Helminth infections are among the neglected tropical diseases affecting billions of people globally, predominantly in developing countries. Helminths’ effects are augmented by coincident tuberculosis disease, which infects a third of the world’s population. The role of helminth infections on the pathogenesis and pathology of active tuberculosis (T.B.) remains controversial. Parasite-induced suppression of the efficacy of Bacille Calmette-Guerin (BCG) has been widely reported in helminth-endemic areas worldwide. T.B. immune response is predominantly proinflammatory T-helper type 1 (Th1)-dependent. On the other hand, helminth infections induce an opposing anti-inflammatory Th2 and Th3 immune-regulatory response. This review summarizes the literature focusing on host immune response profiles during single-helminth, T.B. and dual infections. It also aims to necessitate investigations into the complexity of immunity in helminth/T.B. coinfected patients since the research data are limited and contradictory. Helminths overlap geographically with T.B., particularly in Sub-Saharan Africa. Each disease elicits a response which may skew the immune responses. However, these effects are helminth species-dependent, where some parasites have no impact on the immune responses to concurrent T.B. The implications for the complex immunological interactions that occur during coinfection are highlighted to inform government treatment policies and encourage the development of high-efficacy T.B. vaccines in areas where helminths are prevalent. Schistosoma mansoni, Trichuris trichuria (whipworm), Ascaris lumbricoides (roundworm), Necator americanus and Ancylostoma duodenale (hookworms) [Intestinal helminths are parasitic worms infecting over 1.5 billion people globally . Climateokworms) .Mycobacterium tuberculosis (Mtb) complex [Tuberculosis (T.B.) is an infectious bacterial disease caused by different strains of acid-fast bacilli belonging to the  complex . The T.B complex . T.B. co complex . Approxi complex . Globall complex . Further complex .T.B. exposure results in the initiation of an immune response to fight the infection. The immune response to T.B. involves the interaction of innate and adaptive immune responses. It is dependent on the cellular immune response, which is mediated by proinflammatory T-helper type 1 (Th1) and Th17 cells ,9,10. ThT.B. commonly overlaps geographically with soil-transmitted helminths, especially in developing countries ,14,15,16Given the current evidence on potential immunologic implications, such as those that could influence T.B. vaccination, treatment and diagnosis, more research is needed to determine the influence of helminth coinfection on T.B. control and how to negate any adverse effects. As a result, this review will summarize what is currently known about T.B. and helminths’ immune responses in human and experimental studies, both separately and in the context of coinfection. The review will also elucidate the effects of T.B. and helminth coinfections on vaccine efficacy and the implications for long-term health care.An electronic search of online databases such as Google Scholar, Google, PubMed, Science Direct, online library sources, and Web of Science were utilized to extract research and review articles using phrases and words: helminth, tuberculosis, helminth and tuberculosis coinfection, helminth and tuberculosis vaccine and helminth and tuberculosis diagnosis in humans, animals and in vitro studies. A PRISMA flow diagram of the search strategy and research design process for this review is presented in Helminths are parasitic and multicellular organisms that coevolved with their hosts . These pHelminths mature within the infected subject and lay eggs for transfer to another host, exposing them to multiple stages of parasite development, each of which elicits a unique immune response . HelmintTissue injury activates the production of “alarmins” ) and the identification of invaders by pattern recognition receptors (PPRs) in the host . The Th1Helminths stimulate increased mucin synthesis, smooth muscle contractility and epithelial cell turnover as a host defense to eliminate the infection. There is also increased IgE and IgG1 production in mice and IgE and IgG4 production in humans ,28. All The Th2 immune response induced by helminths includes interleukins , broad or localized eosinophilia and hyperplasia of goblet and mucosal mast cells ,28. The Tregs maintain the Th2 dominance, IL-10 and TGF-β, which mediate the suppression of competing Th1 and Th17 cell populations . Tregs mHelminth-induced suppression of immunopathology also involves CD4+ Tregs (Foxp3+ or Foxp3), CD8+ Tregs, regulatory B cells (Bregs), IL-4-responsive cells, TGF-β, and IL-10 . Since aAccording to the hygiene hypothesis, in developed countries where sanitation is good, and helminths have been eliminated, there is an increase in allergic diseases such as asthma and allergic rhinitis, and autoimmune diseases such as Crohn’s disease . This hyHelminths induce various immune and physiologic modifications to survive the hostile immune response directed against them and their general survival. These survival mechanisms include this modified Th2 response . These pT.B. enters the body via inhaled droplets to the alveoli. It interacts with the alveolar macrophages, infecting and multiplying inside them, thus making these cells the first line of defense against infection . In immuMtb is, therefore, a pathogen that can cause both latent and active disease [In some cases, the disease is controlled and kept in an inactive or latent state in distinct foci known as granulomas bacteria ,15,38,39 disease .The initial stages of T.B. infection include phagocytosis of the bacteria by macrophages . ReceptoMtb, which is aided by the creation of peptide cathelicidin [When phagocytic cells encounter T.B., they get activated and generate cytokines, including proinflammatory cytokines such as TNF-, IL-1, IL-6, IL-12 and IFN-γ . Increaselicidin .The presentation of T.B. antigens by dendritic cells in lymph nodes, possibly aided by neutrophils, initiates a local immune response that culminates in pathogen killing by reactive oxygen species (ROS) and antimicrobial peptides .Mtb include monocytes, macrophages, neutrophils, natural killer (NK) cells and dendritic cells. Together, these cells form a primary granuloma, which may allow Mtb growth while containing the infection until T cells are recruited to the infection site, a response process that takes weeks [Mtb [Cells required in the host’s defense against es weeks . Phagolyeks [Mtb .Mtb through the activation of perforin, where the antimycobacterial factor granulysin binds to the bacterial cell surface and disrupts the membrane, resulting in bacterial osmotic lysis [Mtb replication to a minimum. Phagocytic cell apoptosis may prevent the spread of disease, diminish the viability of intracellular mycobacteria and reduce the risk of infection [NK cells may eliminate intracellular ic lysis . Apoptosnfection .Adaptive immunity develops after exposure to mycobacterial antigens or vaccination with BCG. This part of the immune system is triggered when the innate immune response is insufficient to suppress T.B. infection. The control of T.B. requires Th1 immune responses  and Th17 responses (IL-17 and IL-23). Th1 responses are proinflammatory and develop a cell-mediated reaction . Th1 celT-bet binds to and affects the expression of Th1-specific genes and Th1 and Th17 cell expression . This isT.B. immunity involves many cells, such as T cells, B cells and natural killer (NK) cells, with CD4+ T cells being the primary cell type in T.B. control . The CD4IFN-γ is regarded as the primary cytokine that regulates T.B. infection and eradication. It works by activating the infected macrophage, resulting in the production of reactive oxygen and nitrogen species, which have a microbicidal role . In termTh17 was first described as a distinct population of the T helper cells controlled by the transcription factor RAR-related orphan receptor gamma (RORyt) . They deMtb infection are still poorly understood, owing to the limitations in examining pulmonary-specific immunity. On the other hand, human innate immune responses to Therefore, understanding the interaction of innate and adaptive immune cells in human T.B. is crucial for identifying new immunomodulatory targets and clarifying protective immunity processes. The immune response profiles to tuberculosis infection are presented in Innate and Th1-dominant adaptive immune responses interact to produce granulomas. Innate and adaptive immune responses are critical for microorganism eradication.The geographic distributions of helminths and T.B. overlap substantially, particularly in underdeveloped countries, resulting in an increased likelihood of coinfection with both pathogens ,16. ThisAn Ethiopian study investigated the association between intestinal helminths and active T.B. and found that helminth infection increases the likelihood of developing active T.B. . This anMtb killing.One school of thought suggests that helminths create an environment that weakens the host’s defenses against T.B. By activating the IL-4 receptor pathway, a preexisting helminth infection inhibits an innate pulmonary anti-T.B. defense . In coinMtb [Mtb resistance in helminth-infected mice is promoted by AAMs. This major cellular pathway compromises the helminth-infected host’s ability to limit Mtb growth [On the other hand, alternatively activated macrophages (AAMs) induce arginase, which competes with iNOS for L-arginine, thereby reducing nitric oxide production for the intracellular killing of Mtb . Mtb resb growth .Mtb control illustrated that AAMs might inhibit the macrophage killing of Mtb [Nippostrongylus brasiliensis (Nb) revealed that Mtb colonies were reduced in the lungs of Nb-infected mice. The stimulation of pulmonary CD4+ T cells and Th1 and Th2 cytokines, neutrophils and alveolar macrophages was elevated. This suggests that Nb infection triggers a macrophage response, which protects the host throughout the early phases of mycobacterial disease and subsequent illness [A review in support of this proposed role of the Th2-dominant phenotype on g of Mtb . Convers illness .Both helminths and T.B. have independent mechanisms for initiating the host immune response, with significant consequences for the immunology of each infection ,16. The In Ethiopia, some studies reported an increase in the prevalence of helminth coinfection in T.B. patients, where one study found a higher risk of parasites among active T.B. patients than in healthy community controls ,57,58. LS. mansoni was also a risk factor for T.B. infection, and it altered the clinical presentation and pathogenesis of T.B. in Tanzania [Tanzania . The autTanzania .Toxoplasma gondii (T. gondii) seropositivity and having tuberculosis, with T. gondii seropositivity, which indicates chronic infection, being relatively common among tuberculosis patients [A systematic review suggested that health education be implemented to help prevent intestinal helminth infection. It further added that screening for helminths should be possibly included in the treatment strategies for tuberculosis patients . Anotherpatients .Strongyloides stercoralis coinfection with pulmonary T.B. was implicated in the cause of the skewed immune response to mycobacterial disease [S. mansoni in Kenya, on the other hand, discovered that the expression of T.B.-specific Th1 cytokines was maintained. Individuals with latent tuberculosis and S. mansoni infection had more CD4+ Th1 cells than those who were only latently T.B.-infected [lumbricoides infection had no impact on Th1, Th2 and Th17 responses or the T cell populations [ disease . The pro disease . A studyinfected . There wulations .Mtb antigens [A Th1 immune response observed during persistent filarial infection was characterized by a reduction in Purified Protein Derivative (PPD)-specific IFN-γ and IL17 responses . The stuantigens . Elias aantigens . Lastly,antigens .Mtb. By contrast, the induction of type 2 immunity, e.g., Th2 and Treg cells (as seen in helminth infections) and their anti-inflammatory cytokines, were reported to suppress the efficient immune response against T.B. [Furthermore, enhanced Treg function is associated with helminth infection and may suppress Th1 responses against unrelated antigens ,67. Thisnst T.B. .Mtb [A mouse model study of Schistosoma mansoni showed a reduced protective efficacy of BCG vaccination against Mtb . AnotherMtb . During Mtb . In compMtb . AnotherMtb .In Egypt, it was determined that hookworm infection was one of the risk factors for the failure of T.B. therapy . HoweverAlthough HIV is not covered in this review, there is evidence of a concurrent distribution of triple disease burden involving tuberculosis, helminths and HIV, particularly in Sub-Saharan Africa. This necessitates a greater focus on disease management strategies by various policymakers .BCG is currently the only T.B. vaccine available; it celebrated its 100th anniversary in 2021. Alternative vaccines are being developed . The BCGThe BCG vaccine is administered to more than 80% of all newborns and babies in countries where it is included in the national childhood immunization program; however, it does not prevent the development of latent tuberculosis or the reactivation of pulmonary disease in adults . BCG hasAn Ethiopian study found that helminth infection influenced BCG vaccination outcomes, and PPD-specific cellular immune responses improved in helminth-treated individuals compared to untreated controls . DewormiS. mansoni was found to reduce the protective efficacy of BCG vaccination against Mtb, possibly by attenuating protective immune responses to mycobacterial antigens and polarizing general immune responses to a Th2 profile [Furthermore, in another study,  profile . Th2-like IL-10 responses elicited by intestinal helminths may interfere with Th1-like IFN responses induced by BCG, altering the protective immune response to BCG vaccination . The impFurthermore, helminth infection during pregnancy has been shown to persist into childhood and shift immunity away from Th1 responses, which are required in T.B. infection and vaccination . ChronicWhile there is mounting evidence that helminth prophylaxis could have a role in combating the HIV/AIDS and T.B. pandemics , observaThe typical immune response to helminths, characterized by decreased IFN-γ, reduced T cell proliferation and IL-2 as a result of increased Th2/Treg cytokines, attenuates a potent anti-tuberculosis IFN-γ immune response and therefore uncontrolled T.B. pathology . FurtherThe effects of deworming can be used to determine the impact of helminth infections. It was shown that the use of anthelminthic drugs to treat patients with helminths resulted in increased T cell proliferation and IFN-γ production of PBMC stimulated with PPD. The study showed that T cell responses to PPD were improved in filarial-infected patients treated with diethylcarbamazine ,65.The treatment of helminth-infected patients with albendazole during BCG vaccination increased proliferative and IFN-γ responses to PPD, suggesting that persistent helminth infection during BCG vaccination may contribute to a decreased T cell response to mycobacterial antigens. This meant that removing helminths via anthelminthic treatment would reduce Th2 cell and cytokine inhibitory effects on Th1 responses .Toulza et al. found that anthelminthic therapy altered antimycobacterial immune responses in U.K. migrants. Patients with helminth infection had a higher frequency of CD4 + Fox P3 + T cells (Tregs) and a lower frequency of CD4 + IFN-γ + T cells, but these effects were reversed after treatment .Schistosoma infection resulted in a significant decrease in CD4 + Fox P3 + T cells after treatment [Another study in Gabon found that anti-helminth treatment with praziquantel against reatment . Since hreatment . Mtb infection. The virulence and infection route of the mycobacterial strain may also contribute.From the above, it is apparent that concurrent helminth and T.B. infections have demonstrated various effects on the host. These reactions could be due to different helminth species, their location in the body, different life cycles, variable (excretory/secretory) E/S products and Mtb infection in terms of immune response and disease severity, but the clinical and treatment outcome is unknown, possibly due to underpowered studies, the type or intensity of the infecting helminth and the various methodologies used to detect helminth infection [Some in vitro studies have been reported to have shown that helminth infection affects nfection .Concurrent helminth infection and T.B. both produce antagonistic immune responses. Helminths have the potential to impair the host’s ability to respond to bystander infections such as T.B. Helminth and T.B.’s spatial overlap may impair the host’s ability to respond to mycobacterial conditions. Th1 responses are required for T.B. immunity, whereas helminths mount an opposing Th2 response, which tends to dominate and thus skew the immune response. Furthermore, chronic helminth infections impair innate and adaptive immune responses to T.B. and induce immunoregulatory responses, lowering T.B. immunity even further. However, whether these opposing immune responses in helminth and T.B. coinfection affect pathological outcomes is unclear.In helminth-endemic areas, it is suggested that chronic helminth infections reduce the efficacy of BCG, the currently available T.B. vaccine. There is conflicting evidence regarding the effectiveness of regular anti-helminth medication in the treatment of T.B., and this requires further investigation. Clarification of the effect of deworming in concurrent helminth-T.B. infections may aid in the development of government treatment policies. Since vaccines can prevent T.B. infection, the co-occurrence of helminths and T.B. must be considered when developing new vaccines and conducting research on them. Finally, more research is needed to understand better the effects of multicellular coinfecting pathogens on immune responses.


Trifolium pratense L. applying amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses. We observed low levels of genetic and epigenetic differentiation among populations and between habitat types. Genetic and epigenetic variations were not interdependent. Thus, genetic variation was significantly isolated by habitat dissimilarity, whereas epigenetic variation was affected by environment. More specifically, we observed a significant correlation of epigenetic diversity with soil moisture and soil pH . Genetic variation was, therefore, affected more strongly by habitat-specific environmental conditions induced by land use-related disturbance and gene flow patterns, while epigenetic variation was driven by challenging environmental conditions.Central European grasslands, such as calcareous grasslands and oat-grass meadows, are characterized by diverse environmental conditions and management regimes. Therefore, we aimed to determine potential differences in genetic and epigenetic variation patterns between the contrasting habitats and to identify the drivers of genetic and epigenetic variation. We investigated the genetic and epigenetic variation of the ecologically variable plant species  For instance, T. pratense occurs in both calcareous grasslands (CGs) and oat-grass meadows (OMs). Semi-natural CGs developed from grazing over thousands of years and represent nutrient-poor grasslands with relative dry soil conditions [T. pratense in CGs and OMs appeared as a promising model system for studying genetic and epigenetic variation patterns within contrasting habitats.Hutchinson  defined nditions . Howevernditions . AlthougPrevious studies showed that environmental conditions may affect the genetic code of a plant species indirectly –8 indicaet al. [The potential reversible DNA methylation of cytosine represents the most studied epigenetic mechanism with important effects on ecologically relevant traits ,14. Cytoet al. , allow tet al. . These met al. , since oet al. ,9,18,19.Changes in DNA methylation were observed to increase in response to biotic and abiotic stressors –22 such During the last few decades, numerous studies on various plant species observed profound effects of environmental conditions on both genetic and epigenetic variation patterns e.g. –7,9,25).,9,25.7,9T. pratense: (i) are populations genetically and/or epigenetically differentiated among contrasting grassland habitats or are they isolated by distance? (ii) does genetic and/or epigenetic diversity differ between CG and OM populations? (iii) what is the impact of environment on genetic and/or epigenetic diversity levels? and (iv) is genetic and epigenetic variation of T. pratense populations interdependent?We asked the following questions to gain a better understanding about the impact of contrasting environmental conditions on genetic and epigenetic variation in . 2. 2.1Arrhenatherum elatius (L.) J. Presl & C. Presl) meadows  flowers between June and September [T. pratense is an essential species for profitable grassland management due to its high fodder value [The widespread species eptember . It is september . The pereptember . T. prater value  and its er value .et al. [Ellenberg indicator values (EIVs), using plants as bio-indicators, were applied to gain information about environmental conditions . Environmental conditions may often fluctuate in time and space and can, thus, not be estimated in a single measurement . The indet al. , represeet al. . EIVs shet al. . Therefoet al. . These iFor molecular analyses, we identified the species in the field and took leaf samples from 16 individuals per population and species to cover more than 90% of the total (epi)genetic diversity . Samples. 2.22O.All 160 individuals were analysed genetically and epigenetically. DNA was extracted following the CTAB protocol from Rogers & Bendich  modifiedGenetic variation within populations was determined using genome-wide genotyping with amplified fragment length polymorphisms (AFLPs) . The AFLet al. [MSeI by two isoschizomers HpaII and MspI. These restriction enzymes attach at the same tetranucleotide (5′ CCGG) sequence with differing sensitivity to cytosine methylation states and cover, thus, the most frequent methylation types in the CG and CHG  sequence context [MSAP analyses were performed in accordance to the technique of Schulz et al. . Thus, M context ,16. TherHpaII and MspI) were analysed per run, respectively. Samples without a clear banding pattern between 60 and 420 bp were repeated and only strong and clearly defined fragments were taken into account for further analyses. Fragment data were analysed manually with the software Bionumerics 7.6.2 .The fluorescence-labelled DNA fragments were separated by capillary gel electrophoresis using an automated capillary electrophoresis machine . Ninety-six samples and 48 individuals  fragments are present in both profiles (unmethylated state/u-type), (ii) fragments are present only in EcoRI/MspI profiles , (iii) fragments are present only in EcoRI/HpaII profiles , and (iv) complete absence of fragments in both profiles (uninformative state).After fragment detection, we applied the ‘mixed scoring 2’ by Schulz et al.  to score [et al.  defined The reproducibility of the AFLP and MSAP analyses was tested by calculating the genotyping error rate . Ten per. 2.3Genetic and epigenetic differentiation within and among populations as well as between habitat types were partitioned with hierarchical analyses of molecular variance (AMOVA). AMOVAs were calculated based on pairwise Euclidean distances among samples using the software GenAlEx 6.41 .ΦPT values; electronic supplementary material, table S4). Pairwise geographic distances (km) were calculated from coordinates, and for habitat types, a habitat dissimilarity matrix was constructed by coding pairs of CG/OM populations by ‘1′ and pairs of equal habitats by ‘0’ . A correlation between genetic and epigenetic distance matrices was examined applying a simple Mantel test. Geographic and habitat dissimilarity matrices were also checked for correlation patterns. Genetic and epigenetic isolation by distance (IBD) and isolation by habitat dissimilarity (IBH) were tested performing simple and partial Mantel tests with 9999 permutations applying the ‘vegan’ library in R [Epigenetic and genetic distance matrices were calculated within the AMOVA (ary in R .doi:10.5061/dryad.kt71r).Although simple and partial Mantel tests are suitable to test dissimilarity hypotheses ,52, e.g.pi · log2pi, where pi is the frequency of the (epi)genetic marker score ‘1’ within the population. The acronyms ‘SIgen’ and ‘SIepigen’ stand for the mean Shannon's information index and will be substituted by the terms ‘genetic diversity’ and ‘epigenetic diversity’ in the discussion.Genetic and epigenetic diversity within populations were determined using the R script ‘MSAP_calc’ . Applyingen, SIepigen, and environmental parameters  between CG and OM populations.Two-sided T-tests (and Wilcoxon–Mann–Whitney tests if necessary) were calculated to examine differences of SIgen and SIepigen with light, soil moisture, soil pH, and soil nitrogen were analysed with correlation tests (Pearson correlation coefficients) applying the ‘PerformanceAnalytics' [Possible correlations of SIalytics'  and ‘Hmialytics'  librariegen and SIepigen were examined with paired T-tests. Additionally, SIgen and SIepigen were tested for interdependence applying the correlation tests as mentioned above. Unless otherwise stated, the R environment [Differences between SIironment  was used. 3. 3.1ΦPT of 0.07 with a differentiation between habitat types of 3% and a differentiation among populations of 4%. The hierarchical AMOVA of the combined epigenetic dataset resulted with 0.05 in a lower ΦPT. 1% of epigenetic variance resided between habitat types and 4% among populations. Values of epigenetic differentiation for h-, m- and u-subepiloci are given in Hierarchical AMOVA of genetic data  revealedr = 0.30; p = 0.069). Geographic distance (IBD) and habitat dissimilarity (IBH) were also not correlated .A simple Mantel test revealed no correlation between genetic and epigenetic differentiation across all populations  and partial  Mantel tests   (el tests  as well = 0.010) . Epigene. 3.2epigen) of 0.46   was significantly associated with soil moisture and soil pH (epigen decreased with increasing drought and soil pH.SIitrogen) . However soil pH . Thus, Sgen revealed significantly lower values than SIepigen (p < 0.001). Moreover, SIgen and SIepigen were not significantly correlated across populations .SI. 4. 4.1Genetic differentiation levels were higher than epigenetic ones indicating that genetic variation may be more strongly structured than epigenetic variation . Some pret al. [T. pratense.Neither epigenetic nor genetic differentiation correlated with geographic distance among populations (IBD). Kloss et al.  showed tT. pratense populations showed higher genetic than epigenetic differentiation among habitat types. This result complies with the findings of Lele et al. [Scabiosa columbaria. Management practices like mowing and grazing differ strongly in intensity and time of application [T. pratense was shown to depend on plant weight, stem length, leaf size and further traits [per se seem to shape genetic differentiation.However, even common and outbreeding species may reveal increased differentiation among populations through reduced abundance, spatial isolation, different land use regimes, and thus lowered gene flow . T. prate et al. , who obse et al.  observedlication ,61. Earllication . These alication . Flowerir traits  shaped b. 4.2T. pratense complied with genetic diversity levels previously reported for common grassland species [Mean genetic diversity of  species . The com species ,3,9,19.Previous studies about genetic diversity patterns in common CG  and OM pT. pratense populations significantly decreased with increasing drought and soil pH. Therefore, challenging environmental conditions may affect epigenetic diversity in different ways.However, OM populations showed higher epigenetic diversity levels than CG populations. The difference of epigenetic diversity between CG and OM populations was significant only for m-subepiloci. Therefore, changes of methylation in the CG context (m-subepiloci) may play a more important role for habitat adjustment than regulation of gene function in the CHG context (h-subepiloci). As mentioned above, the pattern and amount of DNA methylation in plants is sensitive to biotic and abiotic stressors ,24,69. Oet al. [T. pratense populations in OMs, while they could not observe any impact of local habitat quality. Therefore, genetic diversity of T. pratense may be affected more by landscape structure, related management, and/or gene flow patterns than by local environmental conditions.Pearson correlation revealed no significant association between genetic diversity and environment. Pagel et al.  postulatT. pratense populations seemed to be associated with environment, while genetic diversity was not. These results accompany the assumption that DNA methylation and demethylation at a genome-wide scale are induced by environmental changes [et al. [T. pratense produced less yield under drought stress. Furthermore, Labra et al. [T. pratense populations may decrease with increasing drought. Additionally, epigenetic diversity decreased with increasing soil pH. Soil pH influences the amount of plant available nutrients. Since T. pratense is a nitrogen-fixing legume [T. pratense populations may show limited productivity and decreased epigenetic diversity as reaction to phosphorus limitation. However, the correlation with soil moisture and soil pH was not significant for h-epiloci indicating that the regulation of gene function by (de-)methylation in the CHG context may not be an issue for adaptation to different environmental conditions.However, several studies reported correlations between environmental factors and epigenetic characteristics of plant populations ,12,13. I changes  and cons changes ,69. More changes –22. Labr [et al.  emphasiz [et al. , who obsa et al.  postulata et al. . Thus, eg legume , its perg legume . In calcg legume  and thusT. pratense, epigenetic diversity was even significantly higher than genetic diversity indicating that these natural populations seem to vary more in DNA methylation than in DNA sequence [Previous studies observed higher levels of epigenetic than genetic diversity especially in natural plant populations ,13,17,18sequence .et al. [T. pratense populations as facilitated or pure rather than obligatory. This finding is in accordance with the results of previous studies on wild plants, which also observed epigenetic variation to be largely autonomous from genetic variation [Furthermore, neither correlation nor simple Mantel tests revealed a significant association of epigenetic with genetic diversity or distance. In this context, Richards  defined et al.  stated tet al. ,74 and cariation ,75.. 5Our results revealed an impact of different environmental conditions on genetic and epigenetic variation. Genetic variation was affected by habitat-specific environmental conditions induced by management-related disturbance as well as gene flow patterns. Epigenetic variation was driven by challenging environmental conditions in two ways. It increased with rising necessity for niche establishment, but decreased under drought and high pH, the latter potentially resulting in phosphorus limitation.Nevertheless, MSAP markers reveal only a limited number of anonymous loci, which are difficult to link to functional genomic elements. Therefore, future studies should apply next-generation-based bisulfite sequencing approaches to evaluate the effects of challenging environmental conditions on methylation patterns more precisely .


Pistacia lentiscus is a wild-growing shrub and has been utilized for medicinal applications as well as for culinary purposes. The antibacterial and antioxidant activities of P. lentiscus bark in vitro, as well as the phytochemical composition, are the focus of this inquiry. The bark extract of P. lentiscus showed significant antimicrobial activity in experiments on bacteria and yeast isolated from human and food sources. The exposure time for the complete inhibition of cell viability of P. aeruginosa in the extracts was found to be 5% at 15 min. Phytochemical inquiry of the methanol extract demonstrates the existence of carbohydrates, flavonoids, tannins, coumarins, triterpenes, and alkaloids. Deep phytochemical exploration led to the identification of methyl gallate, gallic acid, kaempferol, quercetin, kaempferol 3-O-α-rhamnoside, kaempferol 3-O-β-glucoside, and Quercetin-3-O-β-glucoside. When tested using the DPPH assay, the methanol extracts of P. lentiscus bark demonstrated a high free radical scavenging efficiency. Further, we have performed a molecular modelling study which revealed that the extract of P. lentiscus bark could be a beneficial source for novel flavonoid glycosides inhibitors against SARS-CoV-2 infection. Taken together, this study highlights the Pistacia lentiscus bark methanol extract as a promising antimicrobial and antiviral agent.Foodborne infections and antibiotic resistance pose a serious threat to public health and must be addressed urgently.  There is a growing need for novel compounds to replace antibiotics because of increasing resistance to the treatments and their unwanted side effects . There hMalva spp., Thymbra capitate (L.) Cav., and Olea europaea L. . Co. CoP. leamnoside . Compounlucoside . Water a the oil . It has  the oil . P. lente unique ,41. In ae unique . The exte unique . As longe unique .P. lentiscus bark. As indicated in P. lentiscus bark has antibacterial properties against a variety of micro-organisms in vitro. The extract demonstrated substantial antimicrobial activity with width inhibition zones ranging from 10 to 25 mm for all studied micro-organisms. Comparing the susceptibility of the extract to different food and clinical pathogens, Serratia marcescens appears to be more resistant to P. lentiscus extract. Moreover, our results found that the P. lentiscus extract has a greater antibacterial effect on Gram-positive bacteria than Gram-negative bacteria. P. lentiscus bark extract has been shown to have antimicrobial activity in several investigations, which aimed to scientifically explain their widespread use in treating infectious disorders. P. lentiscus terpenoids, which have been shown to prevent the growth of drug-resistant micro-organisms that are difficult to treat even with conventional antibiotics, served as the starting point for the research. The superior resistance among Gram-negative bacteria could be attributed to the phospholipidic membrane, which is almost impermeable to lipophilic compounds [We have next evaluated the antimicrobial activity of methanolic extracts of ompounds . When thompounds .P. lentiscus bark methanol extract might be attributed to the presence of triterpenes and flavonoids. Several studies have shown the antibacterial properties of these components [P. lentiscus bark [The antibacterial properties of mponents ,46. Phencus bark .P. lentiscus bark methanolic extract demonstrated potent antimicrobial activity [The presence of phenolic chemicals in the plant extract is primarily responsible for its antibacterial effect . As repractivity ,50,51. Eactivity .P. lentiscus bark methanol extract, Pseudomonas aeruginosa appears more susceptible than other reported infectious pathogens. Furthermore, the effect on the cell capabilities of P. aeruginosa confirmed the acquaintance of extract at 0.25, 0.5, and 1% concentrations. P. lentiscus bark methanol extract had a potential antibacterial effect on the capabilities of P. aeruginosa. The contact times for the comprehensive embarrassment of cell viability of P. aeruginosa in extracts was found to be 5% at 15 min. Infections caused by the bacterium Pseudomonas aeruginosa are common in severely unwell individuals. Additionally, the mortality rate linked with P. aeruginosa infection is substantially higher than that associated with other bacteria [P. aeruginosa have recently been classified as invasive or cytotoxic [P. lentiscus bark extract. The extracts may have penetrated the Gram-negative P. aeruginosa outer membrane by pre-disruption of the membrane [In comparing microbial sensitivity to bacteria . Differeytotoxic . This cyytotoxic . In thismembrane .P. lentiscus bark demonstrated a moderate total antioxidant activity compared to that of ascorbic acid. The antioxidant capacity was assessed utilizing the revised equation based on the concentration versus optical density of the investigated compound and the reference control. The extraction process was performed by employing methanol to afford 54.34 ± 2.3 µg of dried P. lentiscus bark. P. lentiscus bark by investigating DPPH radical scavenging activity. As the IC50 value decreases, this indicates a high antioxidant activity. In our investigations, the reference controls (BHA and ascorbic acid) demonstrated values 3.1 and 3.7 times greater than the P. lentiscus bark methanolic extract, respectively. Notably, the methanolic extract demonstrated a considerable antioxidant activity at low concentrations. The DPPH free radical assay has been extensively employed as a tool to assess antioxidant activity by evaluating free radical-scavenging activity [P. lentiscus bark extracts [The methanolic extract of activity . The reaactivity . The keyactivity ,59. Baseactivity , plant eactivity . Polypheactivity . In propP. lentiscus bark possesses potent antioxidant and antimicrobial activities. To further explore the activity of this extract to act against SARS-CoV-2 infection, we have investigated the potency of the identified compounds 3–6 as inhibitors against the 3CL-protease activity. Toward this aim, we have performed in silico molecular docking studies to investigate the binding affinity of compounds 1–5 toward the binding pocket of 3CL-protease protein. There are several 3D X-ray crystal structures for 3CL-protease protein with different substrates and inhibitors [6m2n), since compounds 1–5 have similar flavone scaffolds to baicalein  . Similarnfection ,69. FurtP. lentiscus bark methanol extract phytochemically demonstrates the existence of carbohydrates, flavonoids, tannins, coumarins, triterpenes, and alkaloids. Deep phytochemical exploration led to the identification of methyl gallate, gallic acid, kaempferol, quercetin, kaempferol 3-O-α-rhamnoside, kaempferol 3-O-β-glucoside, and Quercetin-3-O-β-glucoside. When tested using the DPPH assay, the methanol extracts of P. lentiscus bark demonstrated a high free radical scavenging efficiency. The antibacterial activity of the P. lentiscus bark methanol extract was shown in this investigation, which might be attributable to its antioxidant properties. The antimicrobial activity is remarkable and more effective toward Gram-positive bacteria. P. lentiscus bark methanol extract had a potential antibacterial effect on the capabilities of P. aeruginosa. The P. lentiscus bark extract demonstrated a total antioxidant activity similar to that of ascorbic acid. Moreover, Quercetin 3-O-β-glucoside is the most effective inhibitor of SARS-CoV-2 infection, but a synergy from other constituents can work.P. lentiscus bark with potentially active antimicrobial characteristics could be deliberated as an expected source for the conservation or postponement of the shelf life of food. The identified chemicals are predicted to be useful in the future for the research of antibacterial agents.Furthermore, our detailed in silico molecular docking study revealed that the extract constituents have high binding affinity scores toward the 3CL-protease protein, suggesting that this extract could be used against SARS-CoV-2 infection. The industry is disposed to lower the custom of chemical additives in foodstuffs; accordingly, methanolic extract of


Diaz-Arce; Scientific Reports 10.1038/s41598-022-26720-y (2023).1 discuss the utility of barcoding using best-match criterion for the identification of fish species from commercial samples. Focusing on tuna, the evidence provided is a phylogenetic tree containing six problem mitochondrial control region sequences of Blanco-Fernandez et al.2 and 21 reference sequences taken from databases: 8 Thunnus thynnus, 5T. thynnus with T. alalunga introgression, 7T. alalunga, 1T. albacares. Three problem sequences  identified as T. thynnus from best BLAST hit2 clustered together with the T. alalunga references and only one T. thynnus reference, while the rest of T. thynnus were in other clades. This tree poses doubts about the species status of those three samples barcoded as T. thynnus, questioning the conclusions obtained from barcoding. However, with a few references per species it is very difficult to capture the variation of the control region in T. thynnus and T. alalunga. The results can vary depending on the references selected from GenBank to reconstruct the tree. As a proof of this we produced a tree adding three more references to the sequences employed by Diaz-Arce and Rodriguez-Ezpeleta1, with the same parameters and testing the best-fit evolutionary model. In the resulting tree  comes from a T. thynnus voucher specimen identified as introgressed with T. alalunga3. AY699942 and AY99946 correspond to T. thynnus sampled for population genetics5. EU562888 belongs to an individual morphologically identified as T. thynnus used in a study of population genetics in this species6. From this tree the problem sequences MW557512, MW557513 and MW557514, assigned to T. thynnus from barcoding, can be interpreted as belonging to introgressed T. thynnus. Alternatively, we could treat them as T. alalunga, although the best match in BLAST was T. thynnus6 in the three cases2.Diaz-Arce and Rodriguez-Ezpeleta10. However, the reasonable doubt that arises from Fig. T. thynnus has haplotypes corresponding to T. alalunga mtDNA3, while the reciprocal introgression of T. thynnus mitochondrial DNA in albacore has not been detected11. Nuclear markers such as the first internal transcribed spacer (ITS) within the nuclear rDNA could be employed to solve this ambiguity. This marker has been already used to distinguish between T. alalunga and T. thynnus12, and would allow to confirming T. thynnus issued from former hybridization events involving T. alalunga females.BLAST-based barcoding with mitochondrial markers has been employed alone to identify hakes, monkfish, tunas, catfish, and many other fish species using best-match criterionThunnus thynnus in Blanco-Fernandez et al.2, although supported from barcoding and not rejected from phylogenies, should be validated employing nuclear markers. This can be extended to the rest of cases where interspecific introgression occurs. Finally, we have renamed the problem sequences reported in Blanco-Fernandez et al.2 including the Open Nomenclature qualifier cf.  before the species name, stating in their description that they were obtained from seafood samples (see Accession numbers MW557511-MW557516). This way we hope to reduce the noise in databases commented by Diaz-Arce and Rodriguez-Ezpeleta1.In summary, we recognize that the assignation of problem sequences to


Complex interactions between marine Gram-positive pathogens and fish hosts in the marine environment can result in diseases of economically important finfish, which cause economic losses in the aquaculture industry. Understanding how these pathogens interact with the fish host and generate disease will contribute to efficient prophylactic measures and treatments. To our knowledge, there are no systematic reviews on marine Gram-positive pathogens. Therefore, here we reviewed the host–pathogen interactions of marine Gram-positive pathogens from the pathogen-centric and host-centric points of view.Renibacterium salmoninarum, Mycobacterium marinum, Nocardia seriolae, Lactococcus garvieae, and Streptococcus spp. cause economic losses in marine fish aquaculture worldwide. Comprehensive information on these pathogens and their dynamic interactions with their respective fish–host systems are critical to developing effective prophylactic measures and treatments. While much is known about bacterial virulence and fish immune response, it is necessary to synthesize the knowledge in terms of host–pathogen interactions as a centerpiece to establish a crucial connection between the intricate details of marine Gram-positive pathogens and their fish hosts. Therefore, this review provides a holistic view and discusses the different stages of the host–pathogen interactions of marine Gram-positive pathogens. Gram-positive pathogens can invade fish tissues, evade the fish defenses, proliferate in the host system, and modulate the fish immune response. Marine Gram-positive pathogens have a unique set of virulence factors that facilitate adhesion , invasion , evasion , and proliferation and survival  in the fish host. After infection, the fish host initiates specific innate and adaptive immune responses according to the extracellular or intracellular mechanism of infection. Although efforts have continued to be made in understanding the complex interplay at the host–pathogen interface, integrated omics-based investigations targeting host–pathogen–marine environment interactions hold promise for future research.Marine Gram-positive bacterial pathogens, including  Actinobacteria, with high guanine and cytosine (G + C) contents in their genomes, and the phylum Firmicutes, with low (G + C) contents . R. RR. salytoplasm ,108,109.R. salmoninarum are attributed to its cell wall resistance to lysozyme and slow growth rate . O. OR. sal factors ,110,111.M. marinum uses two methods to evade phagocytosis, it escapes from phagosomes  peritoneal macrophages and rainbow trout primary macrophages [M. marinum inhibited phagosome–lysosome fusion in fish monocytes and resided within unfused vacuoles of the carp leucocyte culture cells infected with M. marinum, which did not acidify [M. marinum intracellular survival and virulence have been linked to the Pkng and SecA2 pathway [Bacteria that survive intracellularly either multiply and spread to cells in the infected tissues or migrate to adjacent tissues from the primary site of colonization. agosomes A and/or agosomes B. M. marl spread . The latl spread . Evidencrophages ,115. Therophages ,115. In  acidify . Mycobac acidify ,119,120. pathway ,122. For pathway . On the M. marinum in a zebrafish model [ex vivo resuscitation-promoting factor addition [Investigating the insights of bacterial mechanisms related to the survival of mycobacteria within phagolysosomes will be noteworthy. For instance, Parikka et al. (2012) demonstrated the latency mechanism of sh model . Mycobacaddition . Thus, rR. salmoninarum cannot survive for extended periods of time outside of its host, and the survival times for R. salmoninarum in environmental samples ranged from 4 to 21 days at 10–18 °C, which is relatively a short time and suggests that this pathogen replicates within the host rather than in the environment [R. salmoninarum genome may have contributed to the apparent decrease in many anabolic pathways. Simultaneously, the number of bacterial proteins involved in energy metabolism, transcription, and signal transduction in the genome of R. salmoninarum was lower than that of its environmental relative, Arthrobacter spp. [R. salmoninarum depends on the host for its unique requirements. As a result, R. salmoninarum should have evolved to exploit the fish host’s intrinsic machinery.After entry, pathogens commandeer and use the host cells, not only for their own replication and survival but also for thriving in the host . Pathogeironment . The patironment . For insNutritional immunity is a mechanism used by the host to restrict the availability of essential nutrients in their tissues and fluids, such as iron and vitamins, and prevent the proliferation of potential pathogenic invaders ,127,128.R. salmoninarum, including NADPH reductase, siderophore production, and heme utilization [R. salmoninarum strain (H-2) with a high siderophore production capability was grown under the iron-limited condition and showed significant over-expression of the iron-acquisition-related genes compared to the bacteria grown under normal conditions. On the other hand, R. salmoninarum H-2 displayed higher virulence in terms of cytotoxicity, cytopathic effects and induced the expression of pro-inflammatory cytokines in Atlantic salmon kidney cell lines than a strain with lower siderophore production capacity. A proteome analysis of R. salmoninarum H-2 grown under iron-limited conditions indicated that the iron homeostasis pathway and critical virulence factors related to iron deprivation were significantly enriched [Three iron-acquisition mechanisms have been reported in lization ,136. Undlization ,138. Sidlization . Followilization ,140. In enriched .R. salmoninarum conducted by Wiens et al. (2008) and Bethke et al.  revealed important facts about R. salmoninarum iron homeostasis [R. salmoninarum has gene clusters that encode for a ferric siderophore import system [R. salmoninarum could be similar to the other Gram-positive bacteria based on the genes that encoded for heme uptake in R. salmoninarum  ABC transporter system) [R. salmoninarum genome were acquired via horizontal gene transfer during species divergence [R. salmoninarum grown under iron-limited conditions suggests protection from oxidative stress during intracellular survival [R. salmoninarum falls under high G + C content (56.3%) Gram-positive bacteria, the presence of an additional iron-dependent repressor belonging to the DtxR/IdeR family and their binding sites upstream of important iron-acquisition-related genes were observed [Genomic analyses of t al. 200 and Betheostasis ,141. R. t al. 200 and Beth system) ,142,143.t al. 200 and Bethsurvival . As R. st al. 200 and Bethobserved ,145.S. phocae isolate of Atlantic salmon secretes siderophores and can acquire heme directly through binding receptors [S. phocae expressed an unknown iron-regulated protein (95 kDa) under iron-limited conditions, which could be a receptor for siderophore–iron complexes/heme groups or interact with host-iron-carrying components  [S. phocae’s ability to sense iron availability in the host. Therefore, the bacterium could develop strategies for bacterial adherence, which leads to successful colonization within the host.The eceptors . Interessferrin) . In addiR. salmoninarum grown under iron-limited conditions identified important virulence factors related to their iron acquisition mechanisms , which could aid in designing therapeutic approaches targeting these essential bacterial proteins [More is known about Gram-negative than Gram-positive iron acquisition systems . Therefoproteins . For insMarine Gram-positive bacterial pathogens use diverse mechanisms to infect and manipulate fish host cells and evade immune responses. In contrast, the fish host will mount an immune defense to control the infection and subsequently eliminate it from the system to maintain its homeostasis A. Fish iIn fish, TLRs (Toll-like receptors), NLRs (NOD-like receptors), CLRs (C-type lectin receptors), and PGRP (peptidoglycan recognition proteins) are the four main types of pattern recognition receptors (PRRs) . Only TLL. garviae [N. seriolae [S. dysgalactiae [tlr2 was observed in grey mullet  in response to L. garviae [tlr1 and tlr2 were upregulated in zebrafish following M. marinum infection, which agreed with the known functions of mammalian TLR1 and TLR2 in sensing acid-fast/Gram-positive cell wall components [tlr1 showed a similar expression pattern as ifnγ (interferon gamma)  in the Atlantic salmon kidney cell line in response to R. salmoninarum [TLRs are the innate immune receptors that recognize conserved pathogen molecules , flagellin, and the cell wall)  and ther garviae , N. seriseriolae , and S. alactiae  were repalactiae . The upr garviae . Concurrmponents . Though oninarum ,150. Thioninarum .S. iniae and R. salmoninarum, tlr5 was upregulated in turbot (S. maximus) and Atlantic salmon, respectively [Fish TLR5 recognizes bacterial flagellin . Howeverectively ,162,163.tlr4 encoding gene in soiny mullet (Liza haematocheila) was upregulated in spleens upon Gram-positive S. dysgalactiae infection, suggesting an alternative role of TLR4 in the fish immune response to Gram-positive bacteria [Mammalian TLR4 recognizes Gram-negative LPS . Teleostbacteria .S. iniae infection increased tlr14 expression in Japanese flounder kidney at 1 dpi [tlr20 and tlr22 in zebrafish infected with M. marinum suggest a role of fish-specific TLR clusters in recognizing bacterial infections [Among six non-mammalian fish-specific TLRs  , TLR14, at 1 dpi . Moreovefections .Although the functions of fish-specific TLRs have yet to be reported , TLR intil-6) [hamp) [fpn1), an iron exporter that plays an important role in iron homeostasis [fpn1 expression, the iron release is blocked, and iron uptake is decreased [The host-mediated withholding of essential nutrients to limit bacterial colonization or nutritional immunity is one of the first lines of defense against bacterial infection. The most significant form of nutritional immunity is iron sequestration in host proteins because iron is essential for bacterial proliferation and virulence . Like inil-6) , which i) [hamp) . Increaseostasis ,174. As ecreased ,176,177.ecreased . In otheecreased .hamp 1) is the iron metabolism regulator, and type 2 hepcidin (hamp 2) presents an antimicrobial role [S. iniae [R. salmoninarum [hamp 1 and hamp 2 were observed in response to S. parauberis and L. garviae infection [hamp 1 showed no antibacterial activity, similar to what was reported for Japanese flounder hepcidins against L. garviae and S. iniae infection [hamp 2 exhibited significantly stronger antibacterial activity, especially against Gram-positive bacteria, compared to Gram-negative bacteria. Future studies will be required to unravel the prophylactic use of fish-derived hepcidins to control Gram-positive bacterial infections and to better understand the insights into their antimicrobial properties.Hepcidin is an antimicrobial peptide with iron regulatory properties . Two funial role . SignifiS. iniae  and R. soninarum ,163, resnfection . Here, hnfection ,182. HowR. salmoninarum i.p. infection [R. salmoninarum-resistant salmon population showed higher transferrin expression than the susceptible population. Moreover, differential resistance among the three transferrin genotypes of coho salmon (O. kisutch) was observed after injection with R. salmoninarum [Mycobacterium chelonei significantly increased their nitric oxide (NO) production [R. salmoninarum and M. chelonei) has not been explored and could reveal disease resistance mechanisms in fish and improve brood stock selection based on transferrin allelic variation.Transferrin is one of the serum proteins capable of binding and transporting iron and creating an environment where low levels of iron restrict the growth of pathogens . The bionfection . Interesoninarum . On the oninarum ,196. In oduction . The rolAeromonas hydrophila infection. Developing research to understand the immune effects of Lcn2 and Lcn2-mediated resistance against marine Gram-positive pathogens might be useful.The second layer of iron nutritional immunity involves Lcn2, which binds to bacterial siderophores and sequesters ferric siderophore complexes away from bacterial siderophore receptors ,197. A rS. phocae [The first protective barrier against infection is the fish mucus, which has bactericidal properties. It is also the first interaction site between skin epithelial cells and the pathogen . Lysozym. phocae . In addi. phocae ,199,200.R. salmoninarum, rainbow trout macrophages activated inflammatory responses  at 2 hrs post-infection [R. salmoninarum survived initial contact with macrophages by avoiding/interfering with the TNF-α-dependent killing pathways of fish [ifnγ and mx1-3 upon R. salmoninarum infection in chinook salmon and rainbow trout may be linked to the priming of adaptive immunity [R. salmoninarum strain with reduced p57 suggests these genes as possible immune indicators in vaccine design [In response to nfection . TNF-α, nfection . R. salm of fish . Moreove of fish . Interfe of fish . Interfe of fish ,204,205.immunity ,201,202.e design ,207.R. salmoninarum, there is no clear correlation between this antibody response and protective immunity [The teleost adaptive immune system is subdivided into humoral immunity, which involves antibodies to neutralize pathogens in body fluids, and cell-mediated immunity, which kills and eliminates pathogen-infected cells . Extraceimmunity ,111. Morimmunity ,209,210.immunity ,211.R. salmoninarum [R. salmoninarum by using a 44 K salmonid microarray platform in a transcriptome profiling study [R. salmoninarum differentially regulated the Atlantic salmon adaptive immune responses, including B- or T-cell differentiation, function, and antigen presentation. Moreover, R. salmoninarum infection levels have an impact on the JAK-STAT signaling pathway during host–pathogen interactions [Few studies reported the cell-mediated immune response of oninarum ,213,214.oninarum . Khalil ng study . For insractions .Streptococcus spp. bacterin [Streptococcus spp.Sakai et al. (1989) demonstrated the protective immune response against streptococcal infection in rainbow trout immunized with β-hemolytic bacterin . Here, tR. salmoninarum and Mycobacterium spp) in fish [The cell-mediated immunity involving CD8+ T cells is effective in killing and eliminating intracellular Gram-positive pathogens  observed an acquired immune resistance in yellowtail (Seriola quinqueradita) when the fish that had recovered from experimental infection with S. iniae were reinfected [Resistance is the ability to limit a pathogen in terms of its replication or spread. On the other hand, tolerant fish would show less pathology when comparing high- and low-tolerance fish populations with equivalent pathogen burdens . Metzgerhallenge . The WI infected . Here, tR. salmoninarum [R. salmoninarum infection in low water temperatures (11 °C) [As water temperature affects both the rate of bacterial multiplication and the fish immune response, the rapid shifts in marine water temperature could alter host-pathogen interactions and reduce host resistance . For insoninarum . Moreove (11 °C) .R. salmoninarum has primarily adapted to infect and persist in salmonids [Taken together, this article provides an overview of the host-centric and pathogen-centric approaches at the host–pathogen interface between economically important marine fish and Gram-positive pathogens. Marine Gram-positive pathogens developed a unique set of machinery/strategies to interact with their exclusive fish host cells and modulate the complex molecular and cellular networks of these cells to allow bacterial proliferation and spread while counteracting fish defenses. The pathogenicity of marine Gram-positive pathogens strongly depends on the host it is trying to infect. For instance, almonids .R. salmoninarum may pose a lesser risk to chinook salmon since low temperatures (8 °C) favor its infection compared to higher temperatures (12 °C and 15 °C) [Knowledge of how the host and pathogen interact is crucial for a true understanding of disease and is key to developing control or prevention strategies. Studies in marine Gram-positive pathogens that have been conducted so far focused mostly on bacterial virulence and fish immune responses. A few studies considered how environmental stressors  affect host-pathogen interactions and alter disease progression ,219. DynArthrobacter davidanieli, which is closely phylogenetically related to R. salmoninarum, is commercially licensed for BKD control, and elicits cross-immunity [S. iniae infection [Yersinia pestis, a Gram-negative pathogen and the causative agent of the bubonic plague [R. salmoninarum virulence. Thus, identifying or designing the variants of p57 and p22 with reduced immunomodulatory properties and using them as immune protective antigens will be an attractive vaccine design for BKD control in mariculture. Although the area of functional genomics of fish pathogenic bacteria has been slowly progressing, most of the economically important marine Gram-positive bacterial genomes were sequenced [As prophylaxis design engages in either disabling the bacterial virulence or boosting the host system, vaccines and immunostimulants are effective and sustainable in aquaculture ,222,223.immunity . Howeverimmunity . The pronfection ,227,228.nfection . These tc plague . Variantc plague ,231,232.equenced . These aequenced . Vaccineequenced ,236. ResAs aquaculture continues to grow globally, applying bioinformatics to expand our knowledge on the host-pathogen-environment interactions of marine Gram-positive bacteria will be valuable in solving emerging fish health issues.


Drosophila melanogaster Nora virus (DmNV) is a novel picorna-like virus first characterized in 2006. Since then, Nora virus has been detected in several non-Drosophila species, including insects in the Orders Hymenoptera, Lepidoptera, Coleoptera, and Orthoptera. The objective of this study was to determine if DmNV could infect individuals of other species of invertebrates besides D. melanogaster. The presence of DmNV in native invertebrates and commercially available stocks was determined. Laboratory-reared D. yakuba, D. mercatorum, Gryllodes sigillatus, Tenebrio molitor, Galleria mellonella, and Musca domestica were intentionally infected with DmNV. In addition, native invertebrates were collected and D. melanogaster stocks were purchased and screened for DmNV presence using reverse transcription-polymerase chain reaction (RT-PCR) before being intentionally infected for study. All Drosophila species and other invertebrates, except M. domestica, that were intentionally infected with DmNV ended up scoring positive for the virus via RT-PCR. DmNV infection was also detected in three native invertebrates  and all commercially available stocks tested. These findings suggest that DmNV readily infects individuals of other species of invertebrates, while also appearing to be an endemic virus in both wild and laboratory D. melanogaster populations. The detection of DmNV in commercially available stocks presents a cautionary message for scientists using these stocks in studies of virology and immunology. Drosophila melanogaster Nora virus (DmNV) is a positive-sense single-stranded RNA virus that is spread via the fecal-oral route. DmNV has genetic similarity to viruses of the Picornaviridae and Iflaviridae families, specifically regarding the helicase and RNA polymerase regions, respectively. The DmNV genome has 4 open reading frames (ORFs), with ORF2 encoding a helicase-protease-replicase cassette, characteristic of picornaviruses [ORF1 encodes an RNAi inhibitor [ORF4 encodes a polyprotein that, after proteolytic cleavage gives rise to 3 mature capsid proteins. Finally, ORF3 appears to encode a capsid-stabilizing protein [Picornavirales [aviruses . Meanwhi protein . The stravirales .Picornaviridae family [Nasonia vitripennis virus 3 in Nasonia parasitoid wasps [Haematobia irritans [Spodoptera exigua Nora virus [Agrotis ipsilon Nora virus [Apis mellifera Nora virus [Helicoverpa armigera Nora virus. These viruses are all genetically similar to the DmNV and appear to cluster together in a previously unknown family of insect viruses distinct from Picornaviridae [D. melanogaster is possible.While DmNV is picorna-like, it does not appear to fall neatly within the e family . In factid wasps , an unnairritans , the Spora virus , Agrotisra virus , Apis mera virus  and Heliaviridae . Given tD. melanogaster research, as unrecognized DmNV infection in laboratory stocks could, in theory, confound experimental results. For this reason, stocks ordered from a stock center were tested to investigate the prevalence of DmNV in laboratory-reared D. melanogaster.Finally, the only overt pathogenic effects of DmNV are minimal decreases in survivability and a locomotor deficiency that is apparent through geotaxis assays with large sample sizes . HoweverGryllodes sigillatus (crickets), Tenebrio molitor , Galleria mellonella (wax moths), and Musca domestica (house flies) in the laboratory setting via an established infection protocol. Further, to investigate the potential for DmNV cross-species infection in the wild, insects were collected and tested for the presence of DmNV infection via RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for DmNV ORF1. Finally, the presence of DmNV infection in laboratory stocks of D. melanogaster from a stock center was performed to raise awareness of the prevalence of DmNV in D. melanogaster populations within the research laboratory.The purpose of this study was to investigate the ability of DmNV to infect individuals of other species of Drosophila, and other insects, including RelE23  D. melanogaster were maintained at 25 °C on standard cornmeal, molasses and torula yeast medium with diurnal light. Flies were either infected fecal-orally to establish Nora virus-infected (NV+) stocks or maintained uninfected for further analysis. Once adequately established, stocks were expanded for fly collection by transferring flies into new bottles. The Witi RelE23 stocks were checked weekly via RT-PCR for NV infection via Witi 2·H2O, 182 mM KCl, 46 mM NaCl, 10 mM Tris base, pH adjusted to 7.2, and was sterilized by autoclaving), placed on fresh food, and reared under standard conditions. Once the adults had developed, they were subjected to testing for NV infection via Stock bottles were routinely checked to verify uninfected status. If a stock became NV+, embryos were collected on apple juice agar plates and dechorionated in 2.7% hypochlorite for 2–5 min. The dechorionated embryos were washed with 1X Drosophila Ringer’s solution . After 96 h, the NV+ D. melanogaster males were removed and replaced with 5 Nora virus negative (NV−) males and 5 NV− females of each species: D. yakuba, D. mercatorum, and D. melanogaster. Flies were collected after 5 days and stored at −80 °C. Three biological replicates with ten flies each were tested in duplicate for NV infection via ablished ,16. For ® Instant Drosophila Medium . The males were allowed to defecate on the food for 96 h to ensure adequate transfer of Nora virus to the surface of the food. After 96 h, 10 M. domestica pupa  and 10 G. mellonella larva  were added to individual vials and allowed to eclose at room temperature. The mature M. domestica and G. mellonella remained on the infected food for 7 days to allow for a productive infection. Collected M. domestica and G. mellonella were stored at −80 °C. Three biological replicates with ten M. domestica or G. mellonella each were tested in duplicate for NV infection via Ten NV+ D. melanogaster males were placed into 10 vials with Formula 4-24D. melanogaster were placed into vials containing a 1:1 combination of Formula 4-24® Instant Drosophila Medium  and Instant Dry Cricket medium . The males were allowed to defecate on the food for 96 h to ensure adequate transfer of Nora virus to the surface of the food. After 96 h, 5 nymph G. sigillatus  were added to pre-prepared bug containers with sterile sand and the infected food was placed inside. The G. sigillatus remained on the infected food for 7 days. Collected G. sigillatus were stored at −80 °C. Three biological replicates with ten G. sigillatus each were tested in duplicate for NV infection via Ten NV+ T. molitor  were placed into each container. Every third day, the T. molitor were fed 30 NV+ D. melanogaster for 14 days. After 14 days, the T. molitor were collected and stored at −80 °C. Three biological replicates with ten T. molitor each were tested in duplicate for NV infection via Ten small plastic insect containers were prepared with soil and potato pieces. Five Native insects were randomly collected from counties Hamilton, Butler, Buffalo, Merrick, Hall, and Polk in Nebraska. Invertebrates were placed in sealable baggies and placed on ice. Once collected, insects were stored at −80 °C. Native insects were individually tested in duplicate for NV infection via D. melanogaster. Canton Special wild-type , Oregon-R wild-type , vestigial , and apterous  D. melanogaster stocks were purchased. Upon receipt of the stocks, they were quarantined in a separate room isolated from the other stocks maintained for research purposes that were known to be infected with Nora virus. Ten emergents from each stock vial were collected within 24 h of eclosion and tested in duplicate for Nora virus infection per Drosophila stocks were purchased from the Bloomington Drosophila Stock Center (BDSC) to investigate the prevalence of DmNV in commercially available/laboratory-reared ® per manufacturer’s instructions . Each sample was quantitated using a NanoDropTM ONE spectrophotometer (ThermoFisher Scientific) to assess RNA purity (260/280 ≈ 2.0) and concentration. Samples were analyzed for the presence of Nora virus using Nora ORF1 55–844  primers and qScript XLT 1-Step RT-PCR  according to manufacturer’s instructions. The positive controls were an RNA extraction that previously tested positive for Nora virus. Reactions using 250 ng of total RNA were set-up under the following conditions for Nora virus: 50 °C for 30 min, 94 °C for 2 min,  for 30 cycles, 68 °C for 5 min, and hold at 4 °C. Samples were analyzed on a 1.0% agarose gel in a TAE buffer solution at 50 V for 3 h. A positive reaction yielded a product at approximately 790 bp for DmNV [https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 1 June 2022) to determine sequence identity. All sequenced products were identified as DmNV glycoprotein 1 .Total RNA extraction was performed using TRIzolfor DmNV ,14,15. DORF1. A 790 bp product for ORF1 verified DmNV infection  was selected because it is a non-Drosophilidae Dipteran species. G. sigillatus (house cricket), G. mellonella (wax moth), and T. molitor  were selected because they are commercially available, easy to rear, and easily adaptable for the infection protocol. M. domestica were not able to be infected with DmNV  and/or are currently being used in the laboratory . Using RT-PCR, DmNV infection was found in all stocks purchased , G. sigillatus (Order: Orthoptera–cricket), and T. molitor (Order: Coleoptera–beetle) . The viral connections between some of these species and D. melanogaster have been shown in past studies with different viruses. For example, G. sigillatus is susceptible to Cricket Paralysis virus (CrPV) [D. melanogaster, and G. mellonella [D. melanogaster, G. mellonella [T. molitor [G. sigillatus can be infected by a related iridovirus, Cricket iridovirus (CrIV) [H. irritans  [S. exigua (Order: Lepidoptera) [A. ipsilon (Order: Lepidoptera) [A. mellifera (Order: Hymenoptera) [H. armigera (Order: Lepidoptera) [S. virginica from Order Lepidoptera and O. disjunctus from the Order Coleoptera, as well a non-Insecta class species, Diplopoda (Class: Diplopoda). This indicates that invertebrates in the Orders Lepidoptera and Coleoptera, may share similar virus receptors to Drosophila, specifically when considering DmNV. This also demonstrates the breadth of organisms that can be infected by DmNV, both in the laboratory and in nature. These data corroborate the hypothesis that DmNV may represent the first member of a widespread family of viruses [M. domestica was not able to be infected in the laboratory setting . Therefore, not being able to infect individuals of this species with DmNV is not surprising.Not only was DmNV able to infect individuals of other Drosophila species, it was also able to infect laboratory-reared insects. DmNV was able to infect –beetle) , but nots (CrPV) , which illonella . In addillonella , and T.  molitor , whereass (CrIV) . Additiouscidae) , S. exigdoptera) , A. ipsidoptera) , A. mellnoptera)  and H. adoptera) . Further viruses . Interes setting , nor was setting . Upon anORF1 of these stocks indicates that DmNV is present within all 4 stocks purchased  identified as candidate markers of DmNV infection [D. melanogaster [D. melanogaster, especially those examining gene regulation or innate immunity. The data presented suggests that DmNV has the ability to cross-infect not only individuals of other Drosophila species but other organisms, and unknown contamination of stocks from either stock centers or other laboratories. It is important to quarantine stocks upon receipt and subsequently test for infection before carrying out experiments. Fortunately, since DmNV is horizontally transferred, infected stocks can be easily cured by dechorionation of embryos and transferring these to fresh food.As noted earlier, the only observed phenotype with DmNV infection is a geotaxis defect detectable when large sample sizes are analyzed via a geotaxis assay . What isnfection , similarnogaster ,26. Thisnogaster ). All ofD. melanogaster. Lastly, this investigation offers a cautionary tale for those who perform research in the realm of virology and immunology using stocks from a stock center or donated from other laboratories, as DmNV infection appears to be endemic to many laboratory-reared stocks.In conclusion, this study demonstrates that the host range of DmNV is perhaps larger than previously expected. Namely, DmNV has the ability to infect not only Drosophila species but other invertebrates as well. Further examination of DmNV or DmNV-like viruses in these native invertebrate species needs to be performed. In addition, the receptor that gives DmNV access to host cells needs to be elucidated. This would give a better understanding as to how DmNV is able to infect other organisms besides