Porphyromonas gingivalis and three P. gulae (as an ancestor) to nine antibiotics  was evaluated by E-testing of minimal inhibitory concentration (MIC) according to international standards. The results were compared with 16 international studies reporting MICs from 1993 until recently. In addition, 77 currently available P. gingivalis genomes were screened for antimicrobial resistance genes. E-testing revealed a 100% sensitivity of P. gingivalis and P. gulae to all antibiotics. This was independent of the isolation year (1970 until 2021) or region, including rural areas in Indonesia and Africa. Regarding studies worldwide (675 strains), several method varieties regarding medium, McFarland inoculation standards (0.5–2) and incubation time (48–168 h) were used for MIC-testing. Overall, no resistances have been reported for amoxicillin + clavulanate, cefoxitin, and imipenem. Few strains showed intermediate susceptibility or resistance to amoxicillin and metronidazole, with the latter needing both confirmation and attention. The only antibiotics which might fail in the treatment of P. gingivalis-associated mixed anaerobic infections are clindamycin, macrolides, and tetracyclines, corresponding to the resistance genes erm(B), erm(F), and tet(Q) detected in our study here, as well as fluoroquinolones. Periodical antibiotic susceptibility testing is necessary to determine the efficacy of antimicrobial agents and to optimize antibiotic stewardship.The in vitro antimicrobial susceptibility of 29 strains of the major periodontal pathogen  Porphyromonas gingivalis is a non-motile, Gram-negative, rod-shaped/filamentous (pleomorphic), anaerobic bacterium forming black-pigmented colonies after 3–4 days of incubation on supplemented  blood agar plates. It is an opportunistic pathogenic bacterium, commonly found in the human body and especially in the oral cavity, where it is associated with periodontal diseases. Together with Tannerella forsythia and Treponema denticola, it forms the red complex of etiologically important bacteria and is regarded as THE key periodontal pathogen [P. gingivalis increases with age [P. gingivalis induces an inflammatory response upregulating the nutrient-rich flow of sulcus fluid causing bacterial overgrowth/proliferation and subsequently dysbiosis. Simultaneously, it impairs the bactericidal innate host defenses by blocking Toll-Like-Receptor 4 (TLR4) recognition, assuring its survival [P. gingivalis and age-related macular degeneration [pathogen ,2. The dsurvival ,6. Besidsurvival , vaginalsurvival , amniotisurvival , synoviasurvival , and togsurvival . Recentlneration , adverseneration , Alzheimneration ,15, atheneration , and canneration  need conFrom the clinical perspective and following appropriate antibiotic stewardship, systemic antibiotics should only be used in well-selected patients and cases. In periodontology, their application should be restricted to specific conditions of severe progressive periodontitis defined by international recommendations ,19. MetrP. gingivalis strains, isolated from periodontal pockets in adult patients with advanced forms of periodontitis in Germany and worldwide, to all antibiotics mentioned above and by standardized methods. We selected strains according to the following criteria: of local importance (Germany) or to fill gaps of regions under-represented in MIC testing so far . We also included three P. gulae strains  as the probable ancestor of P. gingivalis [P. gingivalis genomes and searched for resistance genes. By discussing our results with respect to 16 international studies addressing MIC data from 1993 until recently, we intended to meta-analyze the global antimicrobial susceptibility of P. gingivalis over time, as a snapshot from its origin until 2021.The present study aimed to test the in vitro susceptibility of clinical ngivalis  and knowngivalis . In addiP. gingivalis and three P. gulae isolates to nine antibiotics are presented in P. gulae , for strains from rural regions, or for recently isolated strains of Western countries  with access to antibiotics. Comparing five pairs of isolates from the same Java-Indonesian patients in 1994 and 2002 , no tenP. gingivalis showed very different phenotypes in terms of time to pigmentation, encapsulation (apparent by slimy colonies), or colony size, partially explaining the difficulties in obtaining MIC readings.In general, P. gingivalis . For all other antibiotics tested, very few resistant strains were recognized worldwide as the principal reason for the range-extension.After carrying out an extensive search for global data on the in vitro antimicrobial susceptibility of ngivalis , here, wngivalis . InterespgpB gene producing lipid A 4′-phosphatase, which is responsible for polymyxin B (a cationic polypeptide antibiotic) resistance and, as dependent on complex formation with similar peptides called LPS binding protein (LBP), for evading TLR4-sensing and killing of P. gingivalis [erm(F) gene. In contrast, the German ERX1066730 genome carried the following four resistance genes: erm(B) and erm(F) conferring resistance to macrolide, lincosamide, and streptogramin B (MLSB); the cat(A1) gene for the chloramphenicol-resistance (not of interest as no application in anaerobic infections); and the tet(Q) gene encoding for a ribosomal protection protein conferring resistance to tetracycline. These results were confirmed by an alternative pipeline and database. Resistance to fluoroquinolones in Gram-negative bacteria mostly occurs by two mechanisms: first, through mutations in the target enzyme DNA gyrase and topoisomerase IV and second, by reducing intracellular fluoroquinolones through efflux pumps. However, we did not detect any fluoroquinolone-resistance determinants among the 77 genomes investigated here. Resistance to metronidazole in Gram-negative anaerobes is known to be associated with nitroimidazole-resistance genes (nimA–J isoforms). These genes  encode a 5-nitroimidazole reductase that converts nitro-imidazole to amino-imidazole, thus preventing the generation of bactericidal nitroso-residues [nim isoforms here.The presence/absence of resistance genes was detected by various approaches that are described in the Materials and Methods section. In addition, we used two different databases, ARG-ANNOT  and CARDngivalis ,49. Becaresidues . Of noteP. gingivalis MIC range, MIC50, and MIC90 data measured in our very limited number of strains were all within the range found by other studies conducted worldwide. We did not observe any trend of increased antibiotic resistance comparing data from P. gulae  and 4% (Bacteroides) from quite a few regions such as Pakistan, North India, and Kuwait, but also the USA and Europe [P. gulae strains with MICs > 512 µg/mL were already isolated from dogs [P. gingivalis was also demonstrated [nim genes. Of note, nim genes were never found here according to the limited literature addressing this topic [ monkeys ), isolat monkeys ,41,51,52020 also . Althougd Europe . As a marom dogs . Developrom dogs . After instrated . On the nstrated , seems tis topic , our gentet(Q) genes underlines this risk  was reported applying M.I.C.Evaluator strips . As two tudy and ).P. gingivalis-MIC50 over all studies and about 700 strains , but MICs as high as 8 mg/L for erythromycin  or betwP. gingivalis isolates from 2008–2015 by agar diffusion only [P. gingivalis , which is not seen worldwide and might be due to its widespread use in German dental practices  [P. gingivalis strains among Swiss periodontitis patients from different decades [ermF-positive and had MIC values higher than 8 mg/L, 2 mg/L, and 0.25 mg/L for clindamycin, azithromycin, and moxifloxacin, respectively. The second isolate had a high MIC value of 4 mg/L for moxifloxacin. Sequence analysis of the quinolone resistance-determining region (QRDR) of the gyrA gene confirmed a gene mutation, namely Ser-83 ≥ Phe substitution. Dahlen et al. revealed antibiotic susceptibility against seven antibiotics among 67 consecutive fresh isolates of P. gingivalis in a Swedish population, with data given only in figures but not as tables [P. gingivalis/P. endodontalis strains were examined but results not sorted by species [P. gingivalis) in Japan was recently confirmed, but with 4.9% moxifloxacin and 22.8% clindamycin resistance as exceptions and high MIC90 values (64–128 mg/L) for macrolides [Because of a reason given below, some important studies were not included in our analysis in the first instance. However, the results of these six studies will be discussed below for further comparison. Jepsen et al. tested antibiotic susceptibility of 5323 ion only . The aveiptions) . All 532 decades . Becauses tables . The str species . Howevercrolides .Striking for many studies is the delay between the isolation of strains and publication of susceptibility data, which can be more than 5 years. Assuming that the antibiotic resistance would increase by the same rate as measured by Jepsen et al. , presentPorphyromonas species also, and it must be concluded that P. asaccharolytica, P. levii, and P. uenonis were less susceptible to antibiotics [P. asaccharolytica and two P. levii strains from California were resistant to clindamycin (>32 mg/L). There were seven isolates  with levofloxacin MICs of 4 mg/L and three with MICs of 8 mg/L [Porphyromonas sp. with MIC < 256 mg/L was reported from Greece [Of final note, during this study, we came across susceptibility data of other ibiotics ,66. For f 8 mg/L . Metronim Greece .P. gingivalis and 3 P. gulae isolates to nine antibiotics, including amoxicillin, amoxicillin/clavulanate, clindamycin, metronidazole, moxifloxacin (fluoroquinolone), doxycycline (tetracycline), and azithromycin (macrolide), as well as imipenem and cefoxitin (the latter two for severe infections only) were determined by E test method . Bacterial strains were grown on Brucella blood agar plates , supplemented with 5% sheep blood, hemin (5 mg/L), and vitamin K1 (10 mg/L), for up to 5 days. The test strains (a few fresh colonies) were suspended in sterile phosphate-buffered saline equivalent to a 1.0 McFarland standard and streaked confluently over the surface blood agar plates with the aid of a sterile swab. Plates were incubated anaerobically for 2 to 5 days. Inhibition zones were measured at 48 h according to the recommendations of the manufacturer and the Clinical and Laboratory Standards Institute (CLSI), but for slow-growing strains, a longer incubation time was necessary before reading was possible. Percentages of resistant isolates were calculated using breakpoints advised by the CLSI  and by the European Committee on Antimicrobial Susceptibility Testing . For comparison with the literature, an advanced PubMed and Google Scholar search was performed combining MESH terms ), with results illustrated in The strains tested in our laboratory and their origin are summarized in P. gingivalis genomes that are listed in Bactopia pipeline version 1.6.5 [Shovill pipeline version 1.1.0  and the default setting for the SKESA assembler version 2.4.0 [ARIBA pipeline version 2.14.6 [CARD 2020) [ABRicate pipeline  on the assembled genomes using default settings, and here, both the ARG-ANNOT  [https://card.mcmaster.ca/, accessed on 10 October 2021) [https://www.mediterranee-infection.com/acces-ressources/base-de-donnees/arg-annot-2/ (accessed on 10 October 2021).In addition to phenotypic testing, we retrieved the sequence reads of 77 publicly available on 1.6.5 . Within on 2.4.0 . Bactopin 2.14.6  and the RD 2020)  as the der 2021)  and CARDer 2021)  public dP. gingivalis is not yet emerging but an increase of MIC data of tetracyclines, macrolides, lincosamide, and fluoroquinolones, has been recognized since the end of the 20th century. Only very few relevant genes, such as erm(B) or erm(F) conferring MLSB-resistance and tet(Q) encoding resistance to tetracyclines, were detected in the 77 publicly available genomes. Of note, neither phenotypic metronidazole resistance (with very few exceptions needing confirmation) nor corresponding nim genes were reported for P. gingivalis. However, a resistance-transfer from related Bacteroides, Prevotella, or other Porphyromonas species (including pet isolates) could emerge, as these are increasing in many regions worldwide. Thus, adjunctive antimicrobial usage in the treatment of periodontitis must be restricted and antibiotic stewardship and resistance gene screening extended.Fortunately, antimicrobial resistance of


Schistosoma sp. infection. In the Democratic Republic of Congo (DRC), and particularly in north-eastern Ituri Province, little is known about morbidity associated with Schistosoma mansoni infection. For this reason, we aimed to assess intestinal and hepatosplenic morbidity associated with S. mansoni infection in Ituri Province.Reducing morbidity is the main target of schistosomiasis control efforts, yet only rarely do control programmes assess morbidity linked to S. mansoni infection was assessed with a Kato-Katz stool test (2 smears) and a point-of-care circulating cathodic antigen (POC-CCA) urine test. A questionnaire was used to obtain demographic data and information about experienced intestinal morbidity. Each participant underwent an abdominal ultrasonography examination to diagnose hepatosplenic morbidity. Of the 586 study participants, 76.6% tested positive for S. mansoni. Intestinal morbidity reported in the two preceding weeks was very frequent, and included abdominal pain (52.7%), diarrhoea (23.4%) and blood in the stool (21.5%). Hepatosplenic morbidity consisted of abnormal liver parenchyma patterns (42.8%), hepatomegaly (26.5%) and splenomegaly (25.3%). Liver pathology  was positively and significantly associated with S. mansoni infection. Hepatomegaly  and splenomegaly  were positively but not significantly associated with S. mansoni infection at the individual level. At the village level, S. mansoni prevalence was positively associated with the prevalence of hepatomegaly and splenomegaly. High-intensity S. mansoni infections were associated with diarrhoea, blood in the stool, hepatomegaly, splenomegaly, and liver parenchyma . Four study participants were diagnosed with ascites and five reported hematemesis.In 2017, we conducted a cross-sectional study in 13 villages in Ituri Province, DRC. S. mansoni infection status in Ituri Province. The findings call for targeted interventions to address both S. mansoni infection and related morbidity.Our study documents a high burden of intestinal and hepatosplenic morbidity associated with  Schistosoma mansoni is of great public health importance in sub-Saharan Africa. The World Health Organization (WHO) recommends that control efforts aim to reduce morbidity through large-scale intervention programmes. However, intestinal and liver morbidity is rarely assessed in such control programmes. Hence, little is known about (i) the magnitude of the intestinal and liver morbidity burden in the community, or about (ii) the morbidity associated with S. mansoni infection, specifically. We conducted a cross-sectional study in which we assessed intestinal morbidity by questionnaire and liver morbidity by abdominal ultrasonography. Additionally, we determined the infection status of the study participants using standard diagnostic procedures (Kato-Katz technique and point-of-care cathodic circulating S. mansoni antigen [POC-CCA] urine test). Among the 586 study participants, aged six years and older, from 13 villages in Ituri Province, DRC, we observed a high degree of intestinal  and hepatosplenic morbidity . S. mansoni infection was associated with liver and spleen enlargement. Likewise, S. mansoni infection intensity was linked to diarrhoea, to liver and spleen enlargement and to pathological changes in the liver parenchyma. At village level, we observed that the prevalence of an enlarged liver and spleen among patients increased with the prevalence of S. mansoni infection. We conclude that the population of Ituri Province carries an alarming burden of intestinal, liver and spleen morbidity associated with S. mansoni infection. A comprehensive control programme to address this infection and disease burden is urgently required.Schistosomiasis caused by  Schistosoma and is one of the so-called neglected tropical diseases. It is a major cause of morbidity and mortality around the globe ) ,25. Live2), and four categories were set: underweight (<18.5 kg/m2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2) and obese (≥30 kg/m2). Infection prevalence was expressed as the number of S. mansoni-positive individuals divided by the total number of participants examined. Infection intensity was estimated based on helminth egg counts per gram of stool (EPG) when examined with the Kato-Katz technique [S. mansoni infection intensities were classified as light (1–99 EPG), moderate (100–399 EPG) and heavy (≥400 EPG) [Data was entered in Excel and cross-checked against the data sheet. STATA version 14.2 software  was used to manage and analyse data. Only participants with a complete dataset were retained in the analysis . Seven aechnique . S. mansweight <1.5 kg/m2,2) test was used to compare frequency distributions. A univariate logistic regression analysis was carried out to identify associations between S. mansoni infection status (outcome) and morbidity indicators (predictors) and/or demographic factors . Predictors with a significance level of 20% or less, and age and gender variables were included in the multivariable logistic regression models. Odds ratio (OR), adjusted OR (aOR) and corresponding 95% confidence intervals (95% CI) were calculated. Only p-values <0.05 were considered statistically significant. In this study, we combined the results of the Kato-Katz technique and those of the POC-CCA test to diagnose infection status. For comparison, the same analysis was repeated for Kato-Katz and the POC-CCA diagnostic approaches, separately.Arithmetic mean infection intensity was calculated. Categorical variables were presented as frequencies and percentages. Pearson’s chi-square  was not common among participants, with only eight participants diagnosed with an STH infection. In contrast, intestinal symptoms were very common, with 52.7%, 23.4% and 21.5%, reporting abdominal pain, diarrhoea, and blood in the stool within the two weeks preceding the survey, respectively. Five participants (0.9%) had experienced hematemesis at least once in his/her life. Abdominal ultrasound examinations revealed that 26.5% of participants had hepatomegaly, 25.3% had splenomegaly and 42.8% had liver pathology; 36.4% had mild PPF (C and D patterns), 6.4% had severe PPF (E and F patterns), 1.0% presented fatty liver (Y pattern), 0.2% had an unidentified abnormality (Z pattern) not linked to schistosomiasis and 0.7% had ascites. Only 56.0% of the participants had a normal and starry sky liver parenchyma (A and B patterns). More details on liver parenchyma patterns are shown in Of those with a complete dataset, 342 (58.4%) were females, 330 (56.3%) were under 20 years of age and 268 (45.7%) were underweight . The preS. mansoni, but the increased risk was not statistically significant . S. mansoni infection was observed more frequently in younger age groups, with prevalence peaking among young adults (P = 0.024).The results of the univariable risk analysis of the combined diagnostic approach are presented in g adults . ParticiS. mansoni infection status; of these, the association with hookworm infection was statistically significant . Participants who reported an episode of diarrhoea within the two weeks preceding the study had an increased risk of S. mansoni infection .Intestinal helminth coinfections were negatively associated with p = 0.168), splenomegaly  and E/F liver parenchyma patterns  were positively but not significantly associated with an S. mansoni infection.Diagnosed hepatomegaly ; and, unlike splenomegaly (p = 0.017), hepatomegaly (p = 0.079) was not significantly associated with S. mansoni infection.The univariable risk analyses of the Kato-Katz and POC-CCA diagnostic approaches are given in supplementary S. mansoni infection levels, with corresponding peaks in the adolescent and adult age groups . This was also the case when the results of the Kato-Katz diagnostic approach were analysed . However, the association was not significant  for the results of the POC-CCA diagnostic approach. Other indicators, including the presence of blood in the stool, abdominal pain, and history of hematemesis, showed no association, regardless of the diagnostic approach used. Second, hepatomegaly was not significantly associated with an S. mansoni infection when analysing the results of the combined diagnostic approach , the POC-CCA diagnostic approach , or the Kato-Katz diagnostic approach . Splenomegaly was significantly associated with an S. mansoni infection when analysing the Kato-Katz diagnostic results . The association was not statistically significant for the combined  or the POC-CCA  diagnostic approaches. Third, an abnormal liver parenchyma pathology (combined E/F patterns) was significantly associated with S. mansoni infection when analysing the Kato-Katz diagnostic results . The association was not significant for POC-CCA  or the combined approach .The risk analysis took into account the results of the combined diagnostic approach . The resS. mansoni infection , while gender was not significantly associated with S. mansoni infection .Ten variables were included in the multivariable logistic regression analysis, the results of which are displayed in p = 0.053) and hepatomegaly  were associated with S. mansoni infection with a borderline significance level. Patients with abnormal liver parenchyma patterns  did not have an increased risk for S. mansoni infection.Of the morbidity indicators investigated, diarrhoea . The age distribution of the infection intensity levels followed the age-infection prevalence curve. Heavy-intensity infections were mostly found (12.9%) among adolescents, aged 10–14 years, while no one in the oldest age group (50 years and older) had a heavy-intensity infection (p = 0006). Heavy-intensity infections were significantly higher among patients co-infected with Ascaris lumbricoides  and underweight participants .p = 0.004) and blood in the stool  among patients in the heavy-intensity infection group compared to the other infection intensity groups.There was a significantly higher prevalence of reported diarrhoea , while the prevalence of hepatomegaly (38.1%) was not statistically different from other infection intensity groups (p = 0.073). When stratified by age, patients with an enlarged liver and/or spleen bore a higher infection intensity burden compared to those with a normal-sized liver and spleen in the same age group  was significantly higher than among other infection intensity groups (ge group . In geneS. mansoni infection intensity varied considerably among patients with different liver parenchyma pathologies (p = 0.107). When stratified by age, a clear association emerged between increased number of high-intensity infections and increasingly abnormal liver pathologies  and monitor morbidity data. Instead, they largely rely on monitoring infection intensities, which are linked to morbidity and much easier to assess than intestinal and hepatosplenic morbidity. Consequently, little is known about the morbidity burden of schistosomiasis .Control programmes were conducted successfully in colonial times, even with means that today may be considered outdated . FollowiWe found a high degree of intestinal and hepatosplenic morbidity. About one quarter of the study participants reported diarrhoea and blood in the stool. Upon ultrasonography examination, almost one-quarter was diagnosed with hepatomegaly; almost two-thirds had splenomegaly, and more than half had an abnormal liver parenchyma (C–F patterns). Five patients reported an experience of hematemesis and four patients had ascites.S. mansoni infection was high among the study population. As the POC-CCA diagnostic approach does not provide information about infection intensity, the Kato-Katz technique was used to analyse infection intensity. Light-, moderate- and heavy-intensity infections were diagnosed in high frequencies. Only a few cases of soil-transmitted helminths were diagnosed during this study and likely had a very small impact on the morbidity findings. S. mansoni infection prevalence and intensity was highest in the adolescent and young adult age groups. The prevalence of intestinal and hepatosplenic morbidity indicators showed a very similar age distribution ; at village level, hepatosplenic morbidity prevalence increased with infection prevalence. Both observations suggest a close link between morbidity and S. mansoni infection. Furthermore, at individual level, we found an increased risk of hepatomegaly and splenomegaly in S. mansoni-infected patients, consistent with the association found at village level and the aforementioned age distribution. The findings are also consistent with documented hepatosplenic morbidity associated with S. mansoni infection [S. mansoni infection is a major contributor to the overall observed morbidity.The prevalence of nfection ,29, hencS. mansoni infection; second, pathological changes of the liver parenchyma were associated with S. mansoni infection as was hepatomegaly; and third, splenomegaly was not associated with S. mansoni infection. Using the Kato-Katz test alone to diagnose S. mansoni infection reduces the overall sensitivity of the diagnostic approach due to the low sensitivity of the technique itself [S. mansoni infection via the KK test are more likely to have a higher-intensity infection compared to those diagnosed with the combined approach. From this observation, we see that subtle increases in morbidity—such as reported diarrhoea and pathological changes in the liver parenchyma—become statistically significant. Indeed, for both morbidity indicators, we observed an association with S. mansoni infection intensity. Patients with diarrhoea had the highest prevalence of heavy-intensity infections  and a more sensitive (POC-CCA) diagnostic approach, we considered the results of the combined diagnostic approach. First, reported diarrhoea was significantly associated with e itself ,31. Hencfections  and thosfections . HoweverS. mansoni infection intensity and risk compared to those with E pattern. The highly echogenic bands and streaks corresponding to F pattern extend from the main portal vein and its bifurcation to the liver surface. Most commonly, this pattern manifests itself in very advanced cases of liver fibrosis and is frequently accompanied by other changes [We found that patients with abnormal liver parenchyma pattern F displayed lower  changes ,23. Pati changes ,32–34.Schistosoma infection is unspecific. Hence, the morbidity pattern observed might have been provoked partially by or in combination with other pathogens, such as other helminth species, protozoa, bacteria, and viruses. Given that multiple infections are frequent in tropical Africa, a combination of infections is most likely responsible for the observed morbidity. In Ituri Province, other parasitic infections, such as malaria , and other infections with hepatosplenic affinity, such as viral hepatitis, are prevalent [Quantifying schistosomiasis morbidity is a challenging ,35 and crevalent –37 and mrevalent –40.S. mansoni infection. However, ultrasonography devices are rarely available in poor-resource settings. In Ituri, the device may be found only in the provincial hospital, and in some district hospitals and private clinics. During our field work, we found that the use of portable devices was feasible in the villages. However, in the most remote rural areas, usage is challenging due to the unavailability of electricity. Additional equipment, such as solar panels and rechargeable batteries, are required. Despite these shortcomings, abdominal ultrasonography yields crucial information about liver morbidity in the community and hence, indispensable information about the public health burden of S. mansoni infection [Nevertheless, these findings do not in any way reduce the value of abdominal ultrasound in diagnosing liver pathologies associated with nfection . Severe S. mansoni infection, which further underscored the importance of the infection’s morbidity burden. Four people (0.9%) reported a history of hematemesis and two people (0.5%) reported ascites. The finding appears to corroborate the health service’s statistics report from the Angumu health district (on the shore of Lake Albert), which declares that hematemesis is a frequent medical emergency and that adults in this area have died after vomiting blood. Oesophageal varices remain silent until they rupture and irreversible damage occurs [We encountered patients with severe complications from e occurs . Angumu S. mansoni endemic communities outside of the DRC present similar morbidity levels [S. mansoni infection.The morbidity levels we observed are consistent with those measured by Ongom and Bradley , who fouy levels –42. Veryy levels ,43. Our S. mansoni infection, the examined population is not representative of the entire province, but rather of high transmission areas. Furthermore, ongoing civil unrest in Ituri creates a challenging security situation, which only afforded us a short time in each village. For this reason, only one stool sample could be collected from each study participant. Finally, limited available resources did not allow us to examine participants for parasitic, bacterial, and viral coinfections, which could have helped to better explain the degree of morbidity linked to S. mansoni infection.The study presents some limitations. As the study was conducted in purposively sampled villages known for their high prevalence of S. mansoni infection in the hepato-splenic pathology.Other limitations include a lack of blood coinfection diagnosis and a body mass index (BMI) cut-off that did not take into account variations among the study population, as recommended . Concern2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2) and obese (≥30 kg/m2). This clustering may have introduced a selection bias in our results. However, in the risk analysis, we used the BMI as a continuous variable rather than a categorical one.The BMI cut-off values were defined by four categories, including underweight Click here for additional data file.S1 TableStudy conducted in 13 purposively selected villages in Ituri province (n = 586). Only diagnostic results of Kato-Katz (KK) tests have been considered.(DOCX)Click here for additional data file.S2 TableSchistosoma mansoni infection among participants from 13 villages in Ituri province (n = 586). Only diagnostic results of Kato-Katz (KK) tests have been considered.Results of the multivariable analysis of risk factors for morbidity due to (DOCX)Click here for additional data file.S3 TableStudy conducted in 13 purposively selected villages in Ituri province (n = 586). Only diagnostic results of the point-of-care circulating cathodic antigen (POC-CCA) tests have been considered.(DOCX)Click here for additional data file.S4 TableSchistosoma mansoni infection among participants from 13 villages in Ituri province (n = 586). Only results of the point-of-care circulating cathodic antigen (POC-CCA) diagnostic tests have been considered.Results of the multivariable analysis of risk factors for morbidity due to (DOCX)Click here for additional data file.S5 TableResults from 13 purposively selected villages of Ituri province (n = 586). Prevalence derived from combined diagnostic approach and intensity determined by Kato-Katz test results.(DOCX)Click here for additional data file.S6 TableResults from 13 purposively selected villages in Ituri province (n = 586). Prevalence derived from a combined diagnostic approach.(DOCX)Click here for additional data file.S7 TableStudy conducted in 13 purposively selected villages in Ituri province (n = 586). Only results of the Kato-Katz diagnostic tests have been considered in this analysis.(DOCX)Click here for additional data file.S8 TableResults from 13 purposively selected villages of Ituri province (n = 586). Analysis of Kato-Katz results, POC-CCA results, and the combined diagnostic approach results.(DOCX)Click here for additional data file.


Ixodes ricinus feeding. Our data indicate that tRNA synthetases represent attractive, anti-tick targets warranting the design of selective inhibitors. Further, we tested whether these severely impaired ticks were capable of transmitting Borrelia afzelii spirochaetes. Interestingly, biologically handicapped I. ricinus nymphs transmitted B. afzelii in a manner quantitatively sufficient to develop a systemic infection in mice. These data suggest that initial blood-feeding, despite the incapability of ticks to fully feed and salivate, is sufficient for activating B. afzelii from a dormant to an infectious mode, enabling transmission and dissemination in host tissues.It has been demonstrated that impairing protein synthesis using drugs targeted against tRNA amino acid synthetases presents a promising strategy for the treatment of a wide variety of parasitic diseases, including malaria and toxoplasmosis. This is the first study evaluating tRNA synthetases as potential drug targets in ticks. RNAi knock-down of all tested tRNA synthetases had a strong deleterious phenotype on  Currently, there is no human vaccine to prevent borreliosis. The traditional approaches target antigens on the surface of Borrelia, such as OspA [Borrelia requires for effective transmission to the mammalian host have also been proposed. As ticks are armed with a unique repertoire of salivary molecules displaying anticoagulation, anti-inflammatory and immunomodulatory activities [Borrelia spp.) exploit the pharmacological activity of tick saliva to colonize and persist in the vector, and support the transmission from the vector to the vertebrate host [Borrelia transmission through the primary neutralization of tick salivary proteins [Ixodes scapularis salivary proteins has been tested. This vaccine elicited an antibody response, which consequently induced an immediate inflammatory reaction in the guinea pigs in response to tick attachment. Such a response could help to alert the host shortly after tick attachment, thus facilitating early removal of the tick and preventing transmission of Borrelia [Lyme borreliosis is the most common vector-borne disease in Europe and the USA. It is caused predominantly by the spirochaete  as OspA –3, but ativities –6, it waate host . Therefoproteins –10. ReceBorrelia .Borrelia transmission is enhanced in the presence of salivary proteins [Borrelia with homogenates of tick salivary glands. However, most of these experiments either overestimated the amount of homogenate used, exceeding physiological amounts of saliva produced during in vivo feeding [Borrelia transmission [Borrelia transmission.It has been documented that proteins ,12,13. T feeding , or used feeding ,16, timesmission , indicatDuring the past decade, it has been demonstrated that impairing protein synthesis using drugs targeted against tRNA amino acid synthetases presents a promising strategy for treating a wide variety of parasitic diseases . Inbred. 2.2Total DNA was isolated from murine tissues using a NucleoSpin Tissue Kit  according to the manufacturer's protocol.Total RNA was extracted from nymphs and adult females using a NucleoSpin RNA Kit (Macherey-Nagel) according to the manufacturer's protocol. Isolated RNA was reverse transcribed into cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit . All cDNA preparations were prepared in biological triplicates.. 2.3I. ricinus midgut transcriptome [IrAsn-RS; GenBank JAP73321), Valyl-tRNA synthetase , Leucyl-tRNA synthetase  and Lysyl-tRNA synthetase . A 328 bp fragment of IrAsn-RS, a 560 bp fragment of IrVal-RS, a 420 bp fragment of IrLeu-RS and a 380 bp fragment of IrLys-RS was each amplified from I. ricinus midgut-specific cDNA using primers listed in electronic supplementary material, table S1, and cloned into plasmid pll10. Respective dsRNA fragments were synthesized as described previously [–1) was injected (345 nl or 32.2 nl) into the haemocoel of I. ricinus adult females or nymphs, respectively, using Nanoinject II (Drummond). After 3 days of rest in a humid chamber at room temperature, ticks were fed on rabbits or C3H/HeN mice. The level of gene silencing was measured by RT-qPCR.Four tRNA synthetases were identified as RNAi targets in our previously described criptome , namely eviously . Each ds. 2.4Borrelia afzelii-infected nymphs injected with dsRNA were fed on naive C3H/HeN mice (three nymphs per mouse) for 3 days and then forcibly removed. Each experiment was carried out with five mice per dsRNA group. Engorgement weights and feeding successes were recorded for each individual nymph.6 murine ß-actin copies.Four weeks after the challenge by infected tick nymphs, the total spirochaete load in murine tissues was determined by quantitative real-time PCR (qPCR) as described previously . The spiIrAsn-RS, IrVal-RS, IrLeu-RS and IrLys-RS were normalized to I. ricinus elongation factor 1 using the ΔΔCt method [cDNAs from silenced nymphs and adult females served as templates for the quantitative expression analyses by relative qPCR. Relative expressions of t method .. 2.5Ixodes ricinus nymphs were fed on mice for 48 h. Forcibly detached nymphs were opened by micro-dissection, and a pair of salivary glands was removed, washed in ice-cold PBS and placed into ice-cold PBS containing a tablet of protease inhibitor mix (Thermo Scientific A32963). Seven pairs of salivary glands were homogenized in 50 µl of the buffer using a 29G insulin needle and three freeze/thaw cycles. The homogenate was clarified by centrifugation at 15 000g, 20 min, 4°C. The quantity of soluble protein present in the extract was determined in a Bradford assay by absorbance at 595 nm (A595), with a dilution of the sample to a value of A595 ∼ 0.2.. 2.6I. ricinus nymphs were fed on mice for 72 h. A pair of salivary glands from 10 nymphs was dissected, mounted in DABCO mounting medium and immediately inspected under the Olympus BX53 microscope . The area of 10 representative acini from individual salivary glands was measured using the ImageJ program. The size of the acini in silenced nymphs was compared with the GFP control group.Silenced . 2.7t-test was used for evaluation of statistical significance. A p-value of less than 0.05 was considered statistically significant. Error bars in the graphs show the standard errors of the means.Data were analysed by GraphPad Prism 6 for Windows, v. 6.04, and an unpaired Student's . 3. 3.1I. ricinus females. Mutant ticks were produced by RNAi-mediated silencing of four transcripts encoding enzymes involved in proteosynthesis, namely IrAsn-RS, IrVal-RS, IrLeu-RS and IrLys-RS. Only 0–6% of ticks injected with respective dsRNAs were able to finish feeding, and their average weights were 25.5, 8.4, 20.8 and 11.8 mg, respectively. In comparison, 90% of gfp-dsRNA injected females completed feeding, and their average weight was 303.2 mg (a). These transcripts were clearly good RNAi targets, producing biologically handicapped ticks with severe feeding disabilities. While these ticks remained attached to the host for days and were viable, they did not display any feeding progression typical for ticks (slow and rapid feeding phases). Therefore, we denoted these ticks as ‘zombie ticks’.Tick feeding is mediated by immense metabolic transformation from a ‘dormant’ state into a highly processive state . To enab303.2 mg a. These . 3.2I. ricinus nymphs are sensitive to RNAi silencing of tRNA synthetases, we injected dsRNAs into unfed nymphs and monitored their capacity to feed on a host. Indeed, nymphs, a key developmental stage of I. ricinus ticks that transmit Lyme borreliosis, were severely impaired by silencing tRNA-synthetases transcripts (b). While RNAi of asn- and val-tRNA synthetase reduced the capacity of ticks to engorge only partially , no nymphs engorged in leu-, and only 6.6% of lys-tRNA synthetase silenced nymphs managed to engorge. On average, RNAi silenced nymphs had 2.3, 1.5, 0.3 and 0.7 mg, compared to 3.8 mg of gfp control (b). These ticks thus clearly imbibed a reduced amount of blood. To inspect whether RNAi of tRNA synthetases has any impact on tick salivation, we analysed salivary gland morphology of the RNAi-silenced nymphs and quantified soluble protein content within. Indeed, disrupted proteosynthesis mediated by RNAi of tRNA synthetases resulted in reduced saliva production. A pair of salivary glands from silenced I. ricinus nymphs fed for 48 h contained approximately 3–12 fold lower concentrations of soluble proteins than control ticks . Whether these ‘zombie’ tick nymphs would be capable of transmitting Borrelia spirochaetes was further addressed. By feeding tick larvae on B. afzelii-infected mice, infectious nymphs were produced with a greater than 90% infection rate [asn-dsRNA, val-dsRNA, leu-dsRNA, and lys-dsRNA or gfp-dsRNA), were populated by quantitatively identical amount of B. afzelii spirochaetes in the mouse ear, bladder and heart  virus and Lyme borreliosis spirochaetes in Europe. While the TBE can be successfully controlled by vaccination, a human vaccine against borreliosis is not currently available. In 1998, the FDA approved a new recombinant Lyme vaccine, LYMErix, targeting the outer surface protein A (OspA). Nevertheless, low demand for the vaccine, combined with mounting lawsuits from patients, led to the manufacturer pulling out of the market in 2002. Although the second generation of OspA-based vaccines is currently being tested [Borrelia spirochaetes require for mammalian host infection have been proposed as promising targets [Borrelia, during their transmission. First studies demonstrated that injection of Borrelia spirochaetes into mice, together with I. ricinus or I. scapularis salivary gland extract (SGE), increased the level of spirochaete burden in mice and enhanced the infection of feeding ticks [B. afzelii by macrophages in vitro [in vitro data, new reassessments regarding a systemic in vivo framework might reveal further details. Most of the early studies employed SGE, or pilocarpine-induced saliva from half-fed adult females. It has been reported that the pilocarpine concentration in tick saliva is much higher than the minimum required for the activity of this agent. Due to the known effects of pilocarpine on smooth muscle and immune cells, caution should be taken when interpreting results obtained with this type of preparation [Ticks possess very complex feeding biology similar to other hematophagous arthropods. However, they have distinct features. Their ability to imbibe a vast amount of blood over a long period of time explains their remarkable success as disease vectors. g tested ,29, alte targets ,31. Tick targets . Many inng ticks ,32. The in vitro . Despiteparation .I. ricinus nymph fed for 48 h contains about one microgram of soluble protein , complement , release of histamine (tHRF) and other components of the host immune system have been described [Borrelia transmission.While the pioneering works on salivary-assisted transmission used 10–50% of species-specific SGE, representing 50–250 ng of protein , later S protein . Therefoescribed . RNAi-siB. afzelii and European Lyme disease vector I. ricinus [Borrelia spirochaetes suggested by Willy Burgdorfer and others [I. ricinus saliva in the infectivity and survival of B. afzelii spirochaetes. We suggested that the main requirement for B. afzelii survival and successful host colonization is a complex remodelling of surface antigens and not the immunomodulatory effect of tick saliva. B. afzelii was depicted as an independent, motile bacterium that actively migrated from the tick midgut towards the host tissues [Recently, we have challenged the salivary route of transmission for the  ricinus  and provd others ,38. Our  tissues .I. ricinus salivary glands and saliva proteins do not play a decisive role in B. afzelii transmission, suggesting that transmission of spirochaetes happens independently of continuous tick feeding . RNAi-mediated knock-down of four essential tRNA synthetases resulted in severe phenotypes regarding the progression of I. ricinus feeding. However, B. afzelii spirochaetes were transmitted in comparable quantities to the normally feeding dsRNA-mock control. We argue that, during tick feeding, I. ricinus ticks serve as a mere vehicle that needs to (i) mediate the connection with host tissues using hypostome mechanics and early saliva, and (ii) take up initial blood meal to trigger the infectivity of B. afzelii spirochaetes. This all facilitates an essential bridging of tick midgut with vertebrate bloodstream, enabling Borrelia to transverse between these two hosts. We suggest that spirochaetes then actively transverse into the host independently of concomitant processes inherent to tick feeding. We argue that the success of B. afzelii dissemination in the host is mainly determined by their ‘infectivity mode’, a shift undertaken while in the tick midgut, rather than continuous support of tick salivation.The current study provides an additional piece of evidence that Borrelia species should be conducted to answer the question of whether our data are specific to the European case of B. afzelii and I. ricinus, or a general phenomenon that tick saliva is dispensable for successful Borrelia transmission.We suggest that similar studies involving other tick and . 5 (1)I. ricinus. Using RNA interference, we have demonstrated that tRNA synthetases represent promising anti-tick targets, which warrant the design of selective inhibitors.Identified and validated four tick tRNA synthetases across two developmental stages of  (2)I. ricinus feeding and retards the normal function of tick tissues, including salivary glands, which is best exemplified by leu-tRNA KD ticks. The leu-tRNA KD nymphs gained virtually no weight from blood-feeding (0.3 mg compared to 0.24 mg of unfed nymphs). Interestingly, these severely handicapped nymphs did not lose their capacity to transmit B. afzelii spirochaetes. These data indicate that tick physiology, including salivary gland products, is dispensable for the dissemination of B. afzelii spirochaetes.Demonstrated that silencing of tRNA synthetases severely impairs  (3)B. afzelii transmission. This, to a large extent, negates the ambition to prevent Lyme borreliosis by antibody-mediated targeting of I. ricinus salivary proteins.Expressed our opinion that arrest in tick feeding is not a sufficient phenotype to prevent In this study, we have:


Vibrio genus have had considerable significance upon human health for centuries. V. cholerae is the causative microbial agent of cholera, a severe ailment characterized by profuse watery diarrhea, a condition associated with epidemics, and seven great historical pandemics. V. parahaemolyticus causes wound infection and watery diarrhea, while V. vulnificus can cause wound infections and septicemia. Species of the Vibrio genus with resistance to multiple antimicrobials have been a significant health concern for several decades. Mechanisms of antimicrobial resistance machinery in Vibrio spp. include biofilm formation, drug inactivation, target protection, antimicrobial permeability reduction, and active antimicrobial efflux. Integral membrane-bound active antimicrobial efflux pump systems include primary and secondary transporters, members of which belong to closely related protein superfamilies. The RND (resistance-nodulation-division) pumps, the MFS (major facilitator superfamily) transporters, and the ABC superfamily of efflux pumps constitute significant drug transporters for investigation. In this review, we explore these antimicrobial transport systems in the context of Vibrio spp. pathogenesis and virulence.Infectious diseases caused by bacterial species of the  Vibrio genus  is one of the most important food pathogens of human health significance in seafood. Vibrios are naturally present in freshwater, marine, and estuarine environments and are found in variable numbers in all kinds of seafood [Vibrio species have been isolated from humans [Vibrio spp. usually ensue from exposure to contaminated water or consumption of raw or undercooked contaminated fish and shellfish [Vibrio infections [Vibrio infections in humans can be categorized into two groups: cholera and non-cholera infections. Vibrio cholerae causes a severe diarrheal illness, cholera, through ingestion of contaminated food or water. Non-cholera Vibrio species such as V. parahaemolyticus and V. vulnificus are widely distributed in coastal-marine waters. They can cause various infections such as gastroenteritis, wound infections, septicemia, etc., and are responsible for the majority of seafood-borne infections worldwide [V. parahaemolyticus and V. vulnificus outbreaks are also common in developed countries.The  seafood . Their nm humans , represehellfish . Howeverfections . Vibrio orldwide . CholeraV. cholerae bacterium is the causative agent of the global disease cholera, with an estimated 1.3–4 million cases and 21,000–143,000 deaths worldwide every year [V. cholerae is associated with chitinous organisms such as copepods, crustaceans, and microalgae, like zooplankton and fish [V. cholerae bacteria are classified broadly as O1 and non-O1 serovars based on their observative agglutination in the presence of O1 antiserum. O1 V. cholerae and a non-O1 V. cholerae  have the potential to cause epidemic or pandemic cholera, and all other non-O1 and non-O139 V. cholerae do not infect humans or only cause mild illnesses [V. cholerae O1 and O139 Bengal is primarily due to their ability to produce cholera toxin CTX and cause epidemic diarrhea [V. cholerae O1 serogroup has two biotypes  and three serotypes . Amongst them, the highly rampant serotype is Ogawa, whereas Hikojima is sporadic, rare, and unstable in the environment [V. cholerae strains habitually inhabit rivers and estuarine areas against O1 and O139 strains. Studies have shown that non-toxigenic environmental strains could be switched to toxigenic strains through transduction with cholera toxin (CT)-encoded phage CTXϕ [The ery year . V. choland fish . V. cholllnesses . The virdiarrhea . These tironment . Non-O1 age CTXϕ .V. parahaemolyticus is widely distributed worldwide in coastal waters and is commonly found in seafood, sometimes in numbers as high as 103–104/g in oysters and <102/g in tropical shrimp [V. parahaemolyticus is free-swimming or attached to organisms such as zooplankton, fish, shellfish, and sediment. Food-borne infections with V. parahaemolyticus occur when shellfish such as oysters and clams are consumed raw or minimally cooked. Severe watery diarrhea, abdominal cramps, vomiting, nausea, and fever are some of the symptoms of V. parahaemolyticus infection. It can also cause infections in open wounds exposed to contaminated water. However, all V. parahaemolyticus are not pathogenic, but those manufacturing either a thermostable direct hemolysin (TDH) factor or a TDH-related hemolysin (TRH) are pathogenic [tdh and trh genes with about 70% nucleotide sequence similarity [V. parahaemolyticus seafood isolates are tdh+, while the incidence of trh+ V. parahaemolyticus is considerably higher, with some investigations reporting as high as 60% [tdh gene but not trh. V. parahaemolyticus can be isolated from farmed shrimp, and recently, early mortality syndrome (EMS) has been ascribed to V. parahaemolyticus in Litopenaeus vannamei farms [The halophilic pathogenic microorganism l shrimp . V. paramilarity . Studiesh as 60% . Since th as 60% . These sei farms .V. vulnificus is another important human pathogenic vibrio associated with fish and shellfish. V. vulnificus is known to cause serious infections in people with compromised immunity, liver diseases, and iron overloaded conditions, with a mortality rate as high as 30–75% [V. vulnificus infections are linked with eating raw molluscan shellfish, which accumulate this pathogen from surrounding waters. V. vulnificus is responsible for about 95% of deaths associated with the consumption of seafood. V. vulnificus can cause fatal wound infections because the microorganism can enter the circulatory system and cause septicemia. Low salinities (5 to 25 ppt) and warm temperatures (20 to 35 °C) have been reported to be favorable for this organism [V. vulnificus infection are reported from tropical and subtropical regions. Based on their biochemical characteristics, V. vulnificus are classified into three biotypes, with Biotype 1 responsible for severe human infection and are found naturally in marine and estuarine waters, and biotype 2 are eel pathogens. Biotype 3 is a known hybrid of biotypes 1 and 2 found in freshwater fish in Israel [s 30–75% . V. vulnorganism ,16. Mostn Israel .Vibrio spp. can be problematic in treating severe cases of clinical infections [Vibrio spp. is a widely recognized resistance mechanism [The recalcitrant nature of specific toxigenic and non-toxigenic strains of fections . In genefections . These mfections . Biofilmfections . The altfections . A well-fections . The enzfections . These rfections . These rechanism ,26. ThesVibrio species where the cellular targets for antimicrobial agents reside, the growth inhibitory effects of such intracellularly located drugs are diluted by various solute drug efflux systems [Inside bacterial cells of  systems . These s systems . Active  systems .Vibrio genus harbor these primary active drug pump systems, which are described below. Another active transport system involves the biochemical modification of substrate during transport across the membrane, as exemplified by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), also known as group translocation [Vibrio microorganisms [In primary active transport systems, the energy stored in ATP is utilized by its hydrolysis to actively accumulate their water-soluble substrates on one side of the membrane . Severallocation . While srganisms ,32,33, sVibrio species possess secondary active transport systems for drug efflux, which use the energy stored in the form of ion gradients across the membrane [Likewise, membrane . These imembrane ,36. TogeV. parahaemolyticus [Regarding the active extrusion of antimicrobial agents, several large superfamilies of efflux pumps have been characterized in bacteria . Of partolyticus . The ATPolyticus .+), while the MFP is an adaptor protein that connects OMP with the IMP [Escherichia coli [Pseudomonas aeruginosa [The resistance-nodulation-cell division (RND) superfamily of membrane transporters is composed of a tripartite system with an outer membrane protein (OMP), an inner membrane protein (IMP), and a periplasmic membrane fusion protein (MFP) . Differe the IMP ,47. As a the IMP ,47,48,49hia coli ,51 and truginosa ,53,54 haruginosa . RND pumruginosa ,55. Catiruginosa ,55.V. cholerae O1 include VexLM, VexIJK, VexGH, VexEF, VexCD, as well as VexAB and contribute to bile acid and antimicrobial resistance [vexAB and vexCD genes in the presence of bile substantiates the role of these efflux pumps in bile resistance, and this might positively contribute to successful colonization of the small intestine by V. cholerae [V. cholerae O1 and non-O1 serovars revealed the presence of all but one (VexE) efflux pump in the genome of non-O1 strain PS15 [E. coli  background with the help of the TolC outer membrane factor from V. cholerae (TolCvc) [vexH gene in a VexB-deficient V. cholerae resulted in higher susceptibility to these antibiotics [+ ions, unlike all other RND pumps energized by H+, and was the first such RND efflux pump described from bacteria [Some of the well-characterized RND efflux pumps from sistance ,57,58. Hain PS15 . The Tol(TolCvc) . Of thesbacteria .V. cholerae in the host and the environment. It has been shown that VexB, VexD, VexH, and VexK are necessary for intestinal colonization and production of virulence factors such as the cholera toxin (CT) and toxin co-regulated pilus (TCP) [ompT, which has a negative role in bile salt resistance [ompU, and the OmpU-producing strains are more resistant to bile [V. cholerae mutant strain lacking all RND efflux pumps exhibited increased susceptibility to bile, decreased cholera toxin production, and an inability to colonize infant mouse small intestines [V. cholerae.No antibiotic substrates have been identified so far for VexIJK and VexLM pumps ,60. Thesus (TCP) . A majorus (TCP) , the rolsistance . At the  to bile . The inctestines ,64. TogeV. vulnificus has 11 putative RND efflux pump-encoding genes, three of which  have been characterized by gene deletion studies [norM gene in whole-genome sequences of clinical V. vulnificus isolates [V. vulnificus lacking the vexAB homolog was more susceptible to erythromycin, acriflavine, ethidium bromide, and bile acid, deletion of acrAB homolog resulted in increased sensitivity of acriflavine alone, and vexCD deletion did not have any effect on the susceptibility to any of the antibiotics, dyes or bile acid [V. vulnificus, have been involved in resistance to antibiotics and inhibitory dyes. V. vulnificus mutants lacking TolCV1 and TolCV2 exhibited increased susceptibility to antibiotics novobiocin, erythromycin, and tetracycline, while TolCV1 mutant was also susceptible to DNA intercalating dyes  and detergents  and exhibited reduced motility [tolCV1 and tolCV2 increased when the bacterium was exposed to antibiotics and other chemicals [E. coli by interacting with the AcrA protein of the AcrAB-TolC efflux system of E. coli [The genome of  studies . A studyisolates . While aile acid . Two Tolmotility . The exphemicals . TolCV1  E. coli . Althoug E. coli ,70.V. cholerae (TolCvc) in an E. coli background, compared to the MICs of VexEF with TolC of E. coli (TolCEC) itself, although both share 47% identity and 7% similarity at the amino acid level [E. coli could not function with the TolC from V. parahaemolyticus, suggesting a species-specific preference for the TolC component [V. cholerae O1 El Tor strain has been reported to play an essential role in the transcription of the ToxR regulon, a finding that emphasizes the importance of efflux pump-mediated regulation of virulence in pathogenic bacteria [The outer membrane component TolC is generally essential for the function of RND efflux pumps irrespective of bacterial species. In certain instances, however, the TolC components of one species cannot functionally replace the TolC of the other. For example, the MICs of antibiotics for VexEF were much higher with TolC from id level . Similaromponent ,72. TolCbacteria .V. parahaemolyticus was the first Na+/drug antiporter to be reported from bacteria with no affiliation to any known transporter protein groups known at its time of discovery [+ unlike all other known H+/drug antiporters and shared high homology with YdhE protein of E. coli, as well as similar protein sequences in diverse bacterial groups [+-dependent drug efflux activity in NorM [+ electrochemical gradients, such as DinF-BH, NorM-PS, and VcmN [V. cholerae, by contrast, has been reported to be coupled to both Na+ and H+ [The norfloxacin efflux pump NorM of iscovery ,75. NorMl groups ,75. Thusl groups . Subsequ in NorM . Howeverand VcmN ,79,80. N+ and H+ . The MAT+ and H+ .V. parahaemolyticus [+ and H+ induced conformational changes in TM1, Thr200 is essential for Na+-mediated conformational transition alone [Based on mutational and molecular dynamics simulation studies, amino acid residues Asp36, Glu255, and Asp371 in NorM-Vc are presumed to be important for the substrate binding and transport activity, corresponding to Asp32, Glu251, and Asp367 in NorM from olyticus ,83,84,85on alone .+/drug antiporter of the MATE family in V. cholerae non-O1 and is capable of effluxing multiple antibiotics, including hydrophilic quinolones  and aminoglycosides (streptomycin and kanamycin), but not hydrophobic quinolones such as sparfloxacin and nalidixic acid [V. parahaemolyticus and YdhE of E. coli [V. cholerae O1 and non-O1 [+/drug antiporter-type multidrug efflux pump VcrM has been reported from Vibrio cholerae non-O1 [+ or Li+ [VcmA is a Naxic acid . This 45 E. coli . The pred non-O1 ,88. Anote non-O1 . This 44+ or Li+ . However+-dependent MATE efflux protein PfMATE from Pyroccocus furiosus [V. cholerae [E. coli [+-dependent NorM-Vc from V. cholerae [Neisseria gonorrhoeae [The X-ray crystallographic structures of Hfuriosus , VcmN frcholerae , ClbM of[E. coli , and thecholerae , and Nororrhoeae  have beeorrhoeae   but not both, in a single conformation state [+ binding site in the N-terminal lobe, which induces conformational changes in the protein upon binding with Na+ [+ or the substrate, and not both simultaneously, and the associated rearrangement of hydrogen bonds around Asp35 disallows re-binding of the substrate after extrusion [The MATE transporters adopt an “alternating-access model”, switching between the substrate-bound outward-facing (OF) or ion-bound inward-facing (IF) conformations, which allows binding of either the substrate or the ion  appeared to be under the control of a transcriptional regulator called MfsR [More recently, the five determinants denoted acycline . Interesled MfsR , a homolled MfsR ,112.Allium sativum and its bioactive agent allyl sulfide inhibited the antimicrobial efflux activity of EmrD-3 from V. cholerae [A. sativum extract exhibited profound synergy when combined with clinically critical antimicrobial agents, like lincomycin, vancomycin, and others [Vibrio species. Lastly, it remains to be determined whether allyl sulfide itself is another substrate or gene expression regulator of the EmrD-3 multidrug efflux pump.In 2017, our laboratory discovered that garlic extract from cholerae , indicatcholerae ,114,115.d others . These s+ antiport process [The multidrug efflux systems of the MFS are known to share similar amino acid sequences and protein structures, predicting that the MFS transporters share a common mechanism of solute transport across the membrane ,116,117. process .A detailed postulated catalytic mechanism for multidrug efflux pump systems is thought to involve several steps, as predicted by kinetic studies of secondary active drug transport ,122. FirIn the MFS antimicrobial exporters, the highly conserved amino acid sequence motif C (antiporter motif) has been shown to play a molecular hinge role during the conformational alterations in drug antiport across the membrane ,124. FurV. cholerae genome has several ABC transporters predicted to be involved in the transport of amino acids, vitamins, peptides, sugars, lantibiotics, etc. [V. cholerae [The ABC (ATP-binding cassette) transporters derive energy from the hydrolysis of ATP to move diverse compounds such as sugars, amino acids, antibiotics, anticancer drugs, peptides, etc., across the membranes and are widely distributed among both eukaryotes and prokaryotes . A typiccs, etc. . VcaM wacholerae . This efcholerae . More recholerae .V. cholerae coded by six operons: vexAB, vexCD, vexEF, vexGH, vexIJK, and vexLM operate with the same chromosomal element, tolC, encoding an outer membrane protein. These efflux pump systems have significant roles to play in the V. cholerae infections by colonizing the small intestine [V. cholerae, such as cholera toxin (CT) and the toxin-coregulated pilus (TCP), is regulated by RND efflux pump systems. The toxT gene encodes a transcriptional activator responsible for transcription of the genes encoding for CT. The efflux systems VexM and VexF are also implicated in supporting the expression of virulence factors [V. vulnificus [The RND efflux system in ntestine . This contestine ,132,133. factors . The mec factors . The evi factors . The outlnificus .V. cholerae associated with zooplankton and other sediment-dwelling organisms in the aquatic environment could also be correlated with the significance of a few RND efflux systems that have no role in other resistance mechanisms [Nevertheless, it has been proposed that removing redundant function systems results in the accumulation of a low molecular weight compound. This agent acts as a negative effector molecule functioning as a regulator of the expression of the important virulence factors. The life cycle of chanisms .Vibrio sp. in biofilm for intercellular communication. Quorum sensing is the prominent mechanism by which bacteria regulate physiological activities by detecting and responding to the levels of these extracellular compounds. Bacteria produce biofilm once the bacterial cells reach a specific density as detected by quorum sensing, which also regulates the expression of virulence factors [Vibrio sp. could be associated with the expression of virulence factors regulated by the quorum-sensing mechanism. The compounds involved in the bacterial quorum-sensing mechanism require membrane transporters similar to efflux pumps. Hence, the inhibition of efflux pumps could reduce biofilm production and other virulence factors [Biofilm is an aggregation of surface-attached bacterial cells associated with biotic and abiotic surfaces embedded in an extracellular polymeric matrix . The bac factors . Multidr factors .Vibrio species by contributing to the bacterial ability to survive antimicrobial substances and high osmolarity, biofilm formation, quorum sensing, and even virulence. Although the whole genome sequences have allowed the identification of new efflux pumps in the genome of Vibrio bacteria, some of which do not contribute to resistance to any known antimicrobials, their exact functions have remained elusive. Crystallographic structures of some efflux proteins of Vibrio species and other closely related bacteria have allowed an understanding of the structure-function relationships of these efflux pumps to a greater extent. Much remains to be understood regarding the roles of efflux pumps in the natural environment of Vibrio species, their substrate profiles, regulation, and transport mechanisms.Membrane efflux pumps play a critical role in the survival physiology of marine


N.grossedentata). DHM possesses many pharmacological effects, including anti-inflammatory , antioxidant, improving mitochondrial dysfunction, and regulating autophagy and so on. In this review, we consulted the studies in the recent 20 years and summarized the mechanism of DHM in inflammation-related diseases. In addition, we also introduced the source, chemical structure, chemical properties, and toxicity of DHM in this review. We aim to deepen our understanding of DHM on inflammation-related diseases, clarify the relevant molecular mechanisms, and find out the problems and solutions that need to be solved urgently. Providing new ideas for DHM drug research and development, as well as broaden the horizons of clinical treatment of inflammation-related diseases in this review. Moreover, the failure of clinical transformation of DHM poses a great challenge for DHM as an inflammation related disease.Inflammation plays a crucial role in a variety of diseases, including diabetes, arthritis, asthma, Alzheimer’s disease (AD), acute cerebral stroke, cancer, hypertension, and myocardial ischemia. Therefore, we need to solve the problem urgently for the study of inflammation-related diseases. Dihydromyricetin (DHM) is a flavonoid mainly derived from Nekemias grossedentata (Hand.-Mazz.) J.Wen and Z.L.Nie ( N.grossedentata)  is a flavonoid mainly derived from Nekemias grossedentata (Hand.-Mazz.) J.Wen and Z.L.Nie (dentata) . Flavonodentata) . DHM exiant food . There aant food , diabetiant food , endotheant food , neurodeant food , and canant food  as well ant food , and so ant food , especiaant food . This suN. grossedentata has been used as an herbal medicine belonging to the Parthenocissus inserta f. dubia (Rehder) Rehder J. Arnold Arbor  Schrad. tea. It has been used for herbal tea and traditional Chinese medicine for hundreds of years. Modern pharmacological studies have shown that N. grossedentata has a variety of pharmacological effects, including antioxidative, anti-inflammatory, and antiviral  Makino (AMP)  was first discovered from  in 1940   . The cons 30–40% . DHM wasr plants . In addir plants . DHM wil ethanol . The eff ethanol . The ani ethanol . The mai ethanol . DHM was ethanol . And for ethanol .Following sterile tissue injury, the positive response of the host to pathogens is called “inflammation” . Inflamm20.Some studies have confirmed that DHM is closely related to palmitic acid (PA)-induced human umbilical vein endothelial cells (HUVECs). Hu et al. have clarified that DHM ameliorated pyroptosis by activating the Nrf2 (NF-E2-related factor 2) signaling pathway. DHM inhibited the activation of NLRP-3 by down-regulating mitochondrial reactive oxygen species (ROS) in PA-induced HUVECs . DHM treResearch confirmed that DHM down-regulated the expression of the NF-κB signal pathway by directly binding to IκB kinase (IKK), thereby inhibiting IKK phosphorylation. To sum up, DHM exerted an antiarthritic effect in collagen-induced arthritis rats through down-regulation of NF-κB . Tang etLiu et al. have confirmed that DHM improved the inflammatory response of the liver and aorta by inhibiting the expression of TNF-α as well as Interleukin (IL)-6 in LDL receptor-deficient mice . DHM redNeuroinflammation plays a significant role in many neurological diseases, such as depression , AD Cal, stroke Staphylococcus aureus by destroying the integrity of the cell wall and increasing the permeability of the cell membrane 1  pathway 1 . DHM proHEI-OC)1 . Studies pathway . DHM sig pathway . Studies pathway . In addi pathway . DHM imp pathway . In addi pathway  (Figure  pathway .DHM improved TNF-α-induced endothelial dysfunction by inhibiting miR-21 . We specWe consulted the studies on DHM in the most recent 20 years, and we found that the clinical reports on DHM were very limited. There were few studies about the anti-inflammatory effect of DHM in the clinic. Sixty adult patients with nonalcoholic fatty liver disease underwent a randomized double-blind experiment. Following DHM treatment, the level of TNF-α and cytokeratin-18 in serum decreased significantly in this group . In a rain vivo or clinic. To better apply DHM in the clinic, improving the bioavailability and gastrointestinal absorption of DHM is not only an urgent problem to be solved but also difficult and challenging for the clinical application of DHM.Studies have shown that DHM is only 0.2 mg/ml at 25°C, the solubility of DHM is very low, so DHM cannot be completely absorbed from the intestine . Xiang eA.grossedentata. It can also be combined with other related drugs to make compound preparations to improve the curative effect and bioavailability.Numerous studies have shown that inflammation plays a vital role in a variety of diseases, including diabetes, arthritis, asthma , AD, acuThe clinical transformation of candidate drugs or active compounds is an urgent problem to be solved in future research, which is meaningful and valuable life science research .


Aspergillus niger is a prolific secretor of organic acids, proteins, enzymes and secondary metabolites. Throughout the last century, biotechnologists have developed A. niger into a multipurpose cell factory with a product portfolio worth billions of dollars each year. Recent technological advances, from genome editing to other molecular and omics tools, promise to revolutionize our understanding of A. niger biology, ultimately to increase efficiency of existing industrial applications or even to make entirely new products. However, various challenges to this biotechnological vision, many several decades old, still limit applications of this fungus. These include an inability to tightly control A. niger growth for optimal productivity, and a lack of high-throughput cultivation conditions for mutant screening. In this mini-review, we summarize the current state-of-the-art for A. niger biotechnology with special focus on organic acids , secreted proteins and secondary metabolites, and discuss how new technological developments can be applied to comprehensively address a variety of old and persistent challenges.The filamentous ascomycete fungus  Aspergillus niger has been gradually harnessed and modified into a multipurpose cell factory capable of converting a variety of cheap substrates into a range of valuable molecules , suggesting bioproduction of this product may soon be economically feasible [A. niger product portfolio, as evidenced by engineering itaconic acid producing isolates by heterologous expression of A. terreus biosynthetic and transport genes [Other useful organic acids may soon be produced by nhydride . Althougfeasible . Additiort genes .A. niger is enzyme production, which broadly began in the late 1950s when advances in chromatography purification technology enabled biotechnologists to study a diverse repertoire of useful proteins. This included lactoferrin, lipase, arabinase, asparaginase, catalase, cellulase, β-galactosidase, glucoamylase, pectinase, phytase, proteases, xylanase, glucose oxidase and hemicellulase, amongst others [A. niger reaches titres of 30 g/l [The other major application of t others . The most others . Fermentf 30 g/l .A. niger has also emerged as a producer of secondary metabolites (SMs) with titres of several g/l [Fusarium oxysporum non-ribosomal peptide synthetase (NRPS) encoding gene produced g/l titres of enniatin [In the last decades, eral g/l ,18. For enniatin , which wenniatin . Excitinenniatin . Additioenniatin .A. niger biotechnology has emerged, as applications of this fungus have looked to earth’s orbit and beyond. A. niger is one of the few fungi known to grow robustly inside the international space station [A. niger is an outstanding candidate for use in a variety of space biotechnological applications, from generating useful products during extended space flight to terraforming efforts [Most recently, a new frontier in  station , and met station . It is t efforts .A. niger research has determined precise abiotic growth conditions necessary for optimal product titres, ranging from pH, carbon source, spore inoculum density, oxygenation, among many other parameters . More rA. niger are metabolites of the tricarboxylic acid cycle (TCA), which are produced under specific growth stages and in response to various abiotic cues . One, low pH .A. niger secretes citric acid into the external media is unknown, but this may occur after a diauxic shift to phosphate limited growth [citA [ctpA does not impact productivity [Citric acid is formed from precursors oxaloacetate, acetyl-CoA and water at the final stage of the TCA by the mitochondrial citric acid synthase CitA . Citric d growth , and to d growth . Geneticth [citA  and otheth [citA  does notuctivity . In contuctivity ,36 or hiuctivity .A. niger due to the exchange of malic acid and citric acid across the mitochondrial membrane [ctpA mutants display comparable levels of both citric and malic acid in culture filtrates compared with progenitor controls – possibly due to functional redundancy of this transporter – thus limiting applications for genetic engineering of CtpA [Fermentation of the emerging value product malic acid is highly interconnected with that of citric acid due to the role of malic acid the TCA cycle. Additionally, as noted above, cytosolic malic acid is thought to be imported into the mitochondria by the malate/citrate shuttle CtpA. Increase of cytosolic malic acid has therefore been hypothesized to initiate citric acid fermentation in membrane . However of CtpA .A. niger membrane transporters, in a comparable approach to that of CexA and citric acid. The major A. niger malic acid exporter was identified by homology searches using the sequence from the fission yeast malate/succinate proton symporter Mae1 [A. niger orthologous gene, named dctA, malic acid titres could be increased by over 20% compared with controls [cexA can be used to hyperproduce malic acid [cexA concomitantly reduces titres of citric and malic acid, with the mutant having reduced expression of genes for glucose transportation and the glycolytic pathway [cexA disruption mutant, including a low affinity glucose membrane transporter mstC, hexokinase hxkA and pyruvate kinase pkiA, among other genes, malic acid titres could be increased by ∼23% compared with the progenitor control [A. niger biotechnology, which may soon be applied to generate organic acid hyper-producing isolates at an industrial level, especially when combined with other recent genetic strategies for maximizing productivity [A. niger organic acid organelle and plasma membrane transporters, and their detailed mechanistic functions, are crucial future objective for biotechnologists.Despite these challenges, malic acid titres can be successfully increased by genetic engineering of ter Mae1 . By overcontrols . Elsewhelic acid . Disrupt pathway . By over control . Taken tuctivity ,38. MoreA. niger is a potential chassis host for production of itaconic acid, an emerging bulk chemical commercially produced by A. terreus [cis-aconitate decarboxylase CadA [A. terreus by the membrane transporter mfsA [A. niger lacks a known cadA orthologue, and expression of a codon optimized cadA and mttA was sufficient for proof of principle production of itaconic acid, with export from the cell presumably performed by a native membrane transporter [A. niger cytosolic citric acid synthase citB [It is also notable that  terreus . In thisase CadA . Ultimatter mfsA . A. nigensporter . By overase citB , and by ase citB , titres The classical protein secretion route involves trafficking of cargo-loaded vesicles along microtubules and actin cables from the endoplasmic reticulum (ER)/Golgi to the hyphal tip . VesicleA. niger protein secretion. There is growing evidence that proteins may exit hyphae at intercalary regions, most notably septal junctions [A. niger aspartic protease PepN is secreted by non-classical routes, which are independent of autophagic processes necessary for unconventional secretion in Saccharomyces cerevisiae [Cryptococcus neoformans [A. nidulans 40 years earlier [A. niger. In other fungi they can contain proteins, nucleic acids, carbohydrates, and lipids , and coA. niger SMs are produced during low or zero growth [Most o growth  by biosyo growth . These eo growth . EnzymesA. niger genome sequence was published in 2007 [A. niger BGCs by specialized (co)cultivation conditions are promising for new bioactive molecule discovery [The first  in 2007 , with 78 in 2007 , the hig in 2007 . Consequiscovery . Moleculiscovery .Penicillium chrysogenum (which can be considered one of the best characterized SM biosynthetic pathways) includes storage of amino acid precursors in vacuoles, biosynthesis both in the cytosol and peroxisomes, and secretion by either a hypothetical membrane transporter or in secretory vesicles . In A. d titres , highligA. niger biotechnological research has been separated into protein, organic acid and SM sub-disciplines. There is however a recent trend to holistically study these related fields and to understand primary metabolism including redox and cofactor metabolism as foundational basis for all products formed [A. niger isolate D15 [Traditionally, s formed ,3,67. Onlate D15 . Non-acilate D15 .A. niger strains  in many mutagenized A. niger mutant D15 was due to a SNP in the putative methyltransferase gene encoding a master regulator of secondary metabolism LaeA [cexA promoter of the shochu producer A. luchuensis mut. kawachii [A. niger D15 [A. niger LaeA in regulating hundreds of A. niger SM genes [A. niger, which is dependent from the sugar and organic acid metabolism, with protein and glucoamylase production. It was demonstrated that metabolic engineering through NADPH cofactor engineering can indeed cause a substantial increase in GlaA titers [Intriguingly, this approach demonstrated that non-acidification by ism LaeA . A follokawachii , thus liiger D15 . From SMSM genes . FinallyA titers .A. niger as a maximally efficient cell factory. Two key challenges are discussed below.Many more insights into the interconnections between organic acid, protein and secondary metabolite production are likely to be revealed in future studies, which may enable rational design of hyperproduction strains. Despite the above advances, several challenges exist for (re)engineering A. niger application is crucial for any kind of submerged production process and can happen on two basic levels, micromorphology and macromorphology, describing the structure of individual hyphae and the characteristics of an entire aggregate of mycelium, respectively , clumps  and pellets  [A. niger, both morphological forms can be favourable, depending on the desired product [The control of filamentous morphology in an industrial iewed in ). Whereauctivity ), a signchanism) ,74. Whilchanism) . Due to  product ,76. As c product .aplD), results in a hyperbranching phenotype with defects in pellet formation, and could thus give a possible target for morphology engineering [The predominant macromorphology present in a culture is classically controlled by process parameters like inoculum, medium composition, pH, temperature or mechanical forces . In addiineering .A. niger pellet in relation to the hyphal fraction [While dispersed mycelia can be analysed comparatively easily using light microscopy, this technique is not sufficient to image the complex, 3D structure of filamentous pellets, and especially their cores. To overcome this problem, pellet-slicing and subsequent light microscopic investigation has become a common tool . The obvfraction . Remarkafraction . With thA. niger process as follows: (i) depending on the desired product, a suitable overall macromorphology is selected (dispersed/pellet). If the chosen form is pelleted, optimization is needed: (ii) nutrient gradient calculation to reverse engineer a theoretical pellet structure, optimized in nutrient and oxygen supply for the production of the specific substance; (iii) genetic and process engineering techniques to adjust the macromorphology to the calculated optimum; (iv) X-ray microtomography or other methods to examine the resulting macromorphological structures. Consequently, morphology engineering of A. niger chassis isolates with a pre-programmed macromorphology in submerged culture will drastically reduce screening time for putative hyperproduction mutants , shake flask (100 ml) and 96 well (100 µl) cultivations [A. terreus pellets (∼100 µm diameter) probably enabled comparable productivity across cultivation platforms. Are such scale down methods possible for A. niger, where pellets in shake flask/bioreactor models of either protein or citric acid fermentation reach over several mm in diameter [Despite these advances, a significant bottleneck to screening such ser/week . MicrotiA. niger . Howevereveloped . High-theveloped . Therefoivations . As the diameter ,94?A. niger pellet size with growth in microtiter wells [A. giganteus was combined with online measurements of fungal growth using scattered light in the BioLector system [2 [A. carbonarius that included an automated pipeline of sample processing, injection, image acquisition and morphology analysis [A. niger, a limitation to this approach is that certain products which are favourably or exclusively secreted by large pellets will not be detectable [An innovative approach combines microparticle based reduction in er wells . By modier wells . In anotr system . This apystem [2 . This apanalysis . These stectable . AddressA. niger is a microbial cell factory used to produce proteins, organic acids, and secondary metabolites.The molecular, cellular and metabolic basis of product biosynthesis and secretion are currently being revealed, suggesting they are highly interconnected.A. niger toolkit and available resources promise a rapid period of genetic, metabolic, and morphology engineering for efficient cell factories.The existing


Spodoptera frugiperda, is a notorious agricultural pest worldwide, causing great damage to a wide variety of crops. This pest exhibited a remarkable field-evolved resistance to multiple insecticides. According to the evidence, a few cuticular proteins (CPs) participate in the insecticide resistance of several insects. This study was designed to explore whether CP genes of the FAW exhibit functional roles in responding to insecticides stress. There are a set of CP genes significantly regulated in response to the exposure to different insecticides, implying that CP genes play an important role in the FAW against insecticides stress. The results inspire further functional validation of CP genes in FAWs to gain a better understanding of its resistance to insecticides.The fall armyworm (FAW), Spodoptera frugiperda, is a serious pest of crucial crops causing great threats to the food security of the world. It has evolved resistance to various insecticides, while the underlying molecular mechanisms remain largely unknown. Cuticular proteins (CPs), as primary components in cuticle, play an important role in insects’ protection against environmental stresses. Few of them have been documented as participating in insecticide resistance in several insect species. In order to explore whether CP genes of the FAW exhibit a functional role in responding to insecticides stress, a total of 206 CPs, classified into eight families, were identified from the genome of the FAW through a homology-based approach coupled with manual efforts. The temporal expression profiles of all identified CP genes across developmental stages and their responses to 23 different insecticides were analyzed using the RNA-seq data. Expression profiling indicated that most of the CP genes displayed stage-specific expression patterns. It was found that the expression of 51 CP genes significantly changed after 48 h exposure to 17 different insecticides. The expression of eight CP genes responding to four insecticides were confirmed by RT-PCR analysis. The results showed that their overall expression profiles were consistent with RNA-seq analysis. The findings provide a basis for further functional investigation of CPs implied in insecticide stress in FAW.The fall armyworm (FAW),  Spodoptera frugiperda (Lepidoptera: Noctuidae), is a notoriously destructive pest that feeds on more than 350 host plants, causing major damage to economically important crops such as corn, rice, cotton, sorghum, soybean and vegetables [https://www.cabi.org/isc/fallarmyworm, accessed on 28 August 2021). Due to its bioecological aspects such as extreme polyphagy, high reproductive capacity, strong migratory behavior and rapid adult dispersion, the FAW poses a global threat to agricultural production and food security. For instance, FAWs have caused maize yield losses of 50% from Africa and southern Asia since 2016 [Bacillus thuringiensis (Bt) toxins and synthetic insecticides [The fall armyworm (FAW), getables ,2. The Fnce 2016 , and it cticides ,8,9. TheCuticular proteins (CPs) are critical constituents of the insect cuticle, the exoskeleton, as well as the cuticle lining the foregut, hindgut and tracheae. They can be classified into more than ten families according to their conserved protein sequence motifs. The most abundant family is known as CPs with the Rebers–Riddiford motif (CPR) that can be assigned into three subfamilies, RR-1, RR-2 and RR-3 ,11. The In this study, CP genes have been annotated based on the recently published chromosome-level genome sequences of FAW ,23. They−5 [http://www.insect-genome.com/Sfru/, version WMCG01000000, accessed on 15 May 2021) and CNSA (CNGB Nucleotide Sequence Archive)  with accession no. CNP0000513. Additionally, putative CP genes were identified by seeking the genomic data based on Hidden Markov Models of different CP families by HMMER3 search  [http://bioinformatics.biol.uoa.gr/CutProtFam-Pred/, accessed on 20 August 2021) with default parameters [To exhaustively identify CP genes from FAWs, the corresponding protein sequences were retrieved from Acyrthosiphon pisum, Anopheles gambiae, Apis mellifera, Bombyx mori, Drosophila melanogaster, Manduca sexta, Pediculus humanus, Spodoptera litura and Tribolium castaneum ,15,24. T−5 ,23. The ay 2021) . The redS. litura, B. mori and M. sexta. Amino acid sequences were aligned with MUSCLE in MEGA X [http://microbesonline.org/fasttree, accessed on 26 July 2021) with SH-like 1000 support under the model of WGA and CAT [http://tree.bio.ed.ac.uk/software/figtree/, accessed on 26 July 2021).Phylogenetic analysis was performed using the corresponding protein sequence of CP genes of FAW, n MEGA X . A phylo and CAT . Trees whttp://www.insect-genome.com/Sfru/, accessed on 15 May 2021) [https://db.cngb.org/cnsa/, accessed on 23 May 2021) with accession no. CNP0001020 [RNA-seq data of the FAW from different developmental stages including 1st–6th instar larvae, pupae and adults of both sexes were downloaded from InsectBase (ay 2021) , which cP0001020 . The detP0001020 . These pP0001020 . SignifiP0001020 . Gene exP0001020 .https://irac-online.org/, accessed on 5 March 2019). They were put on moist filer paper in a Petri dish with a diameter of 10 cm for feeding the larvae of the FAW. Ten FAW larvae were added to each Petri dish. The larvae fed on the corn leaves dipped into solution was set as control. Corn leaf pieces were replaced at an interval of 12 h. After 24 and 48 h, ten surviving larvae were put together for one biological replicate. They were transferred into a 1.5 mL Eppendorf tube. After homogenization of samples in liquid nitrogen, their total RNA was extracted with TRIzol regent  according to the manufacturer’s protocol. Its quantity and integrity were accessed by Nano Drop ND-1000 spectrophotometer  and 1% formaldehyde agarose gel electrophoresis, respectively. An equal amount of 1 ug total RNA from each sample was used to synthesize cDNA using PrimeScript RT Reagent Kit with gDNA Eraser  following the manufacturer’s protocol. A total of eight CP genes were selected for validating their expressions after exposure to the above four insecticides by RT-PCR with Dream Taq Green PCR Master mix . Gene-specific primers were designed based on their sequences derived from the genome of the FAW using Primer Premier 5. Actin genes were used as an internal reference gene for normalization. Primers used here were listed in FAW used in the experiments were a colony maintained in our lab. Its larvae were fed on corns. The 3rd larvae of the FAW exposed to four insecticides including 20% dinotefuran soluble granules (P10), 33 g/L avermectin and bifenthrin emulsifiable concentrate (P14), 40% bifenthrin and thiacloprid suspension concentrate (P16) and 35% chlorantraniliprole water dispersible granule (P23) were treated as described by Gui et al. . In brieS. litura, B. mori and M. sexta CPs, phylogenetic tree of major CP families or subfamilies was constructed separately  were highly induced by azinphosmethyl in adult of Leptinotarsa decemlineata [Cimex lectularius). The expression of CPAP3-A1, CPAP3-C1, CPAP3-D1 and CPAP3-E2 genes were significantly induced in the oriental fruit fly  by malathion, the main pesticide used against this pest [A. sinensis and Culex pipiens pallens, and pyrethroid-resistant strains of A. stephensi, A. gambiae and A. sinensis [C. pipiens pallens was higher in the deltamethrin-resistant strain than in the susceptible strain [A. stephensi, part of them were down regulated [As crucial components in insect cuticular, CPs functioned in environmental stresses such as insecticides, extreme temperature and ultraviolet by mediating cuticular permeability . By compmlineata . Koganemmlineata  found thhis pest . Interessinensis ,39,40. Te strain , and knoe strain . The ovee strain ,21,36,42e strain . For insegulated . Since segulated ,35, the Genome-wide analysis with exhaustive homology-based searches and manual efforts in FAWs led to the identification of 206 CP genes. Their expression profiles during different development time points and in response to the exposure of 23 different insecticides were investigated. A set of them, presumed to be responsive to a range of insecticides, were identified and validated using RT-PCR analysis. Our results provide insights for the further functional investigation of CP genes of FAW involved in insecticide stress.