Anopheles mosquitoes collected in various localities of the region. Land cover types were characterized using orthorectified aerial photographs to estimate landscape metrics. The relationship between landscape fragmentation and species diversity was evaluated by regression analysis. The correlation between species abundance and land cover types was determined using canonical correspondence analyses. Results showed a statistically significant tendency for a lower diversity of the Anopheles community in landscapes with higher patch number, patch density and effective mesh size. For most species, there was evidence of a significant relationship between species abundance and land covers modified by anthropic activities which generate forest loss. These results indicate that activities that modify the landscape structure and land cover composition generate changes that affect the spatial distribution and composition of epidemiologically-important Anopheles species, which may impact malaria distribution in a region. This information is useful to guide control interventions that promote unfavorable landscapes for malaria vector propagation.Landscape structure influences the distribution and abundance of anopheline mosquitoes and has an indirect impact on malaria transmission. This work aimed to determine the effect of land cover and landscape fragmentation on anopheline mosquito abundance and diversity in an important Colombian malaria endemic area, the Bajo Cauca region. Diversity indices were calculated for  Malaria is a problem of public health in Colombia. In 2018, the country occupied the third position in the number of cases among the countries in Latin America . The BajAnopheles species, indicate that land cover composition is one of the most influential factors affecting species abundance and distribution [Anopheles composition, distribution and behavior, aspects known to affect malaria transmission [Anopheles darlingi [Anopheles gambiae and Anopheles funestus [Landscape structure determination includes the description of the spatial pattern of elements (land covers) and their connections ; a relevribution , 14–16. ribution . Regardiribution –20. Few smission , 21–24. darlingi , and witdarlingi . Similarfunestus .Anopheles species composition, natural infection, and behavior [Anopheles community has not yet been established. Therefore, this study was conducted to test the hypothesis that in the Bajo Cauca region, Anopheles species abundance is related to land covers derived from anthropic activities and that species diversity is influenced by landscape fragmentation. This information will contribute to the understanding of malaria transmission dynamics; in addition, it provides the bases for control interventions that include epidemiologically responsible landscape management for malaria prevention.In the Colombian malaria endemic Bajo Cauca region, mining, livestock and farming are the main economic activities , 27; thebehavior , 31, genbehavior , 33; howAnopheles mosquitoes were collected in the malaria endemic Bajo Cauca region in Antioquia Department, Colombia . An area of 1.5 Km of radius from the collection site was characterized; this distance corresponds to the maximum average dispersion range reported for nopheles –41. The nopheles  and labenopheles  , Shannon-Weaver index (H’), Simpson (1-D), Equitability (J), and Dominance (D). Simple linear regression was used to evaluate the relationship between Anopheles diversity (Shannon-Weaver index) with landscape fragmentation indices and landscape diversity, using the PAST software v. 3.15 [Species accumulation curves for the V. 9.1.0 . Estimat v. 3.15 .Anopheles species abundance and land cover types were determined for the entire region by Canonical Correspondence Analysis (CCA); this multivariate gradient analysis is widely used to relate species with environmental variables [Anopheles [The relationships between ariables . For thinopheles , 40. To nopheles , 50.Anopheles mosquitoes corresponding to 10 species were collected in six sampling sites of each of the five localities visited, during 180 hours of sampling. Anopheles braziliensis , An. nuneztovari  and An. darlingi  were the most abundant species and were present in all localities, except An. braziliensis in La Lucha-BAG. Other species collected were An. albitarsis s.l. (10.6%), An. triannulatus s.l. (5.7%), An. punctimacula (2%), species near An. peryassui (1.3%) first described in this region [An. oswaldoi (0.8%), An. rangeli (0.1%) and An. pseudopunctipennis (0.04%) (p< 0.05), except for An. darlingi (W = 0.86 p> 0.05), An. triannulatus s.l.  and An. albitarsis s.l. .A total of 2,458 s region , An. osw (0.04%) . SpeciesAnopheles community showed low diversity (Shannon-Weaver H’< 2). The highest species richness was registered in Puerto Triana-CAU with eight species and the lowest in La Lucha-BAG and Villa Grande-BAG with five species each. The highest Anopheles diversity was found in Cuturú-CAU and Puerto Triana-CAU with a Shannon-Weaver index of 1.424 and 1.382, Simpson 0.71 and 0.70 and Equitability (J) of 0.73 and 0.66, respectively; in accordance, these localities also had the lowest Dominance (D) value with 0.29 and 0.30, respectively, which indicates the uniformity of the Anopheles community. The locality with the lowest species diversity was La Lucha-BAG (H’ = 0.49) that showed the highest Dominance D value (0.76), with An. nuneztovari as the dominant species , except for crop . La Lucha-BAG and Villa Grande-BAG had similar mean patch sizes ; they also had similar land cover compositions, except for the presence of crop cover in Villa Grande-BAG (< 1Ha); forest was the matrix coverage defined for both localities. Cuturú-CAU, Puerto Triana-CAU, and Puerto Astilla-NEC presented landscapes without a defined matrix, as a result of the landscape interventions by mining activities. These localities also showed higher mean patch sizes and land cover types than those observed in La Lucha-BAG and Villa Grande-BAG . A larger number of land cover types with uniformity of area indicate higher landscape diversity in the CAU and NEC localities      (The simple linear regression models showed a significant negative relationship for  p<0.05)  and withp< 0.05) ; also, tp< 0.05) . No sign p>0.05) .p<0.05). According to the CCA analysis, a higher abundance of An. nuneztovari was related to forest cover and to a lesser extent with shrub. Anopheles darlingi was located in the center of the canonical axis, showing a slight tendency of greater abundance related with wetland, water body and grass covers. While, An. albitarsis s.l. showed a tendency of being more abundant in landscapes composed by wetland and water body covers, and slightly with the grass cover. The abundance of An. braziliensis was strongly related to landscapes mainly composed of bare soil and showed a mid-to-low relationship with shrub covers. Finally, An. triannulatus s.l. was found in higher abundance in landscapes consisting mainly of grass cover . According to the permutation test applied to the CCA, the model and the first two canonical axes explained a higher variance of data than the expected by chance; the results of these tests were statistically significant   , is simimunities –58, whicmunities , 59. TheAnopheles diversity and the effective mesh size, number and density of patches . This species is recognized for its ability to colonize anthropically impacted areas [An. nuneztovari with forest cover and slighter with shrub cover , where open-pit mining and livestock were the main economic activities [An. darlingi, An. albitarsis s.l. showed a tendency of being more abundant in landscapes composed by wetland, water body and grass covers  Bajo Cauca region. (B) By locality.(TIFF)Click here for additional data file.S1 TableLand cover types were label according to categories of national land cover legends by the Instituto de Hidrología, Meteorología y Estudios Ambientales of Colombia (IDEAM).(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


As an alternative to traditional chemical processes, bio-based MA production has progressed to the establishment of de novo MA pathways in several microorganisms, such as Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, and Saccharomyces cerevisiae. Redesign of the metabolic pathway, intermediate flux control, and culture process optimization were all pursued to maximize the microbial MA production yield. Recently, MA production from biomass, such as the aromatic polymer lignin, has also attracted attention from researchers focusing on microbes that are tolerant to aromatic compounds. This paper summarizes recent microbial MA production strategies that involve engineering the metabolic pathway genes as well as the heterologous expression of some foreign genes involved in MA biosynthesis. Microbial MA production will continue to play a vital role in the field of bio-refineries and a feasible way to complement various petrochemical-based chemical processes. An entation .S. cerevisiae by removing Ric1, which is a transcriptional repressor. In silico modeling and pathway analysis confirmed the production of 2705 mg/L of MA and its precursor using the BY47471-MA4 strain [In addition to an engineering strategy at the metabolic site, such as gene deletion or overexpression of the structural genes, another study produced MA and shikimate from 4 strain .S. cerevisiae could produce MA from lignocellulosic biomass hydrolysate using recombinant xylose-fermenting yeast. In addition to the exogenous MA biosynthetic pathway, the xylose isomerase gene from Bacteroides vulgatus and pentose phosphate pathway genes from S. cerevisiae were overexpressed in the yeast strain, and the overexpression of the Aro1 gene (with a stop codon of AroE) and a feedback-resistant Aro4opt mutant gene from S. cerevisiae were also applied. Under aerobic conditions, the MA titer reached 424 mg/L, and 1286 mg/L MA was produced with the supplement of 1 g/L catechol. Fermentation of an oil palm empty fruit bunch hydrolysate resulted in 31.3 g/L ethanol and 53.4 mg/L MA [Recently, it was reported that  mg/L MA .Amycolatopsis spp. also tolerate lignin-based aromatics, such as catechol, guaiacol, phenol, toluene, p-coumarate, and benzoate, and even favor these aromatics as carbon sources over sugar. The aqueous phase, obtained through hydrothermal conversion as a lignin treatment, contained a large quantity of guaiacol of 7 g/L. Research on the metabolically engineered Amycolatopsis sp. ATCC 39,116 revealed 3.1 g/L of MA production from the guaiacol, while 1.8 mM of MA was produced with the lignin hydrolysate [rolysate .P. putida has excellent tolerance towards organic solvents and was the first soil microbe among Gram-negative bacteria to be recognized as a safety strain from the Recombinant DNA Advisory Committee [P. putida has attracted attention as a metabolic engineering chassis for applications in the industrial bioengineering field. Moreover, P. putida can be used as a carbon source for the growth and energy production of lignin-related aromatics, such as vanillin [ommittee ,40,41. Tvanillin ,43,44,45P. putida to produce MA from lignin without glucose through metabolic engineering. The precursor was induced to accumulate by the removal of pcaG/H and catB from the β-ketoadipate pathway using the same strategy as with C. glutamicum. The PCA decarboxylase gene was overexpressed to complete the MA pathway. A medium containing 25 nM each of aromatic compound 4-hydroxybenzoic acid (4-HBA) and vanillin produced MA with a yield of 19.0%, whereas 0.11 mM MA was biosynthesized through softwood lignin [Sonoki et al. conducted an experiment on d lignin ,47.P. putida KT2440. To block the synthesized MA, P. putida KT2440-CJ103 was established, in which a catBCA operon (metabolism operon of MA through β-ketoadipate pathway) was substituted with Ptac_catA_dmpKLMNOP (encoding phenol monooxygenase), and 13.5 g/L of MA was produced from the p-coumarate through fed-batch fermentation conducted for 78.5 h [Vardon et al. substituted protocatechuate 3,4 dioxygenase (PcaG/H) with protocatechuate decarboxylase (AroY) to convert aromatics from the catechol and protocatechuate branches to MA with r 78.5 h .P. putida KT2440 ΔcatRBC::Ptac:catA ΔpcaHG::Ptac:aroY:ecdB:ecdD) strain expressed a codon-optimized AroY, EcdB (subunit of decarboxylase AroY) and EcdD (subunit of decarboxylase AroY) from E. cloacae, and produced 15.59 g/L of MA in bioreactor cultivation that contained p-coumarate, which is an aromatic lignin monomer, and glucose [Two subunit genes, which play important roles in PCA decarboxylase and decarboxylase activity, were expressed as a genome-integrated gene to reduce PCA accumulation, which is a bottleneck in MA production, resolve the conversion of PCA to MA, and increase MA production. The KT2440-CJ184  strain was used to produce MA; 12 g/L of MA was produced through a fed-batch mode bioreactor cultivation from glucose. This is a practical example of the use of microbial strains for the production of chemically diverse molecules as building blocks [Furthermore, a pathway was engineered that could catabolize a range of aromatic compounds based on g blocks .E. coli or S. cerevisiae, it is endogenous to aromatic degrading organisms, such as P. putida. This is because P. putida has been engineered extensively to produce MA directly from pretreated biomass as well as from lignin-derived monomers. Although the β-ketoadipate pathway is not present in E. coli.Recently, information on the metabolic pathways of numerous microbes has become readily available, owing to the development of extensive genomic analysis, well-established molecular tools, system biology techniques, and fermentation techniques. Therefore, with such technologies, it has become possible to build new metabolisms that did not exist in nature or to reinforce existing ones. MA synthesis via PCA and catechol as starters in the shikimate pathway has been applied to various microbes, starting from primary research with E. coli has been diversified with microbes, such as C. glutamicum and P. putida, which have resistance to the toxicity of aromatic compounds. These aromatic-tolerant microbes possess abundant pathways and enzymes that can produce MA with aromatic compounds that are being discarded. This paper summarizes the use of microbes with strong potential as microbial cell factories at an industrial level for MA production. Finally, various other aromatics should be applied to obtain higher MA titers considering the economic and industrial aspects in the mass production of MA and the metabolic engineering approaches and optimized process operations using a range of microbes.In addition, MA production with


Prevalence of GAD is between 3 to 5% with onset in the early to mid-twenties . The Penn State Worry Questionnaire (PSWQ) is a 16-item self-report instrument assessing generalized anxiety symptoms . Brown (2003) and Olatunji et al. (2007) conducted Confirmatory Factor Analyses identifying a two factor model of Worry Engagement and Absence of Worry. No published studies have examined the factor structure of the PSWQ across age groups. The current study presents data from 612 people across three groups: 221 young adults , 283 middle-age adults , and 108 older adults . An ordinal confirmatory factor analysis (CFA) using robust weighted least squares (WLSMV) tested for invariance across groups. Results showed CFI/TLI values of .983/.981, 984/.983, and .981/.984 for Configural (CI), Metric (MI), and Scalar (SI) models. The RMSEA for CI, MI, and SI models was .064, .061, and .059. Based upon Cheung and Rensvold (2002), Sass (2011), and Chen (2007), a cutoff of ΔCFI ≥ 0.01 was established as evidence of invariance. The ΔCFI between CI and MI models was < .01 so analysis continued with the SI test. The ΔCFI between MI and SI models was < 0.01 and did not justify rejection of the null hypothesis. These analyses suggest PSWQ scores are valid across age groups and provide additional support for the multidimensional nature of the PSWQ.


Background: Microbial dysbiosis is closely associated with visceral hypersensitivity and is involved in the pathogenesis of irritable bowel syndrome (IBS), but the specific strains that play a key role have yet to be identified. Previous bioinformatic studies have demonstrated that Fusobacterium is a shared microbial feature between IBS patients and maternal separation (MS)-stressed rats. In this study, we assessed the potential role of Fusobacterium nucleatum (F. nucleatum) in the pathogenesis of IBS.Methods: Fecal samples of patients with diarrhea predominant-IBS (IBS-D) and healthy controls were obtained. An MS rat model was established to receive gavage of either F. nucleatum or normal saline. Visceral sensitivity was evaluated through colorectal distension test, and fecal microbiota was analyzed by 16S rRNA gene sequencing. F. nucleatum-specific IgA levels in fecal supernatants were assessed by western blotting. The antigen reacted with the specific IgA of F. nucleatum was identified by mass spectrometry and the construction of a recombinant Escherichia coli BL21 (DE3).Results: IBS-D patients showed a lower Shannon index and a higher abundance of Fusobacterium. The F. nucleatum-gavage was shown to exacerbate visceral hypersensitivity in MS rats, with both the F. nucleatum-gavage and MS causing a decreased Shannon index and a clear segregation of fecal microbiota. In addition, specific IgA against F. nucleatum was detected in fecal supernatants of both the F. nucleatum-gavaged rats and the IBS-D patients. The FomA protein, which is a major outer membrane protein of F. nucleatum, was confirmed to react with the specific IgA of F. nucleatum in fecal supernatants.Conclusion:Fusobacterium increased significantly in IBS-D patients, and F. nucleatum was involved in the pathogenesis of IBS by causing microbial dysbiosis and exacerbating visceral hypersensitivity in a colonization-independent manner. Meanwhile, F. nucleatum was found to induce an increase in specific secretory IgA through FomA. Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder that primarily manifests as abdominal pain and stool irregularities . AlthougDue to the importance of the gut microbiota and viscera in the microbiome–gut–brain axis, both microbial dysbiosis and visceral hypersensitivity have been considered to be associated with the severity of IBS symptoms , 2018. IFusobacterium was subsequently identified as one of the shared microbial features between IBS patients and MS-stressed rats in our previous research , a typical strain of the genus Fusobacterium, is a gram-negative, obligate anaerobic bacterium that constitutively colonizes the oral mucosa  -stressed rats were observed to form independent clusters that differed from the normal control group, and research . Thus, Fl mucosa . F. nuclse (IBD) . In addil cavity . NeverthPatients with diarrhea predominant-IBS (IBS-D) (diagnosed as per the Rome IV criteria) and healthy controls (HC) aged 18–65 years were recruited from Qilu Hospital of Shandong University between August 2016 and March 2018. This study was approved by the ethics committees in Qilu Hospital of Shandong University. Patients suspected of being postinfectious IBS were excluded due to the heterogeneity in pathophysiological mechanisms. The exclusion criteria included pregnancy; lactation; previous surgeries within half a year; vaccination within 3 months; organic diseases or mental diseases ; treatment with systemic corticosteroid, antipsychotics, and immunosuppressant agents; and taking antibiotics, probiotics, and laxatives within a month. The severity of symptoms was assessed by the IBS symptom severity scale (IBS-SSS) . The SelPregnant Sprague Dawley rats were purchased from Beijing HFK Bioscience Co., Ltd, and fed in the Experimental Animal Center of Shandong University. All animals were maintained under pathogen-free conditions . All experiments performed on animals were approved by the Ethical and Institutional Animal Care and Use Committee of Qilu Hospital of Shandong University.F. nucleatum  or normal saline (1 ml/100 g) once a week between the 4th and 8th weeks by oral gavage. Based on animal modeling, four groups were used in this study: group MS with F. nucleatum, the MS and F. nucleatum-gavage treatment group; group F. nucleatum, the normal-breeding and F. nucleatum-gavage treatment group; group MS, the MS and normal saline-gavage treatment group; and control group, the normal-breeding and normal saline-gavage treatment group. Fecal samples were collected at the end of weeks 3, 8, and 12. Visceral sensitivity was evaluated at week 12.The experimental procedure is shown in F. nucleatum strain ATCC 25586 was purchased from the General Microbiological Culture Collection Center . Escherichia coli (E. coli) BL21 (DE3) was purchased from TransGen Biotech . F. nucleatum was cultured anaerobically at 37°C for 18 h in Brain Heart Infusion Broth  before harvesting, while E. coli BL21 (DE3) strain was cultured aerobically at 37°C for 8 h in Luria-Bertani (LB) infusion broth.Fecal samples collected from the IBS-D patients, HC, and rats were rapidly frozen in liquid nitrogen and stored at -80°C. These samples were then shipped to Majorbio  for high-throughput sequencing .A FastDNA SPIN Kit  was used to extract stool DNA. Polymerase chain reaction (PCR) amplification of the V3–V4 regions of the bacterial 16S rRNA gene was performed with barcode-indexed primers  and TransStart FastPfu DNA Polymerase  in an ABI GeneAmp instrument . The amplicons were subsequently purified by gel extraction  and quantified using a QuantiFluor-ST instrument . The purified amplicons were pooled in equimolar concentrations, and paired-end sequencing was performed using an Illumina MiSeq System .Visceral sensitivity was evaluated by colorectal distension (CRD) test . A urinaF. nucleatum gene was detected by PCR. Total DNA was extracted from the stool of rats using TIANamp Stool DNA Kit . The detection of F. nucleatum was performed using specific primers  in a Mastercycler® pro S instrument . Positive and negative controls were standard strains of F. nucleatum (ATCC 25586) and E. coli BL21 (DE3), respectively. The V4 region of the bacterial 16S rRNA gene was amplified by primers    as interg, 4°C, 10 min), filtered using 0.22-μm micron syringe filters , and stored in a fridge at -80°C.The fecal samples of the IBS-D patients, healthy volunteers, and rats were diluted in normal saline  supplemented with 1% PMSF  and homogenized on ice . The supF. nucleatum, E. coli, and FSN was determined by BCA assay . For quantification of total IgA in FSN, the ELISA assay  was performed in a 96-well flat-bottom plate. Both of the BCA and ELISA assays were performed according to the manufacturer’s instructions.The total protein concentration of F. nucleatum or E. coli was collected, resuspended in normal saline, and the total protein was extracted by hypothermic ultrasonification. The F. nucleatum and E. coli proteins were denatured and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk at room temperature for 5 h, the membrane strips were incubated at 4°C for 20 h with FSN from either human or rats that had been diluted to a specific concentration in normal saline. After washing, the PVDF membrane strips were incubated with either horseradish peroxidase (HRP)-labeled goat anti-human IgA alpha chain  or HRP-labeled goat anti-rat IgA alpha chain  for 60 min at room temperature. Immobilon Western Chemiluminescent HRP Substrate  was used to detect the bands.The process was based on the protocols described previously . F. nuclThe proteins were extracted from the gels after SDS-PAGE (10%), and the samples were treated , cleanedF. nucleatum) was recognized by the specific IgA (F. nucleatum fomA gene (GenBank Accession Number: X72582) was amplified using a forward PCR primer (5′-TTTC ATATGGAAGTTATGCCTGCACCTAC-3′) containing an NdeI site  and a reverse PCR primer (5′-CGGCTCGAGTTAAGTAACTTTCATACC AG-3′) containing a XhoI site . The primers were synthesized by the Sangon Biotech Company . The generated fragment was inserted into a pETDuet-1 vector that was subsequently transferred into E. coli BL21 (DE3) and plated onto LB agar medium containing ampicillin (50 μg/ml). Single colonies were selected and cultured in LB broth at 37°C for 8 h with shaking. The culture was diluted (1:100) with LB broth and incubated at 37°C, and isopropyl-β-D-thiogalactoside (IPTG)  was added at a final concentration of 1 mM when the optical density at 620 nm reached 0.5. After induction, bacteria were harvested by centrifugation  and then washed twice with normal saline. The total protein was extracted by hypothermic ultrasonification, after which SDS-PAGE was used to assess FomA expression, and then the reaction between the recombinant FomA and specific IgA of F. nucleatum in FNS was detected by western blotting as described above.The following procedures were performed to confirm that FomA  was used to analyze data. A 95% confidence level, i.e., The raw sequences were processed using Usearch version 8.1.1861_win32. The raw reads were dereplicated, and the singletons or chimeras were removed. The sequences were then clustered into operational taxonomic units (OTUs) based on a 97% similarity and analyzed using the RDP classifier Bayes algorithm to calculate the community composition at each taxonomic level. The OTU tables were imported into Mothur v 1.38.1  for the A total of 71 IBS-D patients and 39 HC were recruited. Demographics between IBS-D patients and HC were well balanced .A total of 110 fecal samples were analyzed using a MiSeq PE 300, with 5,530,955 valid reads obtained. In the analysis, 4,657,712 reads were used after noise reduction and removal of low-quality reads and chimeras. Subsequently, the reads were analyzed using Usearch and the RDP database, resulting in the identification of 2,425 OTUs.P = 0.047, Mann–Whitney test)     . Further Adonis) . Based o Adonis) . The genenriched . From thpatients . The abuey test) . In addielation) .A total of 96 fecal samples were analyzed using MiSeq PE 300, with 3,645,585 valid reads obtained. After noise reduction and removal of low-quality reads and chimeras, 3,008,922 reads were used. Subsequently, the reads were analyzed using the Usearch and the RDP database, with 1,239 OTUs obtained.2 = 2.27, df = 1, P = 0.13, KW)          . The Sha.02, KW) , while g.02, KW) . Moreove024, KW) . The con008, KW) , while t997, KW) . For the090, KW) , and all005, KW) , whereas990, KW) . These rP < 0.01). A significant difference also existed across the four groups at both weeks 8 and 12 (P < 0.001). No significant difference in cohousing was observed.The OTU data was clustered through an NMDS analysis. At week 3, no visible distinction was observed . HoweverF. nucleatum were less than those of the other three groups at week 12   . This re2 = 20.313, df = 8, P = 0.009, KW). Group MS with F. nucleatum and group MS had higher VHI values than group F. nucleatum and the control group . The VHI of group MS with F. nucleatum was higher than that of group MS . These results showed that F. nucleatum-gavage exacerbated visceral hypersensitivity in MS rats.The visceral sensitivity of each group was evaluated by determining AWR scores based on graded CRD . The VHIF. nucleatum was not detected from 16s rRNA gene sequencing or PCR  occurred at different positions, confirming the specificity of IgA toward F. nucleatum  and 40 kDa   and 40 kDa   , at week001, KW) . Thus, FF. nucleatum-specific IgA in FSN and the microbial community was analyzed by RDA. As shown in F. nucleatum and group F. nucleatum, both of which received the F. nucleatum-gavage, whereas group MS and the control group exhibited a negative correlation  and body mass index (BMI)  were observed. The IBS-D patients showed more notable anxiety and depression based on SAS  and SDS . The severity of intestinal symptoms was assessed using the IBS-SSS, with all IBS-D patients exhibiting severe IBS .To further confirm the association of F. nucleatum-specific IgA was performed by western blotting. Ponceau S staining was used as loading control         and 130 ey test) . Furtherelation)  and 40-kelation) . Similarelation)  and 40-kelation) . TherefoF. nucleatum was performed by combining the protein molecular weight and functional category annotated in eggNOG. By filtering the results, 57 proteins in the M category    with an genesis) , which wE. coli strain harboring the expression vector. A strong reactive band with a molecular mass of 40 kDa was observed in the western blotting test using the recombinant FomA and the F. nucleatum-reactive FSN, whereas no band was observed at 40 kDa using the FomA-negative E. coli  database with accession code PRJNA511738.The datasets generated for this study can be found in the National Center for Biotechnology Information (NCBI) database with accession code The studies involving human participants were reviewed and approved by the ethics committees in Qilu Hospital, Shandong University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Ethical and Institutional Animal Care and Use Committee of Qilu Hospital, Shandong University.XG, ML, and XZ designed the project. ML conducted the bioinformatic data mining. XG, LS, TL, and MZ conducted key experiments. XG and ML performed statistical analysis. LL, ZL, and PW provided vital expertise and advice. XG and LS wrote the manuscript. All authors read the manuscript and approved the final draft that was submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptococcus mutans (S. mutans), and non-cariogenic Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii). Biofilm colony-forming-units (CFUs), lactic acid and polysaccharide production were measured. TaqMan real-time-polymerase-chain reaction was used to determine the percentage of each species in biofilm. The rnc gene-knockout for S. mutans with DMAHDM composite reduced biofilm CFU by five logs, compared to control (p < 0.05). Using parent S. mutans, an overwhelming S. mutans percentage of 68.99% and 69.00% existed in biofilms on commercial composite and 0% DMAHDM composite, respectively. In sharp contrast, with a combination of S. mutans rnc knockout and DMAHDM composite, the cariogenic S. mutans percentage in biofilm was reduced to only 6.33%. Meanwhile, the non-cariogenic S. sanguinis + S. gordonii percentage was increased to 93.67%. Therefore, combining rnc-knockout with bioactive and therapeutic dental composite achieved the greatest reduction in S. mutans, and the greatest increase in non-cariogenic species, thereby yielding the least lactic acid-production. This novel method is promising to obtain wide applications to regulate biofilms and inhibit dental caries.The objectives were to investigate a novel combination of gene-knockout with antimicrobial dimethylaminohexadecyl methacrylate (DMAHDM) composite in regulating oral biofilm from a cariogenic state toward a non-cariogenic state. A tri-species biofilm model included cariogenic  Streptococcus mutans (S. mutans), and noncariogenic Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii) ), where load P divided by the related displacement d is the curve’s slope in the linear elastic region . Con. ConS. mntration . Based op < 0.05.Statistical analyses were performed using Statistical Package for the Social Sciences . All data were expressed as the mean value ± standard deviation (mean ± SD). One-way analyses of variance (ANOVA) were performed to detect the statistical significance of the variables. Tukey’s multiple comparison test was used to compare the means of each of the groups. The differences in the means of data were considered significant if n = 6) are presented in p < 0.05), and the load-bearing capabilities were not negatively affected with the different mass fractions of DMAHDM (p > 0.1).The load-bearing properties of the four composites  on DMAHDM composites were reduced with higher DMAHDM mass fraction (p < 0.05). Combining rnc knockout for S. mutans with 3% DMAHDM composite achieved the greatest biofilm inhibition. Compared to the control group, the CFUs of the rnc gene-knockout biofilms in 1.5% and 3% DMAHDM groups was reduced by about three logs and five logs, respectively (p < 0.05). Similarly, rnc gene-knockout for S. mutans with DMAHDM composite led to the least biofilm metabolic activity (p < 0.05).As shown in 490nm against the polysaccharide content was expressed as y = 0.0207x + 0.3082, and the coefficient of determination (R2) was 0.9836. A linear curve for the standard lactic acid  was obtained within the concentration range of 0–3.5 mM. The equation of the regression was y = 8.467x − 1.973, and the coefficient of determination (R2) was 0.9942. In n = 6). Biofilm polysaccharide amount was greatly reduced with increasing DMAHDM mass fraction, compared to control composites (p < 0.05). In addition, the decreasing trend was more profound in biofilms with rnc-knockout S. mutans than that of parent S. mutans (p < 0.05). The lactic acid secretion (p < 0.05). The combination of rnc-knockout in S. mutans with the 3% DMAHDM composite led to the biggest decrease in lactic acid secretion by the Sm + Ss + Sg biofilms.The standard curve of ODecretion B was dect value versus bacterial numbers of S. mutans, S. sanguinis and S. gordonii were constructed. The equation of the regression for S. mutans was y = −3.3043x + 43.794, and the coefficient of determination (R2) was 0.9997. The equation of the regression for S. sanguinis was y = −3.2962x + 48.733, and the coefficient of determination (R2) was 0.9964. The equation of the regression for S. gordonii was y = −3.3246x + 48.305, and the coefficient of determination (R2) was 0.9988.Standard curves of Cn = 3). With parent S. mutans, an overwhelming S. mutans proportion of 68.99% and 69.00% existed in the Sm + Ss + Sg biofilm on commercial control composite and 0% DMAHDM, respectively. In contrast, the patent S. mutans proportion decreased with increasing the mass fraction of DMAHDM, accounting for 36.67% in 1.5% DMHADM group, and 13.56% in 3% DMHADM group.The TaqMan real-time PCR results demonstrated the ratio variation of bacteria in 2-day multispecies biofilms with different DMAHDM mass fractions   The cariogenic S. mutans proportion, (B) non-cariogenic S. sanguinis proportion, and (C) lactic acid secretion . These results demonstrate that: (1) The rnc-knockout for S. mutans regulated the Sm + Ss + Sg biofilm species composition shift toward a healthier state and decreased the lactic acid secretion, even on composites without DMAHDM; (2) DMAHDM composite had the ability to regulate biofilm equilibrium and reduce the lactic acid secretion; (3) The combination of rnc-knockout in S. mutans with DMAHDM composite led to the biggest reduction in cariogenic S. mutans percentage in Sm + Ss + Sg biofilm, gained the most substantial increase in non-cariogenic species percentage, yielding the least lactic acid secretion.rnc gene knockout in S. mutans on species proportional shift in a tri-species biofilm model, and furthermore, on the effect of combining S. mutans rnc knockout with DMAHDM composite in regulating the biofilm from a cariogenic state toward a non-cariogenic state. The S. mutans rnc gene-knockout alone significantly decreased the cariogenic S. mutans percentage in the Sm + Ss + Sg biofilm and increased the non-cariogenic species percentage. The DMAHDM composite alone caused a shift in biofilm species from a cariogenic composition toward a non-cariogenic composition. Most dramatically, by combining the rnc gene-knockout technology with the antimicrobial DMAHDM composite, the S. mutans percentage in the Sm + Ss + Sg biofilm was decreased from an overwhelming 69% of the parent S. mutans on control composite to only 6.3% of rnc-knockout S. mutans on the 3% DMAHDM composite. Meanwhile, the corresponding non-cariogenic S. sanguinis proportion in the Sm + Ss + Sg biofilm was increased from 12% to 61%. Therefore, the dual strategy of rnc-knockout for S. mutans + DMAHDM composite is promising to be a viable method for regulating the biofilm composition to promote non-cariogenic species and suppress cariogenic species, thereby inhibiting biofilm acids and recurrent caries.This present study represents the first report on the effect of + and a long alkyl chain with a chain length of 16 [+ can contact the negatively-charged cell membrane of bacteria and alter the electric balance, leading the bacteria to explode under its own osmotic pressure [QAMs are promising for incorporation into composites, primers and adhesives due to their antimicrobial activity and low toxicity ,47,48. Dth of 16 ,50. N+ cpressure ,50. Meanpressure . DMAHDM pressure ,50. ThisS. mutans, S. sanguinis and S. gordonii [S. mutans is the primary exopolysaccharides (EPS) producer in the oral cavity and is responsible for dental caries [S. mutans is an important player in caries pathogenesis, its role in caries is not etiological [S. mutans has been controversial and not well-established [S. mutans and the carious lesion, whereas other studies revealed no clear association [S. mutans [Interactions within oral microbial communities are major factors affecting the development of biofilm, and the cariogenic potential of the oral microbial consortia depends on the environmental conditions and the composition of the bacterial flora ,51. For gordonii ,8,44. Asl caries ,15. The l caries ,53. Althological . The corablished ,55,56. Sociation . In some. mutans .S. sanguinis and S. gordonii can antagonize S. mutans [S. mutans was always found in the caries lesion, which indicated its acidogenicity and cariogenicity [S. sanguinis and S. gordonii in dental plaque were associated with sound tooth surfaces without caries [Oral streptococci account for 20% of all supragingival microorganisms, but they account for nearly 80% of the initial colonizers during the early stages of biofilm formation . Further. mutans . Accordigenicity ,61. In ct caries ,61.rnc knockout for S. mutans disrupted the polysaccharide synthesis in the Sm + Ss + Sg biofilm. The fewer polysaccharides indicated an easier exposure to antimicrobial agents [rnc knockout for S. mutans reduced the virulence factor and rendered the biofilm to become more sensitive to DMAHDM composite. This synergistic effect from the combination of rnc-knockout for S. mutans with DMAHDM composite achieved the greatest biofilm-inhibition efficacy, reducing biofilm CFUs by five logs.The exopolysaccharides (EPS) matrix is a key virulence factor. It serves as nutrients and helps form the core of the matrix scaffold . In addil agents . Therefornc-knockout for S. mutans with the 3% DMAHDM composite achieved the greatest reduction in the lactic acid secretion of the Sm + Ss + Sg biofilm by an order of magnitude. The substantial reduction in acid secretion helped the non-cariogenic species to survive and grow, thus maintaining a healthy non-cariogenic biofilm composition.Biofilms can produce organic acids, lower the pH, and induce the selection for an acidogenic and aciduric flora, thus shifting the biofilm composition from a healthy state to a cariogenic state . In the rnc knockout in S. mutans and DMAHDM composite on biofilm modulation was remarkable. With only 6.3% S. mutans in the biofilm, the non-cariogenic species can grow and thrive. Therefore, rnc knockout in S. mutans plus DMAHDM composite demonstrated the ability to bring the biofilm species balance back toward a healthy state for the first time. This percentage shift of bacteria was attributed to the dual pressure of both the competition among bacteria and the antimicrobial agent. This was because: (1) the antimicrobial effect from DMAHDM exerted an acid-inhibition effect to weaken the competitiveness of S. mutans in the Sm + Ss + Sg biofilm; (2) rnc knockout for S. mutans produced less polysaccharide in Sm + Ss + Sg biofilm models which decreased the biofilm drug-tolerance and induced greater sensitivity to DMAHDM; (3) S. mutans rnc-knockout plus DMAHDM composite achieved the greatest reduction in lactic acid secretion, which helped the non-cariogenic and non-aciduric species to survive and grow [rnc gene-knockout in S. mutans plus the bioactive and therapeutic composite caused the greatest decrease in biofilm acid secretion, which provided an environment in favor of the non-cariogenic species, which would be clinically beneficial to prevent caries. A previous study [S. mutans rnc gene-knockout effect in an oral biofilm model in vivo with animal experiments. Further studies are needed to investigate the dual strategy of rnc gene-knockout in S. mutans plus DMAHDM in regulating biofilm development tendency to a healthy composition by testing more species to mimic the complexity of oral biofilms.Indeed, the combined contribution of and grow ,12. Therus study  reportedrnc gene knockout in S. mutans and a bioactive and therapeutic composite in Sm + Ss + Sg biofilm modulation to suppress the cariogenic species percentage and increase the non-cariogenic species percentage for the first time. The S. mutans rnc gene-knockout alone reduced the cariogenic S. mutans percentage in the Sm + Ss + Sg biofilm. The DMAHDM composite induced a shift in biofilm species from a cariogenic composition toward a non-cariogenic composition. The dual strategy of combining rnc gene-knockout with DMAHDM composite yielded the greatest reduction in S. mutans percentage in the Sm + Ss + Sg biofilm, and the greatest increase in the non-cariogenic species percentage, thereby resulting in the lowest amount of biofilm acid. The dual strategy reduced the cariogenic S. mutans proportion from 69% on commercial composite, to 6% on 3% DMAHDM. Combining the rnc knockout for S. mutans with DMAHDM composite reduced the biofilm CFUs by five logs. Therefore, the novel dual strategy of bacterial gene-modification and bioactive and therapeutic dental resin has the potential to modulate biofilm shift from a cariogenic state to a non-cariogenic state. This novel dual strategy is promising for a wide range of applications in preventive and restorative dentistry.This study demonstrated the high efficacy of


Cryptosporidiumfelis is an important cause of feline and human cryptosporidiosis. However, the transmission of this pathogen between humans and cats remains controversial, partially due to a lack of genetic characterization of isolates from cats. The present study was conducted to examine the genetic diversity of C. felis in cats in China and to assess their potential zoonotic transmission. A newly developed subtyping tool based on a sequence analysis of the 60-kDa glycoprotein (gp60) gene was employed to identify the subtypes of 30 cat-derived C. felis isolates from Guangdong and Shanghai. Altogether, 20 C. felis isolates were successfully subtyped. The results of the sequence alignment showed a high genetic diversity, with 13 novel subtypes and 2 known subtypes of the XIXa subtype family being identified. The known subtypes were previously detected in humans, while some of the subtypes formed well-supported subclusters with human-derived subtypes from other countries in a phylogenetic analysis of the gp60 sequences. The results of this study confirmed the high genetic diversity of the XIXa subtype family of C. felis. The common occurrence of this subtype family in both humans and cats suggests that there could be cross-species transmission of C. felis. Cryptosporidium spp. are important apicomplexan parasites inhabiting the gastrointestinal tract of humans and other vertebrates, causing severe diarrhea [Cryptosporidium species, but C. hominis, C. parvum, C. meleagridis, C. felis, and C. canis are the most common ones [C. felis mainly infects cats and is therefore considered a host-adapted species [C. felis infections, however, are common in developing countries [C. felis between a household cat and the owner has been reported [C. felis infections [diarrhea . Human cmon ones . Among t species . Human Countries ,5,6,7, areported . Neverthfections .gp60) gene have been developed for human-pathogenic Cryptosporidium spp. to track infection sources [gp60-based subtyping tools are available for C. hominis, C. parvum, C. meleagridis, C. ubiquitum, C. viatorum, Cryptosporidium skunk genotype, and Cryptosporidium chipmunk genotype I [Cryptosporidium spp. in humans and animals [Subtyping tools based on sequence analysis of the 60-kDa glycoprotein  worldwide [C. felis has been identified in children and immunocompromised patients in China [C. felis isolates from Guangdong and Shanghai for assessment of their zoonotic potential.A C. felis . Thus faorldwide ,18. Mostin China ,20,21,22C. felis based on nested PCR analysis of the small subunit (SSU) rRNA gene, 20 (66.7%) generated the expected products in the gp60 PCR. Among them, 9 were from cats in pet shelters, 5 were from cats visiting animal hospitals, 4 were from cats in pet shops, and 2 were from stray cats  over the partial 60 genes . Among tAltogether, 15 subtypes were identified, with two subtypes in samples SCAU392 and SCAU754 having nucleotide sequences identical to the XIXa-39 and XIXa-40 subtypes (GenBank reference sequences MH240852 and MH240853) from humans in the United Kingdom, respectively . Accordigp60 sequences from the 20 C. felis isolates were all placed in the XIXa subtype family in the phylogenetic tree  formed a subcluster with several subtypes identified from humans and cats in several European countries and another subtype (XIXa-82) formed a subcluster with several subtypes from humans and cats in the United Kingdom and Sweden. In addition, two cat-derived subtypes (XIXa-87 and XIXa-88) in 4 isolates from this study formed their own subcluster. As expected, the known subtypes (XIXa-39 and XIXa-40) identified in the present study and previously in humans from the United Kingdom and Indonesia formed a subcluster  being identified [C. felis in healthy cats could be one of the reasons for the relatively low PCR amplification efficiency.This represents the first subtyping study of entified ,18. In tgp60 gene confirmed the high genetic diversity of C. felis isolates. Like observations in previous studies [C. felis isolates successfully analyzed from cats. However, all C. felis isolates in this study belonged to the subtype family XIXa. As 13 of the 15 XIXa subtypes identified were novel, there could be geographic isolation among some of the XIXa subtypes, as previously suggested by us [An analysis of the  studies ,18, 15 sC. felis could be possible. In this study, among the 15 XIXa subtypes, two were identical to subtypes previously found in humans, while others clustered with human-derived subtypes from other countries. Importantly, one of the subclusters formed included two sequences from an owner and the household cat (GenBank reference sequences MH240883 and MH240884) with possible zoonotic transmission [C. felis isolates from one child in Shanghai (XIXa-14) and one rhesus macaque (XIXa-12) in Guizhou, China [gp60 sequences were different with the 15 XIXa subtypes identified in the present study. They clustered together in a large clade within the XIXa subtype family in the phylogenetic tree  rRNA gene was used to detect ium spp. . DNA pre in cats ,28.gp60 gene was employed to identify the subtypes of C. felis in this study [C. felis DNA) and negative (reagent-grade water) controls in each PCR run. The positive products from the secondary PCR were identified by 1.5% agarose electrophoresis.The newly developed nested PCR targeting the conserved region of the is study . Brieflyis study . Each DNhttp://technelysium.com.au/wp/) and edited using BioEdit 7.1.3.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). These and the reference sequences from GenBank were aligned with each other using the MUSCLE program implemented in MEGA 6 (https://www.megasoftware.net/). Tandem Repeats Finder 4.09 (http://tandem.bu.edu/trf/trf.html) was used to identify repetitive sequences within them. A maximum-likelihood tree was constructed using MEGA 6 based on substitution rates calculated using the general time reversible model and gamma distribution. The bootstrap method with 1000 replicates was used to assess the reliability of the phylogenetic clusters formed. Representative nucleotide sequences of the C. felis subtypes identified in the present study were deposited in the GenBank database under accession numbers MW351820-MW351832.All positive products of the expected size were sequenced bidirectionally on an ABI3730 autosequencer by the Sangon Biotech  using secondary PCR primers. The DNA sequences generated were assembled using ChromasPro 2.1.6 (C. felis isolates from cats in China for the first time. The results of the phylogenetic analysis suggested the potential zoonotic transmission of this pathogen. More isolates from diverse areas and hosts should be analyzed to confirm this conclusion.The present study reported the subtype characteristics of


The changes of contractility proteins and the relative molecules are in a non-linear fashion. Specifically, the expression and activation of cMLCK increase first then fall during ischemia and reperfusion (I/R). This change is responsible for the nonmonotonic contractility alteration in I/R which the underlying mechanism is still unclear. Contractility recovery in I/R is also attenuated by age. The age-related change in cardiac contractility influences the therapeutic effect and intervention timepoint. For most cardiac ischemia therapies, the therapeutic result in the elderly is not identical to the young. Anti-aging treatment has the potential to prevent the development of ischemic injury and improves cardiac function. In this review we discuss the mechanism underlying the contractility changes in the aged heart and age-induced ischemic injury. The potential mechanism underlying the increased susceptibility to ischemic injury in advanced age is highlighted. Furthermore, we discuss the effect of age and the administration time for intervention in cardiac ischemia therapies.Cardiac function of the human heart changes with age. The age-related change of systolic function is subtle under normal conditions, but abrupt under stress or in a pathogenesis state. Aging decreases the cardiac tolerance to stress and increases susceptibility to ischemia, which caused by aging-induced Ca Patientpression ,10-12. RThe therapeutic effect of ischemic cardiac dysfunction varies in old patients. Patients older than 80 have worse survival rate than 70-year-old patients after cardiac ischemic therapy, but the survival rate for patients who undergo coronary artery bypass grafting is not affected by age . Early iThis review focuses on cardiac contractility changes and the underlying mechanism during aging. The potential mechanism of ischemia intolerance and the mechanism leading to a acute contractility dysfunction after ischemia in aging patients are also discussed. Finally, an overview of cardiac ischemic therapies and the effect of aging on those therapies are also provided in this review.Cardiac aging is accompanied by complex morphological and functional changes, involving compensatory left ventricular wall thickening to cardiomyocyte senescence and number loss . The oveet al. [et al. [Previously, EF and FS are the main indices to evaluate cardiac contractility; however, EF and FS alterations in advanced age are disputable. Fiechter et al.  reported [et al.  and Rans [et al.  showed c [et al. . The gen [et al. . Therefo [et al. -29. A su [et al. . A decre [et al. . PreciseSevere contractility dysfunction is easily identified under pathologic states with irregular cardiac contraction and decreased EF, FS, dp/dt, LVSP, and LVDP in the elderly -33. Inte2+. Ca2+ transient initiates L-type Ca2+ current influx and subsequently triggers Ca2+ release from the sarcoplasmic reticulum (SR) through the Ca2+ release channels and ryanodine receptors (RyRs) [2+ will activate the myofilament protein, then undergo reuptake back into the SR to achieve excitation-relaxation coupling [2+ homeostasis, which is maintained by Ca2+ influx and SR Ca2+ storage [2+ transient, is significantly prolonged in aged cardiomyocytes [2+ ATPase 2 (SERCA2a) and over-activation of RyRs are responsible for the prolonged SR Ca2+ transient in the aging heart. However, an opposite result of SERCA2a expression was recently reported on atrioventricular junction of 24-month-old Wistar rats [2+ transient might be different in each part of the heart during aging, which may involve aging contractility compensatory mechanisms. The overall increase in protein kinase A (PKA) and phospholamban (PLB) can also lead to SERCA2a dysfunction and slow Ca2+ re-uptake in 24-month old rats [2+ diminishes SR Ca2+ storage during recovery and results in Ca2+ transient impairment; Schmidt et al. [et al. [2+ release, is reported to have an age-associated reduction [2+ leak and decreased SR Ca2+ storage [et al.[2+ homeostasis and repaired RyR function by inhibiting ROS production in cardiomyocytes. Therefore, the attenuated recovery of Ca2+ homeostasis by ROS-induced over activated RyR is one of the potential reasons for AVV1/SERCA2a therapy inefficiency.Cardiac contraction is activated by a transient rise in intracellular free Cas (RyRs)  Fig. 1)2+. Ca2+ coupling . Cardiom storage ,38. One myocytes . Reducedtar rats . This fiold rats ,41. The t et al.  and Dele [et al.  confirme [et al. -47 Altho [et al. ,45, no s [et al. . Hence, eduction ; however storage . Togethe storage  and fina storage ,50. Intee [et al. rebuilt Reduction of ATP production in the aging heart, which is caused by mitochondrial dysfunction, was observed in 2005 . The caret al. [et al. [2+ uptake in mitochondria [2+ overload [2+ coupling proteins, such as CaMKII and RyR2, resulting into abnormal excitation-contraction coupling. Treating the aged heart with MitoTEMPO, a ROS expression suppressor, increase contractile performance of senescence hearts [2+ sensitivity of myofilament proteins and then induce systolic dysfunction [Excessive endogenous ROS caused by mitochondrial dysfunction is the main source of oxidative stress, which is related to multiple age-related cardiac morphologic changes, such as cardiac fibrosis, cell apoptosis, DNA damage, and cardiac remodeling . Fernandet al.  demonstr [et al. observed chondria . And thioverload . Over-ace hearts . Age-inde hearts . low-grae hearts . The profunction . Excessiet al.[2+ sensitivity during I/R. Treating cardiomyocytes with rhNRG-1, a growth factor inducing MLCK expression, can improve the EF% and FS% in murine hearts with cardiac I/R injury [The cMLCK/MLC-2 pathway directly regulates cardiac contractility in aging. Cardiac light chain kinase (cMLCK) is a Ca2+/calmodulin-activated protein kinase that phosphorylates the 20 kD regulatory light chain (MLC-2) of myosin ,62. The et al. demonstrR injury . Old MLCR injury , so aginet al. [Accumulating evidence has shown that miRNA, small non-coding RNAs regulating mRNA translation, is an important regulator of the cardiac aging process and susceptibility to ischemia. Previously, miRNA-34a, miR-1, and miR-133a were shown to accelerate cardiac cell death and promote cardiac aging -72. Receet al.  indicateet al. . miR-21 et al. . The miRet al. . AlthougLong non-coding RNAs (lncRNAs) also regulate mRNA expression in aging. LncRNA MALAT1 and MIAT regulate the aging process by controlling the cell cycle . LncRNA Histone modification recently is found to play a crucial role in cardiac aging. Histone modification-related proteins, such as histone deacetylases (HDACs), are closely related to regulation of cardiac contractility. HDAC6 is associated with a-tubulin degradation  and preset al. [ROS, as a therapeutic target, has been shown to suppress the cardiac aging process and to improve cardiac I/R injury. Roshni et al.  reportedet al. .2+ transient are closely interdependent. The ER Ca2+ leaking is mediated by the XO/ROS/mPTP pathway. Thus, inhibiting xanthine oxidase activity has been shown to reduce ROS production and restore cardiac ability in MI. SERCA2a function and Ca2+ transient are enhanced after administering a xanthine oxidase inhibitor [Meanwhile, ROS production and Canhibitor . Therefonhibitor .2+ transient is more effective in improving heart failure. Transient receptor potential vanilloid 4 (TRPV4), an ion channel involved in Ca2+ homeostasis, improves contractility by over-expression in both young and aged MI mice [2+-channel-dependent therapy and ROS-related therapy on aged MI animal models are warranted.Compared to ROS therapy, regulation of Ca MI mice . Both co MI mice . Howeveret al. [Stem cell therapies have been studied for a long time to mend MI. Cardiac regeneration could be initiated by transplantation or eliciting innate cardiomyocyte proliferation. Non-cardiomyocytes are used, such as mesenchymal stem cells (MSCs), bone marrow-derived mononuclear cells/mesenchymal stem cells (BM-MCs/MSCs), and pluripotent stem cells, as transplant material . In a stet al.  also demet al. . Not onlet al. , but alsExosomes of cardiosphere-derived cells (CDCs) and MSCs, nano-extracellular particles carrying protein and RNAs from host cells, are both found to improve cardiac function after acute and chronic myocardial infarction ,94. Exosmax and developed pressure were well-preserved compared to the vehicle group, but still lower than control [111 reported that mocetinostat, a class I and IV HDAC inhibitor, is able to increase LVEF and +dp/dt in ischemia-induced heart failure and decrease infarction size. Since the crucial work of HDAC in the cardiac aging process was reported, HDAC inhibitors have recently been shown to have a promising effect on anti-aging [et al. [et al. [HDAC inhibitors have recently been shown to effectively improve cardiac function and to reduce infarction size. Applying trichostatin A (TSA) and SAHA, FDA-proved HDAC pan-inhibitors, during reperfusion reduces infarction size and improves FS% in both mice and rabbits . Interes control . Furtherti-aging . Therefo [et al.  applied  [et al.  reported [et al. . Moreove [et al. .Healthy aging will cause approximately the loss of one-third of cardiomyocytes in the human heart. Senescence of long-lived differentiated myocardial cells may induce apoptosis to consequent on severe impairment following myocardial infarction . TherefoRapamycin, another found senolytic, is able to rejuvenate aging stem cells by targeting the mTOR/PI3K pathway. Resveratrol is also a well-known drug that benefits cell function by targeting mitochondrial biogenesis. The combination of these two drugs has been shown to prevent cell senescence and improve cardiac output following myocardial infarction . These s2+ transient, myofilament reaction and epigenetic regulation are responsible for the poor recovery and adjustment on contractility after ischemia in advanced age. However, the aging process is not regulated by an isolated molecular pathway. Interestingly, the cell organelle interaction, specifically the SR-mitochondrial interaction, are involved into the myocardial function impairment in advanced age. This study reminded us the significant engagement of inter-organelle communication in cardiac aging process which indeed is worthy for more attention.Contractility dysfunction is not as apparent as diastolic dysfunction in heathy aging. But, systolic dysfunction is stimulated by cardiac stress or a pathogenic state in advanced age . Complicet al. [Epigenetic regulation has also presented a crucial role in both cardiac aging and pathology of MI. Independent study of miRNAs and lncRNAs in aging process has reveal their regulatory mechanisms, but the role of interaction between those non-coding RNAs in aging and aging-induced MI are still poorly investigated. Further more, Ounzain et al.  has founThe effectiveness of stem cell therapies is always influenced by aging-related environmental changes. For example, ageing-induced low grade systematic inflammation cause poor survival rate of injected stem cells ,111. SimSurprisingly, administration time point during I/R is also crucial to the therapeutic effect of a therapy. TSA has clearly shown a difference between pre-treatment and post-reperfusion administration. To optimize the administration timepoint of each therapy, molecular and cellular alteration during I/R need to be further investigated. Previous study has reveal a non-linear change of proteome in duration of I/R . Therefo2+ transient impairment and myofilament alternations has been closely studied in aging researches, but epigenetic regulation, especially histone modification, is still full of unknowns. A research gap on the function and effect of ageing-induced histone modification in aging-induced ischemic injury is worthy to pay more attention. Further study on the aging induced molecular and cellular alterations will provide scientific basis for precision medicine for the elderly and novel biomarkers for cardiac aging and aging related cardiac dysfunction in the future. The effect of aging on clinical myocardial infarction treatments are also in an urgent need. Those studies could help to improve the therapeutically effects and provide appropriate therapeutic regimens for the elderly.Aging researches on MI are largely limited by the difficulties of animal model establish, because the high mortality of old animals after surgery. Therefore, the different pathological differences between young and old are not explicit. The mitochondrial disfunction, Ca


Cangshanaltica fuanensis sp. nov. is described; the morphology of the adult and immature stages is described and illustrated. The life history and remarkable biological features of this species are revealed. Females deposit one large egg at a time; egg length equals 0.4–0.5 times the female body length. Females lay and hide each egg under a spoon-shaped moss leaf. There are only two ovarioles on each side of the ovary in the female reproductive system, which has not been reported before in Chrysomelidae. Besides, a modified fan-driven Berlese funnel is designed for faster extraction of moss inhabiting flea beetles. We suggest this improved device could also be useful for collecting other ground-dwelling arthropods.The immature stages and the biology of the moss inhabiting flea beetles are poorly understood. In this study, a new species of moss-eating flea beetles—Cangshanaltica fuanensis sp. nov. is described; the morphology of adult and immature stages is examined and illustrated. Its life history and biology are studied. The remarkable and unique biological features are revealed: (1) females deposit one large egg at a time, egg length equals 0.4–0.5 times the female body length, these are unusual in Chrysomelidae; (2) females have only two ovarioles on each side of the ovary, which has not been reported in other Chrysomelidae species; (3) females lay and hide each egg under a spoon-shaped moss leaf; (4) cannibalism of a second instar larva on an egg was observed. Both adults and larvae feed on moss and are polyphagous; their feces mainly consist of un-digested moss fragments; high humidity is essential for the survival of eggs and larvae and expedites the hatching. In addition, a modified fan-driven Berlese funnel is designed for faster extraction of moss inhabiting flea beetles. This device could also be used for collecting other ground-dwelling arthropods. Its working diagram is illustrated and described.The biology of the moss and leaf litter inhabiting flea beetles is poorly understood. In this study, a new species of moss-eating flea beetles  Nanomela, Zeaphilon, Aphilon and Maurodus) are associated with mosses and liverworts . ADPC: personal collection of A. Damaška. IZCAS: Institute of Zoology, Chinese Academy of Sciences, Beijing, China. SZPT: School of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, Shenzhen, Guangdong, China.Abbreviations for insect collections. Mosses were identified using “Flora Bryophytorum Sinicorum” . A bryopCangshanaltica fuanensis sp. nov. are hard to observe in nature; most of the biological data were collected based on lab-reared individuals. A rearing container was designed for rearing C. fuanensis sp. nov. Transparent plastic containers (15 cm × 7 cm × 5 cm) were selected and placed in a north-facing room to avoid direct sunlight. Two rectangle openings were carved in the sides of the container; they were sealed by non-woven fabrics using adhesive, allowing for air circulating and preventing other organisms from coming into the container. A thick layer of moist paper towel was placed at the bottom of the container in order to maintain proper humidity and avoid larvae from drowning in water drops; a thin layer of soil was placed above the paper towel in order to provide nutrition for host plant and site for larvae to pupate; fresh host plant moss was collected and placed loosely above the soil layer. Distilled water was sprayed on the moss once a day to maintain humidity using a small spraying device.The activities of 0) adults were collected from wild and raised until they were dead. Their first filial generation (F1) was also maintained alive in the same container for the remainder of their lives. An F2 generation was not successfully bred. The biology and behavior of numerous individuals of different stages were observed .Adults extracted from moss were transferred to the rearing container . Approximately 30 parent . We used cox1 barcoding primers: forward LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and reverse HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) ; (2) PARATYPE Cangshanaltica fuanensis sp. nov. Des. Ruan et al. 2019. • 1♀ (IZCAS), labels: (1) China, Fujian Prov., Fuan, Shuyang, 250 m, 27.159° N 119.677° E, 27-IV-2019, sifted from moss; (2) PARATYPE Cangshanaltica fuanensis sp. nov. Des. Ruan et al. 2019. • 2♂2♀ (IZCAS), labels: (1) China, Fujian Prov., Fuan, Shuyang, 290 m, 27.1611° N 119.6763° E, 16-VIII-2019, Extracted from moss, Leg. Y. Ruan; (2) PARATYPE Cangshanaltica fuanensis sp. nov. Des. Ruan et al. 2019. • 1♂ (ADPC), labels: (1) China, Fujian Prov., Fuan, Shuyang, 290 m, 27.1611° N 119.6763° E, 3-X-2019, Extracted from moss, Leg. Y. Ruan; (2) Cangshanaltica fuanensis des. Ruan, 2019; (3) PARATYPE Cangshanaltica fuanensis sp. nov. Des. Ruan et al. 2019; (4) VOUCHER SPECIMEN A. F. Damaška coll., AFD-265.• 1♂ (ADPC), labels: (1) China, Fujian Prov., Fuan, Shuyang, 290 m, 27.1611° N 119.6763° E, 3-X-2019, Extracted from moss, Leg. Y. Ruan; (2) Cangshanaltica fuanensis des. Ruan, 2019; (3) PARATYPE Cangshanaltica fuanensis sp. nov. Des. Ruan et al. 2019. • 3♀5♂ (SZPT), labels: (1) China, Fujian Prov., Fuan, Shuyang, 290 m, 27.1611° N 119.6763° E, 3-X-2019, Extracted from moss, Leg. Y. Ruan; (2) PARATYPE Cangshanaltica fuanensis sp. nov. Des. Ruan et al. 2019. • 3♂10♀ (SZPT), labels: (1) China, Fujian Prov., Fuan, Shuyang, 290 m, 27.1611° N 119.6763° E, 13-II-2020, Extracted from moss, Leg. Y. Ruan; (2) PARATYPE Cangshanaltica fuanensis sp. nov. Des. Ruan et al. 2020.Paratypes: 1♀ (IZCAS), labels: (1) Host plants (see “Biology” section for more information). Adults feed on Hypnum plumaeforme (Hypnaceae) and Racopilum cf. aristatum (Racopilaceae) in nature, they also casually feed on Bazzania tridens (Lepidoziaceae), Hylocomiaceae sp. and Thuidiaceae sp. In a lab environment; moss spores were found in their intestinal tract. Larvae: feed on both Hypnum plumaeforme and Racopilum cf. aristatum in a lab environment.Remarks. By the external habitus, Cangshanaltica fuanensis sp. nov. is similar to other non-metallic species of Cangshanaltica .Cangshanaltica fuanensis sp. nov. may be distinguished from C. nigra by the following characters: dorsum dark brown throughout ; protrusion at distal part of antennomere VII barely visible ; in lateral view, apex of median lobe of aedeagus very slightly bent dorsally .Cangshanaltica fuanensis sp. nov. may be distinguished from C. siamensis by the following characters: body length 1.35–1.70 mm, width 1.10–1.35 mm ; middle-longitudinal ridge on first abdominal ventrite long and strongly developed, extending to near posterior margin ; in lateral view, apex of median lobe of aedeagus very slightly bent dorsally .Cangshanaltica fuanensis sp. nov. may be distinguished from C. sprynari in by the following characters: surface of pronotum and elytra smooth, with fine punctures ; middle-longitudinal ridge on first abdominal ventrite almost extending to posterior margin ; median lobe of aedeagus in ventral view, apical fourth of median lobe gradually narrowed, without an abruptly narrowing step ; median lobe of aedeagus in lateral view, apex slightly bent dorsally .Cangshanaltica, e.g., in C. nigra: male does not have larger first pro- and meso- tarsomeres; apex of last visible abdominal ventrite has the same shape in female and male [C. fuanensis sp. nov.: females are almost indistinguishable from males by external characters, except for the slightly larger body size. However, in C. fuanensis sp. nov., the female tignum could be seen through abdominal ventrites when they are alive or preserved in ethanol (but hardly visible when the specimens are dry).The main sexually dimorphic features present in other genera of flea beetles are absent in some and male . This coCangshanaltica are very similar to each other in external characters, the shape of genitalia is useful to separate species, especially the female vaginal palpi , head brown, eye spots black; legs blackish, semi-transparent; pronotum and body tubercles grey-black; ovoid micro sculpture present on body surface . Larvae preserved in ethanol: body “C”-shaped, pale yellow to whitish.Head  present behind antenna (inside head capsule).Head . Head noeyespots A: Eys prEp): with six pairs of long (e1–e6) and one pair of short (e7) setae; e5–e7 situated at posterolateral part of epicranial halves. The epicranium has 12–13 pairs of sensilla (Sen), including four pairs of larger ones near frontal sutures. Epicranial suture (Eps) short, approximately half as long as endocarina (En).Epicranium A: Ep: wiFr): with three pairs of long setae (f1–f3) and one pair of sensilla; frontal sutures (Frs) reaching antennal sockets; median endocarina narrow ridge-shaped, extending from base of frontal sutures to clypeus.Frons A: Fr: wiCly): sclerotized, transverse, band-shaped, bearing 1 pair of long setae and three pairs of sensilla. Clypeus and frons divided by epistomal sulcus; epistomal ridge strongly sclerotized.Clypeus A: Cly: sLbr): sclerotized, transverse; bearing one pair of setae medially, one pair of setae laterally and one pair of sensilla near midline; anterior edge deeply incised , bearing numerous microtrichia; posterior edge with middle part produced posteriorly.Labrum C,H: Lbr:Md): symmetrical, palmate, mesal membranous lobe absent (present in adults); each mandible with four sharp teeth, one sensillum near base and two setae on lateral edge, penicillus with 7–8 stiff setae, first tooth largest, remainder decrease in size.Mandibles F,J: Md: Antenna E,G: weakCa) small, triangular, bearing one seta, with a longitudinal band-shaped sclerotization (Scl); stipes (St) elongate with a long and curved sclerotization (Scl), bearing two long setae near lateral edge. Mala with galea  and lacinia (Lac) not fused; apical part of galea with approximately 5–6 setae arranged around a stout pedunculate seta (appearing two-segmented); apical part of lacinia bearing a row of peg-shaped stout setae; maxillary palpus (Mxp) three-segmented: first palpomere with two setae, second palpomere with two setae and one sensillum, third palpomere with one digitiform sensillum (Dsen).Maxilla B,D: cardLbi): submentum (Smen) trapezoid, membranous, bearing two pairs of long setae; mentum not well defined; prementum short and transparent, with prominent horseshoe-shaped mental sclerite, bearing one pair of setae at base; ligular membranous, not separated from prementum, anterior edge broadly rounded to straight, bearing numerous microtrichia; labial palpi small, two segmented; five pairs of sensilla present near labial palpi.Labium B,D: Lbi:Thorax. The tubercular pattern on the right side of the thorax is illustrated in D-DL-EPa bearing seven long setae and about five sensilla, well sclerotized. Tubercles on epipleural, pleural, and sternal regions weakly sclerotized, with poorly defined edge. Epipleural region with EPp bearing one long seta (not capitate). Pleural region with Pa and Pp each bearing one spine-shaped seta. Sternal region with ES and SS obsolete, each bearing one spine-shaped seta.Prothorax: dorsum with large Da and Dp each bearing one long capitate seta and one sensillum. Dorso-lateral region with DLi bearing one long capitate seta and one egg-burster; DLe bearing two capitate setae, and two sensilla. Epipleural region with EPa bearing one spiracle and EPp bearing one long seta (not capitate). Pleural region with P bearing one spine-shaped seta. Sternal region with ES and SS obsolete, each bearing one spine-shaped seta.Mesothorax: tubercles weakly sclerotized, with poorly defined edge. Dorsal region with Epa and spiracle; one egg-burster present on DLi.Metathorax has the same tubercular pattern as mesothorax, except for the absence of Leg  and one setose sensillum. Pleural region with P bearing one long and one short spine-shaped setae. Sternal region with SS-PS bearing one long and one short spine-shaped setae; ES obsolete, bearing one spine-shaped seta.Segments I–VIII: with same tubecular pattern and chaetotaxy, tubercles weakly sclerotized, with poorly defined edge. Dorsal region with Segment IX. Pygidium D moderatPd).Segment X not visible in dorsal view, bearing prominent eversible pygopod C: Pd.Remarks on larval characters. The general appearance of Cangshanaltica fuanensis sp. nov. is similar to some free-living flea beetle larvae described by previous studies ; Epa present on mesothorax and absent on metathorax; pygidium with four long setae; only two tubercles (DLi and DLe) present on dorso-lateral region of mesothorax, metathorax, and abdomen.dies see , such asGeoffroy , Ivalia a Jacoby , Distigmra Blake . HoweverCangshanaltica Konstantinov et al. is close to Ivalia Jacoby [C. fuanensis sp. nov. also shows extreme similarity with Ivalia korakundah Parathapan et al., especially in the body shape, tubercular pattern, and chaetotaxy. Both larval and adult morphology suggests that they are very close lineages. Despite the resemblance in general morphological features, the larvae of Cangshanaltica fuanensis sp. nov. and Ivalia korakundah can be differentiated by the following characters: in C. fuanensis sp. nov. body tubercles with weak sclerotization and poorly defined edge (tubercles with moderate sclerotization and well-defined edge in I. korakundah), tubercle EPa of mesothorax absent (EPa of mesothorax present in I. korakundah), maxilla with triangular cardo (cardo narrow and transverse in I. korakundah).The adult morphology of a Jacoby . Larval C. fuanensis sp. nov. is very similar to that of adults in the general palmate shape, sharp teeth, decreasing in size from the first to last tooth. The significant difference is that the adult mandible has one more tooth, and a mesal membranous lobe with dense microtrichia, which are absent in the larvae. The resemblance of their mandible shape may be related to similar feeding behavior, both are eating the leaves of the same host plant.The larval mandible of Body length 1.60–1.75 mm; width 0.90–1.10 mm; bent ventrally in lateral view. Body white–yellow to yellow–brown, bearing long, brownish, and spine-shaped setae. Head invisible from above, bearing 3 pairs of long setae. Mesothorax and abdominal segment I–V bearing one spiracle each. Pronotum bearing seven pairs of long and one pair of short setae; meso- and metanotum each bearing one pair of setae near middle. Abdominal segments I–VIII each bearing one pair of dorsal and one pair of lateral setae. Abdominal segment IX bearing two pairs of setae and one pair of distal urogomphi. Abdominal segment X invisible in dorsal view, bearing prominent eversible pygopods B,F.Length 0.68–0.74 mm, width 0.36–0.40 mm. Egg large, egg length to female adult body length ratio: 0.4–0.5; egg width to female adult body width ratio: 0.29–0.33. Egg oval and lemon yellow. Surface with minute reticulated pattern consists of irregular polygonal cells  .Agasicles hygrophila Selman and Vogt (observed in current study), and the hexagonal cells further subdivided into 4 to 6 smaller ones in Disonycha leptolineata Blatchley [Remarks. The polygonal pattern on egg chorion resembles those in some other flea beetles, e.g., the polygonal pattern in latchley .0 parent adults, 35 F1 eggs, 30 F1 larvae, 10 F1 pupae, and 6 F1 adults were observed in this study . The following biological data were collected based on these lab-reared individuals .Egg stage  against egg chorion, after several times of contraction, the egg-bursters slit the chorion. Newly hatched larvae usually stay at the base of moss, they feed on both rotten and fresh leaves of the host plant. Two larvae were spotted feeding on adult feces . As the adult feces consist of un-digested moss leaf fragments . Furthermore, the host plant tissue in their intestinal tract is only partially digested and highly visible externally, which makes their color much closer to that of moss.Agasicles hygrophila Selman and Vogt).First instar. Duration: ca. 4–7 days. Head width 0.27–0.30 mm; body length 0.90–1.50 mm, body width 0.30–0.40 mm. Newly hatched individuals appear unsclerotized C: body bSecond instar. Duration: ca. 4–6 days. Head width 0.34–0.37 mm. Head width of second instar to that of first instar ratio: 1.2–1.3. Body length 1.50–2.30 mm, body width 0.45–0.61 mm. The newly molted individual with body transparent and soft. Old individual: body lemon yellow; pronotum, pygidium, and tubercles light black; mandibles reddish-brown to dark brown. Egg-bursters obsolete.Third instar. Duration: ca. 8–13 days . Head width 0.42–0.46 mm. Head width of third instar to that of second instar ratio: 1.2–1.3. Body length 2.00–2.80 mm, body width 0.50–0.80 mm. With similar coloration as second instar. Egg-bursters obsolete.Prepupal period (pupating period): 3–5 days, body color turned milky white. In the first 1–2 days: prepupal larvae stop feeding, crawl down to soil surface near the base of moss, excrete residual contents in the intestinal tract, and search for pupation location. Day 2–3: they build an earthen chamber, use legs to collect moist earth patches, bite off minute earth granules one by one using mouthpart, then attach them to the chamber wall; by this means, they construct the oval and smooth inner wall of the earthen chambers. Day 3–5: earthen chambers are completed, they cease movement and are not responding to minor stimuli.Pupal stage , with yellow and unsclerotized integument, and are unable to jump upon stimulation. After a few days (ca. 2–3 days), their integuments gradually turn brown, and they can perform minor jumps. After approximately 5–6 days, adults can perform explosive jumps.lt stage H,I. AdulCangshanaltica fuanensis sp. nov. were dissected in this study , which is a common moss in East Asia (including both north and south China) and grows in various habitats at different altitudes (50–4000 m above sea level) [Racopilum cf. aristatum (Racopilaceae) in nature. However, at the type locality, Racopilum cf. aristatum is rather scarce compared to the dominant moss species Hypnum plumaeforme. In a lab environment, when Hypnum plumaeforme and Racopilum cf. aristatum are absent, adults also casually feed on Bazzania tridens (Lepidoziaceae), Hylocomiaceae sp. and Thuidiaceae sp. Larvae feed on both Hypnum plumaeforme and Racopilum cf. aristatum in a lab environment; their feeding behavior in the natural environment was not studied.Both adults and larvae are polyphagous; they can feed on different host plants of different moss families. In nature, adults primarily inhabit and feed on a level) . Adults C. fuanensis sp. nov. An investigation was conducted on a 30 m2 sample plot at the type locality in July 2020. It turned out only 15 adults were discovered on the surface of moss from 9–11 p.m. in a single night. Based on our field investigation, the host plant Hypnum plumaeforme is abundant, and the feeding injury on the host plant  is scarce. All these indicate the population density of this species may be low.The food source is abundant to Adults usually feed on the distal end of moss branches, the end of young shoots are usually chopped off by their feeding, which is destructive to the host plant. In a lab environment, adults can feed on other parts of the host plant after most of the end of young shoots are eaten. Dissection of the intestinal tract of adults collected from nature shows that they also consume moss spores E.C. fuanensis sp. nov., another flea beetle species-Benedictus sp. was discovered. In both natural and lab environment, it feeds on H. plumaeforme, and has very similar feeding habits, eating the distal end of young branches. The population density of Benedictus sp. is probably lower than C. fuanensis sp. nov. based on the number of specimens collected (less than 10). The competition between the two species in nature is still unknown.At the type locality of Feces of larvae and adults mainly consist of undigested fragments of host plant leaf see B,D, whicMost specimens collected in this study were extracted from moist moss in a north-facing valley with a creek flowing nearby at the type locality  A. The suCangshanaltica fuanensis sp. nov. is diurnal and nocturnal, they could be discovered in nature in both day and night as long as the mosses are moist. We searched for the individuals in a 30 m2 sample plot at the type locality in July 2020. The number of adults presented on the mosses at different times of a single day was counted: 5 adults were discovered in the early morning ; 3 adults were discovered in the late morning ; 10 individuals were discovered in the dusk ; approximately 15 adults were found in the night . The valley we investigated was exposed to the sunlight from 9 a.m. to 5 p.m. The mosses in the open area appeared dry, and no specimens were found on them. Some individuals could be discovered on these same mosses in the night. This is because the valley became damp even in clear nights, the dew could revive the dry mosses quickly after sunset. The behavior of the larvae in nature is still unknown, as they were not found in this investigation.The adult of C. fuanensis. This was tested on two first instar larvae and one prepupal larva: (1) we placed the first instars in an open plastic container with 40% ± 5% air humidity, they were dead and entirely exsiccated within 2 h; (2) an earthen chamber containing a prepupal larva was taken from moist soil and placed in an open plastic container with 40% ± 5% air humidity, the prepupal larvae inside the chamber was exsiccated and killed within 8 h. Furthermore, based on our observations, the egg stage is much shorter when the moss has more moisture (see “Egg stage” section above). These observations show that the high humidity of the air, host plant, and earth is essential for the survival of immature stages.Based on the laboratory observation, the arid environment is lethal for the immature stages of However, an extremely wet environment (flooding environment) could also be lethal for both larvae and adults. When reared in the lab, larvae were frequently drowned dead in the minute condensed water drops on the lateral walls of rearing containers. A water immersion experiment was conducted on one adult: it was completely immersed in the distilled water, after approximately 1.5 min, it ceased movement and was not responding to stimuli.C. fuanensis was not found at the type locality . Hypnum plumaeforme still grows in winter in Fujian province. Numerous adults were extracted from the moss in February at the type locality ; these adults were still active after transferring them to a rearing container with the same temperature as the type locality. Adults of C. fuanensis are probably active all year round in nature, as they were collected from the field in February, May, July, August, and October. We have collected the larvae in February and October; it is unknown if they also present in other months. In a lab environment with similar air temperature as the type locality, adults and larvae could be reared throughout the year.Dormancy of Cangshanaltica fuanensis sp. nov. in nature.Fungus gnats (Diptera: Sciaroidea sp.). In this study, one prepupal larva in the pupation chamber was attacked by two fungus gnat larvae, its first abdominal segment was ruptured by them, which led to its death. Larvae of Mycetophilidae were frequently discovered in moist moss in our study; it is still unknown if they can affect the survival of immature stages of Cangshanaltica fuanensis sp. nov. for 5 min , adults of ground beetles , thrips , spiders and mites , centipedes , etc.When stimulated with a brush, the larvae would curl into ‘C’ shape for 30 s to 3 min before they started to move. Adults do not show prominent death feigning; instead, they can conduct several continuous jumps upon stimulation.Maximal jumping distance: 29 cm . Adults could perform continuous jumps upon stimulation in a lab environment. In the natural environment, when the mosses are gently disturbed, they tend to crawl away and hide; they only jump when the mosses are shaken severely, or our hands are very close to them.Traditional Berlese funnel (using a light bulb as light and heating source) was frequently used for extracting the soil, leaf litter and moss inhabiting arthropods, including flea beetles . These sCangshanaltica fuanensis sp. nov. were successfully extracted from moss and kept alive. This may be the second most successful (based on the number of specimens) documented moss inhabiting flea beetle collection event after the collection of 83 specimens of Kiskeya elyunque Konstantinov and Konstantinova in Puerto Rico [Benedictus sp., Chaetocnema constricta Ruan et al., Minota sp.1, Minota sp.2, Parathrylea septempunctata, Jacoby, Philopona vibex (Erichson)). Apart from beetles, various other arthropods were also collected from moss cushions.The modified fan-driven Berlese funnel was proven suitable for collecting moss inhabiting flea beetles. Approximately 30 adults and 15 larvae of We tested the modified Berlese funnel for different samples: such as moist humus soil and leaf litter. In 2019, approximately 10 samples of either humus soil or leaf litter were collected under subtropical evergreen broad-leaved forest from different localities in Guangdong and Yunnan Provinces, China. Various and numerous arthropods were successfully collected.We further conducted 10 tests using moist humus soil in Fuan, Fujian Province, then calculated arthropod specimens of each sample (volume of sample: 4–5 L), an average of ca. 400 specimens for each sample were counted. The average specimen numbers of different arthropod groups for each sample were counted as follows: Collembola ≈ 200, Arachnida ≈ 160, Insecta ≈ 30, Diplopoda ≈ 3 Chilopoda ≈ 2; Crustacea ≈ 2 D. It shoChaetocnema constricta , anditex sp. ).C. fuanensis sp. nov., previous studies pointed out that cannibalism may be advantageous to the individuals in the acquisition of essential nutrients, remove future competitors for food, and regulate population density [Although the moss cushions allow for the comparatively cryptic lives of flea beetles, there were various potential natural enemies discovered in this study and in . In addi density .C. fuanensis sp. nov. feed together on the leaves of Hypnum plumaeforme. A similar condition was previously found in Distigmoptera borealis [C. fuanensis sp. nov. and D. borealis have the same feeding habit as adults. It is also interesting that adults of C. fuanensis sp. nov. and D. borealis are able to feed on moss spores. It is unknown if the spores could be a source of nutrients, and the cause of the spore feeding behavior remains unknown.It occurs that adults and larvae of borealis . BryophyC. fuanensis sp. nov. is a unique feature in leaf beetles. Suzuki  summ summC. f Suzuki , and Hydroscapha (Hydroscaphidae) also produce a single large egg at a time [C. fuanensis sp. nov. are adaptations to the unique habitat environment or are related to the small body size.Another distinct biological feature in y volume ; Sphaerit a time ,40. It iCangshanaltica sprynari Damaška and Aston [Taiwanoshaira spp. [C. fuanensis sp. nov. is both nocturnal and diurnal in the lab environment and nature.Recent studies show that some of the moss-eating leaf beetles are nocturnal and were only found on moist moss at night, for example, nd Aston  and Taiwira spp. . Based oHigh-power heating elements had been used in modified Berlese funnels previously e.g., ,42,43). ,43. 42,4In this study, we proposed a fan-driven high-power Berlese funnel for collecting moss inhabiting flea beetles. Apart from flea beetles, this method may have broader applications in collecting other organisms inhabiting soil or leaf litter. Although this method is time-saving , it is still unknown if this could lead to incomplete sampling, which is also not known for some other extracting techniques. We have not used a light source in the modified device as it may lead to a failure of collecting some small photophobic arthropods. A recent study also shows that the heating temperature could be vital for the efficiency of extracting small animals from soil . Therefo