CgMYB58 was further identified as a critical R2R3 MYB TF involved in lignin overaccumulation owing to its high transcript levels in Huanong Red-fleshed pummelo  fruits. Transient expression of CgMYB58 led to an increase in the lignin content in the pummelo fruit mesocarp, whereas its stable overexpression significantly promoted lignin accumulation and upregulated 19 lignin biosynthetic genes. Among these genes, CgPAL1, CgPAL2, Cg4CL1, and CgC3H were directly modulated by CgMYB58 through interaction with their promoter regions. Moreover, we showed that juice sac granulation in pummelo fruits could be affected by indole-3-acetic acid (IAA) and abscisic acid (ABA) treatments. In HR pummelo, ABA significantly accelerated this granulation, whereas IAA effectively inhibited this process. Taken together, these results provide novel insight into the lignin accumulation mechanism in citrus fruits. We also revealed the theoretical basis via exogenous IAA application, which repressed the expression of CgMYB58 and its target genes, thus alleviating juice sac granulation in orchards.In citrus, lignin overaccumulation in the juice sac results in granulation and an unpleasant fruit texture and taste. By integrating metabolic phenotyping and transcriptomic analyses, we found 702 differentially expressed genes (DEGs), including 24 transcription factors (TFs), to be significantly correlated with lignin content.  In woody plants, a high lignin content in vascular bundles is a positive trait for woody products; however, lignin accumulation is undesirable in the fruit flesh of crop species, such as loquat (Eriobotrya japonica)6, pear (Pyrus bretschneideri)8, and citrus10. Therefore, understanding the regulatory mechanism underlying lignin biosynthesis and lignin accumulation is of great importance, and such knowledge could potentially be applied to assist fruit breeding programs.Lignin, which normally accumulates in secondary cell walls, has been reported to play a critical role in maintaining mechanical strength and facilitating the transport of water and nutrients in plantsO-methyltransferase (CCoAOMT), cinnamoyl CoA reductase (CCR), caffeic acid O-methyltransferase, cinnamyl alcohol dehydrogenase, and ferulate 5-hydroxylase. Through reactions catalyzed by these enzymes, plants produce three types of lignin monomers, p-hydroxyphenyl (H) monomers, guaiacyl (G) monomers, and syringyl (S) monomers, which are then polymerized by laccases (LACs) or peroxidase11.In plants, the lignin biosynthetic pathway involves a series of enzymes, including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), hydroxy cinnamoyl CoA, shikimate/quinate hydroxy cinnamoyl transferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeoyl CoA 3-Arabidopsis thaliana12, Populus trichocarpa13, and switchgrass (Panicum virgatum)14. In Arabidopsis, AC elements bound specifically by MYBs are widely distributed in the promoters of PAL1, PAL2, 4CL1, 4CL2, HCT, C3H1, CCoAOMT1, CCR1, and CAD52. AtMYB58 and AtMYB85 selectively bind AC elements in the promoter of 4CL1, thereby regulating developmental lignification in vascular tissues16. Evidence has confirmed that MYBs can regulate lignin biosynthetic genes involved in secondary cell wall formation, including PvMYB58/63, PvMYB42/85, and PvMYB4, in switchgrass14. EjMYB1, a homolog of AtMYB58 in loquat, has been reported to modulate lignin biosynthesis during fruit storage5. In the stone cells of pear fruits, PbrMYB169 was found to upregulate lignification by activating the promoters of lignin biosynthetic genes7. In addition to MYBs, other TFs have also been revealed to play important roles in fruit lignin biosynthesis; e.g., EjNAC1, EjAP2-1, and EjHSF3 are involved in lignin formation in loquat18.Transcription factors (TFs) regulating lignin biosynthetic genes have been well studied in various plant species, such as 20, volatile compounds21, alkaloids22, and carotenoids26. However, in citrus fruit juice sacs, overaccumulation of lignin  often occurs28. This granulation causes a “gritty” texture and dryness of the juice sacs, reduces consumer acceptance of the fruits, and is a severe problem in sweet orange (Citrus sinensis)10, Ponkan mandarin (C. reticulata)28 and Guanximiyou pummelo (C. grandis)9. Granulation is affected by disorders involving mineral nutrition, phytohormones, growth temperature, and genetic factors29. Previous findings have revealed that TFs play a crucial role in sweet orange granulation. For example, CsMYB330 and CsMYB85, which are homologs of AtMYB58/AtMYB63 and AtMYB85, respectively, have been reported to interact with CsMYB308 through the binding of the Cs4CL1 promoter30 to regulate juice sac granulation.Citrus fruits, which are the most popular fruits worldwide, provide an assortment of functional components that are beneficial to human health, including flavonoids9. However, our knowledge of the regulation of lignin accumulation and thus granulation is limited in pummelo fruits. In the present study, we investigated juice sac granulation and evaluated lignin accumulation in four pummelo cultivars, Huanong Red-fleshed (HR), Hirado Buntan (HB), Fenghuang (FH), and Kao Pan (KP), throughout different fruit developmental stages. We found that indole-3-acetic acid (IAA)- and abscisic acid (ABA)-responsive CgMYB58 played a critical role in lignin biosynthesis. This TF can interact with the promoter regions of CgPAL1, CgPAL2, Cg4CL1, and CgC3H. These results contribute to our understanding of the genetic mechanism controlling juice sac granulation and provide a potential practical approach to inhibiting this phenomenon in citrus orchards.Among citrus fruits, pummelo fruits are especially prone to granulation during ripening and storageWe investigated juice sac granulation in four pummelo cultivars, HR, HB, FH, and KP, at 205 days post anthesis (DPA) and found that HR exhibited the most severe granulation Fig. . Moreove3 A280 kg−1 at 205 DPA . The lignin content of the juice sacs gradually increased in HR during all developmental stages and the lignin content in HR was much higher than that in KP, FH, and HB at 175 and 205 DPA Fig. . The ligDPA Fig. . Moreove31  at 55, 85, 115, 145, and 205 DPA. The RNA sequencing (RNA-seq) approach produced ~6 Gb of data for each sample and ~92% of the reads were mapped to the pummelo genomeCgPAL1, −2, −3, −4, and −5; CgC4H; Cg4CL1; CgC3H; CgCCoAOMT3 and −4; and CgLAC4, −5, −7, −11, −13, −15, and −22 , and mandarin by two indels  construct. The combined analyses of the levels of CgMYB58 expression and lignin contents in eight CgMYB58-OE lines CC-3′ or 5′-ACCTAAC-3′) were identified in the promoter sequences at −383, −248, and −210 bp from the translational start codon for CgPAL1; at −500 and −295 bp for CgPAL2; at −224 bp for Cg4CL1; and at −1966, −1954, −1415, −457, −405, and −15 bp for CgC3H  to verify these interactions. The purified pMAL-c5x-CgMYB58 protein could directly bind to the 25–27 bp probes containing the AC elements in the promoters of CgPAL1, CgPAL2, Cg4CL1, and CgC3H 36. Several types of TFs, such as MYBs, NACs, and ERFs, have been reported to regulate lignin biosynthesis genes in plants13. In Arabidopsis, AtMYB58 and AtMYB85 selectively bind to AC elements to regulate lignification in vascular tissues16. In this study, by integrating transcriptome and metabolite analyses, we found that CgMYB58 promoted the expression of four upstream core monolignol biosynthetic genes by directly binding to AC elements in the promoters of these genes. A homolog of CgMYB58 in sweet orange, CsMYB330, was also identified as being abundant in granulated flesh, together with its target gene Cs4CL10. Our LUC assays and EMSAs showed that CgMYB58 could regulate not only Cg4CL1 but also three other genes  involved in lignin biosynthesis . In addition, fruits of nine pummelo cultivars at the commercially mature stage (~200 DPA) were also harvested  with a ×0.5 optical adapter. The images were captured and exported using ZEN 2.3 software (Zeiss).The reagents used for paraffin sectioning were analytically pure and purchased from Dingguo, Co. (China). The method was performed according to the methods of Li et al.5 (with minor modifications), the juice sacs  were ground into powder and 8 g  of each sample was homogenized in 15 ml of washing buffer . The mixture was washed on a shaker  at room temperature at 250 r.p.m./min for 30 min and then centrifuged  for 10 min . Finally, the supernatant was discarded. Washing buffer was added three times, as described above. Next, the precipitates were washed with 20 ml of 100% methanol four times. The precipitates were subsequently dried at 60 °C in a vacuum rotary evaporator  overnight. Fifty milligrams (mg) of the lyophilized powder of the juice sac  was dissolved in 5.0 ml of 2.0 M HCl and 0.5 ml of thioglycolic acid. The mixture was then boiled within a water bath at 100 °C  for 8 h, cooled on ice for 5 min, and centrifuged at 8000 × g for 20 min at 4 °C . The precipitates were washed with distilled water three times and dried thereafter at 60 °C in a vacuum rotary evaporator overnight. The sample was resuspended in 1.5 ml of 1.0 M NaOH and subsequently placed on a shaker at 100 r.p.m. at room temperature for 18 h. The solution was centrifuged at 10,000 × g for 20 min. Five hundred milliliters of the supernatant of was transferred to a new tube that contained 0.1 ml of concentrated HCl. The tubes were incubated at 4 °C for 4 h to precipitate the lignin thioglycolic acid, followed by centrifugation at 13,000 × g for 20 min at 4 °C . The precipitate was then dissolved in a 1 : 100 ml volume of 1.0 M NaOH. Absorbance was then measured at 280 nm using ultraviolet spectrophotometry , with 1.0 M NaOH used as a blank. The data were expressed on a fresh weight basis and three biological replicates were used for each sample.According to the methods of Xu et al.40, while RNA from calli was isolated with TRIzol reagent . The RNA from the HR, HB, KP, and FH fruit juice sacs at 55, 85, 115, 145, and 205 DPA was extracted, with three biological replicates included per sample. The following procedures were performed by Novogene Bioinformatics Technology, China. RNA integrity was assessed using a RNA Nano 6000 Assay Kit of a Bioanalyzer 2100 system . A total amount of 3 µg of RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using a NEBNext Ultra RNA Library Prep Kit for Illumina  following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The constructed RNA libraries were sequenced on an Illumina HiSeq 2500 platform in paired-end mode, with a read length of 150 bp.The total RNA from the juice sacs was isolated according to the methods of Liu et al.31. Read counts for each gene in each sample were extracted with the edgeR program. The FPKM of each gene was calculated based on its definition41. Differential expression analysis was performed using DESeq v1.18.0 in R v3.5.142. We then used KOBAS software to test the statistical enrichment of DEGs in KEGG pathways. Gene Ontology (GO) enrichment analysis of DEGs was implemented by GOseq v1.38.0, in which gene length bias was corrected. GO terms with corrected P-values (α = 0.05) were considered significantly enriched for those DEGs43. For correlation analyses, Spearman’s rank correlations of the lignin content and FPKM of all the genes expressed in the KP, HB and HR pummelo juice sacs at the five developmental stages was calculated using the pspearman package. Genes with a correlation coefficient to lignin content ratio higher than 0.75 and a significant P-value (α = 0.05) were selected.Quality control of the raw sequencing data was performed with FastQC. Adapters were removed from reads and the data were cleaned based on phred scores using fastp. The clean data were mapped to the pummelo genome44. The primers designed for qRT-PCR are listed in Supplementary Table 31. The actin gene was used as previously reported26. The qRT-PCR data were analyzed using the 2-ΔCt analysis method.A total of 0.5 μg of RNA was used for cDNA synthesis with a RevertAid First Strand cDNA Synthesis Kit . qRT-PCR was performed with a Roche LightCycler 480 system in conjunction with 23 LightCycler 480 SYBR Green Master Mix , as described by Lu et al.CgMYB58 and the promoter of lignin biosynthetic genes were amplified from the cDNA and DNA of various citrus cultivars. The sequences of the primers used are listed in Supplementary Table The full-length coding DNA sequence (CDS) and 2.1 kb promoter region of https://www.genome.jp/tools-bin/clustalw) was utilized for multiple alignment of the amino acid sequences of CgMYB58 and the corresponding sequences of other species. Conserved domains were extracted using gblocks (http://www.phylogeny.fr/one_task.cgi?task_type=gblocks). MEGA 7.0 was subsequently used to construct a phylogenetic tree according to the maximum likelihood method. There were 1000 bootstrap replications and values higher than 50 for each node are presented in the tree.The online version of ClustalW (CgMYB58 CDS without a stop codon was fused to green fluorescent protein (GFP) within a pM999 vector. Citrus (C. limon Burm “Eureka lemon”) mesophyll protoplasts were extracted carefully for transient transformation. Plasmids of 35S:CgMYB58-GFP and 35S:OsGhd7-RFP (nuclear marker) and pM999 empty vector plasmids were mixed together equally. These plasmid mixtures were then transferred into separate protoplasts. After 24 h, the florescence images were scanned via a confocal laser-scanning microscope .The CgMYB58 overexpression construct (vector: pH7WG2D) and a pH7WG2D empty vector used as a control were introduced into Agrobacterium tumefaciens strain GV3101 separately. The GV3101 cells containing the constructs were suspended in infiltration buffer  at a concentration of OD600 = 0.8, and then injected into the mesocarp of KP and HR pummelo fruits. GFP signals were captured by an inversion fluorescence microscope  equipped with a light source (H-150). Twelve fruits were transformed per treatment.The CgMYB58 was isolated from HR and cloned into a pH7WG2D overexpression vector. The vector was then introduced into the A. tumefaciens strain EHA105 before it was transformed into calli of RM grapefruit (C. paradisi). The citrus callus transformation and growth conditions were performed according to the methods of Li et al.45, with minor modifications. Each transgenic callus was cultured in 500 ml of MT (Murashige and Tucker) media with vitamins , supplemented with 80 μl of 50 mg ml−1 hygromycin B, whereas without hygromycin B was used for RM (the WT control). Each CgMYB58 overexpression line and RM callus were subcultured six to seven times before analysis.The CDS of 44. DNA sequences upstream of the translational start codon were amplified by PCR from genomic DNA of HR to generate 1062, 1101, 1298, and 939 bp promoter fragments for CgPAL1, CgPAL2, Cg4CL1, and CgC3H, respectively. The amplified fragments were subsequently inserted into a pGreenII 0800-LUC reporter vector, which was then introduced into A. tumefaciens GV3101 (pSoup-p19) competent cells. The effector vector was a CgMYB58 overexpression (vector: pH7WG2D) construct, and a pH7WG2D empty vector was used as a control. The constructs were also introduced into GV3101 (pSoup-p19). The GV3101 cells containing effectors and reporters were mixed to a proportion of 4:1 and then injected into leaves of Nicotiana benthamiana. LUC activities were analyzed according to the methods of Lu et al.44 at 2.5 days after injection.The dual LUC transcriptional activity assay procedure was modified from that of a previous study44. The promoter fragments of CgPAL2 and CgC3H were cloned into a pAbAi vector to produce pAbAi-CgPAL2P and pAbAi-CgC3HP bait constructs, respectively. The bait plasmids were then linearized and integrated into Y1H Gold yeast, after which they were selected with synthetic dextrose media lacking uracil. The CgMYB58 coding sequences were ligated into pGADT7 to generate an AD-CgMYB58 construct. pGADT7 empty vectors (AD-pGADT7), serving as negative controls, were then transferred separately into yeast cells containing bait constructs. The transformed yeast cells were diluted with 0, 400×, and 500× dilution series of aureobasidin A (AbA) and dotted on SD plates lacking leucine. The cells grew on both types of media containing prey proteins to allow the interaction of bait sequences.Y1H assays were carried out using a Matchmaker Gold Yeast One-Hybrid system kit  and modified according to the methods of Lu et al.46. pMAL-c5x-CgMYB58  without a stop codon was expressed and purified as described by Lu et al.44. The 25–27 bp probes containing the AC elements in the promoters of CgPAL1, CgPAL2, Cg4CL1, and CgC3H were extracted and used as reference sequences to synthesize probes. 5’-FAM-labeled oligonucleotide probes were synthesized and labeled by Shanghai Sangon Company . The same oligonucleotides without labels were used as cold competitors. To perform binding reactions, the binding solution ), 2 μl (0.5 mg ml−1) of purified maltose binding protein tag fused CgMYB58 and 1 μl of the 5’-FAM-labeled probe (10 μmol l−1) were mixed together and incubated at 4 °C for 45 min. For competition assays, the unlabeled oligonucleotides were incubated with protein and binding buffer at 4 °C for 45 min. Afterward, 1 μl of the 5’-FAM-labeled probe (10 μmol l−1) was added and incubated at 4 °C for 45 min. The samples were then loaded onto a prerun 6% polyacrylamide gel. Electrophoresis was performed at 4 °C using 0.5× Tris-borate-EDTA as an electrophoresis buffer in the dark for 1 h. Gel images were acquired using an Amersham Imager 600 .EMSA assays were performed as described previouslyhttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The juice sacs of the KP, HB, and HR pummelo cultivars were collected at 145 DPA in 2019 and cultured in murashige and skoog media , with IAA and ABA at a final concentration of 0.5 mM. After being cultured for 50 days on the media, the samples were collected for the evaluation of gene expression and lignin content. High-performance liquid chromatography-grade IAA and ABA standards were purchased from Coolaber Science & Technology .The ABA- and auxin-responsive elements of gene promoters were predicted by the PlantCARE online database .Metabolite and gene expression profiles were processed with Excel and GraphPad Prism 7. Heatmaps were processed by R with the gplot and pheatmap packages. Duncan’s multiple comparison test and Student’s Supplementary FiguresSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S6Supplementary Table S4Supplementary Table S8Supplementary Table S5Supplementary Table S7


Sitophilus granarius. Adult contact toxicity was found 24 h after treatment with hop EO (LD50/LD90 13.30/40.23 µg/adult), and its three most abundant components, α-humulene, β-myrcene, and β-caryophyllene ; negligible variations at 48 h, except for α-humulene (LD50/LD90 26.83/49.49 µg/adult), were found. The fumigant toxicity of the EO and terpenes was also checked: in the absence of wheat grains, β-myrcene showed the highest inhalation toxicity (LC50/LC90 72.78/116.92 mg/L air), whereas α-humulene, β-caryophyllene, and the EO induced similar values (LC50/LC90 about 130/200 mg/L air); with the exception for EO, the wheat presence increased (30–50%) LC50/LC90 values. Moreover, EO and terpenes were perceived by insect antennae and elicited repellent activity. Only β-caryophyllene showed an anticholinesterase effect, this suggesting that different mechanisms of action should be responsible for hop EO toxicity. Therefore, hop EO appears suitable for developing control means against this pest.Besides its use in the brewing industry, hop cones appear as a powerful source of biologically active compounds, already checked for their putative anticancer, antimicrobial, and other bioactivities. Conversely, hop use in pest control remains to date under-investigated. Therefore, the biological activity of hop essential oil (EO) and its main constituents was investigated here against  Humulus lupulus (L.), is a flowering and perennial plant belonging to Cannabacea family, whose female flowers, clustered in inflorescences, are used in the brewing process of beer. However, the aerial part of this plant produces several compounds as bitter resins, essential oil (EO), tannins, and terpenes, proved to have biological activities. For instance, xanthohumol was reported to exert anticancer [Sitophilus granarius (L.) , develop within grains, limiting the efficacy of these pesticides and leading to a more intense application [Hop, ticancer ,2, antioticancer , and antticancer  effects;ticancer ,6,7,8; iticancer . Moreoveticancer ,11), theticancer , mites [ticancer , and insticancer ,15. Thisticancer . Howeverticancer . In partticancer  (for othticancer ). Moreovlication . For thelication ,22.S. granarius. Moreover, the electrophysiological and the anticholinesterase activities of the EO and individual compounds were also determined to contribute to the characterization of their bioactivity towards granary weevil adults.Thus, in the present study, the attention is focused on the wild hop of the southern Italian Apennines by investigating the contact and inhalation toxicities and the repellent effects of hop EO and its main components against the granary weevil, 3 37.26%), with a very low content of available phosphorus (P2O5 5.14 mg/kg) for plants. A voucher specimen was deposited in the herbarium at the Department of Agricultural, Environmental, and Food Sciences of the University of Molise. β-Myrcene, α-humulene, and β-caryophyllene  were purchased from Sigma-Aldrich .Aerial parts of hops were collected during the flowering stage in Bojano  at 482 m a.s.l. The area (N 41°47’840” E 14°49’428”) has an average annual rainfall of 700 mm, and the mean annual temperature of 14–15 °C. The soil where hop plants were harvested has a neutral reaction (pH 7.25), a sandy texture , a low organic carbon content (10.7 g/Kg), and a low C/N ratio (5.9). It is also a strongly calcareous soil  closed by a metallic net (1 mm) and maintained in the dark in a climatic chamber set at 25 ± 2 °C and 60 ± 5% r.h. Adults beetles, 2–4 weeks old, were used for the experiments.n = 3) and EO density was 0.875 g/mL.Hop female flowers were oven-dried (36 ± 2 °C for 72 h), ground, and aliquots of powder (50 g) were used to obtain essential oil (EO) by hydro-distillation in a Clevenger-type apparatus for 3 h according to the method recommended in the current European Pharmacopoeia (2010). The oil was dried over anhydrous sodium sulphate and stored at 4 °C in the dark until analyzed and tested. The yield obtained was 0.82 ± 0.13%  and a 1 μL sample was injected in the gas chromatographic system. Three samples were analysed. A 7890B series gas chromatograph (Agilent Technologies) with an Agilent 5977 mass selective detector (MSD) and equipped with a HP-5MS capillary column  was used. The carrier gas was helium at a flow rate of 1.25 mL/min.p > 90%) and wherever possible by comparing retention times and mass spectra with those of commercially available standards. Moreover, a mixture of a continuous series of straight-chain hydrocarbons, C8-C20  was injected into an HP-5MS column under the same conditions previously described for the oil samples to obtain the linear retention indices (RIs). The RIs were calculated according to the RI Van den Dool and Kratz equation [The injection was made in the splitless mode, and the injector temperature was 250 °C. The column oven temperature was initially held at 60 °C, then it was programmed to 220 °C at 3.0 °C/min and to 250 °C at 15.0 °C, with a final holding time of 20 min. Spectra were recorded in the electron impact mode  in the range of 30–550 amu at 2.9 scans/sec. A solvent delay time of 5 min was used to avoid overloading the mass spectrometer with the solvent. The identification of volatile compounds was achieved by comparing mass spectra with those of the data system library  was applied on the pronotum of S. granarius adults in thanatosis through a Hamilton’s syringe . Each dilution was applied on 5 unsexed adults (mean body weight 1.983 ± 0.018 mg) of S. granarius and an equal number of individuals was treated with n-hexane as a control. Insects were confined to each Petri dish within metal rings (Ø 4.0 × 2.5 cm), covered with a metallic net (mesh 1 mm) to prevent insects escape, with 5 wheat kernels and maintained under controlled condition (26 ± 2 °C and 60 ± 5% r.h.) in the dark. Three replicates of each dose and control were set up. The mortality of adults was observed after 24 and 48 h. The percentages of mortalities were transformed to arcsine square-root values to meet assumptions of normality and submitted to analysis of variance (ANOVA). Treatment means were compared and separated by Tukey’s HSD test. Statistical analyses were performed with SPSS  v.23 for Windows .The contact toxicity of hop EO and individual compounds to granary weevil adults was determined using topical application, as reported in previous studies ,25. All 50) and 90 (LD90) values, the confidence upper and lower limits, regression equations, and chi-square (χ2) values were calculated using probit analysis [The Lethal Dose 50  inserted in a Pasteur pipette (15 cm long) after solvent evaporation. Using a disposable syringe, the vapor stimuli (3 cm3) were blown for 1 s into a constant stream of charcoal-filtered humidified air (500 mL/min) flowing in a stainless steel delivery tube (1 cm i. d.) with the outlet positioned ~1 cm from the antenna. Control (10 μL of hexane) and standard  stimuli were also applied at the beginning of the experiment and after each group of 3 test stimuli. The intervals between stimuli were 1 min. Each concentration of the EO and individual compounds was tested on three antennae from different males and females.Hop EO, β-myrcene, α-humulene, and β-caryophyllene solutions  in hexane  were prepared. The test stimulus (10 µL of an EO or compound solution) was adsorbed onto a filter paper (Whatman No. 1) strip . Levene’s test was used to assess the homogeneity of variances.EAG responses were evaluated by measuring the maximum amplitude of negative polarity deflection (-mV) elicited by a stimulus . The ampElectrophorus electricus, SIGMA) were incubated in phosphate buffer  plus 5,5′dithio bis(2-nitrobenzoic) acid , either in the absence or in the presence of different aliquots of EO or pure compounds. The reaction was started by the addition of a saturating concentration (2.5 mM) of acetylthiocholine iodide and the rate of absorbance change was obtained as a tangent to the initial part of the progress curve. Results were expressed as % of the control (reaction rate measured in the absence of plant EO/pure compounds). Data were submitted to ANOVA followed by Tukey’s HSD test for mean comparisons.The anticholinesterase activity of hop EO and its main components was investigated as in Reference  by detecIn order to characterize the wild hop ecotype used in this study, the EO composition was investigated by means of GC-MS analysis. In the EO, 29 constituents were identified, accounting for about 98% of the whole EO . The maiS. granarius adults 24 and 48 h after treatment are reported in 50 and LD90 values of 13.30 and 40.23 µg/adult 24 h after application, slightly decreasing to 11.77 and 36.80 µg/adult after 48 h, respectively  in the presence of grains , β-myrcene , β-caryophyllene , and α-humulene  starting from the first one. Negative and significant RIs, indicating actual repellency, were elicited by EO, α-humulene, β-caryophyllene, β-myrcene at the 0.54, 1.11, 2.26, and 4.00 mg dose, respectively  and dose-dependent EAG responses in both males and females and without significant differences between sexes. The mean EAG response to EO was significantly higher than those to individual compounds at the 0.5 mg , 1 mg , and 2 mg  doses.The sensitivity of granary weevil antennae toward increasing doses of the hop EO and individual compounds is reported in α-humulene, β-caryophyllene, and β-myrcene on AChE were checked  collected from plants growing wild in the Molise Region  mainly contained α-humulene 37.01%), β-myrcene, (26.85%), and β-caryophyllene (13.74%). A predominant presence of these three compounds was recorded in the fresh EOs of a large number of wild and commercially available hop varieties but in very different proportions .01%, β-m,39.Despite the extensive use of hop cones as a bittering agent in the brewing industry and clear evidence on the content of compounds with anticancer, antimicrobial, and other positive health effects, the bioactivity of hop cone EO and their main constituents towards stored-product insect pests was little investigated.Rhyzopertha dominica (F.) and S. granarius [Aedes albopictus (Skuse)  and freshwater snail Physella acuta (Draparnaud)  [Spent hops EO exerted a strong repellent activity against ranarius , whilst hysidae) .50 value (13.30 µg/adult), hop EO was less toxic for adult granary weevils than Carum copticum L. (0.009 µg/mg body wt) and Cuminum cyminum L. (0.016 µg/mg body wt) EOs [Hyptis suaveolens (L.) (LD50 = 0.252 µl/adult) and Hyptis spicigera Lamarck (LD90 = 0.292 µl/adult) [Lavandula angustifolia Miller (LD50 = 58.3 μg/adult) [In this study, good acute contact toxicity was demonstrated for the EO obtained from wild hop cones against the granary weevil adults. In fact, topical application of the EO to adult insects induced high and dose-dependent contact mortality 24 h after treatment. On the basis of the 24 h contact LD wt) EOs  but morel/adult)  and the 50 value (41.87 µg/adult) compared to the other major EO components, β-myrcene (75.91 µg/adult) and β-caryophyllene (138.51 µg/adult). Moreover, 48 h after treatment, the LD50 value of α-humulene markedly decreased (26.83 µg/adult), unlike those of β-myrcene and β-caryophyllene that showed only negligible reductions over time. These results are in line with previous researches showing α-humulene as one of the most active compounds contributing to the high contact toxicity of cinnamon and clove EOs to adult granary weevils [α-humulene, also known as α-caryophyllene, was almost 5-fold higher than that of its β-isomer, β-caryophyllene, further confirming the importance of isomerism on the biological activity of compounds. Molecules with the same chemical composition but different spatial orientation of their substitutes may have totally different interactions with a specific bind site and, therefore, different biological effects [This high contact toxicity of hop EOs may be at least in part attributed to its main component α-humulene. Indeed, this compound showed the lowest 24 h contact LD weevils . It is w effects .50 = 132.41 mg/L air) was lower than those reported for EOs of many other plants against the same insect pests, including, for instance, Mentha spicata L. (about 1 µL/L air) [Artemisia species (about 6 µL/L air) [Foeniculum vulgare Gaertner (27.30 µL/L air) and Satureja hortensis L. (52.96 µL/L air) [Salvia leriifolia (Benth) (79.17 μL/L air) [L. angustifolia (1.6 mg/L air) [The 24 h inhalation toxicity of hop EO to adult granary weevils in the absence of wheat grains (LCL/L air) , some ArL/L air) , FoenicuL/L air) , Salvia L/L air) , L. angug/L air) .S. granarius and, in some cases, even more toxic than the corresponding EO. None of these compounds were identified in detectable amounts in the hop EO tested in this study, which may account for its poor fumigant toxicity compared to EOs of other aromatic plants. This was also confirmed by the low toxicity of its three major components. Even in the absence of wheat grains, β-myrcene, the most abundant monoterpene in the hop EO, showed the lowest 24 h LC50 value (72.78 mg/L air) whereas for the two sesquiterpenes, α-humulene and β-caryophyllene, the LC50 values were higher than 100 mg/L air. Moreover, in the presence of wheat grains, an increase in the LC50 values was observed for the hop EO and individual compounds tested, suggesting the sorption of compounds to the wheat grains or reduced diffusion of the compound through the interstitial spaces of grains [Studies on the toxicity of some essential oils related to their major components showed that the monoterpenes 1,8 cineole ,49, eugef grains ,52.Different from what was recently reported for water and ethanol extracts obtained by hop cones , hop EO The behavioural responses of adult granary weevils to hop EO and their main components were evaluated in two-choice pitfall bioassays in the presence of wheat grains in order to point out possible attractant or repellent effects ,29. All S. granarius antennae, demonstrating the capability of the insect olfactory system to perceive them. An electroantennogram is the summation of receptor potentials evoked by an olfactory stimulus from various olfactory neurons on the insect antennae [Among different stimuli, the EO and α-humulene were the most effective repellents since, compared with the other compounds, they elicited a negative and significant RI at lower doses. In electrophysiological recordings, EO and individual compounds evoked EAG dose-dependent responses in adult antennae . TherefoConsidering the toxicity of hop EO and its major compounds to granary weevils, showed by contact and fumigation tests in this study, we can speculate that the decreasing granary weevil attraction to wheat grains might be due to the need to move away from a source of toxic compounds .Such repellent compounds could be used to treat empty stores to flush out hidden infestations before grain storage, to create protective chemical barriers around cereal bulks to disrupt host location by granary weevil adults, or incorporated into packaging materials to prevent insects from entering packaged cereal products ,60.The protection of stored cereals and derived products with EOs or their active components is one of the most promising research areas for the development of new and sustainable options to be implemented in integrated pest management strategies. However, proper formulations of these substances must be developed to overcome some drawbacks such as the need to guarantee a long-lasting release of the active ingredients at a constant and effective concentration and to limit the impact of their strong odours on food products.S. granarius, which can be in part attributed to α-humulene, β-caryophyllene, and β-myrcene, the three most abundant constituents of the EO. Thus, the high contact and the moderate inhalation toxicities, together with the negligible anticholinesterase of EO and its compounds, makes wild hop an excellent low-cost resource for the production of eco-friendly alternative to synthetic insecticides. In addition, hop EO showed a good repellent activity able to disrupt S. granarius orientation to a very attractive host substrate, indicating also a possible application as an insect behaviour-modifying compound to prevent granary weevil infestation of stored cereals and derived food products. Therefore, the use of hop cones for purposes different from beer production is increasingly gaining interest.This study clearly shows that wild hop EO possesses an interesting biological activity against the pest


Litopenaeus vannamei, including BigPEN, PEN2, PEN3, and PEN4, were significantly induced in hemocytes during the early stage of WSSV infection. Knockdown of each penaeidin in vivo via RNA interference resulted in elevated viral loads and rendered shrimp more susceptible to WSSV, while the survival rate was rescued via the injection of recombinant penaeidins. All penaeidins, except PEN4, were shown to interact with several envelope proteins of WSSV, and all four penaeidins were observed to be located on the outer surface of the WSSV virion. Co-incubation of each recombinant penaeidin with WSSV inhibited virion internalization into hemocytes. More importantly, we found that PEN2 competitively bound to the envelope protein VP24 to release it from polymeric immunoglobulin receptor (pIgR), the cellular receptor required for WSSV infection. Moreover, we also demonstrated that BigPEN was able to bind to VP28 of WSSV, which disrupted the interaction between VP28 and Rab7 – the Rab GTPase that contributes to viral entry by binding with VP28. Taken together, our results demonstrated that penaeidins interact with the envelope proteins of WSSV to block multiple viral infection processes, thereby protecting the host against WSSV.Emerging studies have indicated that some penaeidins restrict virus infection; however, the mechanism(s) involved are poorly understood. In the present study, we uncovered that penaeidins are a novel family of antiviral effectors against white spot syndrome virus (WSSV), which antagonize the envelope proteins to block viral entry. We found that the expression levels of four identified penaeidins from  The host innate immune system plays a significant role in protecting the organism from pathogenic invasion, particularly in invertebrates, which lack adaptive immunity. Antimicrobial peptides (AMPs) establish a first line of defense against a large spectrum of pathogens, such as bacteria, fungi, parasites, and viruses . In geneMarsupenaeus japonicus (MjsvCL) shrimp [Penaeus monodon (PmRab7) shrimp [M. japonicas (MjpIgR) as the transmembrane receptor for WSSV infection, which interacts with the envelope protein VP24 and facilitates viral entry [White spot syndrome virus (WSSV) is an enveloped virus with a dsDNA genome of approximately 300 kbp . WSSV ha) shrimp  and the ) shrimp . Recentlal entry . In addial entry . All staal entry . AMPs aral entry . Thus, aLitopenaeus vannamei, play a significant role in antibacterial immunity [PmPEN5 from P. monodon was significantly induced upon viral infection, suggesting its possible role in shrimp antiviral immunity [Penaeidins, which belong to an AMP family initially characterized in the shrimp immunity . Penaeidimmunity . Based oimmunity . The mRNimmunity . HoweverIn this study, we examined the mechanism of inhibition of WSSV infection by penaeidins. We found that penaeidins have potent anti-WSSV activity. These molecules block WSSV infection by disrupting the interactions of viral envelope proteins with essential host factors, such as VP28–Rab7 and VP24-pIgR, thereby preventing virus-mediated entry into host cells. These studies reveal the antiviral mechanism of penaeidins, which highlight a new antiviral strategy that has the potential to be employed to control WSSV infection.L. vannamei shrimp, excluding the hepatopancreas, were collected on ice. Ten grams of infected tissues were homogenized in 500 ml of TNE buffer  containing a combination of protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, and 1 mM Na2S2O5) and then centrifuged at 5000×g for 5 minutes at 4°C. After filtering with a nylon net (400 mesh), the supernatant was centrifuged at 30,000×g for 30 minutes at 4°C. Then the upper loose pellet was rinsed out carefully, and the lower white pellet was suspended in 10 ml TM buffer . After centrifugation at 5000×g for 5 minutes, the virus particles were sedimented by centrifugation at 30,000×g for 20 minutes at 4°C and then resuspended and stored in 1 ml TM buffer containing 0.1% NaN3. The degree of isolated virus purity was evaluated by negative-staining transmission electron microscopy (TEM) .Virions were isolated from WSSV-infected shrimp following a previously published method . The tisL. vannamei (approximately 4–6 g each) were purchased from a local shrimp farm in Zhanjiang, Guangdong Province, China, and cultured in a recirculating water tank system filled with air-pumped sea water with 2.5% salinity at 27°C. The shrimp were fed to satiation three times per day with a commercial diet . Before all experimental treatments, shrimp  were analyzed and confirmed to be free of white spot syndrome virus (WSSV) and V. parahaemolyticus by PCR or RT–PCR methods according to published standard operating procedures [V. parahaemolyticus  were cultured in Luria broth (LB) medium  overnight at 37°C. Bacteria were quantified by counting the colony-forming units (CFU) per milliliter on LB agar plates. The final injection concentration of V. parahaemolyticus was approximately 1 × 105 CFU/50 μl. The WSSV  was extracted from the WSSV-infected shrimp muscle tissue and stored at −80°C. Before injection, muscle tissue was homogenized and prepared as a WSSV inoculum with approximately 1 × 105 copies in 50 μl PBS . In the pathogenic challenge experiments, each shrimp received an intraperitoneal injection of 50 µl WSSV or V. parahaemolyticus solution in the second abdominal segment using a 1-ml syringe.Healthy ocedures . The GraTotal RNA was extracted from different tissues of shrimp using the RNeasy Mini kit . The genomic DNA was extracted from shrimp tissues using a TIANGEN Marine Animals DNA Kit , according to the manufacturer's instructions. First-strand cDNA synthesis was performed using a cDNA Synthesis Kit , following the manufacturer's instructions.BigPEN was obtained from transcriptomic sequencing of L. vannamei [PEN2 (accession no. DQ206401), PEN3 (accession no. DQ206403), and PEN4 (accession no. DQ206402) was obtained through the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In brief, RACE PCR and nested PCR were performed using a SMARTer RACE cDNA amplification kit  in accordance with the manufacturer's instruction. At the same time, the gene-specific primers were used to amplify PEN2, PEN3, and PEN4 (Supplementary Table 1). The final PCR products of BigPEN, PEN2, PEN3, and PEN4 were cloned into the pMD-19T cloning vector , and 8 positive clones were selected and sequenced.The partial cDNA sequence of vannamei , and itsvannamei . The fulBigPEN, PEN2, PEN3, and PEN4 sequences were translated conceptually, and the deduced protein was predicted using ExPASy (http://cn.expasy.org/). Similarity analysis was conducted using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi/) and the domain architecture prediction of the proteins was performed using SMART (http://smart.emblheidelberg.de). The neighbor-joining (NJ) phylogenic tree was constructed based on the deduced amino acid sequences of penaeidins by utilizing MEGA 5.0 software (http://www.megasoftware.net/download_form) [The ad_form) .in vivo. For the tissue distribution assay, shrimp tissues, including eyestalk, epithelium, pyloric ceca, stomach, gill, heart, hepatopancreases, antenna, intestine, and hemocytes, were sampled. Three samples from each tissue were collected from 15 shrimp (5 shrimp pooled together as a sample). For the pathogenic challenge experiments, shrimp were injected with approximately 1 × 105 CFU of V. parahaemolyticus or approximately 1 × 105 copies of WSSV particles in 50 μl of PBS. In addition, a control group received a PBS injection. Hemocytes of challenged shrimp were collected at 0, 4, 8, 12, 24, 36, and 48 hours post-injection, and 3 samples at each time point were pooled from 9 shrimp (3 shrimp each sample). The method of total RNA extraction, cDNA synthesis, and qRT-PCR analysis was performed as described [2O, 5 μl of 2× SYBR Green Master Mix , and 250 nM of each primer (Supplementary Table 1). The cycling programme was as follows: 95°C for 2 minutes to activate the polymerase, followed by 40 cycles of 95°C for 15 seconds, 62°C for 1 minute, and 70°C for 1 second. The cycling programme ended at 95°C with 5°C/second calefactive velocity to create the melting curve. The expression level of each gene was calculated relative to the internal control gene EF-1α by using the Livak (2−ΔΔCT) method.qRT-PCR was conducted to detect the mRNA levels of penaeidin genes for tissue distribution assays, pathogenic challenge experiments, or silencing efficiency assays by RNAi escribed . In briewsv069 (WSSV32678-F/WSSV32753-R), a WSSV single copy gene, and a TaqMan fluorogenic probe (WSSV32706) as described previously [wsv069 (32678–32753 in the WSSV genome) (accession no. AF332093.2) was obtained and subcloned into the pMD19-T plasmid. The plasmid pMD19-T containing the 675-bp DNA fragment was used as the internal standard and serially diluted 10-fold to generate a standard curve. The extracted shrimp DNA and the internal standard plasmid were subjected to ab-qPCR. The PCR reaction mixture and cycling conditions were the same as previously described [The quantification of WSSV copy number was detected by ab-qPCR. The ab-qPCR was conducted with a forward and reverse primer of eviously . The priescribed . Each saBigPEN, PEN2, PEN3, PEN4, Dorsal (accession no. ACZ98167), Relish (accession no. ABR14713), and GFP (as a control), were generated by in vitro transcription with the T7 RiboMAX Express RNAi System kit  using the primers shown in supplementary Table 1. The quality of dsRNA was checked after annealing via gel electrophoresis. The RNAi assay was performed as we described previously [BigPEN, PEN2, PEN3, PEN4, Dorsal, and Relish. Primer sequences are listed in supplementary Table 1. To investigate the effects of Dorsal or Relish on the expression of penaeidins in vivo after WSSV infection, expression of penaeidins in shrimp after receiving Dorsal dsRNA or Relish dsRNA plus WSSV challenge was detected by qRT-PCR. The mRNA levels of penaeidins were also detected by qRT-PCR with specific primers (Supplementary Table 1).The dsRNAs, including eviously . The expBigPEN-FL , BigPEN-R, and BigPEN-PEN were amplified by PCR using corresponding primers (Supplementary Table 1) and subcloned into pET-32a (+) plasmid . The coding sequences of PEN2, PEN3, and PEN4 without N-terminal signal peptides were also subcloned into pET-32a (+) plasmid with specific primers (Supplementary Table 1). Since pET-32a-PEN2 and pET-32a-PEN4 could not be induced by isopropyl β-D-thiogalactoside (IPTG), PEN2 and PEN4 were subcloned into the pMAL-c2x plasmid  with specific primers (Supplementary Table 1). The coding sequence of VP19 (NP_477936.1), VP24 (NP_477524.1), VP26 (NP_477833.1), VP28 (NP_477943.1), and VP16 (NP_477843.1) was cloned into pGEX-4T-1 plasmid  with specific primers (Supplementary Table 1).The coding sequences of E. coli Rosetta (DE3) cells  for expression. After 4 hours of induction with 0.3 mM IPTG at 37˚C, cells were pelleted by centrifugation and sonicated for 30 minutes on ice water. The supernatant from the sonicated proteins was purified by using Ni-NTA agarose , GST-resin , or Amylose Resin  according to the manufacturer's instructions. The purified proteins were checked by Coomassie staining or western blot. The concentration of the purified proteins was determined using a BCA protein assay kit .The recombinant plasmids of BigPEN-FL, BigPEN-R, BigPEN-PRN, PEN2, PEN3, PEN4, VP19, VP24, VP26, VP28, and VP16 were transformed into in vivo. For the WSSV challenge experiments, shrimp were injected with 105 copies of WSSV particles by intraperitoneal injection or mock-challenged with PBS as a control 48 hours post-dsRNA injection. Then another 48 hours later, muscle tissues from 8 shrimp were collected. Muscle DNA was extracted with a TIANGEN Marine Animals DNA Kit  according to the manufacturer's instructions. The quantities of WSSV genome copies were measured by utilizing ab-PCR as described above. The survival rate of each group was recorded every 4 hours. The Mantel–Cox (log-rank χ2 test) method was used to analyze the differences between groups using GraphPad Prism software .To understand the function of penaeidins during WSSV infection, viral titres and survival rates were analyzed after RNAi-mediated knockdown of each penaeidin in vivo or survival rates after the knockdown of BigPEN, PEN2, PEN3, or PEN4 in shrimp. Each rPenaeidin (10 μg) was first incubated with WSSV for 1 hour, and then the mixture was inoculated into the experimental shrimp. The rMBP and/or rTrx proteins were used as controls. Likewise, viral loads and survival rates were analyzed as above.In parallel, a series of rescue experiments were performed to monitor the effect of rPenaeidins on WSSV replication levels Pull-down assays were performed to explore whether the recombinant full-length BigPEN (rBigPEN-FL), rBigPEN-PEN , rBigPEN-R , rPEN2, rPEN3, or rPEN4 could interact with the main envelope proteins of WSSV . For GST pull-down assays, 100 μl of rBigPEN-FL (1 μg/μl), rBigPEN-PEN (1 μg/μl), rBigPEN-R (1 μg/μl), rPEN2 (1 μg/μl), rPEN3 (1 μg/μl), or rPEN4 (1 μg/μl) was incubated with 100 μl of GST-tagged WSSV protein solutions (1 μg/μl) at 4°C for 2 hours, and then the GST-bind resin was added and incubated for 2 hours at 4°C. The resin was thoroughly washed with PBS. The proteins were eluted with elution buffer  and then analyzed using 12.5% SDS-PAGE and western blot analysis. The empty GST-tag vector was used as control. For His pull-down assays, 100 μl of rBigPEN-FL (1 μg/μl), rBigPEN-PEN (1 μg/μl), rBigPEN-R (1 μg/μl), or rPEN3 (1 μg/μl) was incubated with 100 μl WSSV protein solutions (1 μg/μl) at 4°C for 1 hour, and then the Ni-NTA binding resin was added and incubated for 2 hours at 4°C. The resin was thoroughly washed with PBS. The proteins were eluted with elution buffer  and then analyzed using 12.5% SDS-PAGE and western blot analysis. The Trx vector was used as control. For MBP pull-down assays, 100 μl of rPEN2 and rPEN4 (1 μg/μl) was incubated with 100 μl of WSSV protein solutions (1 μg/μl) at 4°C for 1 hour, and then the MBP-bind resin was added and incubated for 2 hours at 4°C. The resin was thoroughly washed with PBS. The proteins were eluted with elution buffer  and then analyzed using western blot analysis. The MBP-tag empty vector was used as control.E. coli Rosetta (DE3) cells and purified with Amylose Resin  according to the manufacturer's instructions. For GST pull-down assays, 100 μl of rPmRab7 (1 μg/μl) and rLvRab7 (1 μg/μl) was incubated with 100 μl of GST-tagged VP28 protein solutions (1 μg/μl) at 4°C for 1 hour, and 100 μl of rLvpIgR (1 μg/μl) was incubated with 100 μl of GST-tagged VP24 protein solutions (1 μg/μl) at 4°C for 1 hour. The GST-binding resin was then added and incubated for 2 hours at 4°C. The resin was thoroughly washed with PBS. The proteins were eluted with elution buffer  and then analyzed via western-blot. The empty GST-tag vector was used as control. For MBP pull-down assays, 100 μl of rPmRab7 (1 μg/μl) and rLvRab7 (1 μg/μl) was incubated with 100 μl of VP28 at 4°C for 1 hour, and 100 μl of rLvpIgR (1 μg/μl) was incubated with 100 μl of VP24 at 4°C for 1 hour. Then the MBP-bind resin was added and incubated for 2 hours at 4°C. The resin was thoroughly washed with PBS. The proteins were eluted with elution buffer  and then analyzed via western blot. The MBP-tag empty vector was used as control.Pull-down assays were also performed to explore whether recombinant PmRab7 or LvRab7 could interact with VP28, and LvpIgR could interact with VP24. The ORF of PmRab7 was synthesized by a sequencing company  and cloned into the pMAL-c2x plasmid using specific primers. The coding sequences of LvRab7 and LvpIgR were cloned into the pMAL-c2x plasmid with specific primers (Supplementary Table 1). The recombinant plasmids were expressed in The pull-down samples were boiled for 10 minutes, and then the proteins were separated on 12.5% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes . After blocking with 3% non-fat milk diluted in TBST buffer  for 1 hour, the membrane was incubated with mouse anti-6× His, anti-GST, or anti-MBP for 2 hours at 25°C. After washing in TBST buffer, membranes were incubated for 1 hour at 25°C with a rabbit anti-mouse IgG (H+L)-HRP secondary antibody. Both primary and secondary antibodies were incubated in TBST buffer with 0.5% BSA. Membranes were developed using an enhanced chemiluminescent (ECL) blotting substrate , and the chemiluminescent signal was detected using the 5200 Chemiluminescence Imaging System .L. vannamei by centrifugation  at 25°C and deposited onto a glass slide in 12-hole microtiter plates for 30 minutes, and then the above virion suspension was added. Subsequently, the glass slices in the wells were washed with PBS three times and fixed with 4% paraformaldehyde at 25°C for 15 minutes. The hemocytes on the glass slides were washed with PBS three times and blocked with 3% bovine serum albumin (dissolved in PBS) for 60 minutes at 25°C. The hemocytes were then incubated with mouse anti-β-actin antibody serum (1:1000 diluted in 2% BSA)  overnight at 4°C. The hemocytes were then washed with PBS three times and then incubated with anti-mouse IgG (H+L) Alexa Fluor 596  for 60 minutes at 25°C in the dark. After washing with PBS three times, hemocytes were incubated with DAPI 33258 (0.5 mg/ml)  for 10 minutes. Finally, the slices were visualized with a confocal laser scanning microscope . Hemocytes (at least 100 cells/slice) were counted at 1000× magnification [Intact WSSV particles were labelled with fluorescein isothiocyanate (FITC) (1 mg/ml) for 2 hours and then washed with PBS three times. The FITC-labelled WSSV was then mixed with 100 μl of rBigPEN-FL, rBigPEN-PEN, rPEN2, rPEN3, rPEN4, or rTrx (1 mg/ml) and then incubated at 25°C for 1 hour. Hemocytes were collected from healthy fication .g. The supernatant was then removed, and the soft pellet was resuspended in 1.5 ml of PBS containing 0.04% PEG and stored at 4°C. The colloidal gold-labelled penaeidin was diluted 1:10 in PBS containing 0.02% PEG. The empty Trx-His tag protein, BSA, or PBS (as controls) were also labelled with gold nanoparticles. The purified virions were absorbed onto carbon-coated nickel grids and incubated with labelled rBigPEN-FL, rBigPEN-PEN, and rPENs  for 10 minutes at 25°C. After washing with distilled water three times, the samples were counterstained with 2% sodium phosphotungstate for 1 minute and then observed under a transmission electron microscopy .To investigate whether penaeidins can bind to WSSV virions, rPenaeidins were labelled with 10-nm-diameter gold nanoparticles , following a previously reported method . Briefly6 copies/ml). After incubating for 1 hour at 28°C, hemocytes were detected using cytometry for the signals of FITC and the forward scatter (FSC) values of the cells. An FSC threshold was determined using the detection of free FITC-labelled WSSV in order to eliminate cell debris and WSSV virions. The fluorescence boundary was set based on the detection of auto-fluorescence of the untreated hemocytes. A total of 10,000 events were detected for each sample.Hemocytes were collected from shrimp, washed with PBS three times, and counted using a BD FACSCalibur flow cytometry , and then mixed with 100 μg of rBigPEN-FL, rBigPEN-PEN, rPEN2, rPEN3, and rPEN4  together with 100 μl of FITC-labelled WSSV . The ORF of LvpIgR without a stop codon was amplified and cloned into the pcDNA3 plasmid. The primers are listed in supplementary Table 1. HEK293T cells were seeded in a 6-well-plate using Fugene HD Transfection Reagent  according to the manufacturer's instructions. Twenty-four hours later, the FITC-labelled WSSV was then mixed with 100 μl of rPEN2 or rTrx (1 mg/ml) and incubated at 25°C for 2 hours. The above virion suspension was then added into the cells and incubated at 37°C for 1 hour. Subsequently, the cells were extensively washed with PBS twice to remove uninfected virus particles, and DNA of the cells was isolated using a TIANGEN Marine Animals DNA Kit . The isolated DNA was then subjected to qPCR assays to detect the WSSV DNA. The primers used in the qPCR assays are listed in supplementary Table 1.BigPEN was cloned by genome walking PCR amplification via the GenomeWalker Universal Kit  according to our previous paper [The 5′ flanking regulatory regions of us paper . Two paiL. vannamei Dorsal and Relish expression vectors  were obtained from our previous studies [BigPEN, PEN2, PEN3 or PEN4, were cloned using primers (Supplementary Table 1) and then linked into pGL3-Basic  to generate pGL3-BigPEN, pGL3-PEN2, pGL3-PEN3, or pGL3-PEN4, respectively. Two putative NF-κΒ binding sites  within the promoter of BigPEN were predicted by JASPAR database (http://jaspardev.genereg.net/). Overlap extension PCR using primers (Supplementary Table 1) was performed to construct three mutants of pGL3-κΒ12 with deletions at the κΒ1 site, κΒ2 site, or both sites, and named pGL3-κΒ12, pGL3-κΒ-M1, pGL3-κΒ-M2, and pGL3-κΒ-M12.The  studies ,35. The Drosophila Schneider 2 (S2) cell line (ATCC CRL 1963) was used instead in order to detect the effects of L. vannamei NF-κΒ on the promoters of L. vannamei BigPEN, PEN2, PEN3, and PEN4. S2 cells were cultured at 28°C in Schneider's Insect Medium  containing 10% fetal bovine serum . For dual-luciferase reporter assays, S2 cells were plated into a 96-well plate, and 12 hours later, the cells of each well were transfected with 0.05 μg of firefly luciferase reporter gene plasmids, 0.005 μg pRL-TK renilla luciferase plasmid , or 0.05 μg proteins expression plasmids or empty pAc5.1A plasmids (as controls) using the Fugene HD Transfection Reagent  according to the user manual. Forty-eight hours post-transfection, the dual-luciferase reporter assays were performed in order to calculate the relative ratios of firefly and renilla luciferase activities using the Dual-Glo Luciferase Assay System kit , according to the manufacturer's instructions. All experiments were repeated six times.Since no permanent shrimp cell line was available, An EMSA was performed using a Light Shift Chemiluminescent EMSA kit  according to a previously published method . Brieflyt test was used to calculate the comparisons between groups of numerical data. For survival rates, data were subjected to statistical analysis using GraphPad Prism software  to generate the Kaplan–Meier plot (log-rank χ2 test).All data were presented as means ± SD. Student's L. vannamei [L. vannamei shrimp and clustered into three major groups , we observed that the four penaeidins were mainly expressed in hemocytes of naïve (uninfected) shrimp , with a slight upregulation and downregulation, respectively  shrimp C–F, and emocytes G–J. In pectively G and H. nfection I. The trt 36 hpi J. Taken in vivo. We designed and synthesized different dsRNAs, namely dsRNA-BigPEN, dsRNA-PEN2, dsRNA-PEN3, and dsRNA-PEN4, which specifically targeted BigPEN, PEN2, PEN3, and PEN4, respectively. As shown in in vivo RNAi experiments coupled with rPenaeidins were performed. Our results showed that shrimp co-injected with rPenaeidins and dsRNA had significantly reduced viral replication levels as compared to the control group  (P < 0.01)  combined with injection of recombinant penaeidin proteins (rPenaeidins) was performed ol group C. These  < 0.05) E. Nevert < 0.01) D–G. Thisin vivo) in hemocytes during WSSV infection. The result showed that rBigPEN could interact with the VP28 from the hemocyte lysates at 24 hours post WSSV infection . To further verify which domain of BigPEN was able to interact with WSSV envelope proteins, two separate domains, including BigPEN-R and BigPEN-PEN, were used in pull-down assays. In both of the GST pull-down and His pull-down assays, we observed that BigPEN-PEN, but not BigPEN-R (the RPT domain), was able to interact with VP26, VP28, and VP16 . These results strongly suggested that preincubation of penaeidins with WSSV can effectively inhibit virus entry into host hemocytes.Successful infection is required for virus entry into host cells . To explin vitro A. The WSin vitro B. Comparin vitro B. In addP < 0.01)  C. FurtheM. japonicus shrimp is a host entry receptor for WSSV, and the envelope protein VP24 is the receptor-binding protein [L. vannamei (LvpIgR) (accession no. MN164612), resulting in an inhibition of the infection process. To determine this, we first investigated whether LvpIgR served as a host entry receptor for WSSV in L. vannamei shrimp. We cloned the full length of LvpIgR from our transcriptome data and found that LvpIgR was highly homologous to MjpIgR with sequence identity up to 91% (data not shown). MBP-tagged LvpIgR was expressed and purified  from  protein . Thus, wpurified A. Using In the previous result, we found that PEN2 could interact with VP24 H. Next, P < 0.01) (in vitro (P < 0.01)  with ectopic expression of LvpIgR. The ORF of LvpIgR without a stop codon was constructed in the plasmid pcDNA3 (Invitrogen) to generate pcDNA3-pIgR that was used to express V5-tagged LvpIgR in HEK293T cells F1. The c < 0.01) F3. To fuin vitro G. The WS < 0.01) H. Taken P. monodon Rab7  has been shown to interact with WSSV protein VP28, which is beneficial for WSSV infection [L. vannamei Rab7  protein and found that LvRab7 shares 100% sequence identity to PmRab7 (data not shown), which suggests that LvRab7 might also be able to interact with VP28. To address this, MBP-tagged PmRab7 (as a control) and LvRab7 were expressed and purified , the downstream transcription factors of Toll and IMD signalling pathways, respectively, were regarded to be the major factors that directly induce the production of AMPs in response to infection [in vivo experiment was performed. By quantitative RT–PCR analysis, the mRNA levels of Dorsal and Relish were effectively suppressed by corresponding dsRNAs (P < 0.01)   were performed. We first obtained the promoter regions of the four penaeidins, including BigPEN, PEN2, PEN3, and PEN4, by a genome walking method (Supplementary data 1) and then cloned them into the pGL3-Basic vectors. We observed that overexpression of L. vannamei Dorsal or Relish could significantly induce the promoter activities of all four penaeidins in Drosophila S2 cells (in vivo and in vitro (P < 0.01). Subsequently, BigPEN was chosen to further confirm these results in detail. We analyzed the 5′ flanking regulatory region of BigPEN and found that it contained two conserved κB motifs located at −349 to −339  and −91 to −81   expressed in E. coli cells. As shown in L. vannamei rDorsal-RHD, but not the control rTrx, effectively retarded the mobility of the bio-labelled probe 1 (Line 5). We further observed that the DNA/protein complex was faintly reduced by the competitive 2× unlabeled probe 1 but markedly reduced by the competitive 100× unlabeled probe 1  A. We the < 0.01) B. To addS2 cells C. The abTTTTTAC) D. Four vTTTTTAC) D, were cegulated E. These  probe 1 F, line 3Drosophila AMPs, attC and dptB, have been demonstrated to restrict Sindbis virus (SINV) infection, but the actual antiviral mechanism is still unknown [L. vannamei, including BigPEN, PEN2, PEN3, and PEN4, were assessed in their potential anti-WSSV activities. The critical role of penaeidins during an innate anti-WSSV response was demonstrated by using RNAi in vivo to silence each individual penaeidin. We observed that knockdown of each penaeidin resulted in higher viral loads, whereas each purified penaeidin could effectively confer protection to shrimp against the WSSV. In addition, we identified that the antiviral mechanism of penaeidins involved blocking viral internalization and disrupting the process of infection. In summary, we show for the first time that penaeidins are a novel class of anti-WSSV effectors in shrimp.In invertebrates, some AMPs are identified as viral responsive effectors; however, the molecular mechanism underlying the antiviral activities of AMPs is poorly understood. For example, two  unknown . Herein,L. vannamei were significantly induced by WSSV infection , which has been shown to contribute to effective infection [The most probable scenario is that the antiviral activity of penaeidins is due to the direct interaction between the WSSV and penaeidins, which would inhibit viral entry into target cells. Our current studies have provided several lines of evidence strongly supporting this notion. First, all four penaeidins were able to interact with the outer surface of the WSSV virion, and all, except PEN4, were shown to bind one or more of the five tested viral proteins. Second, the purified penaeidin proteins inhibited WSSV entry into hemocytes and significantly reduced the infection rate of hemocytes by WSSV virions. Finally, PEN2 and BigPEN directly interacted with VP24 and VP28 of WSSV, respectively, and interfered with the binding of these viral proteins to host factors. The exact antiviral mechanism of penaeidins against WSSV could be their ability to disrupt the processes of viral infection. In this study, the identified penaeidin-binding viral proteins were VP24, VP26, VP28, and VP16 – most of which play significant roles in WSSV infection. VP24 is a chitin-binding protein and deemed to be a key factor involved in WSSV infection . Importast cells . In thisst cells . VP28 isst cells . In addinfection . The intnfection . We alsoIn addition, interactions between envelope proteins are common in enveloped viruses, and they might form complexes that have specific roles in host-viral interactions or the infectivity of viruses . A similin vivo. This observation was further confirmed via an EMSA that showed that Dorsal was able to interact with the canonical κB motif within the promoter region of BigPEN in vitro. It is important to note that one or both κB motifs could also be responsive to Relish, as Relish strongly induced the expression of BigPEN in vitro as determined by dual reporter gene assays (wsv069) has been demonstrated to be dual-responsive – that is, regulated by either Dorsal or Relish [Drosophila, a single κB motif in the promoter of Metchnikowin (Mtk) has been shown to bind both DIF and Relish [A common innate defense mechanism in invertebrates, including shrimps, is that immune signalling pathways, such as Toll, IMD, and JAK/STAT pathways, regulate the production of specific sets of effectors for antiviral defense ,47. Idene assays C. Similar Relish . In Drosd Relish . Additiod Relish . Thus, wIn summary, for the first time we have identified penaeidins as a novel class of innate antiviral factors against WSSV. Based on our results, we proposed a model for the function of penaeidins in the innate antiviral response . Infecti


Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics.Group B  Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs’ mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs.Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B  The development of antibiotics is arguably one of the most important advances in modern medicine. Antibiotics can be organized according to the cellular component or system they engage and whether they inhibit cell growth (bacteriostatic) or induce cell death . Although antibiotics that target cellular viability are effective, these agents impose selective pressures that foster the evolution of resistant phenotypes . Combina2–Streptococcus (GBS)  . GBS is us (GBS) –14. Reces (GBS) ––19. As tTreatment of GBS disease relies primarily on penicillin and ampicillin, followed by first-generation cephalosporins and vancomycin . Alterna21–50), HMO extracts reduced the MICs of certain intracellular targeting antibiotics up to 32-fold  possess antimicrobial and antivirulence properties . Indeed,28–33– 32-fold . Interesdfr genes that encode resistant DHFRs , an antifolate used in the treatment of enteric, respiratory, skin, and urinary tract infections . Mechani45–HMOs were isolated from donor breast milk and pooled to create two HMO cocktails; the first (HMO-1) used milk from 10 donors, while the second (HMO-2) used milk from 7 donors. Prior to potentiation studies, the MIC of the HMOs and TMP were determined separately in each strain of GBS grown in Todd-Hewitt broth (THB) using a broth microdilution assay . HMOs we25s) in CNCTC 10/84 and GB2. The growth of the remaining GBS strains were so rapidly affected by HMO treatment that subsequent IC50 curve fitting yielded immeasurable confidence limits. For these strains, the IC25 from a similar strain having a superior fit dose-response curve was used  was recorded after 24 h of HMO treatment at increasing HMO concentrations. MIC, 2.56 mg/ml; IC50, 1.897 mg/ml; IC25, 1.43 mg/ml; Hill slope, −3.894. (B) IC50 curve of HMO cocktail for GB590 in THB. Bacterial growth (OD600) was recorded after 24 h of HMO treatment at increasing HMO concentrations. MIC = 5.125 mg/ml, IC50 ∼ 2.417 mg/ml, IC25 ∼2.30 mg/ml, hill slope = −22.37. (C) IC50 curve of HMO cocktail for GB CNCTC 10/84 in THB. Bacterial growth (OD600) was recorded after 24 h of HMO treatment at increasing HMO concentrations. MIC = 5.125 mg/ml, IC50 = 2.05 mg/ml, IC25 = 1.42 mg/ml, hill slope = −2.98. (D) IC50 curve of HMO cocktail for GB83 in THB. Bacterial growth (OD600) was recorded after 24 h of HMO treatment at increasing HMO concentrations. MIC = 5.125 mg/ml, IC50 = 3.616 mg/ml, IC25 = 3.51 mg/ml, hill slope = −39.06. (E) IC50 curve of HMO cocktail for GB651 in THB. Bacterial growth (OD600) was recorded after 24 h of HMO treatment at increasing HMO concentrations. MIC = 5.125 mg/ml, IC50 = 2.616 mg/ml, IC25 = 2.33 mg/ml, hill slope = −9.639. Download FIG S1, DOCX file, 0.1 MB.(A) ICCopyright © 2020 Chambers et al.2020Chambers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, checkerboard assays were conducted with GB2 and GB590, stains for which strong and weak potentiation of TMP were observed, respectively, to determine if the HMO-TMP combination was synergistic or additive in nature  and B. S10.1128/mBio.00076-20.2FIG S2FIG S2, DOCX file, 0.1 MB.(A) Checkerboard assay in GB2 showing the synergistic combinations between TMP and the HMO cocktail (bolded). (B) Checkerboard assay in GB590 showing the synergistic combinations between TMP and the HMO cocktail (bold). The combination is considered synergistic when the ΣFIC is ≤0.5, additive or indifferent when the ΣFIC is >0.5 to <4, and antagonistic when the ΣFIC is ≥4. Download Copyright © 2020 Chambers et al.2020Chambers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the de novo biosynthesis of thymidylate and purine nucleotides. However, in addition to de novo synthesis, cells can produce these nucleotides via salvage pathways that use free thymidine or purine bases as precursors for the corresponding nucleotides. Thus, we hypothesized that if HMOs facilitate TMP inhibition of the de novo synthesis pathway, the addition of the preformed nucleotide precursors thymidine or hypoxanthine would dampen the growth inhibitory capabilities of the HMO-TMP combination, as thymidine and hypoxanthine serve as precursors in the pyrimidine and purine salvage pathways, respectively  combination . In bothThe mode of action of an antimicrobial agent cannot accurately be described in terms of a single static target; rather, the complete induced response must be evaluated. In theory, a single chemotherapeutic could have a wide range of direct and indirect targets, simultaneously interfering with multiple enzymes or pathways. Accordingly, the final stage of the study focused on utilizing global, untargeted metabolomic analysis to characterize the early response of GBS to HMO-mediated perturbations. The analysis described provides HMO-mediated perturbations. For this study, strain GB2 was used, as it was most susceptible to treatment with HMOs . Two groups were analyzed and compared, with the first being an untreated GB2 control and the second being GB2 treated with HMOs dosed at 1 mg/ml. This concentration promoted cellular death (ca. 20 to 40%) compared to the untreated controls but also provided enough remaining cellular mass for analysis .Our experimental design from sample collection through data analysis is depicted in 10.1128/mBio.00076-20.3FIG S3x axis). Download FIG S3, DOCX file, 0.1 MB.Global principal-component analysis (PCA) of cellular extracted metabolites for the two biological conditions is illustrating a distinct shift in metabolic profiles between groups. Presenting the abundance data in PC space allows us to separate the run samples based on overall variability and shows a clear separation of untreated samples versus HMO-treated samples across the first PC dimension  identified at confidence level L3 are highlighted in red for the linoleic acid metabolism, generated by the MetaboAnalyst 4.0 software. Download FIG S4, DOCX file, 0.2 MB.Linoleic acid metabolism supplementary images. (A) Heat map visualization of the differently regulated members within the linoleic acid metabolic pathway upon HMO treatment. Linoleic acid metabolism members shown here were detected by HILIC-positive LC-MS/MS analysis. (B) Red nodes reveal individual significant metabolites matched to the linoleic acid metabolic pathway. The KEGG C number is shown within a node. The individual significant metabolites  identified at confidence level L3 are highlighted in red for the glycerophospholipid metabolism, generated by the MetaboAnalyst 4.0 software. Download FIG S5, DOCX file, 0.4 MB.Glycerophospholipid metabolism supplementary images. (A) Heat map visualization of the differently regulated members within the glycerophospholipid metabolic pathway upon HMO treatment. Glycerophospholipid metabolism members shown here were detected by RPLC-positive LC-MS/MS analysis. (B) Red nodes reveal individual significant metabolites matched to the glycerophospholipid metabolic pathway. The KEGG C number is shown within a node. The individual significant metabolites , direct experimental evidence that rapid metabolic changes are causal in facilitating the microbial response to antibiotics is lacking. In fact, little is described about the downstream phenotypic changes induced by antimicrobial agents. In the future, metabolomic experiments will be employed to better describe the phenotypic response of GBS to HMO-induced effects. We hypothesize that metabolomics will enable the characterization of the indirect connections critical to HMOs’ mechanism of action. Since metabolites present the final phenotypic manifestation of an organism and the final endpoint of biochemical reactions reflects the interplay between gene expression, protein function, and the environment, we argue that further metabolomics analyses are necessary to understand the HMO mode of action , 71.Kluyveromyces lactis, at 2,600 units/g, was purchased from Sigma-Aldrich. Acetonitrile , methanol , and water  for the mass spectrometry analysis were obtained from Thermo Fisher Scientific.Trimethoprim lactate 98% was purchased from Alfa Aesar. β-Galactosidase from in vacuo, the HMO-containing extract was dissolved in 0.2 M phosphate buffer (pH 6.5) and heated to 37°C. β-Galactosidase from Kluyveromyces lactis was added, and the reaction mixture was stirred until lactose hydrolysis was complete. The reaction mixture was diluted with roughly 1:0.5 (vol/vol) 180 or 200 proof ethanol, chilled briefly, and then centrifuged for 30 min. The supernatant was removed and concentrated in vacuo, and the remaining salts, glucose, and galactose were separated from the oligosaccharides using size exclusion chromatography with P-2 gel (H2O eluent). The oligosaccharides were then dried by lyophilization. Correspondingly, HMO isolates from donors were combined and solubilized in water to reach a final concentration of 102.6 mg/ml.Human milk was obtained from 17 healthy, lactating women between 3 days and 3 months postpartum and stored between −80 and –20°C. Deidentified milk was provided by Jörn-Hendrik Weitkamp from the Vanderbilt Department of Pediatrics, under a collection protocol approved by the Vanderbilt University institutional review board (IRB no. 100897), or from Medolac. Milk samples were thawed and then centrifuged for 45 min. Following centrifugation, the resultant top lipid layer was removed. The proteins were then removed by diluting the remaining sample with roughly 1:1 (vol/vol) 180 or 200 proof ethanol, chilling the sample briefly, and centrifuging for 45 min, followed by removal of the resulting HMO-containing supernatant. Following concentration of the supernatant 600) using a Promega GloMax-Multi detection system plate reader. Bacterial numbers were determined using the predetermined coefficient of an OD600 of 1, equal to 109 CFU/ml.The bacterial strains are shown in 10.1128/mBio.00076-20.7TABLE S1Streptococcus strains evaluated in the study. *ST, sequence type as defined by multilocus sequence typing (MLST). Download Table S1, DOCX file, 0.1 MB.Group B Copyright © 2020 Chambers et al.2020Chambers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00076-20.8TABLE S2aTHB medium. bShown in μg/ml. c1:4.85 ratio of TMP-sulfadiazine. Download Table S2, DOCX file, 0.1 MB.TMP-sulfadiazine antibiotic combination assay. Copyright © 2020 Chambers et al.2020Chambers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5 CFU/ml. To 96-well tissue culture-treated, sterile polystyrene plates was added the inoculated medium in the presence of increasing concentrations of antibiotic or HMO cocktail to achieve a final volume of 100 μl per well. Bacteria grown in medium in the absence of any compounds served as the controls. The plates were incubated under static conditions at 37°C in ambient air for 24 h. Bacterial growth was quantified through absorbance readings (OD600). The MICs were assigned at the lowest concentration of compound at which no bacterial growth was observed.All strains were grown overnight as described above and used to inoculate fresh THB or THB plus 20 μg/ml thymidine to achieve 5 × 105 CFU/ml. Freshly inoculated medium was then supplemented with HMOs at their IC25. To 96-well tissue culture-treated, sterile polystyrene plates was added the inoculated medium supplemented with HMOs in the presence of increasing concentrations of antibiotic. Bacteria grown in medium in the absence of any compounds served as one control. Bacteria grown in medium supplemented with HMOs in the absence of any antibiotic served as a second control. MICs were determined as previously described.All strains were grown overnight as described above and the subcultures used to inoculate fresh THB or THB plus 20 μg/ml thymidine to achieve 5 × 10Streptococcus strains (GB2 and GB590) were grown overnight as described above and used to inoculate fresh THB to achieve 5 × 105 CFU/ml. One hundred microliters per well of inoculated medium was added to 96-well tissue culture-treated, sterile polystyrene plates. Trimethoprim was 2-fold serially diluted descending down the plate to achieve a final volume of 100 μl per well. The final row was left without any trimethoprim. The HMO cocktail was 2-fold serially diluted going from right to left across the plate. The far-left column was left without any HMO cocktail. Bacteria grown in medium in the absence of either compound served as the controls. The plates were incubated under static conditions at 37°C in ambient air for 24 h. Bacterial growth was quantified through absorbance readings (OD600). The MICs were assigned at the lowest concentration of compound at which no bacterial growth was observed. The fractional inhibitory concentration (FIC) index was used to evaluate synergy. The calculation of the FIC index is as follows: ΣFIC = FIC A + FIC B =  + , where A is trimethoprim and B is the HMO cocktail. The combination is considered synergistic when the ΣFIC is ≤0.5, additive or indifferent when the ΣFIC is >0.5 to <4, and antagonistic when the ΣFIC is ≥4.Group B post hoc Dunnett’s multiple-comparison test comparing growth in the presence of ca. 5 mg/ml HMOs to growth in medium alone. HMO IC50 curves were generated in the GraphPad Prism software v. 7.0c using an inhibition dose-response nonlinear regression curve fit for log(inhibitor) versus normalized response with a variable slope.The data for the HMO antimicrobial and combination assays represent 3 biological replicates, each with 3 technical replicates. The data for the synergy assays represent 3 biological replicates. Data are expressed as the mean biomass ± standard error of the mean (SEM). Statistical analyses were performed in the GraphPad Prism software v. 7.0c. Statistical significance was determined using one-way analysis of variance (ANOVA) with Streptococcus strain GB2 was grown overnight as described above and used to inoculate 10 ml of fresh THB medium to achieve 5 × 105 CFU/ml. Untreated GB2 in 10 ml of medium served as a control, while other GB2 cultures were treated with HMOs at 1.00 mg/ml. After 24 h, the samples were centrifuged at 1,500 rpm for 20 min to generate a bacterial pellet. The medium was removed and the pellet washed with 200 μl of 50 mM ammonium formate buffer. The pellet was then resuspended in 200 μl of 50 mM ammonium formate buffer and transferred to a sterile Eppendorf tube. This was then centrifuged at 1,500 rpm for 10 min to generate a bacterial pellet. The buffer was removed and the pellet flash frozen in liquid N2 and stored until use.Group B in vacuo and stored at –80°C until further UPLC-HRMS/MS analysis.The bacterial cell pellets were lysed using 400 μl ice-cold lysis buffer  and vortexed. Individual samples were sonicated using a probe tip sonicator, with 10 pulses at 30% power and cooling down in ice between samples. A bicinchoninic acid (BCA) protein assay was used to determine the protein concentration for each individual sample and adjusted to a total amount of protein of 200 μg in 200 μl of lysis buffer. Isotopically labeled standard molecules phenylalanine-D8  and biotin-D2  were added to each sample to assess sample preparation reproducibility. Metabolites were extracted from untreated control and HMO-treated cultures using protein precipitation by the addition of 800 μl of ice-cold methanol (4× by volume) and incubated overnight at –80°C. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate precipitated proteins, and the metabolite-containing supernatant was dried Metabolite extracts were analyzed using reverse-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), followed by subsequent mass spectrometry analysis using a high-resolution Q-Exactive high-fidelity (HF) hybrid quadrupole-Orbitrap mass spectrometer  equipped with a Vanquish ultrahigh-performance liquid chromatography (UHPLC) binary system and autosampler . A quality control sample was prepared by pooling equal volumes of each sample. Isotopically labeled standards tryptophan-D3, carnitine-D9 , valine-D8, and inosine-4N15  were added to each sample to assess MS instrument reproducibility.Metabolite extracts (10-μl injection volume) were separated on a SeQuant ZIC-HILIC 3.5-μm, 2.1-mm by 100-mm column  held at 40°C for the HILIC analysis. Liquid chromatography was performed at 200 μl/min using solvent A  and solvent B  with the following gradient: 95% B for 2 min, 95 to 40% B over 16 min, 40% B held for 2 min, and 40 to 95% B over 15 min, and 95% B held for 10 min . For the RPLC analysis, metabolite extracts (10 μl injection volume) were separated on a Hypersil Gold, 1.9 μm, 2.1-mm by 100-mm column (Thermo Fisher) held at 40°C. Liquid chromatography was performed at 250 μl/min using solvent A (0.1% formic acid [FA] in water) and solvent B (0.1% FA in acetonitrile [ACN]) with the following gradient: 5% B for 1 min, 5 to 50% B over 9 min, 50 to 70% B over 5 min, 70 to 95% B over 5 min, 95% B held for 2 min, 95 to 5% B over 3 min, and 5% B held for 5 min .m/z 70 to 1,050 using electrospray ionization positive mode. MS scans were analyzed at a resolution of 120,000, with a scan rate of 3.5 Hz. The automatic gain control (AGC) target was set to 1 × 106 ions, and the maximum injection time (IT) was at 100 ms. Source ionization parameters were optimized, and these include spray voltage, 3.0 kV; transfer temperature, 280°C; S-lens level, 40; heater temperature, 325°C; sheath gas, 40; aux gas, 10; and sweep gas flow, 1. Tandem spectra were acquired using a data-dependent acquisition (DDA) in which one MS scan is followed by 2, 4, or 6 tandem MS (MS/MS) scans. MS/MS scans are acquired using an isolation width of m/z 1.3, stepped normalized collision energy (NCE) of 20 and 40, and a dynamic exclusion for 6 s. MS/MS spectra were collected at a resolution of 15,000, with an AGC target set at 2 × 105 ions and maximum IT of 100 ms. Instrument performance and reproducibility in the run sequence were assessed by monitoring the retention times and peak areas for the heavy labeled standards added to the individual samples prior to and after metabolite extraction to assess sample processing steps and instrument variability  were grouped based on adducts and isotopes, and individual features or metabolites were normalized to all features. Compounds with <25% coefficient of variance (CV) were retained for further analysis. P values were calculated by Progenesis QI using variance-stabilized measurements achieved through log normalization, and metabolites with a P value of <0.05 calculated by one-way analysis of variance (ANOVA) and with a fold change (FC) of >|2| were considered significant.UPLC-HRMS/MS raw data were imported, processed, normalized, and reviewed using Progenesis QI v.2.1 . All MS and MS/MS sample runs were aligned against a quality control (pooled) reference run, and peak picking was performed on individual aligned runs to create an aggregate data set. Following peak picking, unique spectral features . Annotations were further analyzed using pathway overrepresentation analysis using MetaboAnalyst 4.0 (Tentative and putative identifications were performed within Progenesis QI using accurate mass measurements (<5 ppm error), isotope distribution similarity, and fragmentation spectrum matching based on database searches against the Human Metabolome Database (HMDB), METLIN and National Institute of Standards and Technology (NIST) databases, and an in-house database –76. Anno–lyst 4.0 , 78. The