and plasma were analyzed under the same metabolome analysis platform Fig. . The metP < 0.05). Under these criteria, 50 metabolites were identified using the spinal cord tissue samples, which indicated that these metabolites carried out a vital function in SCI. In addition, 48 metabolites were identified in the plasma samples and 46 in the CSF samples. In Fig. In order to investigate the specific metabolites that are associated with SCI, the supervised multivariate analysis PLS-DA, as well as univariate analysis one-way ANOVA, was carried out. The PLS-DA model was able to appropriately categorize all samples across different groups Fig. . Based oThe identified metabolites from the three cohorts were compared. While the spinal cord and CSF exhibited 14 common significantly changed metabolites Table , the spiP < 0.05, using the hypogemeric test. Within the CSF, arginine biosynthesis, alanine, aspartate, and glutamate metabolism and biosynthesis of unsaturated fatty acids pathways were significantly altered, with P = 0.01, 0.02, and 0.04, respectively. Within the plasma and spinal cord tissue, eight and two metabolic pathways were significantly altered, respectively. Among them, the glyoxylate and dicarboxylate metabolic pathway in the spinal cord tissue was most relevant to the SCI response with P = 0.017. Within the plasma, pathways that are associated with amino acid and protein synthesis were affected.The differentially identified metabolites from the different sample types were submitted to KEGG pathway analysis Fig. . The thrHerein, we evaluated metabolic changes of the CSF, plasma, and spinal cord tissue, with respect to SCI. To the best of our best knowledge, this is the first study to investigate metabolite alterations in response to SCI through the use of parallel metabolomic profiling of the CSF, plasma, and spinal cord tissue. The aim of this study is to characterize metabolite changes in these samples, in response to spinal cord injury. Both plasma and CSF samples are an ideal source for the discovery of altered metabolites, which are able to reflect the injury responses within the spinal cord  with spinal cord tissue samples than plasma (five metabolites). Thus, we propose using CSF samples for further spinal cord injury studies. Interestingly, four differentially expressed metabolites, including uric acid, phosphorycholine, pyridoxine, and guanidoacetic acid, were shared among CSF, plasma, and spinal cord. Uric acid, a metabolite that has been shown to have a protective effect on neuronal cells, was augmented in the injured spinal cord . These 24 rats were classified randomly into four groups (6 rats per group), including sham (laminectomy only), light injury, moderate injury, and heavy injury groups . These rats were placed in individual cages at 25 ± 3 °C, with open access to water and food.Fisher  provided the MS-grade acetonitrile, formic acid, methanol, and water. Traceless ammonium formate was bought from Sigma Chemical . Millipore-Q water purification system  was utilized to produce ultra-high purity water. The 2-chlorobenzalanine was acquired from TCI chemicals.Established methods were followed for surgical procedures in order to induce spinal cord injury  MS/MS experiments were carried out using normalized collision energy 30 in order to obtain the ms/ms spectra of metabolites-of-interest. The metabolite identification was achieved by matching the acquired MS/MS spectra with the reference spectra in HMDB and MassBank.Chromatographic separation was conducted on a Dionex Ultimate 3000 system that is endowed with a binary pump, a heated column oven, and a WPS-3000 autosampler. The autosampler temperature was then fixed at 8 °C. Next, 2 μL of each sample was loaded on an ACQUITY UPLC® HSS T3  column retained at a temperature of 40 °C at a flow rate of 0.2 mL/min. A mixture of 5 mM ammonium formate and 0.1% formic acid in the water forms mobile phase A. Similarly, a mixture of 0.1% formic acid and acetonitrile forms mobile phase B. Likewise, 0.1% of formic acid was mixed in water in order to form mobile phase C. However, a mixture of 0.1% formic acid and acetonitrile forms mobile phase D. In a positive mode, a linear gradient was used next: 0 ~ 1 min, 2% D; 1 ~ 9 min, 2% ~ 50% D; 9 ~ 12 min, 50% ~ 98% D; 12 ~ 13.5 min, 98% D; 13.5 ~ 14 min, 98% ~ 2% D; 14 ~ 20 min, 2% D. The gradient that was used in the negative mode was as follows: 0 ~ 1 min, 2% B; 1 ~ 9 min, 2% ~ 50% B; 9 ~ 12 min, 50% ~ 98% B; 12 ~ 13.5 min, 98% B; 13.5 ~ 14 min, 98% ~ 2% B; 14 ~ 20 min, 2% B. The MS acquisition was carried out on a Q-Exactive orbitrap MS. The ESI source parameters were fixed as follows. The spray voltage in negative mode was set at −2.5 kV, while in the positive mode, it was set at 3.8 kV. The auxiliary gas and sheath gas were set at 10 and 30 arbitrary units, while the capillary temperature was kept at 325 °C. The orbitrap mass analyzer scanned over a mass range of m/z, retention time (rt), and peak intensity information of each sample were then exported for further analysis.All the raw LC–MS files were changed to the mzxml format using Proteowizad software  platform . Statistical analysis was conducted by retaining only such LC–MS features, as shared by more than 50% of samples (Cuevas-Delgado et al.


Bacillus species are Gram-positive, aerobic, spore-forming bacteria that are widely spread in soil, dust, and water. One strain, Bacillus sp. strain WL1, was isolated from the surface of the cyanobacterium Nostoc flagelliforme in Yinchuan, Ningxia, China. The draft genome sequence of this strain was 5.62 Mbp. Nostoc flagelliforme is a terrestrial blue-green alga that is distributed throughout the arid and semiarid steppes of the western and northwestern parts of China  solid medium at 37°C. One dominant strain with a uniform shape and size was isolated. The 16S rRNA gene was amplified with the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) as previously described  at −80°C. The strain was streaked onto solid LB medium and cultivated at 37°C. One single Bacillus sp. strain WL1 colony was inoculated into liquid LB medium for shake cultivation overnight at 30°C, and the cell culture was collected by centrifugation for DNA extraction using a DNeasy PowerMax soil kit  . The total number of reads from the paired-end sequencing (2 × 150 bp) was 4,372,767, and a total of 658.84 Mbp of data was obtained. The sequence coverage of this genome was 117×. The draft genome sequences were assembled using SOAPdenovo v2.04 with default parameters, and the resulting assembly was used for further genomic analysis (http://ccb.jhu.edu/software/glimmer/index.shtml) was used for gene prediction. Default parameters were used for all software unless otherwise specified.Germany) . The DNAing 2 × 10 bp was Bacillus sp. strain WL1. The total length of the draft genome sequence is 5,624,893 bp, with a GC content of 35.2%. The length of the largest contig is 226,802 bp, and the N50 contig length is 38,057 bp. A total of 5,697 genes are predicted in the draft genome sequence of Bacillus sp. strain WL1, and the average gene length is 804.9 bp. The gene length composed 81.1% of the whole draft genome sequence. The public version of the draft genome was annotated using PGAP v5.1 through NCBI (B. cereus ATCC 14579.A total of 420 contigs comprised the draft genome assembly of Bacillus sp. strain WL1 was deposited in GenBank under accession number JAGKST000000000, BioProject accession number PRJNA733760, BioSample accession number SAMN19458293, and SRA accession number SRR14099160.The draft genome sequence of


Dear Editor,SupplementaryWe read the recent meta‐analysis by Dai et al. on liver injury caused by oral anticoagulants with great interest.n = 543); a prior diagnosis of liver disease (n = 16); prior liver injury (n = 335) , prior hemorrhagic stroke , and higher levels of creatinine  and urea .Among the 2107 edoxaban users from January 01, 2016 to December 31, 2019 initially identified, 894 patients were excluded for the following reasons: (1) prescriptions of other anticoagulants (The main finding of this study is that there is a low rate of liver injury of 1.5% in patients receiving edoxaban therapy in Hong Kong. This extends previous findings by another local study, which reported an incidence of 1.8% for dabigatran, 2.1% for rivaroxaban, 2.5% for apixaban, with a pooled incidence rate of 2.0% for all three DOACs, as compared to 3.7% for warfarin.None.Appendix S1. Supporting InformationClick here for additional data file.


Lepidium meyenii (maca). A gas chromatographic mass spectroscopy (GC-MS) method was used to determine the levels of three benzyl isothiocyanates and two sterols in maca. Liquid chromatographic separation was achieved on a Waters Acquity UHPLC HSS T3 column  with gradient elution over 15 min. The mobile phase was (B) acetonitrile-(A) 10 mM aqueous ammonium phosphate, and the detection wavelength was 210 nm. The gas chromatographic separation was performed on an SH-Rxi-1 MS column, and the ionization mode was electron ionization (EI). Two methods were confirmed to have desirable precision (RSD < 1.58%), repeatability (RSD < 1.97%), stability (RSD < 1.76%), and good linearity (R2 ≥ 0.999) within the test range. The recoveries were in the range of 96.79–109.99%, with an RSD below 2.39%. We applied the established methods and successfully analyzed 15 compounds in maca processed under different drying conditions, providing a comprehensive reference for maca processing method of development. In summary, this study provided two rapid and effective methods for the quantification of 15 active components, which contributed to the in-depth maca quality control and provided a reference for the development of maca products.In this study, a method using ultrahigh-performance liquid chromatography with photodiode array (UHPLC-PDA) was established and validated for the simultaneous quantification of 10 active components, including eight macamides and two glucosinolates, in  Lepidium meyenii (maca), a well-known nutritious and healthy vegetable, was applied widely in the folk medicine in the South American Andes region, as an enhanced energy agent, improving fertility and sexual function [function . It was function . Variousfunction –6. PlantN-benzyl-hexadecanamide is the active maca ingredient that is responsible for the antifatigue effects [A variety of secondary metabolites, such as alkaloids, glucosinolates and sterols, are thought to be closely related to these unique effects of maca. Among them, a variety of compounds in maca have been identified, including macamides , glucosinolates (benzyl glucosinolate and M-methoxybenzyl glucosinolate), and volatile oils (benzyl isothiocyanate and phenylacetonitrile) –9. Macam effects –12. Furt effects .After being introduced to China as a new health food ingredient, maca has been widely cultivated in Yunnan, Tibet, Xinjiang, and Sichuan . RecentlIn order to ensure the quality and good reputation of maca food and maca product, a systematic raw material quality control is particularly necessary and urgent. The current quality evaluation methods for maca essentially rely on determining the content of a single compound (macamide or glucosinolate), the contents of a few compounds or the total content of a class of compounds as the quality control indicator , 18. TheTo date, a variety of analytical and quantitative methods have been employed for the analysis of maca. UHPLC coupled with PDA, a novel rapid, sensitive and high-resolution technique which has been used to quantify chemical components in several different complex matrices, has been widely applied to analyze and quantify multitarget components in medicinal herbs . GC-MS cLepidium meyenii) plants, and they were sealed with soil after collection and cryopreserved at −27°C.An electronic balance (BSM220.4) was purchased from Shanghai Zhuojing Electronic Technology Co., Ltd. , and an ultrasound machine was obtained from Kunshan Ultrasonic Instruments Co., Ltd. The microtome and drying oven were purchased from Yongkang Bo'ou Hardware Products Co., Ltd.  and the Wujiang Yonglian Mechanical Equipment Factory , respectively. The first batch (6 kg) of fresh maca was collected from Yulong Mountain, Lijiang, Yunnan, China, in mid-February 2019, and the second batch (3 kg) was collected in mid-March 2019. The maca samples were identified by Professor Xue-Yong Wang (Beijing University of Chinese Medicine) as the rhizome of cruciferous maca (N-(m-methoxybenzyl)-octadecadienamide, N-(m-methoxybenzyl)-octadecatrienamide, N-benzyl-9Z,12Z,15Z-octadecatrienamide, N-(3-m-methoxybenzyl)-hexadecanamide, N-benzyl-9Z,12Z-octadecadienamide, N-benzyl-hexadecanamide, N-(m-methoxybenzyl)-9Z-octadecenamide, N-benzyl-heptadecanamide, benzyl glucosinolate, M-methoxybenzyl glucosinolate, benzyl isothiocyanate, 4-methoxybenzyl isothiocyanate, and 3-methoxybenzyl thiocyanate reference standards were purchased from Wuhan Huashite Industrial Biotechnology Development Co., Ltd. . Campesterol and sitosterol were purchased from Nanchang Beta Biotechnology Co., Ltd. . The purity of the above compounds was greater than 98%, meeting requirements for HPLC. The chemical structures of the 15 reference compounds are shown in The μm), and the detection wavelength was 210 nm. The mobile phases consisted of (B) acetonitrile and (A) 10 mM aqueous ammonium phosphate using the following gradient elution: 2%–16% B at 0–6 min, 16%–80% B at 6–8.5 min, 80%–95% B at 8.5–11.5 min, 95% B at 11.5–14 min, and 95%–2% B at 14–15 min. The solvent flow rate was set at 0.3 mL/min. The injection volume was 2 μL. In addition, the column temperature and sample temperature were maintained at 35°C and 4°C, respectively.Analyses were performed by a Waters UHPLC Accuracy system  equipped with a binary solvent system, automatic sample injection, constant-temperature sample manager, photodiode array detector, and Empower 3 chromatography workstation. All separations were performed on a Waters Acquity UHPLC HSST3 column -octadecenamide, N-(3-m-methoxy-benzyl)-hexadecanamide, N-benzyl-hexadecanamide, and N-benzyl-heptadecanamide were accurately weighed 5.86 mg, 7.99 mg, 5.21 mg, and 5.82 mg dissolved in methanol and diluted to 50 mL in a volumetric flask to prepare reference stock solutions with concentrations of 0.117 mg/mL, 0.160 mg/mL, 0.104 mg/mL, and 0.116 mg/mL, respectively. To prepare reference stock solutions with concentrations of 0.179 mg/mL, 0.195 mg/mL, 0.202 mg/mL, 0.152 mg/mL, 0.251 mg/mL, and 0.320 mg/mL, 4.47 mg of N-(m-methoxybenzyl)-octadecadienamide, 4.88 mg of N-(m-methoxybenzyl)-octadecatrienamide, 5.05 mg of N-benzyl-9Z,12Z,15Z-octadecatrienamide, 3.79 mg of N-benzyl-9Z,12Z-octadecadienamide, 6.27 mg of benzyl glucosinolate, and 8.01 mg of M-methoxybenzyl glucosinolate were accurately weighed and dissolved in methanol, and the solutions were brought to 25 mL in a volumetric flask.First, fresh maca samples with no visible damage, decay, or lesions were rinsed with clean water, and the main root and fibrous root were sorted. Next, the washed fresh maca samples were cut into 1-mm-thick maca slices by microtome, weighed, and recorded. Finally, each maca slice was weighed and then dried at room temperature (12–25°C), sampling after 14 d. After getting dried, the maca moisture content was less than 5%.μm filter membrane, and the concentration in the solution was measured.The naturally dried maca samples were crushed into powder by a pulverizer. One gram of accurately weighed powder was mixed with 10 mL of 75% aqueous methanol, in an Erlenmeyer flask, and its weight was recorded. Ultrasonic extraction was performed for 30 min, in a 15 mL Erlenmeyer flask. After that, the sample was cooled at room temperature, and the weight was recorded. Then, 75% aqueous methanol was added to make up the difference in weight. After incubating for 30 sec, the supernatant was filtered through a 0.2 μL of a sample onto an SH-Rxi-1 MS column (30 m × 0.25 mm × 0.25 μm). The carrier gas was helium (purity >99.99%), and the total flow rate was set to 9 mL/min. The column and purge flow rates were set to 1 mL/min and 3 mL/min, respectively. The split ratio was 5 : 1, and the linear speed was 36.5 cm/sec. The program of column temperature was set from 60°C up to 200°C at a rate of 10°C/min and continue up to 280°C with a rate of 30°C/min (keeping for 10 min).Analyses were performed by a gas chromatography mass spectrometry system  with the NIST17 mass spectrometry search database. The analytes were separated by injecting 1 The ionization mode was electron ionization (EI), and the ion source temperature was set at 250°C. Scan was used as the acquisition mode, and the interval was 0.3 sec with a scanning speed of 1666. In addition, the solvent delay was set to 2 min, and NIST 18 was used as the search library.To prepare reference stock solutions with concentrations of 0.457 mg/mL, 0.551 mg/mL, 0.606 mg/mL, 0.501 mg/mL, and 0.522 mg/mL, 4.57 mg of benzyl isothiocyanate, 5.51 mg of 4-methoxybenzyl thiocyanate, 6.06 mg of 3-methoxybenzyl isothiocyanate, 5.01 mg of campesterol, and 5.22 mg of sitosterol were accurately weighed into volumetric flasks, and 10 mL of ethyl acetate was added.μm filter membrane, and the concentration of volatile components in the solution was measured.Approximately 1 g maca powder, which had been dried naturally, was weighed and placed into a 10 mL beaker. Ten milliliters of ethyl acetate was added and the weight was recorded. The mixture was ultrasonically extracted for 60 min, and ethyl acetate was added to make up the difference in weight. The supernatant was removed through a 0.2 100 g maca was placed in a 500 mL round-bottom flask, and 10 volumes of pure water were added. Volatile oil was extracted and collected with a volatile oil extraction device as described in the 2020 edition of the Chinese Pharmacopoeia . The collected volatile oil was blended with 10 mL of ethyl acetate.y-axis) versus the corresponding concentration (x-axis) for each bioactive compound in the corresponding linear range. The limit of detection (LOD) and limit of quantification (LOQ) were determined on the basis of signal-to-noise (S/N) ratios of 3 and 10, respectively.To establish calibration curves, standard stock solutions at a minimum of six concentrations were prepared by serially diluting the mixed standard solution with appropriate volumes of solvent, and all operations were repeated in triplicate. Calibration curves for the 15 bioactive compounds were generated by plotting the average peak area . The repeatability of the method was performed through the extraction and analysis of six replicates of a maca sample obtained. The stability of the samples was checked at predetermined times of 0, 4, 8, 12, 16, 20, 24, and 48 h, and the RSDs were determined for the detected concentrations of each analyte. Method accuracy was tested through spiking experiments. The recovery was calculated using the following equation: recovery = /added amount × 100%.Maca contains two major types of components: liposoluble and water-soluble compounds. To develop an efficient, suitable, and convenient extraction procedure, process parameters, such as the extraction method, extraction time, extraction solvent, and material ratio, were investigated in the article.Currently, there are many methods for the extraction of maca compounds, such as ultrasonic extraction, solvent extraction, and reflux extraction , 23. ComThis study aimed to develop a chromatographic method with simultaneously determining 10 bioactive compounds in maca samples. Methanol and acetonitrile were compared as the organic chromatographic mobile phase. Acetonitrile not only had weak UV absorption at the low wavelengths but also had a weaker effect on baseline fluctuations than methanol. Moreover, acetonitrile could elute both liposoluble and water-soluble compounds. The proportion and composition of mobile phase were investigated, including 0.1% aqueous formic acid, 0.1% aqueous phosphoric acid, 0.1% aqueous acetic acid, and 10 mM aqueous ammonium phosphate. Acetonitrile-10 mM aqueous ammonium phosphate is preferred formula, providing a good resolution. When an Acquity HSS T3 column was used to analyze the maca samples, the chromatographic peaks of each component were well separated, and the peak shapes were sharp. Furthermore, the detection wavelength (scanned from 200 nm to 400 nm) and mobile phase flow rate  were examined, separately. When the detection wavelength was set to 210 nm and the flow rate was set to 0.3 mL/min, the interference from impurity peaks was minimized, as shown in (1) Linearity, LOD, and LOQ. R2 values, and linear ranges for the 10 analytes. Great linear correlations were obtained between the peak areas and concentrations for each analyte. The calibration curves appeared to have good linearity (R2 ≥ 0.999) in the tested concentration ranges. The LODs and LOQs for the 10 bioactive compounds were 0.03–0.11 μg/mL and 0.10–1.48 μg/mL  Precision, Repeatability, Stability, and Recoveries. n = 3) of the 10 bioactive compounds in maca samples were calculated with an external standard method based on their respective calibration curves, as listed in μg/mL, and the LOQs ranged from 0.46 to 0.61 μg/mL  of the 5 bioactive compounds in the maca samples were calculated with an external standard method based on their respective calibration curves, as listed in The variations (RSD) for the intraday and interday analyses were 1.14–1.24% and 1.09–1.21% , respectFirstly, a random sampling of fresh maca slices was taken. The samples were divided into groups and subjected to natural drying (12–25°C) or drying in oven at 40°C, 60°C, or 80°C, and three copies were prepared for each group. The sampling time for different drying methods  were 48 h, 24 h, 8 h, and 14 d, respectively. After getting dried, the maca moisture content was less than 5%. Then, the dried samples were extracted following the procedures described in Sections The contents of 15 compounds in maca dried via different methods are shown in Tables The contents of macamides in fresh maca are low, so traditional processing after harvesting is an important way to enrich the macamide content. Compared with natural drying, drying in an oven at 40°C is much faster and less affected by the external environment. Therefore, drying in an oven at 40°C can be used as a drying method for enrichment of macamides in maca. Glucosinolates mainly exist in plant vacuoles, and their properties are stable. However, when plant tissue is damaged (such as during chewing and backlog), myrosinase is released and hydrolyzes glucosinolates. High temperature decomposes glucosinolates into different chemicals, but also inactivates myrosinase in plants. These results indicated that drying in an oven at 80°C could be the optimum drying method for glucosinolates in maca. Researchers speculate that maca can regulate endocrine functions, improve sexual functions, and increase reproductive abilities without affecting the level of reproductive hormones, which may be due to the phytoestrogen analogs in maca-phytosterols , 26. TheIn this study, two highly sensitive and efficient methods for the detection of 15 active components in maca were established and applied. The method showed good linearity, precision, stability, and recovery for all 15 analytes. In addition, the influence of different drying methods on the contents of the 15 compounds in maca was analyzed. The results showed that the contents of three classes of compounds varied with the drying temperatures. The drying temperature can be optimized according to the characteristics of the chemical components. Consequently, this method can be used to evaluate the quality of medicinal maca and serve as a theoretical reference for the development of medicinal maca products.