Wolbachia was also reported, correlating with its asexual reproduction. Antibiotic treatment of the aphid population resulted in a decrease in its survival rate. The possibility of controlling this pest by altering its microbiota is proposed in the present work.Insects are rapidly adapting to novel niches, such as crops. This is the case of the sugarcane aphid, a pest from Africa that is currently spreading worldwide. In the absence of males, aphids asexually generate many females, causing important economic losses for farmers. The microbiota in the insect was characterized and the presence of Melanaphis sacchari, the sugarcane aphid, native to Africa and currently spreading worldwide. The aphid undergoes successful parthenogenesis, causing important damage to a variety of crops and leading to important economic losses for farmers. A natural M. sacchari population grown in sorghum was studied to identify its microbiome through the sequencing of its 16S rDNA metagenome. A high proportion of Proteobacteria, followed by Firmicutes, Bacteroidetes, and Actinobacteria, was observed. We also detected Wolbachia, which correlates with the asexual reproduction of its host. M. sacchari was challenged in a bioassay with the antibiotics oxytetracycline and streptomycin, resulting in a dose-dependent decay of its survival rate. The possibility of controlling this pest by altering its microbiota is proposed.Insects are under constant selective pressure, which has resulted in adaptations to novel niches such as crops. This is the case of the pest  Melanaphis sacchari Zehntner (Hemiptera: Aphididae), the sugarcane aphid, is distributed worldwide and considered a major pest of sorghum (Sorghum bicolor (L.) Moench) and sugarcane (Saccharum officinarum L.). Furthermore, it has been reported on several important Poaceae crops such as wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), rice (Oryza sativa L.), corn (Zea mays L.), and Johnson grass  Pers.), among others [M. sacchari occurred in sorghum, resulting in significant economic damage to crop growers in North America [Coccinella septempunctata, Insecta: Coleoptera: Coccinellidae) and lacewings (Chrysoperla sp. (Neuroptera: Chrysopidae) as biological controls, with limited success due to the high reproductive rate of these insect species. Indeed, the most devastating impacts of the sugarcane aphid stem from its ability to rapidly reproduce and spread. M. sacchari asexual reproduction is favored under current crop culture. However, M. sacchari has shown sexual forms in Tamaulipas, Mexico, identified as winged adults with an average lifespan of 7.5 days, giving rise to 10.6 nymphs per female [Pentalonia nigronervosa, which is associated with the bacteria Buchnera aphidicola and Wolbachia sp. [Wolbachia is a bacterium infecting the soft tissues of arthropods and is maternally inherited and involved in feminization and parthenogenesis. Wolbachia-infected insect females can asexually reproduce from unfertilized eggs, producing more females as offspring. This reproductive strategy is successful for M. sacchari, as the aphid population can be doubled in 1.7 days [M. sacchari microbiota diversity was assessed using this approach. We identified Wolbachia in these populations by PCR. The effect of the addition of antibiotics on M. sacchari was also evaluated. The use of antibiotics in bioassays significantly decreased nymph emergence, eventually resulting in its death, in a dose-dependent manner. The control of M. sacchari could thus be carried out employing antibiotics directed against bacteria, likely targeting its microbiota, including Wolbachia.Aphids (Hemiptera: Aphididae) feed on the plant phloem sap, challenging photoassimilate allocation and causing stunted growth and yellowing, resulting in a significant impact on plant yield. Additionally, they are vectors for a diverse array of plant pathogenic viruses ,2. Melang others ,4. This g others . In 2013 America ,6. Nowad America . Current America . Anotherr female . This rer female . The elichia sp. . Wolbach1.7 days . To prof1.7 days . ONT pla1.7 days . The mos1.7 days ,18. TherM. sacchari adults were collected from naturally infested sorghum at the National Institute of Agricultural and Livestock Forestry Research (INIFAP), Celaya Experimental Station, Celaya, Guanajuato, Mexico  in December 2019. Fully expanded leaves containing all the insect developmental stages were carefully removed from 20 plants and transferred to an aphid growth chamber. The insect colonies were maintained at 28–30 °C with 16/8 h photoperiod  using aphid-susceptible sorghum . Leaf circles were placed in 80 mm Petri dishes containing 15 mL of 0.8% agar to create a humid environment. M. sacchari adults were then placed on the surface of the sorghum leaves in Petri dishes, and their activities and emergence of immature stages were monitored daily.M. sacchari adults using Biopure Total DNA Extraction kit . Approximately one hundred adults collected from sorghum plants were rinsed with distilled water, homogenized with a sterile pestle, and the purified DNA was used for bacterial 16S rRNA gene amplification. The long-read 16S rRNA region was amplified with the primer set 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) [Total DNA was purified from ACTT-3′) , and PCRhttps://fbreitwieser.shinyapps.io/pavian/#, access date: 30 July 2023). Alpha diversity indices were calculated with krakentools and the R packages vegan and fossil [Base-calling and adapter/barcode trimming were performed using Guppy software V.5.0.11  to generate FASTQ-formatted sequence files. Additional filtering and taxonomic assignment were conducted using the NanoGalaxy platform , a Galaxd fossil ,28.Wolbachia spp., and a local nucleotide alignment was performed (BLAST 2.12.0+) with an E-value of 1 × 10−6 .The quality-filtered FASTQ file was retrieved, and nucleotide sequences were extracted to obtain a FASTA file. Additionally, a database was constructed using available 16S sequences of M. sacchari 16S microbiota showed low abundance reads taxonomically assigned to Wolbachia. To confirm this finding, Wolbachia-specific PCR primers (tpgs2) gene from M. sacchari, along whit three fragments targeting Wolbachia genes: 16S ribosomal RNA gene (wspec), filamenting temperature-sensitive mutant Z (ftsZ), and outer surface protein (wsp81/691) [Full-length metagenomic analysis of  primers  were empp81/691) ,30,31,32Oxytetracycline and streptomycin  were prepared at log concentrations of 0.1–10,000 μg/mL for each antibiotic. Antibiotic solutions were applied by spraying onto detached leaves that had been previously cut to fit the Petri dishes. Each side of the leaf was treated with 0.25 mL of the antibiotic solution, totaling 0.5 mL per leaf. After spray absorption, five active adults were placed on the treated leaves. As positive controls, the chemical pesticide abamectin  was used at concentrations of 0.18%, 0.018%, and 0.0018%. Distilled water was used as a negative control. Each experiment was performed in triplicate. Aphids were monitored daily, and mortality was recorded when they detached from the leaves and changed color from brownish to black, remaining immobile. The offspring were identified by observing the nymphs and adults on the leaf surface using a stereoscope.M. sacchari were counted for each treatment. Lethal concentration 50 (LC50) was calculated using linear regression and PROBIT software from Excel (Microsoft 365 MSO).Dead M. sacchari colony dwelling on sorghum successfully completed its life cycle, which was evident through the emergence of adults alongside their immature nymphal stages. Within five days, the detached leaf retained its turgidity and verdant hue when housed in a humidified chamber. Despite their initial positioning on the adaxial leaf surface, the insects exhibited migratory behavior toward the abaxial side, analogous to their natural habitat across the entire plant. This migration, although consistent with their behavior in the field, remained worthless to the experimental setup as the antibiotic treatment was applied to both leaf surfaces. In the natural ecosystem, these insects reside on the abaxial side of the leaves, utilizing their stylets to suck the sap from the sorghum phloem. In our experiment, aphids capable of transitioning to the abaxial side were selected, whereas those residing on the adaxial surface were excluded in alignment with their typical distribution. Mortality occurred five days after the onset of antibiotic treatment, coinciding with the time frame of leaf viability in the in vitro aphid colony culture.The insect colony was reared within growth chambers designed to replicate the environmental conditions prevalent in sorghum fields. The M. sacchari collected from commercially grown sorghum was characterized by 16S rRNA sequencing. Nanopore sequencing yielded 118,936 raw sequences (83.8 Mbp). After filtering, 53,273 sequences (73.5 Mbp) were retained, with 53,186 sequences (99.8%) successfully assigned to the Bacteria kingdom. Proteobacteria (99.670%), Firmicutes (0.256%), Bacteroidetes (0.040%), Actinobacteria (0.024%), Fusobacteria (0.006%), and Spirochaetes (0.002%) were identified at the phylum level. At the species level, Buchnera aphidicola (98.36%), Salmonella enterica (0.52%), and Escherichia coli (0.31%) were the most abundant  amplicon showed 90.89% homology with 12 Wolbachia sp. accessions . Similarly, the 16S (WSPEC) amplicon, showed 97.44% homology with four Wolbachia sp. accessions found as insect endosymbionts of Bactrocera dorsalis (MK053760.1), Paracorethrura iocnemis (OQ102158.1), Malenia bosnica (OQ102152.1), and Pentastira rorida (OQ102146.1). Additionally, the ftsZ amplicon showed 99.81% homology with five Wolbachia pipiens accessions . Furthermore, tpgs2 amplicon sequence analysis confirmed host identity as M. sacchari (XM_025335778.1) with 100% homology.To validate this finding, a PCR was performed using specific primers for three bacterial loci: wsp81/91 . The resM. sacchari offspring were evaluated by day 5, in which they would have reproduced at least once and the leaves were still turgent. The criterion for considering M. sacchari dead was that they remained motionless, frequently detached from the leaf surface, and in some cases, a black color developed on their body. In the control trials without antibiotics, the presence of offspring was recorded from the third day of incubation (50) at 6.6 μg/mL  increased host insecticide susceptibility via suppression of bacterial symbionts. In our study, the lowest antibiotic concentration of 0.1 μg/mL impacted reproduction, as no progeny was observed, while higher concentrations of antibiotics were lethal for the entire M. sacchari population. The impact on the reproductive rate could be due to the insect’s inability to feed and maintain its metabolism to produce progeny, although selective suppression of Wolbachia by antibiotic treatment as a control strategy needs to be further evaluated.Plants have evolved a range of defense mechanisms to protect themselves from herbivory, including the production of antifeeding compounds that reduce the nutritional value of plant tissues for herbivores, like sap-feeding insects. Ecologically, these compounds play an important role in shaping plant–herbivore interactions, thus reducing crop damage. However, the insect microbiota’s co-metabolism overcomes those limitations by successfully degrading complex compounds . The micrvations , where aM. sacchari microbiome was analyzed through the amplification of the V3 fragment of the 16S rDNA using Nanopore equipment, long used to describe microbiomes in different systems [Buchnera, Staphylococcus, Prevotella, Bacillus, Streptococcus, Granulicatella, Pasteurella, Acetobacter, Rothia, Veillonella, Leptotrichia, Blastobacter, Clostridium, Desulfomicrobium, Bradyrhizobium, and Wolbachia. Wolbachia has been detected before in sorghum in the US [M. sacchari adapted to a sorghum variety widely used in central Mexico (Sumiel II) would not necessarily be the same. Indeed, previous reports on the microbiome of M. sacchari infesting sorghum grown in northeastern Mexico did not report the presence of Wolbachia [In the insect colony maintained on sorghum leaves in Petri dishes, the emergence of nymphs could be observed. This suggests that female reproduction was similar to what is observed in their natural environment; therefore, the in vitro assay was used to assess insect reproductive success and mortality. A limitation of the use of Petri dishes containing aphid colonies is that the leaf loses turgidity despite being in a humid atmosphere. Five-day trials were successfully evaluated. The insect behavior in this microenvironment was similar to that observed in sorghum plants in the field. The composition of the  systems ,16. Each systems . Differen the US . Howevern the US , the bacolbachia , underscolbachia , indicatWolbachia in the M. sacchari microbiome was later confirmed by amplifying three loci previously described for this genus: wspec, ftsZ, and wsp81/91. The homology of these sequences with the Wolbachia genome demonstrated its presence in aphids. As in other insects, Wolbachia could bias the reproductive strategy to parthenogenetic for its transmission. In this case, it would also be beneficial for the aphid since an adult produces 4–12 nymphs during its lifespan [Wolbachia has been observed in a range of insect species, including mites, fruit flies, and wasps [The finding of sequences homologous to lifespan ,9. Parthnd wasps . Howevernd wasps .Wolbachia and other endosymbionts from the aphid microbiota could have an impact on the insect’s reproductive ability, although sublethal concentrations of tetracycline negatively affect the fecundity of the parasitoid Trichogramma pretiosum [Wolbachia influences its host fecundity [M. sacchari infesting Sumiel II sorghum revealed the presence of Wolbachia, the effect of this antibiotic on insect viability was evaluated.It is not clear whether suppression of retiosum . Similarecundity . Given tM. sacchari infestation in sorghum since it has no natural enemies and biological control has had limited success [M. sacchari population in plants.From an applied viewpoint, the use of antimicrobial agents like antibiotics could help solve the problem of  success . DespiteM. sacchari populations in this crop are permanent in agricultural areas, even when sorghum is not planted, because they have adapted to other crops, including weeds. Its persistence at low densities allows it to re-colonize sorghum when the crop cycle begins. In all, the potential alteration of pest insect or vector microbiota offers the possibility of reducing their impact on crops in an agroecological context.


The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control. In diplontic animal organisms, somatic haploidy is generally unstable, causing frequent autodiploidization at the cellular level and severe developmental abnormalities at the organismal level . The halThe mevalonate pathway metabolizes acetyl-CoA to produce sterol isoprenoids, and non-sterol isoprenoids that mediate diverse biosynthetic processes essential for cell construction and proliferation . MevalonIn this study, in an attempt to modulate cell size by an HMGCR inhibitor pitavastatin in human haploid HAP1 cells  supplemented with 10% fetal bovine serum and 1× antibiotic-antimycotic (Sigma-Aldrich). Haploid cells were regularly maintained by size-based cell sorting, as previously described ; U18666A ; Cholesterol ; FTI-277 ; GGTI-298 ; tauroursodeoxycholic acid ; and mevalonate .Antibodies used in this study are listed in To test the effects of inhibition of the mevalonate pathway on cell size control, we treated human haploid cell HAP1 with a competitive HMGCR inhibitor pitavastatin, which has been reported to increase the size of human cell lines such as Jurkat . AlthougNext, we determined downstream branches of the mevalonate pathway crucial for the maintenance of haploid state. Because statins are widely used cholesterol-lowering drugs , we addrNext, we tested the effect of perturbation of cholesterol homeostasis by a non-statin cholesterol inhibitor on haploid stability. An amphipathic steroid U18666A perturbs the cholesterol-mediated bioprocesses by inhibiting both synthesis and intracellular transport of cholesterol . TreatmeAmong the mevalonate-derived metabolites, farnesyl pyrophosphate and geranylgeranyl pyrophosphate are used for the posttranslational prenylation of small GTPases that play crucial roles in the regulation of cell cycle and proliferation, as well as cell size control , 2016. TOn the other hand, treatment with 2 μM GGTI-298 mildly arrested haploid HAP1 cells at the G1 phase within 24 h C, consisSince statins potentially induce ER stress by suppressing dolichol phosphates biosynthesis and inhibiting protein N-glycosylation , we nextFinally, we determined whether ER stress induction is the cause of pitavastatin-mediated destabilization of haploid state in HAP1 cells. For this, we tested the effect of an ER stress-reducing chemical chaperone, tauroursodeoxycholic acid (TUDCA) A–C, presIt is assumed that ploidy differences have pleiotropic effects on intracellular biosynthetic processes and that the altered biosynthesis, in turn, affects cellular physiology at different ploidy states. However, it remains mostly elusive what biosynthetic processes have influences on ploidy-linked cellular phenotypes. In this study, we found that an HMGCR inhibitor pitavastatin destabilized haploid state in human HAP1 cells. This result is consistent with the recent compound screen that identified statins to efficiently promote the expansion of diploidized population over haploids in HAP1 cell culture (Our data further specified the perturbation of mevalonate-mediated ER homeostatic control as a critical cause of the statin-induced haploid destabilization. Interestingly, pitavastatin-induced ER stress caused a drastic transition from haploid to diploid state without affecting the stability of diploid state in HAP1 cells. It remains unknown why the effect of pitavastatin on genome stability was specific to the haploid state. However, a possible reason might be a ploidy-dependent difference in tolerance to ER stress. Haploid cells are half in cell volume than diploid cells (Mevalonate metabolism is an essential process that supports diverse biosynthetic pathways. Though we specified the preservation of ER homeostasis as a critical process underlying statin-mediate haploid destabilization, we cannot rule out other mevalonate-derived biosynthetic processes in haploid stability. For example, we cannot exclude the possibility that specific targets of protein farnesylation play roles in haploid stability, which might have been masked by the gross polyploidization upon the treatment with FTI-277. Comparative metabolome analysis would be a powerful approach to elucidate other biosynthetic processes playing critical roles in determining the physiology of cells at different ploidy states.Conceptualization, K.Ya., and R.U.; Methodology, K.Ya., K. S., D.M., K.Yu., Y.I., G.B., E.M., and R.U.; Investigation, K.Ya, K.S. K.Yo., E.M., and R.U.; Formal Analysis, K.Ya., K.S., and R.U.; Resources, G.B., E.M., and R.U.; Writing—Original Draft, K.Ya., and R.U.; Writing—Review & Editing, K.Ya., D.M., K.Yu., E.M., and R.U.; Funding Acquisition, K.Ya., G.B., E.M., and R.U.


Cistanche deserticola Ma (Orobanchaceae) is a holoparasitic angiosperm that takes life-essential materials from Haloxylon ammodendron (C. A. Mey.) Bunge (Amaranthaceae) roots. Although many experiments have been conducted to improve the quality of C. deserticola, little attention has been paid to the host’s influence on metabolite accumulation. In this study, transcriptomic and metabolomic analyses were performed to unveil the host’s role in C. deserticola’s metabolite accumulation, especially of phenylethanoid glycosides (PhGs). The results indicate that parasitism by C. deserticola causes significant changes in H. ammodendron roots in relation to metabolites and genes linked to phenylalanine metabolism, tryptophan metabolism and phenylpropanoid biosynthesis pathways, which provide precursors for PhGs. Correlation analysis of genes and metabolites further confirms that C. deserticola’s parasitism affects PhG biosynthesis in H. ammodendron roots. Then we found specific upregulation of glycosyltransferases in haustoria which connect the parasites and hosts. It was shown that C. deserticola absorbs PhG precursors from the host and that glycosylation takes place in the haustorium. We mainly discuss how the host resists C. deserticola parasitism and how this medicinal parasite exploits its unfavorable position and takes advantage of host-derived metabolites. Our study highlights that the status of the host plant affects not only the production but also the quality of Cistanches Herba, which provides a practical direction for medicinal plant cultivation.The medicinal plant  Cistanche deserticola Ma or Cistanche mongolica Beck (previously named Cistanche tubulosa) (Orobanchaceae)’. Chinese medicine practitioners prefer using materials derived from C. deserticola because it has been distributed in a broader area including all of northwestern China throughout history and has, thus, been used as a tonic for a longer time. As a holoparasite, this plant loses photosynthetic capacity and takes nutrients as well as water from its host. In previous studies by our group, C. deserticola mostly parasitized Haloxylon ammodendron (C. A. Mey.) Bunge (Amaranthaceae)  plants, and they successfully developed haustoria. In recent years, Atriplex canescens (Pursh) Nutt. (Amaranthaceae) has been reported as a new host for C. deserticola [C. deserticola in different ecotypes [C. deserticola samples collected in spring and autumn also differ in their phenylethanoid glycoside contents [Cistanches Herba is a traditional Chinese medicine. In the Chinese Pharmacopoeia, Cistanches Herba is described as ‘the dried fleshy stem of thaceae) . We alsoerticola . Many stecotypes . Secondlecotypes . C. desecontents .C. deserticola, which cannot survive if it is separated from the host. Studies in other medicinal phytoparasites demonstrate that host plants significantly influence their quality. For example, Thesium chinense Turcz.  plants have higher concentrations of total amino acids when parasitizing Triticum aestivum L. (Poaceae) than when parasitizing other hosts [Scurrula ferruginea (Jack) Danser (Loranthaceae): S. ferruginea stems have different anti-inflammatory properties when parasitizing on three different host plants [However, little attention has been paid to the host plant’s effect on the quality of er hosts . Anothert plants .Lamiales species [Phenylethanoid glycosides (PhGs) are the major active components in Cistanches Herba. PhGs have been reported to have strong antioxidant potential  and hypo species . As irid species , we wondThe aglycons of PhGs are derived from the tyrosine metabolism pathway. Tyrosol, the aglycon of salidroside and osmanthuside H, is derived from tyrosine by two-step or three-step enzymatic reactions . Most PhC. deserticola fleshy stems, their hosts’ roots, and healthy H. ammodendron roots at the metabolomic and transcriptomic levels. By doing so we aimed to (1) explore the defensive changes in H. ammodendron roots caused by C. deserticola parasitism, (2) mark the candidate strategies used by C. deserticola to reduce host-derived damage, and (3) determine how this kind of host–parasite interaction influences C. deserticola metabolite accumulation, especially PhGs. Our research highlights the host’s influence on the quality of Cistanches Herba and proposes a direction for improvement in the field of medicinal plant cultivation.In this study, we compared C. deserticola fleshy stems, their hosts’ roots, and healthy H. ammodendron roots  results showed significantly different accumulation and expression patterns of metabolites from C. deserticola fleshy stems and from the host and healthy H. ammodendron roots. Notably, HcF-vs.-HF and HcS-vs.-HS could be significantly separated by PCA, emphasizing the changes caused by parasites , an orthogonal partial least squares discrimination analysis (OPLS-DA) was performed  was relatively low. 2’-Acetylacteoside was detected in C. deserticola fleshy stems (CF) and their hosts’ roots (HcF) in the fall. The other six PhGs were only detected in C. deserticola fleshy stems. Two PhGs took tyrosol as their aglycon and six PhGs took homovanillyl alcohol. PhGs that took tyrosol as their aglycon  had simpler structures and appeared in C. deserticola fleshy stems (CS), their hosts’ roots (HcS) and healthy roots (HS)  B. CistanC. deserticola fleshy stems and H. ammodendron roots, no hydroxytyrosol or homovanillyl alcohol was detected. Tyrosol was distributed in all samples but maintained relatively lower levels in CF and CS (H. ammodendron roots. The phenylpropanoid groups are often derived from caffeic acid or ferulic acid. Both phenylpropanoids showed relatively lower concentrations in CF and CS.The precursors for PhGs could be classified as precursors for (1) the phenylethanoid aglycon and (2) the phenylpropanoid group modifying the glucose backbone. The former is linked to the tyrosine metabolism pathway and showed significantly different metabolite accumulation patterns between the parasite and host. The latter is produced from the phenylpropanoid biosynthesis pathway. In F and CS . As polyC. deserticola also caused significant transcriptomic changes in H. ammodendron roots. A total of 3291 differentially expressed genes were screened in the HcF-vs.-HF group, with 1280 upregulated and 2011 downregulated , 4CL (1DEG), C3′H (1DEG), CCR (7DEGs), and UGT (11DEGs). In the HcS-vs.-HS group, 54 DEGs were found: 4CL (3DEGs), C3′H (1DEG), CCR (12DEGs), HCT(3DEGs), TyDC(2DEGs), PAO(3DEGs), PPO(5DEGs), and UGT (24DEGs).To investigate the differences in phenylethanoid glycoside biosynthesis caused by the parasitism of together . Nine Phynthesis : C4H  identified in Arabidopsis and 1 novel group (P) identified in maize , and BG  were selected. In H. ammodendron, C3H, HCT, 4CL, TyDC, and PPO were selected. The results were consistent with the transcriptome data  infected by the holo-parasitic broomrape (Orobanche cumana) have larger and thinner leaves because of carbohydrate depletion [Nicotiana spp.) parasitized by O. cernua [Striga spp.) has a significant impact on the head index, leaf biomass, leaf index, root biomass, root index, plant biomass, and root: shoot ratio of sorghum (Sorghum bicolor L. Moench) [Rhinanthus minor has a direct negative influence on host productivity and competitive ability, which helps reconstruct plant communities in grassland [O. crenata, increased phenolic compounds in host plants are related to limited parasitic damage, which can be induced by salicylic acid and indole acetic acid [O. cumana, which is associated with the growth restriction of the parasite [epletion . Reduced. cernua . Though  Moench) . Rhinantrassland . To avoirassland ,19. If trassland . In the tic acid . A similparasite .Phtheirospermum japonicum and Striga hermonthica, the haustorium of S. hermonthica could be initiated by both the S and G-unit, whereas that of P. japonicum was only induced by the S-unit [P. japonicum senses the root of its gymnosperm host which has no tracheary elements and initiates the haustorium. Thus, the consequence of cell wall lignification is contrary to the initial goal of host resistance. Lignin monomers are not the only host-derived resistant chemicals utilized by parasites. In Castilleja levisecta, iridoid glycoside concentrations differ depending on host species and distance. The host-derived diversity leads to different survival rates of Euphydryas editha feeding on C. levisecta, which is partially related to the iridoid glycosides sequestration [Orobanche ramose, which sequestrates pyridine alkaloids from its Nicotiana hosts, and Pedicularis semibarbata, which obtains quinolizidine alkaloids from Lupinus fulcratus [A more interesting phenomenon is the parasite’s response. Lignin monomers in plants can be classified into four types: H-unit, C-unit, S-unit, and G-unit. The S-unit lignifies the cell wall of sclerenchyma fibers and sclereids, whereas the G-unit contributes to the construction of tracheary elements . In resee S-unit . That isstration . The situlcratus ,27. In aC. deserticola parasitism were linked to phenylalanine metabolism, tryptophan metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, fructose/mannose metabolism, and propanoate metabolism. The fructose and mannose metabolism pathway is critical to the success of parasitism. In the case of Orobanche aegyptiaca, the host-induced suppression of the mannose 6-phosphate reductase gene is concomitant with significant mannitol decrease and increased tubercle mortality [H. ammodendron root is a consequence of this counteraction. However, mannitol also acts as an osmoprotectant. The most evident difference between parasitic plants and fungi is that the former takes nutrients from the host mainly by vascular bundles, which may be directed by osmotic potential. Thus, the gap in mannitol levels between C. deserticola and H. ammodendron contributes to the reversed source–sink relationship and the efficient uptake of nutrients. This is another good example of the parasite’s utilization of the host’s resistance. In addition, PhG biosynthesis can be enhanced by mannitol [H. ammodendron roots confirmed this.In our study, the metabolic changes brought about by ortality . In planortality . The relmannitol . The simC. deserticola caused significant changes linked to the phenylpropanoid pathway in H. ammodendron root. The phenylpropanoid pathway is the upstream pathway where lignin, lignan, coumarin, and flavonoid are derived [H. ammodendron’s defense response to the parasite. The mitogen-activated protein kinase (MAPK) signaling pathway is also triggered by parasitism. The MAPK signaling pathway is involved in multiple defense responses, including plant stress/defense hormone biosynthesis/signaling, reactive oxygen species (ROS) generation, stomatal closure, defense gene activation, phytoalexin biosynthesis, cell wall strengthening, and hypersensitive response (HR) cell death [H. ammodendron root parasitized by C. deserticola, most upstream genes in the MAPK signaling pathway were significantly upregulated, whereas downstream ones were inhibited. This indicates that the defense response is suppressed by parasite-derived effectors. The downregulation of genes linked to plant–pathogen interaction also demonstrated this phenomenon.At the transcriptomic level, the parasitism of  derived . These mll death . In H. aOrobanchaceae parasites sequester secondary metabolites from hosts, including polyacetylenes, alkaloids, and iridoid glycosides. These metabolites help in resisting herbivores and other stresses [O. laxissima, the acteoside content is significantly influenced by the host’s acteoside content [Cistanche mongolica, the host species had no significant influence on PhG contents. However, the height and weight of the parasites were significantly affected by the host species, and PhG contents decreased as the parasite grew. No PhGs have been tested in Tamarix, but precursors such as caffeic, coumaric, and ferulic acids exist in Tamarix plants [H. ammodendron and were significantly upregulated by the parasite. The existence of PhGs in healthy H. ammodendron roots also confirmed its capacity to synthesize these metabolites as well as their precursors. The PhGs cistanoside H and osmanthuside H showed relatively low concentrations in the host roots compared with the healthy roots. This may also be the result of the parasite’s depletion of precursors. Further proof of the host’s precursor feeding was significantly lower concentrations of caffeic acid, tyrosol, and dopamine in C. deserticola.stresses . The sit content . In our x plants . In thisC. deserticola changed the structure of host-derived phenols. Phenylpropanoids such as caffeic acid, ferulic acid, and coniferyl-alcohol can damage various cellular components, and their toxicity decreases when bonded to glucose [Philodina acuticornis) reveals that tyrosol is more toxic to salidroside [C. deserticola supported this point, as UGT85A family members catalyze the glycosylation of tyrosol [H. ammodendron, the PhG content was significantly related to one UGT85A and one UGT73B gene expression level. UGT73B is also linked to the glycosylation of tyrosol, though the product can be icariside. This explains the UGT expression in the haustorium, where the parasite builds a vascular connection with the host. Therefore, by improving glycosylation, the parasite detoxifies compounds from the host and protects itself. The PhGs, which are considered as bioactive compounds in C. deserticola, play a vital role in this self-protection process.Then we questioned why  glucose . Consumi glucose . Althougidroside . Dopaminidroside . By reco tyrosol . In H. aC. deserticola fleshy stems, H. ammodendron roots, and haustoria were harvested from the plant using sharp scissors and cleaned using ultra-pure water. Materials from 3 plants were mixed as a biological replicate and each group had 3 replicates. All the samples were rapidly frozen by liquid nitrogen and transported to the Institute for Medicinal Plant Development, Peking Union Medicinal College, Beijing, China, in boxes filled with dry ice. Then the samples were stored at −80 °C for metabolomic, transcriptomic, and gene expression analyses.The plant materials were collected from the Bencaocongrong Planting Base, Ningxia, China. The http://www.anpel.com.cn/ (accessed on 15 March 2021)) for further analysis. To analyze the metabolites, an UPLC-ESI-MS/MS system ; MS: Applied Biosystems Q TRAP 4500, www.appliedbiosystems.com.cn/ (accessed on 15 March 2021)) was used [For this analysis, 6 groups of samples with 3 biological replicates per group were vacuum-dried by vacuum freeze-dryer (Scientz-100F) and mashed with a mixer mill  with a zirconia bead for 1.5 min at 30 Hz. Then, 100 mg of the lyophilized powder was dissolved in 1.2 mL 70% methanol solution, vortexed 6 times for 30 s at intervals of 30 min, and kept at 4 °C overnight. After that, the solution was centrifuged at 12,000 rpm for 10 min and the extract was filtrated through a microporous membrane  [Based on the mass spectrometry data, metabolites were identified using the Metware Database  and quantified according to peak intensity. The quantification data of metabolites were normalized by unit variance scaling and used for PCA and HCA. OPLS-DA was performed after log2 transformation and mean centering. These analyses were carried out using R 3.6.3 software (er 2021) .C. deserticola and its host, differentially accumulated metabolites in the CF-vs.-HcF and CS-vs.-HcS groups were screened using the mass spectrometry data. VIP values were extracted from OPLS-DA results and metabolites with VIP ≥ 1 and absolute log2 (fold change) ≥ 1 were selected as differentially accumulated metabolites [To determine the metabolomic differences between abolites .http://www.kegg.jp/kegg/compound/ (accessed on 25 March 2021)) and mapped to the KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html (accessed on 25 March 2021)) [p-values.The DAMs were annotated using the KEGG Compound database (h 2021)) . Then a C. deserticola parasitism, the same procedure was followed for the HcF-vs.-HF and HcS-vs.-HS groups.To investigate the changes caused by Total RNA was extracted from the plant tissues using a quick RNA isolation kit . To ensure the RNA samples were integrated and DNA-free, agarose gel electrophoresis was performed. RNA purity was then determined by a nanophotometer. Following that, a Qubit 2.0 Fluorometer and an Agilent 2100 BioAnalyzer were used to accurately measure RNA concentration and integrity, respectively. The qualified samples were processed with oligo (dT) beads to enrich the mRNA, which was broken into fragments and used as templates for the cDNA library. To qualify the cDNA library, the fluorometer was used for primary quantification and the bioanalyzer was then used to insert text size. A q-PCR analysis was then performed to accurately quantify the library. The qualified library was sequenced using the Illumina HiSeq 4000 platform.Clean reads were obtained by eliminating low-quality reads and assembled using Trinity 2.6.6 software . The trap-values.The differentially expressed genes were screened based on the thresholds of FDR < 0.5 and absolute log2FC > 1 using DESeq2 1.22.2 software . Then a p ≤ 0.05 were uploaded to Cytoscape 3.9.1 software to draw connection networks between DEGs and DAMs [DEGs and DAMs linked to PhG biosynthesis were selected to perform Pearson’s correlation tests using SPSS 26.0 software. Correlations with Pearson’s correlation coefficient (PCC) ≥ 0.7 and and DAMs .C. deserticola and H. ammodendron were identified following a procedure described by Cheng et al. [http://pfam.sanger.ac.uk (accessed on 1 November 2022)) and the UGTs were identified using HMMER 3.0. Then, the identified unigenes were processed by the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de (accessed on 1 November 2022)) and ORF Finder ) for additional validation. Following that, genes encoding protein sequences < 300 amino acids long were removed. The UGTs were processed by MEGA 10.2 software for further analysis [https://itol.embl.de (accessed on 1 December 2022)) was used to modify the tree [The candidate UGTs in g et al. . First, analysis . Multiplanalysis . The maxanalysis . ITOL . The primers for the 10 unigenes were designed using Primer Premier 6.0, and 2 internal controls  were preC. deserticola parasitism were determined. These metabolites and genes were linked to phenylalanine metabolism, tryptophan metabolism, and phenylpropanoid biosynthesis pathways, which provide precursors for PhGs. C. deserticola makes use of host-derived components to produce PhGs. We highlight the host’s influence on the quality of Cistanches Herba and recommend that C. deserticola growers select suitable host plants.In this study, host metabolites and genes changed by


ATM mutations have been reported in variant A-T cases manifesting isolated generalized or segmental dystonia without any signs of classical A-T.Ataxia-telangiectasia (A-T) is an autosomal recessive disorder with high clinical heterogeneity. A-T may present in complicated variable forms, including classic A-T and milder form of AT. Contrary to the classic A-T, the milder form does not present the cardinal features of A-T such as ataxia and telangiectasia. A few An A-T pedigree with predominant dystonia was collected. Genetic testing was performed by targeted panel of genes involved in movement disorders. The candidate variants were further confirmed by Sanger sequencing. We then reviewed previously published literatures of genetically confirmed A-T cases with predominant dystonia and summarized the clinical characteristics of dystonia-dominant A-T.ATM mutations, p.I2683T and p.S2860P, were identified in the family. The proband presented isolated segmental dystonia without any signs of ataxia and telangiectasias. We reviewed the literatures and found that the patients with dystonia-dominant A-T tend to have a later-onset and slower progression of the disease.Two novel To our knowledge, this is the first report of A-T patient with predominant dystonia in China. Dystonia may appear as one of the predominant manifestations or initial symptom of A-T. Early ATM genetic testing should be considered for those patients with predominant dystonia, despite without accompanying ataxia or telangiectasia. Most peATM mutations have been reported in variant A-T cases manifesting as isolated generalized or segmental dystonia without any signs of classical A-T  mutations in both alleles leading to total loss of ATM kinase activity and milder form of AT  often associated with missense or leaky splice mutations . Patientical A-T .ATM gene which plays a critical role in pathway regulating DNA double-strand breaks (DSBs) repair. To date, more than 400 disease-related ATM mutations have been identified in patients with A-T. Among these ATM mutations, the majority (about 70%) are truncating changes resulting in premature translation termination , immunoglobulin(Ig), and ceruloplasmin) were conducted. An additional 200 normal individuals without any neurological disorders were included as controls. Written informed consents were obtained from all participants. This study was approved by the ethics committee of Wuhan Union Hospital.The pedigree was collected from the Neurology Department of Wuhan Union hospital. DNA samples were obtained from all the participants, including the proband, his affected older sister and their parents. Magnetic resonance imaging (MRI) of brain and cervical, ultrasound of heart and abdominal, electroencephalogram (EEG), and blood biochemical analysis , the Multiplex ligation-dependent probe amplification assay (MLPA) analysis was performed by reference to the method previously reported . A panel was designed to cover 101 genes associated with movement disorders . Deep sereported .2.3.Sanger sequencing was used to validate the candidate variants after data analysis. Forward and reverse primers were designed to amplify the fragments covering the variant sites. The fragments were sequenced on ABI 3530xL DNA Sequencer. Co-segregation analysis was conducted through screening for the confirmed variants in the proband's older sister and parents.3.3.1.GCH1, TH, and SGCE, were found in the proband. About 94.5% of the target bases were covered with at least 50X, and the mean depth of coverage for all target regions was 170. After filtering, two novel missense variants in ATM, c.T8048C (p.I2683T) and c.T8578C (p.S2860P), were identified in the proband (No large deletions or duplications of  proband . The two3.2.II:2), a 15-year-old boy, was delivered by forceps with no birth complications and had a normal childhood development milestones. He started walking around one year of age and had normal speech. He presented at our clinic complaining of neck pain and stiffness since 9 years old, which progressively worsened over the period of 2–3 years. He developed mild head shake at age 12. Then he experienced episodes of involuntary turning of the head to the left and cervical hyperextension, postural hand tremor, and blepharospasm at age 13. On examination at age 15, he had severe dystonia with dominant craniocervical involvement. The jerky right torticollis, postural hand tremor, and choreiform facial movements were present. Eye movements were normal with normal saccades responses and ocular pursuit movements. He had normal muscle power and reflexes, and a slightly increased muscle tone in left upper limb. Sensory examination was unremarkable. There were no cerebellar signs. His cognitive function remains intact, with a Mini-Mental State Exam (MMSE) and Montreal Cognitive Assessment (MoCA) score of 28/30 and 25/30, respectively. Eye ophthalmic examination was unremarkable.The proband (II:1) was a 19-year-old girl and experienced similar symptoms since age 12 years. She noted her first symptom of mild involuntary head shake while taking a photo at the age of 12 years. As the disease progressed, she gradually developed jerky right-sided torticollis, postural hand tremor, oromandibular dystonia. She had recurrent episodes of transient global amnesia, which occurred 2–3 times per year. Her examination was further marked by severe cervical and oromandibular dystonia, slightly increased muscle tone in left upper limb, and postural hand tremor. The deep tendon reflexes were all absent, with flexor plantar responses. She showed normal saccades and ocular pursuit movements. Her cognitive function was unremarkable.His older sister  of A-T patients . The prereviewed , 10–18 (reviewed . The medanalysis . NotablyIn line with previous study , signifiThe neuropathological hallmark of A-T is cerebellar Purkinje cell loss, followed by cerebellar symptoms and degeneration . The exaATM genetic testing should be considered for those patients with predominant dystonia, despite without accompanying ataxia or telangiectasia.In conclusion, our findings expand the clinical and molecular spectrum of dystonia-dominant A-T in China. Dystonia may appear as one of the predominant manifestations or initial symptom of A-T. Isolated dystonia without any signs of ataxia or telangiectasias may result in delay in the diagnosis of A-T. Early


Aeromonas hydrophila infection to aquaculture growth can be prevented through vaccination, but differences among A. hydrophila strains may affect the effectiveness of non-conserved subunit vaccines or non-inactivated A. hydrophila vaccines, making the identification and development of conserved antigens crucial. In this study, a bioinformatics analysis of 4268 protein sequences encoded by the A. hydrophila J-1 strain whole genome was performed based on reverse vaccinology. The specific analysis included signal peptide prediction, transmembrane helical structure prediction, subcellular localization prediction, and antigenicity and adhesion evaluation, as well as interspecific and intraspecific homology comparison, thereby screening the 39 conserved proteins as candidate antigens for A. hydrophila vaccine. The 9 isolated A. hydrophila strains from diseased fish were categorized into 6 different molecular subtypes via enterobacterial repetitive intergenic consensus (ERIC)-PCR technology, and the coding regions of 39 identified candidate proteins were amplified via PCR and sequenced to verify their conservation in different subtypes of A. hydrophila and other Aeromonas species. In this way, conserved proteins were screened out according to the comparison results. Briefly, 16 proteins were highly conserved in different A. hydrophila subtypes, of which 2 proteins were highly conserved in Aeromonas species, which could be selected as candidate antigens for vaccines development, including type IV pilus secretin PilQ (AJE35401.1) and TolC family outer membrane protein (AJE35877.1). The present study screened the conserved antigens of A. hydrophila by using reverse vaccinology, which provided basic foundations for developing broad-spectrum protective vaccines of A. hydrophila.The threat of bacterial septicemia caused by  Aeromonas hydrophila, a rod-shaped Gram-negative bacterium, is an important member of the genus Aeromonas of Vibrionaceae. It is conditionally pathogenic and commonly found in nature. The pathogenic A. hydrophila can infect various species of fish and cause explosive necrotizing fasciitis, resulting in enormous economic losses to the aquaculture industry [A. hydrophila is an important means of clinical epidemiological investigation, which can also provide a scientific basis for cutting off the transmission route and controlling the outbreak of related diseases, and also has certain guiding significance for the preparation and application of more targeted vaccines. It has been found that A. hydrophila shows high intraspecific genetic diversity via the analysis using random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers [A. hydrophila is a genetically heterogeneous species with different pathogenicity potential for humans and other animals [A. hydrophila infection are antibiotic treatment or vaccine immunization. However, the problem of strain resistance affects the effectiveness of antibiotic usage, and the existence of various serotypes also limits the protective effects of vaccines [industry ,2,3. The markers . The dis animals . The maivaccines .A. hydrophila vaccines with OMPs as candidate antigens became a research hotspot and have been immunologically evaluated on various fish species [A. hydrophila [A. hydrophila was found to be able to prevent A. hydrophila infection in Labeo rohita via oral immunization [A. hydrophila [The outer membrane proteins (OMPs) of Gram-negative bacteria are important virulence factors, which are related to the pathogenicity of bacteria . Because species ,10. Wangdrophila . Abdelhadrophila . The outnization . These rdrophila . A. hydrophila. Traditional methods rely on empirical the screening of a few candidate vaccines according to the characteristics of known pathogens. Reverse vaccinology uses high-throughput screening of the whole genome of pathogens to identify genes encoding proteins that exhibit excellent immunogenicity, and then the recombinant proteins can be detected in vitro or in vivo. Reverse vaccinology has been applied to a variety of pathogens, including Neisseria meningitidis, Streptococcus agalactis, Streptococcus pyogenes, Streptococcus pneumoniae, and Escherichia coli [A. hydrophila by using the reverse vaccinology method, which can protect the experimental fish from the infection of different geographical isolates of A. hydrophila [The screening of antigenic proteins is the key approach to preparing a broad-spectrum protective vaccine against hia coli . Previoudrophila . A. hydrophila vaccines were screened by performing signal peptide prediction, transmembrane helical structure prediction, subcellular localization, antigenicity, and adhesion evaluation, as well as interspecific and intraspecific homology comparison of 4268 encoded protein sequences in A. hydrophila J-1 through the use of reverse vaccinology. The categorization of typically identified A. hydrophila strains from diseased fish was accomplished with ERIC-PCR technology, and the conservation of the selected candidate proteins in different subtypes of A. hydrophila was verified by amplifying them through PCR and sequencing. Furthermore, the candidate protein sequences were compared with those of Aeromonas species, including Aeromonas caviae, Aeromonas veronii, Aeromonas salmonicida, and A. hydrophila, and conserved proteins were screened out based on the results of the comparison. This study represents a preliminary step towards screening candidate antigens for the development of a broad-spectrum vaccine against A. hydrophila and will provide the foundation for the therapy and control of diseases resulting from A. hydrophila infection.In this study, candidate antigens for the development of This study was approved by the Animal Care and Use Committee of Jiangsu Ocean University . All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in China.In the present study, reverse vaccinology was applied to provide the basis for the preparation of subunit vaccines by predicting the signal peptides, transmembrane helices, and subcellular localization of candidate proteins .A. hydrophila J-1 strain were downloaded in the FASTA file format from the NCBI database for a total of 4268 sequences (accession number: CP006883.1).The whole genome nucleotide acid coding sequences of the www.cbs.dtu.dk/services/SignalP accessed on 14 December 2021) online software, the 4268 protein sequences of the A. hydrophila J-1 strain were analyzed to predict the presence of signal peptides and the location of hydrolysis sites with Gram-negative prokaryotes as the default setting. Using SignalP 5.0 (http://www.cbs.dtu.dk/services/TMHMM/ accessed on 16 December 2021) and HMMTOP (http://www.enzim.hu/hmmtop/ accessed on 16 December 2021) online software were used to predict the transmembrane helical structures of A. hydrophila J-1 strain-encoding proteins. The lower the number of transmembrane helices in the protein, the less difficult it was for the recombinant protein to be expressed and prepared. TMHMM 2.0 (https://www.psort.org/ accessed on 16 December 2021) is a web-based tool that provides links to the PSORT series of programs to predict subcellular localization. Based on comprehensive analysis, CELLO (http://cello.life.nctu.edu.tw/ accessed on 16 December 2021) can detect specific amino acid composition and predict the subcellular location of the target proteins. Gneg-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/Gneg-multi/ accessed on 16 December 2021) can analyze the subcellular localization on Gram-negative bacterial coding proteins according to the information of gene ontology, functional domains, and sequence evolution. To improve the accuracy of the subcellular localization of candidate proteins, the subcellular location of proteins with less than two TM (transmembrane) helices were predicted on the predictive websites, including PSORTb, CELLO, and Gneg-mPLoc, respectively. PSORTb (http://www.jenner.ac.uk/VaxiJen accessed on 17 December 2021). Candidate vaccine antigens were selected from the adhesion proteins with an adhesion index greater than 0.5, which were assessed using Vaxign (http://www.violinet.org/vaxign/ accessed on 17 December 2021). Vaxign is a web-based system for investigating vaccines. Using Vaxitope, the system accurately predicts MHC class I and II binding epitopes, identifies both known targets, and suggests potential candidates. Proteins with an antigen score of 0.5 or higher are more likely to be recognized by the host immune system, making them strong vaccine candidates for further analysis.Antigenicity and adhesion are important factors to consider when evaluating potential antigens. When bacteria invade, their adhesion structures encounter the immune system of the host. Therefore, immunity against the adhesion structure can help prevent further colonization and infection. The potential antigenicity of candidate proteins was assessed with a cutoff value set to 0.4 with VaxiJen  was performed by using BLAST, including A. allosaccharophila, A. bestiarum, A. caviae, A. dhakensis, A. encheleia, A. eucrenophila, A. fluvialis, A. jandaei, A. lusitana, media, A. piscicola, A. popoffii, A. rivipollensis, A. salmonicida, A. sanarellii, A. sobria, A. taiwanensis, A. tecta, A. veronii, A. aquatica, A. aquatilis, A. australiensis, A. bivalvium, A. cavernicola, A. crassostreae, A. diversa, A. enterica, A. enteropelogenes, A. finlandensis, A. lacus, A. guangheii, A. hydrophila, A. intestinalis, A. molluscorum, A. rivuli, A. schubertii, and A. simiae. To assess the homology of candidate proteins in the genus A. hydrophila strains. Detailed information about all used A. hydrophila strains in this study is listed in A. hydrophila strains isolated from five fish, including Parabramis pekinensis, Megalobrama amblycephala, Carassius auratus gibelio, Ictalurus punctatus, and Carassius auratus were used during this study. Six strains  were isolated and stored by our laboratory, and three strains  were isolated and stored by Neijiang Normal University , and the Ahc001 strain was purchased from the national strain resource center of Wuhan University. All strains were stored in 50% glycerol at −80 °C until use. All strains were activated in LB medium, and cultivated for 8 h at 37 °C, and the bacterial DNA was extracted using a TIANamp Bacteria DNA Kit  according to the manufacturer’s instructions. The extracted DNA was kept at −20 °C until use.The extracted genomic DNA from 10 strains was used as template DNA for the molecular characterization and ERIC-PCR molecular typing of A. hydrophila strains, a pair of primers  were used to amplify the conserved region of the A. hydrophila 16S rDNA gene [2O. The PCR reaction procedure was as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. All PCR products were detected with 1% agarose gel and then sequenced . Finally, the full-length sequences were assembled using DNAStar software (http://www.dnastar.com accessed on 8 April 2022) and sequence alignment was performed by BLAST. To characterize and verify the collected DNA gene . All theA. hydrophila strains [2O. The ERIC-PCR reaction procedure was set up as follows: the temperature of pre-denaturation was 94 °C for 5 min, then denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 68 °C for 8 min by 30 cycles. The last extension was set to 65 °C for 16 min. The amplified ERIC-PCR products were detected using electrophoresis on 1.5% agarose gel.A pair of universal primers  were used in the ERIC-PCR assay, which was performed in triplicate to analyze the fingerprinting patterns of all  strains . The finA. hydrophila strains, specifically including generation of the electrophoresis diagram, calculating the molecular weight of each band according to the DNA markers, and recording the amplified bands of each sample with ‘1′ when the band existed or ‘0’ when it did not exist. NTSYS-pc software  was used to analyze the genetic similarity matrix, pedigree, and distance matrix, and the unweighted paired bucket average (UPGMA) method was used to cluster the fingerprints. Those with similarity ≥0.8 were assumed to be the same subtype, while those with a similarity <0.8 were different genotypes. These software have been widely used in the analysis of gel bands and the molecular typing of species [Quantity One software  was used to analyze the ERIC-PCR fingerprints of  species ,19. A. hydrophila subtypes was detected via PCR amplification of protein-coding sequence (CDS) regions. Starting from the CDS region of the protein, 210 bases were counted both forward and backward. Primers for 39 proteins were designed using Primer Premier 5.0 to ensure amplification of the intact CDS region . Finally, the full-length sequences obtained from sequencing were analyzed and aligned with those of the Aeromonas species in the NCBI database using BLAST.The conservation of candidate proteins in different S region . The finAeromonas species were selected from the NR database of NCBI  using SPSS , with A. hydrophila J-1 strain genome encoded 4268 proteins in total, and 678 proteins with signal peptides in the N-terminal regions. Among them, the number of proteins that possessed common secretory pathway (Sec) signaling peptides, twin-arginine translocation (TAT) signaling peptides, and lipoprotein signaling peptides were 479, 26, and 173, respectively.In the present study, reverse vaccinology was applied to provide the basis for the preparation of subunit vaccines by predicting the signal peptides, transmembrane helices, and subcellular localization of candidate proteins . The A. TMHMM2.0 and HMMTOP analysis showed that 466 proteins had no transmembrane protein α-helix and 158 proteins had one α-helix, which indicated that the structures were mainly β-barrel. β-barrel proteins are mainly porins, which usually exist in the outer membranes of prokaryotes and the organelle membranes of eukaryotes, thereby providing permeability for the outer membrane, participating in the transport of metabolites and proteins, and maintaining the stability of the outer membrane structures. In the prokaryotic expression system, multiple transmembrane helix structures will affect the recombinant expression of proteins; thus, 54 proteins with multiple transmembrane helix structures were excluded, and the remaining 624 proteins could be further analyzed .The pie chart indicates the prediction results of the three software, namely the specific location of the 624 candidate proteins in the cell. The PSORTb software predicts that about 10.6% of the candidates are outer membrane proteins A. The CEThe antigenicity of a candidate antigen to evaluate the ability of the antigen to be recognized by the host immune system was predicted. The potential antigenicity of the candidate proteins was assessed using VaxiJen with the cutoff value set to 0.4, which showed that 64 of 66 proteins scored higher than the critical value (>0.4), indicating that most of the identified outer membrane proteins were potential antigenic proteins. In addition, the adhesion of outer membrane proteins is related to bacterial pathogenicity, determining whether bacteria can adhere and colonize specific sites, which is also a vital indicator in antigen screening. Vaxign was used to predict candidate vaccine antigens based on the criteria of an adhesion index above 0.5, which showed that 39 of 66 outer membrane proteins met the standard and could be selected as candidate antigens for the development of subunit vaccines.Aeromonas species, the candidate protein sequences were compared with 39 other Aeromonas genera via BLAST alignment. In total, 15 of 66 candidate proteins showed high conservation in Aeromonas, with a percent identity greater than 85% and query coverage greater than 40% , and one strain had high sequence similarity with A. veronii (99%). The result was consistent with The size of the 16S rDNA genes sequence was about 1500 bp. These sequences were reversed, aligned, and compared with similar database sequences. BLAST analysis showed that nine strains had high sequence similarity with The gel electrophoresis map showed that the 10 strains could obtain 3–5 bands with molecular weight between 300 and 5000 bp through ERIC-PCR amplification, indicating gene polymorphism. Most strains had obvious main bands at 1000–1500 bp and 2000–3000 bp A. The reA. hydrophila strains were classified into six genotypes. Type I strains had the largest number with three strains, two of which were isolated from different ecological niches (kidney and liver) of the same geographical location (Nanjing) and the same fish source (P. pekinensis). There are two genotypes isolated from different geographical locations (Huai’an and Wuhan) and different fish sources . Each genotype (Ⅲ-Ⅶ) has one strain; the strains were isolated from different geographical locations, fish sources, and ecological niches.The fingerprint cluster analysis showed that 10 strains were divided into seven genotypes B. Nine oA. hydrophila J-1. It was found that 39 proteins were widely distributed in A. hydrophila, with high sequence consistency and good conserved properties. The 24 strains in the comparison results were selected to record the sequence coverage and consistency, and the consistency data with sequence coverage greater than 80% were retained , type IV pilus secretin PilQ (AJE35401.1), TolC family outer membrane protein (AJE35877.1), outer membrane protein transport protein (AJE36247.1), OmpA family protein (AJE37343.1), porin OmpA (AJE37342.1), outer membrane protein OmpK (AJE37487.1), TonB-dependent hemoglobin/transferrin/lactoferrin family receptor (AJE37629.1), TonB-dependent siderophore receptor (AJE38562.1), LPS assembly protein LptD (AJE37705.1), porin (AJE37778.1), peptidoglycan DD-metalloendopeptidase family protein (AJE37804.1), TIGR04219 family outer membrane beta-barrel protein (AJE34989.1), immune inhibitor A (AJE35148.1), and DUF2860 family protein (AJE37735.1). Among them, the sequence consistency of two crucial proteins, type IV pilus secretin PilQ (AJE35401.1) and TolC family outer membrane protein (AJE35877.1), was found to be more than 80% across 30 Aeromonas strains. This indicates that PilQ and TolC are highly conserved in Aeromonas, and suggests that they may play important roles in the biology and pathogenesis of this bacterial genus.NCBI BLAST was used to compare the sequence of 39 candidate proteins of retained . The reswas >80% . In A. vA. hydrophila affects many kinds of fish, and the explosive bacterial septicemia caused by its infection results in serious economic losses to aquaculture. The classification system of A. hydrophila has been very confusing, which has brought great inconvenience to disease control and scientific research exchange. The molecular typing of 120 Aeromonas strains isolated from fecal samples and environmental samples from gastroenteritis patients using RAPD and ERIC-PCR revealed high genetic heterogeneity between these Aeromonas species. ERIC-PCR was shown to be useful for epidemiological investigations and population genetic analysis of Aeromonas and has the same discriminatory power as the RAPD method [The disease caused by A. hydrophila were analyzed using ERIC-PCR. The sample size was small and the prevalence of the strains was complex. Results showed that A. hydrophila strains from the same fish source tended to cluster into one genotype. For example, three A. hydrophila strains isolated from P. pekinensis were classified as genotype Ⅰ. However, strains collected from different ecological niches within the same region did not always cluster into one group, such as strains of genotype II and III that were separated from the gill and liver of P. pekinensis. Additionally, strains from different regions and ecological niches could cluster into the same genotype, as observed with strains collected from P. pekinensis in Huai’an and M. amblycephala in Wuhan classified as the second genotype. This suggests that the genotype of A. hydrophilic may be related to fish source, geographical location, and ecological niche, although it is not absolute. This is consistent with the conclusion reached by Aguilera-Arreola et al. that A. hydrophila is a genetically heterogeneous species [In this study, nine strains of  species . Cross-rB N. meningitidis [Shigella flexneri as potential vaccine candidates [Vibrio parahemolyticus, and conducted protein homology analysis in 32 species of Vibrio spp. According to the positive presence rate of at least 15 species of Vibrio spp, the proteins with high homology were selected as potential multivalent Vibrio vaccine candidates [A. hydrophila J-1 strain whole genome was performed based on reverse vaccinology. The specific analysis including signal peptide prediction, transmembrane helical structure prediction, subcellular localization prediction, and antigenicity and adhesion evaluation, as well as interspecific and intraspecific homology comparison, thereby screened the 39 conserved proteins as candidate antigens for the A. hydrophila vaccine. The conserved properties of 39 proteins were evaluated with the PCR amplification of genes from A. hydrophila isolates, and the positive presence rate of 16 proteins was 100%, indicating a high coverage rate in sequencing strains. However, the low prevalence of vaccine candidates in circulating strains may result in low coverage.The reverse vaccinology approach was first used to identify candidates for vaccines against the serogroup ngitidis . Subsequndidates . Using sndidates . In thisA. hydrophila, which are of great significance for the healthy green cultivation of freshwater fish. In order to identify candidate proteins for vaccine development, bioinformatics analysis was performed, starting with the prediction of the signal peptide. Most secreted proteins of bacteria carry a short peptide at their N-terminal, called the signal peptide [A. hydrophila J-1 have signal peptides in the N-terminal region, identified as three signal peptides, including the secretory pathways (Sec) signaling peptides, twin-arginine translocation (TAT) signaling peptides, and lipoprotein signal peptides. Prokaryotes export unfolded proteins from the cytoplasm through the general secretory Sec system [Many studies have shown that OMPs, as a potential candidate vaccine antigen of bacteria, has the advantages of strong immunogenicity and high safety. Novel OMPs as immunogens were prepared against different subtypes of  peptide . Predict peptide ,26. Our c system . TAT sigc system ,29. To ec system . To crosc system , and Gnec system .A. hydrophila OMPs candidates using BLAST revealed a relatively conserved OMP with the other Aeromonas species, which provides insight into cross-protection with these OMPs. Sixteen broadly conserved OMPs were identified in the A. hydrophila genus based on the overall homology analysis. Many of the conserved proteins are involved in crucial functions. For example, fadl-mediated LCFA and its derivatives play important roles in phospholipid biosynthesis, membrane permeability, and bacterial pathogenicity [A. hydrophila, and the homology of the protein sequences was more than 80%. BamA is the important center component of the complex, and is a member of the Omp85 superfamily [A. hydrophila Gspd family. In Klebsiella pneumoniae, the t2ss component Gspd-k is a relatively stable protein containing signal peptide sequences. It is a secretory protein and exists on the extracellular membrane with a conservative sequence [The homology analysis of the genicity . TolC fagenicity . The Tongenicity ,36. Outegenicity ,38. The genicity . Our reserfamily . It conterfamily . BamC iserfamily . BamE haerfamily . There asequence . Type IVsequence .A. hydrophila biofilm vaccine candidates using reverse vaccinology [Aeromonas at the sequence level. Additionally, it was found in this study that six OMPs , which are highly conserved across Aeromonas spp., could become effective vaccines against A. hydrophila infection due to their strong immunogenicity and cross-protective potential against different fish pathogens. TolC is evolutionarily conservative among Aeromonas, has strong immunogenicity and antigenicity, and may have cross-immune protection against different fish pathogens [A. hydrophila has three TonB energy systems (TBDTs) and they are all involved in the toxic effects of bacteria in zebrafish. With its protective immunogenicity and widespread distribution, tbdt is an excellent candidate for vaccine development [Neisseria gonorrhoeae strains and can serve as potential N. gonorrhoeae vaccine antigens [Pseudomonas aeruginosa, it can be used as a new type of immunogen [P. aeruginosa infection [Legionella pneumophila, PAL/PilE /FlaA DNA vaccine can stimulate strong humoral and cellular immune responses [Aeromonas bacteria, and the homology among different genotypes of A. hydrophila was more than 80%. It was speculated that OmpA protein of A. hydrophila was a cross-reactive common antigen, which could cause protective immunity in fish. Previous studies have shown that OmpA1 can be used as a potential vaccine antigen against the infection of virulent A. hydrophila in channel catfish [In past studies, Basmeet identified 14 proteins including TolC, BamA, LamB, and AH4AK4_2542 as whole-cell cinology . Among tathogens . A. hydrelopment . BamA  expression system to achieve effective expression. Only the first three complexes docked with major histocompatibility complex class I (MHC-I), major histocompatibility complex class II (MHC-II), and TLR-4 receptors were selected as effective vaccine candidates [A. hydrophila. Firstly, the epitope sequences of all selected antigens are predicted, calculated, and screened to determine their immunogenicity, toxicity, sensitization, and solubility. Then, adjuvants and ligands are combined with these sequences to effectively enhance the immune response. Next, the vaccine construction is modeled and docking and molecular dynamics simulations are used to predict the binding affinity of the designed vaccine with different immune receptors. Finally, the stability of the molecule is predicted by estimating the binding free energy of the complex. On this basis, Islam et al. have designed a multi epitope subunit vaccine for A. veronii, but the proposed candidate vaccine still needs to be tested in vivo and in vitro to determine its effectiveness and safety [Individual proteins contain hundreds of epitopes, but not all epitopes can induce T-cell- and B-cell-mediated immune responses. Epitope vaccines are developed and designed based on pathogen epitopes or amino acid sequence characteristics of determining clusters. It has the advantages of low toxicity, high immune targeting, safe and stable removal of antigen except immune-related components and harmful parts, and can improve the immune protection effect through the combination of multiple epitopes . Howeverthe host . Li et aeriments . Sheng elivaceus . Moreovendidates . To enhandidates . In concd safety .A. hydrophila and Aeromonas and showed high protein homology. A total of 16 proteins showed good conservation and they are described as OmpA-, OmpK-, and TonB-dependent transferrin family receptor, outer membrane protein transporter, TolC family outer membrane protein, peptidoglycan DD-metalopeptidase family protein, TIGR04219 family outer membrane barrel protein, immunosuppressant A, and DUF2860 family protein. These results will provide a wealth of candidate antigen materials for subsequent preparation.In this study, 39 outer membrane proteins were selected as vaccine candidate antigens using reverse vaccinology. These candidate proteins were all present in


Malassezia and Staphylococcus are the most dominant genera in human skin microbiome. To explore the inter-kingdom interactions between the two genera, we examined the transcriptional changes in Malassezia and Staphylococcus species induced upon co-culturing. RNA-seq analyses revealed that genes encoding ribosomal proteins were upregulated, while those encoding aspartyl proteases were downregulated in M. restricta after co-culturing with Staphylococcus species. We identified MRET_3770 as a major secretory aspartyl protease coding gene in M. restricta through pepstatin-A affinity chromatography followed by mass spectrometry and found that the expression of MRET_3770 was significantly repressed upon co-culturing with Staphylococcus species or by incubation in media with reduced pH. Moreover, biofilm formation by Staphylococcus aureus was inhibited in the spent medium of M. restricta, suggesting that biomolecules secreted by M. restricta such as secretory aspartyl proteases may degrade the biofilm structure. We also examined the transcriptional changes in S. aureus co-cultured with M. restricta and found co-cultured S. aureus showed increased expression of genes encoding ribosomal proteins and downregulation of those involved in riboflavin metabolism. These transcriptome data of co-cultured fungal and bacterial species demonstrate a dynamic interplay between the two co-existing genera. Malassezia and Candida, when co-cultured, produce stronger biofilms resulting in increased resistance against antifungal reagents [Aspergillus mycelia induces the expression of a gene cluster associated with archetypal polyketides, suggesting that direct physical interactions contribute to the communication among microorganisms and induce gene expression [Pseudomonas piscium inhibits the activity of an acetyltransferase expressed by the fungal species Fusarisu graminearum, resulting in reduced acetylation and repression of gene expression in the fungal pathogen. Thus, the epigenome of a fungal pathogen could be influenced by a chemical produced by co-existing bacteria [Microorganisms interact with each other either directly through physical contact or indirectly via chemical interactions in various ecosystems, such as microbial biofilms , 2, resppression . In planbacteria .Cutibacterium, Corynebacterium, and Staphylococcus are generally the dominant genera [Malassezia is the dominant fungal genus in almost all regions of the skin except the feet, which is colonized by diverse types of fungi [The skin is the largest organ acting as the primary physical barrier against the invasion of pathogens and external substances from the environment , 10. Skit genera , 14. Malof fungi , 15.Malassezia species are known to exist in oily skin environments since they cannot synthesize fatty acids and should acquire them externally. Thus, Malassezia species secrete multiple lipases involved in lipid metabolism and pathogenicity [Malassezia, are also secreted and used to utilize the nutrients present in the skin environment [Malassezia species [Malassezia globosa and Malassezia furfur [M. globosa inhibited the formation of biofilm in S. aureus and the SAP in M. furfur had a wide range of substrates including extracellular matrix [Malassezia restricta expresses a total of 17 secreted proteases, 12 of which are aspartyl proteases [M. restricta.genicity . Similarironment . Proteas species . In preva furfur , 19. Ther matrix , 19. MalM. restricta and two bacterial species of the genus Staphylococcus, which are predominant in skin microbiome. We comprehensively examined transcriptional changes in M. restricta and S. aureus upon co-culturing. We found that the expression levels of ribosomal protein-encoding genes (RPGs) were upregulated, while SAP-encoding genes were downregulated in M. restricta co-cultured with Staphylococcus species. We identified the predominant SAP, MRET_3770, in M. restricta and noticed that MRET_3770 was transcriptionally repressed by co-culturing with Staphylococcus species and by lower pH levels. In bacterial RNA-seq, we observed upregulation of Staphylococcus RPGs and downregulation of genes involved in riboflavin metabolism in co-cultured S. aureus.In the present work, we studied the interplay between the fungus M. restricta KCTC 27527, Staphylococcus epidermidis KCTC 13172, and Staphylococcus aureus NCTC 8325 were obtained from the Korean Collection for Type Cultures . M. restricta KCTC 27527 was maintained in modified Dixon (mDixon) medium  [Staphylococcus cells were maintained in tryptic soy broth  [M. restricta (OD600: 1) and S. epidermidis or S. aureus (OD600: 0.1) were inoculated in mDixon medium and co-cultured at 34°C for 12 h with shaking. To prepare bacterial spent medium, S. epidermidis or S. aureus (OD600: 0.1) cells were inoculated in mDixon medium and cultured at 34°C for 12 h. Then, the bacterial spent medium was separated from bacterial cells by centrifugation. Similarly, M. restricta (OD600: 1) cells were cultured for 12 h, and the M. restricta spent medium was separated by centrifugation.ic acid) , 35. Lowosphate) . For theAspartyl proteases were enriched from the extracellular culture media using pepstatin A-agarose resin . Briefly, prewashed pepstatin A-agarose resins were incubated with the concentrated extracellular media with shaking at 4°C for 1 h. The resins were collected into column with binding buffer (0.1 M citrate and 0.5 M sodium chloride at pH 3.0) and subsequently washed with 0.5 M sodium chloride at pH 3.0 and eluted with elution buffer (0.1 M sodium bicarbonate and 0.5 M sodium chloride at pH 8.7).Diluted cells were seeded and incubated on 96-well cell culture plates  for 12 h. The culture supernatant was removed, and the cells were washed with distilled water (DW) to remove planktonic microbial cells. After the plate was dried, 0.1% crystal violet was added to stain the bacterial biofilm firmly attached to the plate. After 10 min, the plate was washed with DW, and non-specific dye was decolorized using 30% acetic acid. The crystal violet staining was measured using spectrophotometry at OD600.M. restricta KCTC 27525 and GCA_026314335.1 for S. epidermidis KCTC 13172) using hisat2 v.2.2.1 [Total RNA from two biological replicates for each condition was processed to deplete the ribosomal RNA using a NEBNext rRNA Depletion Kit . Sequencing libraries for RNA-Seq were constructed using TruSeq Stranded mRNA Library Prep  following the manufacturer’s instructions. Sequencing was performed using a NovaSeq 6000 instrument (Illumina), following the manufacturer’s protocol, which generated 101-bp-long paired-end reads. The sequencing adapter removal and quality-based trimming of the raw data were performed using Trimmomatic v.0.36 . Cleaned v.2.2.1 . We used v.2.2.1 . Finally v.2.2.1 .M. restricta and Staphylococcus species, M. restricta (OD600: 1.0) and Staphylococcus species (OD600: 0.1) were inoculated in mDixon media and co-cultured for 12 h  were inoculated at the initial step of co-culturing. We tried to measure the growth rate of M. restricta in the co-culturing medium using a hemocytometer, but the number of colonies could not be detected due to the preferential aggregation of M. restricta cells. Instead, the growth rate of M. restricta in the co-culture was indirectly measured by using bacterial spent medium through the personal bioreactor (RTS-1). To prepare bacterial spent medium, Staphylococcus cells (OD600: 0.1) were inoculated in mDixon medium and cultured for 12 h. Then, the bacterial spent medium was separated from the bacteria by centrifugation. Incubation of bacterial species in bacterial spent medium mimics co-culturing conditions. The growth rate of M. restricta was slightly lower in S. aureus spent medium and further decreased in S. epidermidis spent medium  in the expression levels of 378 genes and downregulation of 310 genes in M. restricta  in M. restricta were noticeably elevated after co-culturing with Staphylococcus species, especially with S. aureus  were upregulated more than two-fold compared to M. restricta axenic culture  and M. furfur (FUN_000223) that were previously identified as functional SAPs (M. globosa (MGL_1932) and of M. furfur (FUN_000223) [To identify the major secretory aspartyl proteases (SAP) in ecretory . Genes eecretory . To detenal SAPs . MRET_26_000223) .Malassezia species, MRET_3770 and MRET_2827 were the most abundant putative SAPs in M. restricta.Next, the SAPs present in the culture media were captured by affinity chromatography using pepstatin A-agarose resin, and the proteins were visualized on SDS-PAGE . Then, tM. globosa are known to inhibit the formation of the biofilm generated by S. aureus [M. restricta, we performed a crystal violet assay in both axenic and co-culture conditions. S. aureus at OD600 of 0.5, 0.1, 0.05, or 0.01 were co-cultured with M. restricta at OD600 of 1 or 0.1 on 96-well plates. After 12 h, the amounts of biofilm formed under axenic and co-culture conditions were quantitatively measured through the density of crystal violet. Biofilm formation by S. aureus was significantly reduced upon co-culturing with M. restricta at OD600 of 1, but not altered after co-culturing with M. restricta at OD600 of 0.1 , compared to cultivation in fresh media (OD600: 5.5)  . We notit medium . Under tM. restricta and the bacterial Staphylococcus species. The interplay between the two organisms under co-culturing conditions are schematically summarized in M. restricta, RPGs were upregulated, and putative SAP genes were downregulated upon co-culturing with Staphylococcus species. In cells growing under favorable conditions, a great portion of total cellular energy is dedicated to ribosomal biogenesis [M. restricta co-cultured with S. aureus may experience favorable growing conditions or may be responding to other uncharacterized signals. The growth rate of M. restricta cultured in S. aureus spent media was similar to that observed in fresh media, but the growth rate of M. restricta cultured in S. epidermidis spent media was significantly lower than the growth rate observed in fresh media. Furthermore, we observed differential regulation of RPGs in M. restricta according to sub-cellular localization, motif sequences on the promoters of RPGs, and co-cultured Staphylococcus species.In the present study, we explored the inter-kingdom interactions between two major microorganisms present in skin microbiome, the fungal species ogenesis -22. Tighogenesis -22. ThusM. restricta co-cultured with either Staphylococcus species. However, these three genes were not homologs of the SAP genes previously characterized in M. globosa and M. furfur [M. restricta using the concentrated extracellular media by pepstatin A-agarose resin, which is a reversible aspartyl protease inhibitor [M. globosa and M. furfur [Genes encoding putative SAPs, MRET_3770, MRET_2827, and MRET_4390, were drastically downregulated in . furfur , 19. In . furfur , 19.M. restricta were downregulated in co-cultured conditions. We noticed that the pH of the co-culture medium was reduced from 6.7 to 5–5.5, presumably due to the organic acids emitted by Staphylococcus as a by-product during growth [Staphylococcus species protect the biofilms they form by inhibiting the predominant SAPs by reducing the pH. We further examined the expression of MGL_1932, a previously characterized SAP in M. globosa (MGSAP1) by lower pH or co-culturing with Staphylococcus species [M. globosa. The protein encoded by MGL_3328 might be another functional SAP candidate in M. globosa. Recent interesting study reported that MGSAP1 was upregulated in atopic and seborrheic dermatitis skin. Furthermore, this SAP play a pivotal role in fungal skin colonization and tissue inflammation in diseased skin tissue [We asked how these putative SAPs in g growth  and founn tissue . In our M. restricta and Staphylococcus species, we also performed bacterial RNA-seq of S. aureus co-cultured with M. restricta. We observed that RPGs were upregulated in co-cultured S. aureus. Regulation of bacterial RPGs have been not intensively studied but is known to be linked to a stress response mechanism referred to as “the stringent response”. The expression of RPGs might be repressed as a part of the stringent response by the function of ppGpp. In addition, ppGpp and the transcription factor DksA bind to the promoter regions of RPGs and inhibit transcriptional initiation in Escherichia coli [S. aureus since the growth rate of S. aureus in M. restricta spent medium was retarded, but the expression levels of RPGs were significantly elevated. Our data suggest that S. aureus may have an unknown mechanism to upregulate the expression of RPGs in response to unfavorable growth conditions. Co-cultured S. aureus exhibited downregulated expression of genes involved in riboflavin biosynthesis. Riboflavin is a precursor of co-enzymes FAD and FMN, which function as the prosthetic groups of various oxidoreductases. Bacterial riboflavin homeostasis is regulated by FMN riboswitches [rib genes. Thus, cellular concentration of FMN is critical for controlling the riboswitch, and the FMN riboswitches are among the most sensitive regulators of cellular homeostasis in response to environmental or cellular stressors [S. aureus obtains riboflavin from the host environment or generates it through de novo biosynthesis [M. restricta may produce and secret riboflavin or FMN, which would in turn repress bacterial FMN riboswitches, and the bacteria may use FMN acquired from co-culture environment. It is possible that M. restricta and Staphylococcus species may compete for riboflavin uptake in the co-culture environment.To comprehensively understand the transcriptional interplay between hia coli , 26. In switches -31. FMN tressors . Riboflatressors . S. aureynthesis , 33. TheM. restricta and Staphylococcus species, which co-exist in human skin microbiome. We observed the upregulation of genes encoding ribosomal proteins and the downregulation of genes encoding SAP in co-cultured M. restricta. Then, we noticed that the lower pH is critical factor to control the transcriptional expression of SAP coding genes in M. restricta. We also observed the upregulation of genes encoding ribosomal proteins and downregulation of genes involved in riboflavin metabolism in co-cultured S. aureus. Therefore, we found that the dominant fungus M. restricta and the bacteria Staphylococcus species interact with each other to compete or reconcile in a given co-existing environment.Collectively, we have explored the interplay between http://jmb.or.kr.Supplementary data for this paper are available on-line only at