Rhinella arenarum larvae exposed to the organophosphorus pesticides azinphos-methyl and chlorpyrifos to evaluate the transcriptional effects on a priori selected groups of genes. This approach allowed us to evaluate the effects on hypothesis-selected pathways such as target esterases, detoxifying enzymes, polyamine metabolism and signaling, and regulatory pathways modulating them. We could then compare the responses at the transcriptional level with previously described effects at the enzymatic or metabolic levels to obtain global insight into toxicity–response mechanisms. The effects of both pesticides on the transcript levels of these pathways could be considered moderate, while chlorpyrifos-induced responses were more potent and earlier than those elicited by azinphos-methyl. Finally, we inferred a prevailing downregulation effect of pesticides on signaling pathways and transcription factor transcripts encoding products that modulate/control the polyamine and antioxidant response pathways. We also tested and selected potential housekeeping genes based on those reported for other species. These results allow us to conduct future confirmatory studies on pesticide modulation of gene expression in toad larvae.Transcriptional analysis of the network of transcription regulators and target pathways in exposed organisms may be a hard task when their genome remains unknown. The development of hundreds of qPCR assays, including primer design and normalization of the results with the appropriate housekeeping genes, seems an unreachable task. Alternatively, we took advantage of a whole transcriptome study on  Both OP pesticides have been found in superficial and groundwaters from this region with a high frequency (80% or more) and environmental concentrations reaching 0.02–0.08 mg/L of pesticide4. Usually, the reproductive season of amphibians coincides with the period of pesticide application during Springtime. This is the case for the autochthonous amphibian Rhinella arenarum, whose embryos and larvae are potentially exposed to transient but high concentrations of OP, reaching the order of 1 mg/L5, as well as for other aquatic vertebrates11. Malformations can occur at sublethal concentrations, usually accompanied by swimming alterations and other impairments in movement. Besides the main targets of OP, i.e., synaptic and motor-plate acetylcholinesterases, other targets are involved in sublethal and long-term effects caused by OP. The effects of OP on these secondary biochemical and molecular targets have been described in R. arenarum and other amphibians13. Briefly, AZM and CPF affect the antioxidant defenses, reducing glutathione content, and enzyme activities of such as catalase, glutathione reductase and glutathione peroxidase while superoxide dismutase activity is either reduced or increased depending on toxicant levels17. Organophosphorus pesticides also affect the detoxifying systems metabolizing them, inhibiting carboxylesterase activity, a known buffering system protecting the main target acetylcholinesterase, while increasing glutathione-S-transferase activity in general, as well as mixed-function oxidases activity18. Polyamine metabolism, related to embryonic development, is also differentially affected by OP depending on pesticide concentrations, exposure duration and the chemical structure per se, altering polyamine levels, their synthesis rate through the key enzyme ornithine decarboxylase, and through degrading oxidase enzymes20. Finally, several key regulatory pathways involved in amphibian development, detoxifying and oxidative stress responses are differentially affected by OP at the protein content and/or phosphorylation levels depending on exposure conditions, including Protein Kinase C, Mitogen-Activated-Protein Kinase pathway , cFos and cJun21. These biochemical and molecular targets underlie, at least in part, the morphological and behavioral effects of chemicals on amphibians, leading to their particular susceptibility to contaminants during the embryonic development and metamorphosis as one of the probable causes of their decline in the natural habitats22.The Upper Valley of Rio Negro and Neuquén (North Patagonia) is a region with an intensive fruit production. Because of the massive application of pesticides used for pest control, many aquatic vertebrates and invertebrates living in backwaters and irrigation channels are challenged. Organophosphorus (OP) insecticides have been the pest controllers most frequently and heavily applied in fruit orchards of the region, being azinphos-methyl (AZM) and chlorpyrifos (CPF) the main products successively used over the last two decades23. Molecular targets, effectors, or modulators of toxicant effects are among early-response biomarkers. However, their assessment in autochthonous, nonmodel species such as R. arenarum is difficult due to the lack of sequenced genomes that allow primer design and transcript analysis, as well as the specific antibodies necessary to support protein expression analysis12. In these cases, toxicogenomic and particularly transcriptomic analyses through bulk RNA sequencing (RNA-Seq) are powerful tools that enable screening for effects and responses to a toxicant and therefore allow further molecular studies25. RNA-Seq is a highly sensitive and accurate tool for measuring expression across the transcriptome, allowing researchers to detect changes that otherwise would go unnoticed. It is possible to quantify abundance levels or relative changes for each transcript during a specific developmental stage or under a specific treatment26. In the case of nonmodel organisms such as R. arenarum, RNA-Seq technology allows the obtention of genomic information through a relatively accessible process from an economic point of view and considering the cost-benefit relationship25.When evaluating the risks of exposure to a contaminant for any species, it is desirable to find early-response biomarkers capable of anticipating irreversible damage that may occur at later stages of developmentR. arenarum, focusing on target and detoxifying enzymes, polyamine pathway and signaling, at protein, activity and metabolite levels. However, our advances at the transcript expression level were slow and challenging until we were able to develop a transcriptome study. In the context of this RNA-Seq and transcriptomic analysis performed on R. arenarum larvae exposed to AZM and CPF25, we were able to perform hypothesis-driven data analysis processing on a priori selected mRNA transcripts. This work was done in parallel with the whole transcriptome bioinformatic (big data) analysis, whose results are to be published elsewhere. The aims of the study were a) to compare transcript expression levels in genes from pathways that have been previously recognized as impacted by OP pesticides and that have also been studied at the biochemical, metabolite or physiological level in R. arenarum to obtain a more comprehensive mechanism of toxicity and response based on the involved regulatory pathways and their crosstalk, and b) to test a set of potentially adequate housekeeping genes in R. arenarum larval stages for future quantitative PCR studies.We have been studying the effects of OP pesticides in the development of the common toad R. arenarum published by us in the database from massive RNA transcript sequencing and gene annotation . We selected a group of 14 potential housekeeping genes, considering those currently proposed for mRNA expression normalization in Xenopus laevis29 . A considerable number of annotated genes could be included in the proposed pathways, which were selected on the basis of available information about OP effects at the biochemical level in amphibians10 and other vertebrates: polyamine metabolism genes, ; antioxidant response genes ; OP- metabolizing, primary- and secondary-target enzyme genes ; and gene expression regulators, transcription factors and phosphorylation cascade effectors corresponding to the Mitogen-Activated protein kinase pathway, Transcription factor AP-1, Aryl hydrocarbon receptor (AHR) pathway and Nuclear factor erythroid 2-related factor 2 antioxidant response pathway   to verify that the sequences of the selected transcripts effectively corresponded to the annotated genes. Sequences that produced a match greater than 50% in the vertebrate databases were selected for further analysis. A summary of this sequence validation is shown in Fig. Previously validated transcripts were analyzed to determine whether the transcription levels of treatment replicates were appropriate for statistical analysis. These levels resulted from the massive RNAseq amplification and quantitation of each transcript fragment in the different treatments. Fragments with zero expression levels in any sample and/or replicates with very low and erratic values, were considered as ‘not appropriate’ levels. A summary of the transcripts accepted in each group of selected genes is shown in Fig. R. arenarum. The genes with the least and acceptable variations in group A were: ef1a0, ef1ga, ef1d, ef1b, tuba, tubb, tubb4b, actb and rl8.We carried out a transcriptional stability analysis of the selected potential housekeeping (HK) genes, comparing their variability within and between treatments. The respective TMM means, minimum and maximum expression values, standard deviations, median expressions, and percentual coefficients of variation (CV%) are presented in Table tuba1 and glyceraldehyde 3P dehydrogenase (g3pdh) showed a variability of approximately 22% in their expression levels, roughly in the exclusion limits we considered, and might be considered HK genes if a refined and specific analysis proves better results. In turn, sarcomeric actin acta4 showed a 83% variation in expression, clearly indicating that it cannot be considered a HK gene in R. arenarum larvae for OP pesticide studies. The TMM values of the selected HK genes, rated with respect to their control values, were averaged for each treatment and used to standardize the TMM values of the transcripts corresponding to the genes of groups B, C, D and E (raw and standardized data shown in Supplementary Data File The transcripts belonging to tubulin odc1), S-adenosylmethionine decarboxylase proenzyme , spermidine synthase (srm), and ornithine decarboxylase antizyme (oaz1). The amd transcript 1-a showed a significant increase of nearly 3 times the control values in larvae exposed to both OP pesticides. In turn, the ornithine decarboxylase regulator oaz2 and the antizyme inhibitors azin1 and 2 showed no significant or relevant changes . The aoc3 and 4 transcript isoforms showed variable and non-significant responses to OP pesticide exposure. Acetyl spermidine/spermine oxidase (paox) expression showed an induction trend of 40% with both OP pesticides at 6 h of exposure, while the expression of spermine acetylase transcripts  showed an inhibitory trend with CPF up to 40%. Spermine oxidase (smox) expression was barely inhibited (20%) by AZM and induced by CPF   with respect to the control. On the other hand, sode expression was significantly induced by 60% in larvae exposed to CPF at 24 h. Catalase transcription (cat) was in turn slightly inhibited by AZM exposure at 24 h and by CPF up to 25%.Superoxide dismutase transcripts displayed differential responses to OP pesticide treatments Fig. A. The exgpx1 transcription was considerably induced by AZM at 24 h (3X) and by CPF at both times (up to 2.7X); gpx3 transcription was induced to a lesser extent, but in a significant way, by CPF (1.5X), while gpx4 also showed an induction of approximately 30% by CPF exposure. In turn, the transcript levels of gpx8b showed a tendency towards decrease in larvae exposed to AZM at 24 and CPF at 6 and 24 h, up to 60% of control expression values. Two other gpx transcripts, gpx2 and 7, as well as one GSH-reductase transcript (gsr), showed decreasing but not significant trends due to exposure to OP pesticides (Supplementary Table gss) was downregulated by AZM exposure at 24 h and by CPF at both times, by approximately 25%  were detected, their levels were very low and showed erratic responses . Cytochrome cyp2c19 expression was more markedly inhibited by both OP pesticides after 6 h of treatment, reaching 50–60% inhibition compared to controls.Although some fragments corresponding to cholinesterase transcripts (gst) transcripts were among the most abundant groups, as 15 different isoforms could be identified. The expressions of GST isoforms alpha (gsta3), pi (gstp1 and 2), and theta (gstt3) were downregulated by both OP pesticides, with transcription inhibitions ranging from 20 to 50% with respect to controls  showed a decreasing trend, the effects were not significant (Table S2). On the other hand, the GST Kappa 1 transcript (gstk1) was significantly induced by CPF (45%). Also, gstm3 showed the highest transcription induction values by CPF and by AZM at 24 h (2 X–3.3 X with respect to controls). GST omega 1 transcript (gsto1) also showed a significant increase of 45% in its expression for AZM at 6 h, and for CPF at 24 h.Glutathione-S-transferase  to 70% (CPF-24 h). One mgst3 transcript (mgst3-1) showed roughly similar effects (30–60% increase), while a second transcript (mgst3-2) showed a moderate increase (20%) only for CPF at 24 h of exposure. We also identified two transcripts for mgst2, one of which showed a moderate increase after exposure to CPF , and the other showed slight decreases of approximately 20% in its expression with respect to the controls (mgst2-2).Microsomal GST transcripts were also analyzed, finding increases in the expression levels of isoforms 1, 2 and 3 (map2k) was downregulated; the map2k2 transcript was decreased to 70% of the control values by CPF at 6 h and by AZM at 24 h  was barely affected by both OP pesticides (approximately 12%), while the junk transcript (mapk8) remained unaffected. None of the detected JUN transcription factor transcripts  were significantly affected by exposure to OP pesticides (Table S2). In turn, the transcription factor c-Fos, which associates with Jun as a heterodimer partner of activator protein-1 (AP1), showed a severe reduction in its transcription levels (fos) by AZM at 6 h of exposure (to 35% of control levels) but recovered at 24 h and surpassed control by 25%; CPF caused an inverse pattern, inducing fos transcription at 6 h (40%) but inhibiting it at 24 h (30%) , linked to the antioxidant response through the antioxidant response element (ARE) pathway, showed a decrease in transcription of approximately 30% in larvae exposed to AZM for 24 h and in those exposed to CPF for 6 and 24 h compared to controls  and the aryl receptor repressor (ahrr). Two arnt transcripts were analyzed, the first showing a decrease of nearly 30% in larvae exposed to AZM at 24 h and to CPF at 6 and 24 h compared to controls. The second transcript, arnt2, showed a relevant induction in transcription of nearly 2X after 6 h of exposure to CPF. The ahrr transcript showed an increase in larvae after 24 h of exposure to AZM (80%) and CPF (40%) at 24 h with respect to controls. The ahr transcript itself showed a decreasing but not-significant pattern after exposure to CPF (Table S2). The transcript corresponding to the chaperone HSP90AB1 (hsp90ab1), that binds AhR protein in the cytosol, showed a decrease of 20% after 24 h of exposure to CPF, while the transcript aipl1, corresponding to the cochaperone aryl-hydrocarbon-interacting protein-like-1, was downregulated by AZM (approximately 50%) but increased by CPF (up to 75%). Another isoform, aip, did not show effects, as well as the transcript for the third cochaperone prostaglandin E synthase-3, ptges3 (Table S2).Within the aryl hydrocarbon receptor (AhR) pathway, some differences were found for the nuclear translocator , we could compare the performances with nonparametric analysis in 42 of the 62 a priori selected genes. The coincidences between both statistical approaches extended to 66.7% of the analyzed transcripts, and when the treated- versus control pairs were considered, the result was similar, coinciding in 65.5% of all the cases. A detailed comparison of the transcripts can be found in Supplementary Table We performed both edgeR pipeline statistical analysis on the whole transcriptome data, and nonparametric Kruskal–Wallis ANOVA and Median tests on the hypothesis-selected genes using the filtered database. The main reasons for using both approaches are that edgeR gpx1; cyp2c19; and gstm3; Table S3), while most of the selected transcripts were within the 500th and 1500th ranks or in the NS group . By ranking the selected genes for each group, we verified that the effects of both OP pesticides were predominantly towards the downregulation of transcript expression, that CPF caused noticeable higher effects on gene transcription compared to AZM, and that the effects of CPF became evident earlier, from 6 h on Fig. . The OP oup Fig. . These rR.arenarum was of very good quality and that the annotated gene sequences were adequate for primer design. The general workflow for RT-PCR and qPCR analyses, a detailed description of the methodology, and the corresponding results are presented in the accompanying Supplementary file PCR Methods in Brief.We finally developed PCR assays, with the double purpose of checking the fitness of the fragment sequences determined for the hypothesis-selected genes to design adequate primers, and verifying their response to OP pesticides in ad hoc exposures. We selected 6 genes from the polyamine pathway, 4 genes from the antioxidant and detoxifying pathways, 4 genes from signaling and transcription factors, and 2 HK genes, proceeding to design pairs of primers to develop RT-PCR and qPCR analyses. We succeeded in amplifying 13 of those genes by RT-PCR and could further sequence their products and verify the identities of 8 of them by alignment with the original sequences  for most of the polyamine pathway genes and the R. arenarum larvae exposed to two OP pesticides, with two purposes in mind: (1) to test HK genes currently used in model species and (2) to compare the effects of AZM and CPF on transcript levels of pathways previously described as affected at biochemical or protein expression levels, including an analysis on signaling pathways involved in response regulation. These goals in a native, nonmodel species such as the amphibian R. arenarum are a very good example of what transcriptomics analysis can do, solve, or imply for the advance in molecular toxicology when other tools are not readily available31. Considering the results obtained, it is evident that the transcriptomic expression data allow for a screening analysis into selected pathways to decide for further qPCR studies if necessary. Furthermore, we can confirm that transcriptomic information is an essential tool when developing molecular biology assays in nonmodel organisms. Another interesting conclusion involves the high advantage of transcriptomic data on OP pesticides effects as a tool for massive search of potential biomarkers (“biomarkomics”) in environmental risk analyses, which let us propose a set of “best candidates” within the selected pathways in this case.We succeeded in applying a hypothesis-driven transcript analysis from a transcriptome database of Xenopus laevis. Another approach taking advance of the whole transcriptome data would be to select the most stable genes in terms of constant expression, i.e., those showing the least fold-change values. Even if it were a good choice for our particular conditions  we cannot guarantee that HK genes selected in this way could be used in a generalized way for other situations, even for other OP pesticides. In a comprehensive study conducted with X. laevis, rl8 and g3pdh were stable as reference genes during the first embryonic stages, odc1 was stable in the initial and final stages, and h4 was adequate throughout embryonic development27. In another study in X. laevis, e-ef1a1 and sub1 were identified as the genes whose expression remained more stable, which would allow their use as reference genes28. From a list of 9 genes fulfilling the requirements as HK genes in R. arenarum, we successfully tried actb and rl8 in qPCR assays, suggesting that these are good candidates for future qPCR analyses in different environmental studies involving this native species. Thus, we confirm the potential of RNA-Seq data to identify and/or corroborate suitable HK genes, as suggested by other authors34.Our approach to HK genes selection was to look up on previously tested and recommended candidates, in particular on the amphibian model R. arenarum larvae exposed to sublethal concentrations of AZM and CPF, was that most of the genes from the metabolic and regulatory pathways selected as recognized targets of OP pesticides did not show, in general, remarkable changes in their expression  suggest that Nrf2 protein or activity could also be downregulated.Oxidative stress has been reported in different organisms exposed to OP pesticides. In particular, the effects of AZM and CPF on oxidative stress and antioxidant responses at the metabolite and enzymatic levels have been studied in 43 refer to the alterations in the transcription levels of detoxifying and oxidative stress-related enzymes in the GST and CYP groups in a transcriptomic analysis performed on Rana chensinensis exposed to trichlorfon. This OP pesticide upregulated cyp2c transcripts but downregulated cyp3a and gstk1. We report an increase in gstk1 expression because of CPF exposure in R. arenarum, as well as for other gst isoforms, such as mu and the microsomal isoforms. This upregulation in several gst transcripts is in agreement with the reported increase in GST activity using CDNB as a substrate in R. arenarum embryos and larvae after exposure to both AZM and CPF, among other OP pesticides17. The effect on GST activity was associated with an increase in GST-Pi1 protein in R. arenarum larvae exposed to arsenic23. Other genes under Nrf2 regulation are those belonging to antioxidant defenses, such as sod, glutathione peroxidases , and glutathione reductases (gsr1)44. Accordingly, we observed downregulated levels of sodc and gpx8 accompanied by nfe2 downregulation. Glutathione synthase gss and cat also appeared to be downregulated along with nfe2, while sode, gpx1, 3 and 4 were induced, mainly by CPF. In our experience, the antioxidant enzymatic activity response varies greatly in R. arenarum embryos and larvae exposed to OP pesticides, showing cycles of induction followed by inhibition attributed to catalytic site inactivation due to ROS attack35. These cycles in protein contents and enzyme activity may be in turn triggering induction cycles at the transcription levels to restore the required antioxidant protecting levels.Ma et al.cyp1a1 and cyp2a19 for both AZM and CPF exposures, probably as a negative regulation after a previous detoxification response. The genes cyp1a1/2 are among the genes regulated by the AhR pathway by the binding of activated and nuclear-translocated AhR-Arnt heterodimeric transcription factor to DRE/XRE in their regulatory sequences. Studies carried out with Sparus aurata exposed to PCBs have shown a different expression pattern between Ahrr and AhR-Arnt, in accordance with multiple mechanisms contributing to the downregulation of AhR45. Our findings coincide with these studies, as we observed induced levels of ahrr, while the levels of arnt were decreased in R. arenarum larvae exposed to both CPF and AZM. Furthermore, the decreased levels of ahr and arnt are consistent with the decreased expression found in cyp1a since the entire mechanism is downregulated. A decrease in hsp90 and aip transcripts, supposing reduced chaperone levels, would also contribute to lower AhR protein levels46. The AhR pathway is also involved in the regulation of paraoxonase-1 expression, according to results on exposure to polyphenols and specific inducing ligands47. In turn, the pon2 gene has at least a CRE regulatory sequence for AP-1 regulation linked to the oxidative response and Jnk activation and a PAPR-regulated site related to polyphenol activation and the downregulation of MEK pathway by phosphorylation49. Our results agree with downregulated MEK (map2k), AP-1 (fos) and AHR pathways causing pon2 transcript repression in R. arenarum larvae exposed mainly to CPF. Similarly, the xenobiotic/endobiotic detoxifying carboxylesterase family (CES) is transcriptionally regulated by a series of nuclear factors and pathways: AhR, constitutive androstane receptor (CAR), pregnane X receptor (PXR), and Nrf2 are involved mostly in the upregulation of some of these families50. Although we were unable to annotate putative transcripts corresponding to CAR and PXR pathways in our R. arenarum transcriptome, the downregulation trends followed by AhR and Nrf2 pathways in larvae exposed to AZM and CPF are in line with ces5a transcript downregulation. This may be a surprising response to OP pesticides, in the sense that carboxylesterases are recognized suicide “buffer” enzymes that irreversibly react with OP pesticides to protect the primary target acetylcholinesterase in the nervous system, decreasing their activities. This is, in fact, corroborated in toad embryos and larvae exposed to different OP pesticides, including AZM and CPF, in most of our reports in R. arenarum51. Thus, an induction of carboxylesterase and cholinesterase mRNA and/or protein synthesis would be expected to reestablish normal activity levels. Finally, remarking on the complexity of the responses and crosstalk between pathways, we have reported for different developmental states in R. arenarum and cell cultures exposed to CPF, other OP pesticides, or arsenic, an increase in Mek1/2 and Erk1/2 proteins, their translocation to the nucleus, and Erk phosphorylation, considering that the MAP kinase pathway regulates the Nrf2-mediated response. The enhanced translocation of Mek to the nucleus due to CPF exposure might be a stress response downregulating the pathway. Furthermore, cFos and cJun proteins are increased after oxidative stress in R. arenarum embryos and larvae52. The complex nature of the regulatory crosstalk leading to the prevailing downregulation of biochemical responses in R. arenarum larvae exposed to OP, is depicted in Fig. mek map2k, nfe2, fos, arnt and hsp90ab1 may be good candidates for ‘early-responding’ molecular biomarkers in environmental risk evaluations through conventional qPCR or biomarkomic approaches.We also report here the downregulation of R. arenarum larvae to further analyze gene expression by conventional or quantitative PCR after exposure to pesticides. Second, we were able to visualize the effects of two OP pesticides in a priori selected metabolic and signaling pathways and compare them with previous analyses of metabolite and enzyme activity. In our analysis, we infer a gradient of effects since CPF is more potent than AZM and acts earlier on gene transcription. We found mostly downregulation responses in signaling cascades and transcription factors that act upstream the polyamine metabolism pathway and antioxidant responses, which partially coincides with previously well-characterized responses at the protein and/or activity and metabolite levels. Finally, we were able to deepen the insight on the complex crosstalk of the pathways AhR, MAPK and Nrf2 in the antioxidant response, as well as over detoxifying pathways and the polyamine pathway. From the analysis, several gene transcripts emerge as good candidates for biomarker assessments.In conclusion, we acknowledge the power of a hypothesis-driven transcriptomic data analysis in selected pathways. First, we identified an appropriate battery of potential housekeeping genes in −1 AZM and 1 g L−1 CPF were prepared by dissolving the pesticide standards in acetone. The exact concentrations of AZM and CPF in the standard solution were checked by capillary gas chromatography coupled to a nitrogen-phosphorus detector (GC-NPD).High-purity certified standards of azinphos-methyl (98.3% AZM) and chlorpyrifos  were purchased from Chem Service Inc. . Standard solutions of 18 g LRhinella arenarum (Hensel 1867) is widely distributed throughout Argentina53. Its life cycle includes two fundamental stages before reaching the adult stage: the embryonic and larval stages. The physiological reproduction of the species in the Upper Valley of Rio Negro and Neuquén (North Patagonia) occurs once a year during the spring months in backwaters and irrigation channels. The reproductive season coincides with the period of greatest pesticide application to protect fruit production. Adult females and males of R. arenarum were collected in reference areas, free of pesticide application, in agreement with the corresponding collection permission 040/2020 from the Environment Secretary of Río Negro Province, Argentina. Animals used in this study were maintained and treated with regard to the alleviation of suffering according to recommendations of the Guide for the Care and Use of Laboratory Animals 54. The animals were kept in captivity outdoors for 24–48 h until their use. Female ovulation was induced by intraperitoneal injection of 2500 international units (IU) of human chorionic gonadotropin  and embryos were obtained by in vitro fertilization15. Embryos were maintained until they reached the complete operculum (CO) stage . Ten days after reaching the CO stage, the larvae were used for acute toxicity assays.The amphibian −1, 1/20 96 h-LC50; 6) and CPF (0.1 mg L−1), 1/15 96 h-LC50; 8) were selected to carry out exposures for up to 24 h in glass dishes, maintaining a ratio of 1 larva/10 mL in amphibian Ringer's solution with 0.3% acetone . These concentrations and higher ones in the order of 1 mg L−1 might be transiently found at the irrigation channels in fruit-producing orchards where this species reproduces5. The exact pesticide concentrations were checked by gas chromatography and nitrogen-phosphorus detection. Control acetone treatment was included to discard possible solvent effects. Treatments were carried out in duplicate, and R. arenarum larvae were grown in 10 different glass receptacles to perform AZM/CPF 6 h exposures, AZM/CPF 24 h exposures, and control treatments. From each receptacle, fifteen random larvae were collected and pooled at the corresponding times. Larvae were washed three times with cold Ringer’s solution, placed in 1.5 mL tubes with RNALater® (Thermo Fisher Scientific Inc.) and stored at − 20 °C until processed.The whole protocol was approved by the Faculty Committee for Care and Use of Experimental Animals . Larvae were randomly collected to perform the assays. Sublethal concentrations of AZM  2 × 100 bp readings.RNA extraction, cDNA library generation and massive parallel sequencing were carried out as described by Ceschin et al.25. Briefly, the readings obtained by massive sequencing of the R. arenarum transcriptome were aligned with Bowtie2 v2.3.556 against TSA: GHCG00000000.1 (BioProject PRJNA485066), and transcript expression quantification was performed using RSEM v1.3.058. Expression values were normalized by the TMM method using the R and edgeR packages60. The threshold values currently applied for the selection of DEGs were a fold-change of 2 or one-half, respectively for induction or downregulation, with a p-value ≤ 0.05. Once the appropriate HK transcripts were identified, the TMM values were standardized both by the average of the selected reference genes and by the respective control values. Finally, a nonparametric analysis was performed by the median and Kruskal-Wallis tests to assess significant differences or tendencies using the exact p-values. Raw data, standardization steps and statistical analyses are available in Supplementary Data File A detailed description of the de novo transcriptome assembly, annotation and gene prediction was previously provided by Ceschin et al.R. arenarum larvae exposed to CPF,following the same procedures described above for the treatments and extraction of RNA. The different steps for PCR development are detailed in the “Supplementary file PCR data methods in brief”.The validation of transcript expression results was performed by qPCR analysis on https://arriveguidelines.org).This study is reported in accordance with ARRIVE guidelines (Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


TM) is an ionic assembly of carboxymethyl starch and calcium. The microstructure and chemical properties of the hemostatic powder were analyzed. The hemostatic performance , thromboelastography (TEG), rheology, adhesion force, and C3a complement activation of the OOZFIXTM were evaluated and compared with those of the carboxymethyl starch-based commercial hemostatic powder (AristaTM AH). The in vivo rat hepatic hemorrhage model for hemostasis time and bioabsorption of the OOZFIXTM showed quick biodegradation (<3 weeks) and a significantly improved hemostasis rate (78 ± 17 s) compared to that of AristaTM AH (182 ± 11) because of the reduced desorption. The bioabsorbable hemostatic powder OOZFIXTM is expected to be a promising hemostatic agent for precise medical surgical treatments.In contrast to hemostatic fabrics, foams, and gels, hemostatic spray powders may be conveniently applied on narrow and complex bleeding sites. However, powdered hemostatic agents are easily desorbed from the bleeding surface because of blood flow, which seriously decreases their hemostatic function. In this study, the hemostatic performance of a bioabsorbable powder with decreased desorption was investigated. The proposed hemostatic powder (OOZFIX In recent years, the frequency of surgical operations has considerably increased owing to developments in medical technology and operation convenience . Bleedin®, Ethicon, Somerville, NJ, USA) that promotes hemostasis may be used in packings, gauzes, or compressed sponges; however, it can cause pressure and pain during packing. Therefore, absorbent packings have been developed to compensate for this disadvantage. Representative examples include gelatin sponge , bioabsorbable polyurethane foam , gelatin/thrombin mixture , hyaluronic acid sponge , carboxymethylcellulose mesh , and absorbable collagen sponge  [There are multiple methods to induce hemostasis: mechanical, chemical, cooling, and heating. The mechanical methods include direct pressure with gauze, sutures, clamps, clips, and other devices. Chemical methods include astringents, blood coagulants, and local hemostatic agents. The cooling and heating methods include ice and electric heat-generating devices. Recently, hemostatic agents based on positively charged polymer materials (blood components have a negative charge) have been developed ,12,13,14RI, USA) ,11. HoweIn this study, an effective hemostatic agent addressing the above problems is proposed. A powder-type highly absorbent adhesive hemostatic agent containing blood coagulation factors  and with carboxymethyl starch was designed. Powder-type hemostatic agents are easy to apply even in narrow and complicated areas and generally, do not cause intense foreign body sensations ,16. In aHere, we demonstrated blood plasma absorption and blood coagulations of bioabsorbable hemostatic powder composed of carboxymethyl starch and calcium ions. The developed hemostatic powder is highly promising for use in clinical applications and surgery.2, Daejung, Siheung, Korea) in ethanol  for 2 h. The obtained solution containing precipitate was filtered using a filter paper to eliminate unreacted calcium chloride, which was then rinsed with ethanol three times. Subsequently, the filtered solution was dried in an oven (40 °C for 24 h) and pulverized by cryogenic grinding. The ground hemostatic particles were then sieved using a sieve tray  to obtain a homogeneous powder. The powdered sample was sterilized under UV light (254 nm) and kept in a sealed pack for further use.Carboxymethyl starch  was dissolved and stirred in a solution of calcium chloride  of the polygonal powder particles was determined as the equivalent area diameter  using image analysis software  and a particle size analyzer . The powder particle size was measured in triplicate for each specimen.The morphology of the hemostatic powder was investigated using scanning electron microscopy  with an acceleration voltage range of 11–15 kV after sputter-coating with platinum. The average diameter -Fourier transform infrared (FTIR) spectroscopy  with a germanium ATR crystal. All spectra were recorded in absorption mode with a 4.0 cm2 (0.0125 M) was dissolved in the blood prior to the experiment to avoid any anticoagulant effects. In brief, sample powder (0.1 g) was placed in a watch glass, and then the blood was dropped onto it until saturation. At the moment of saturation, the blood volume and rate (elapsed time) were recorded. The blood absorption was calculated using the following equation:Quantitative analysis of the blood absorption amount, blood absorption rate, and coagulation ability is essential for evaluating the hemostatic performance. The absorption and coagulation characteristics of the blood treated with the hemostatic powder were analyzed using whole blood . CaClBlood coagulation time was measured using the Lee–White method ,21. A gl2 (40 μL) were placed in a TEG cup and analyzed using a plastic TEG instrument (39 °C). The reaction time , clotting time , angle , and maximum amplitude  were recorded. Whole blood (5.4 mL) was collected from other 10 healthy blood donors in 2 tubes (2.7 mL per tube) containing citrate (3.2%); an additional 2 mL of whole blood was collected in a tube containing EDTA-k2. Using these samples, conventional coagulation tests, TEG assays (with 20 mg of each powder), and blood cell counts were performed.The blood-clotting activity of human blood exposed to the hemostatic powder was analyzed using TEG assays. The TEG machine  was calibrated before use according to quality control standards. TEG assays were performed within 2 h of sample collection, in accordance with the manufacturer’s instructions. To study the effect of powder dosage on coagulation function, 5 mL of whole blood were collected in a tube containing citrate (3.2%). The specimen (0.01 g), whole blood , and CaClG’), loss modulus (G″), and delta (δ) of the hemostatic agents were determined using a rheometer . The specimens were analyzed in a gel state obtained by adding 7.5% water to the powder.The storage modulus  of the rheometer . The powder (0.2 g) was placed on the bottom plate of the device, mixed with 1 mL of purified water, and allowed to swell. The swollen sample was applied with an upward force to the pig skin attached to the top of the rheometer.2) for 24 h. Test materials eluate (test group) in L-929 cells were high-density polyethylene (HDPE) film  as a negative control material (negative control group), 0.25% zinc dibuthyldithiocarbamate (ZDBC) polyurethane film  as a positive control material (positive control group), blank test solution (solvent control group), and OOZFIXTM powder. Samples were incubated in an incubator for 48 h.Elution test of bioabsorbable hemostatic powder was performed using L929 fibroblasts (connective mouse tissue) to investigate cytotoxicity . The test substance and control substance were eluted with minimum essential medium (MEM) culture solution containing 10% fetal bovine serum (FBS), 1% penicillin streptomycin in an incubator  was calculated as the following equation.If the viability decreased to less than 70% (<70%) of the blank group, it was judged to be potentially cytotoxic.An enzymatic immunoassay for the quantification of C3a fragments present in the human blood after the interaction with hemostatic powders was performed using the C3a PlusEIA kit from MicroVue  and immunosorbent assay . The powder samples were incubated at 37 °C for 1 h and subsequently processed according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader . The concentration of C3a was expressed in ng/mL and as a percentage of the activation in the control, which was incubated and treated using the same procedure. Zymosan  was used as the positive control. Measurements were performed in duplicate.Outbred Sprague–Dawley rats  were used as experimental models for evaluating in vivo hemostasis and biodegradation. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the Kyungpook National University . The experimental procedures were approved by the Animal Care Committee and performed as follows. The rats were anesthetized using a 4:1 volume ratio of Zoletil  to Rompun . Following stabilization of the rats, their livers beside the hilus were opened, and bleeding was induced using punch biopsy (6 mm) with a cross (+) shape (1 × 1 cm). The bleeding site was covered with cotton gauze, and after the removal of the gauze, the powder samples (0.1 g) were applied. Thereafter, a 50-gram weight block was placed onto the site for 60 s. The site was observed every 15 s and rinsed with a saline solution.TM powder. The powder (0.1 g) was injected intradermally into the backs of the rats (n = 16). The biodegradability of the powder was evaluated at weeks 1, 2, 3, and 4.The absorbable hemostatic powder does not require the removal of the blood-clot complex material. We performed in vivo experiments to evaluate the biodegradability of OOZFIXt-tests and one-way ANOVA with a Tukey’s post hoc test. Statistical significance was set to p < 0.05.All data are presented as mean ± standard deviation. Statistical analyses were performed using KyPlot software version 6.0 . Significance levels were calculated using parametric Student’s TM construct was prepared via ionic assembly of carboxymethyl starch with CaCl2. The dried construct was cryogenically ground and sieved to obtain a homogeneous powder. As shown in TM and AristaTM AH powders are similar, with the mean diameter ranging from 30 to 150 μm. The OOZFIXTM powder (mean diameter: 64.4 μm) consists of spheroidal particles with a relatively smooth surface. The AristaTM AH (mean diameter: 65.5 μm) powder particles are spherical but have a wrinkled surface. Singh reported that irregularities in non-spherical particles of hemostatic powders contained more voids for blood absorption and clot formation [TM and AristaTM AH powder can absorb blood plasma in a similar time.The particle size and surface morphology are the key parameters for assessing the blood absorption and clotting behavior of hemostatic powders with the same base. This is because the specific surface area of the particles and the volume of voids allowing quick blood absorption are proportional to the particle dimensions and surface structure; thus, a high specific area leads to rapid hemostasis. The OOZFIXormation ,23. AlthTM and AristaTM AH powders is carboxymethyl starch. The characteristic spectra of carboxymethyl starch show broad peaks at 3430 and 2930 cm−1, corresponding to the stretching vibrations of –OH and –CH2, respectively. The peaks at 1650 and 1365 cm−1 were assigned to the –OH bending vibration. The peaks at 1000–1200 cm−1 were ascribed to the stretching of the –C–O–C– linkage in the polysaccharide backbone chain. The ATR-FTIR spectrum of the OOZFIXTM powder shows peaks at 1612 and 1425 cm−1, which are not observed in the spectrum of the AristaTM AH powder. We attributed these peaks in OOZFIXTM to the –COO− asymmetric and symmetric stretching vibrations, respectively, caused by the ionic assembly with Ca2+ [The functional groups of the hemostatic agent were confirmed using ATR-FTIR analysis . The basTM and AristaTM AH powders exhibit similar blood absorption amounts and both show a Grade 1 blood absorption rate (less than 5 s). The amount and rate of blood absorption are proportional to the source materials and specific surface area [During in vitro evaluation, the amount and rate of blood absorption until maximum absorption are measured by contacting the blood with a hemostatic powder. As shown in ace area ,4,5. As TM group is significantly lower than that in the AristaTM AH group. Even though the blood absorption amounts of both hemostatic powders are similar, the OOZFIXTM group shows faster blood coagulation than the AristaTM AH group. After blood absorption, OOZFIXTM maintained its network structure because of ionic assembly with Ca2+ that promoted blood coagulation. Therefore, we concluded that Ca2+ on the carboxymethyl starch network structure facilitated hydrophilicity and blood coagulation with gelation at later steps of hemostasis.Shorter coagulation time indicates the effective activation of blood coagulation factors and stronger adhesion of platelets. As shown in R, K, α, and MA) of the OOZFIXTM and AristaTM AH powders are summarized in R and K values for the OOZFIXTM group  are lower than those for the AristaTM AH group . Clotting parameters α and MA for the OOZFIXTM group  are much higher than those for the AristaTM AH group . The TEG data support the results of the blood coagulation time test  in the OOZFIXTM group is higher than that in the AristaTM AH group.The TEG parameters  of 3000 and 1000 Pa, and loss moduli  of 500 and 100 Pa, respectively. The viscoelastic gel properties on plateau region and maximum delta values  of the OOZFIXTM gels maintain higher values than those of the AristaTM AH gels. This means that the OOZFIXTM gels are expected to maintain on the wound skin despite increasing strain after blood absorption and coagulation.Viscoelasticity of hemostatic powders reflects the physical strength of the gelated powder on wound skin site after blood absorption and hemostasis. As shown in TM group is −0.45 N, which is twice that of the AristaTM AH group. The OOZFIXTM powder is a carboxymethyl starch-calcium ion assembly [TM gels.Bioabsorbable hemostatic powders have been widely adopted for the convenience of their use in narrow and complicated sites, particularly during laparoscopy. However, their efficacy is limited because of partial desorption at the bleeding site, which is not observed with other types  of hemostatic agents ,17. A tiassembly , and itsTM showed cell viability of 96.64% and 94.62%, respectively. In addition, qualitative analysis was performed through microscopic observation of a cell monolayer. Compared with the positive control, the negative control and OOZFIXTM were hardly observed to separate the extracellular matrix and inhibit the growth of cells, and showed grades 0 and 1, respectively. Although foreign body reactions of natural carbohydrates are rare, investigating cytotoxicity of a new powder product made of polysaccharides (OOZFIXTM) is important. In both quantitative and qualitative results, the OOZFIXTM powder-type hemostatic agent was judged to be safe to apply as a medical material.Quantitative cytotoxicity of powder-type hemostatic agents was evaluated by counting live cells cultured in the eluate of the sample . CompareTM and AristaTM AH powders reflects the inflammation reaction. As shown in TM AH powder exhibits significant activation.Complement system activation is a major parameter for evaluating the inflammatory reaction to hemostatic agents. The complement system is critical for inducing the immunogenic response to bacteria, viruses, and other foreign bodies. Activation of this system can proceed through three possible pathways, which all involve multistep protein cleavage, but differ by the nature of the triggering element. These three enzymatic cascades have a common enzyme, C3 convertase, which cleaves the C3 protein in anaphylatoxin C3a. The level of C3a detected in the blood incubated with the OOZFIXTM and AristaTM AH powders are 78 ± 17 and 182 ± 11 s, respectively. The OOZFIXTM group showed a significant reduction in hemostasis time. Several hemostatic powders contain both blood absorption materials and blood-clotting reagents, such as thrombin and CaCl2, to facilitate clotting [TM powder was attributed to the synergistic effects of carboxymethyl starch and CaCl2.Hemostatic evaluation through animal model experiments is the most important method for analyzing the performance of hemostatic agents. As shown in clotting ,15. TherTM hemostatic powder was designed as a biodegradable agent for hemostasis without removal [TM powder was investigated in an in vivo environment  by observing the morphology after a set period. As shown in TM powder remained after 1 week, while most of the powder was desorbed with partial debris after 2 weeks. No powder traces were observed in the intradermal areas after 3 and 4 weeks. The biodegradation rates of hemostatic agents vary depending on the base material and type . The OOZFIXTM powder is based on carboxymethyl starch, which is a plant-based bioabsorbable material. Simultaneously, because of the large specific surface area, the hemostatic powder is quickly biodegraded after inducing hemostasis, thus relieving the uncomfortable feelings after laparoscopic surgery in narrow and complicated areas.OOZFIX removal . Thus, tTM) composed of carboxymethyl starch and calcium ions was fabricated via ionic assembly, drying, cryogrinding, and sieving. The OOZFIXTM powder consisted of spheroidal particles. The in vitro blood absorption amount and rate in the OOZFIXTM group were similar to those in the AristaTM AH group. In contrast, the blood coagulation rate and clot strength in the OOZFIXTM group were significantly better than in the AristaTM AH group. The OOZFIXTM group showed lower levels of C3a complement activity than the AristaTM AH group. In vivo rat hepatic hemorrhage model experiment showed remarkable hemostatic ability in the OOZFIXTM group owing to reduced desorption. The biocompatibility and hemostatic properties of the carboxymethyl starch–calcium powder (OOZFIXTM) developed in this study were superior to those of commercialized products. Moreover, incorporating active blood coagulants, such as thrombin, in powder-type hemostatic agents is a challengeable agenda for future works. In order to expand the application area of powder-type hemostatic agents, functional hemostatic properties are required based on this study.A hemostatic powder (OOZFIX


China has been experiencing severe ozone pollution problems in recent years. While a number of studies have focused on the ozone-pollution-prone regions such as the North China Plain, Yangtze River Delta, and Pearl River Delta regions, few studies have investigated the mechanisms modulating the interannual variability of ozone concentrations in Shandong Province, where a large population is located and is often subject to ozone pollution. By utilizing both the reanalysis dataset and regional numerical model (WRF-CMAQ), we delve into the potential governing mechanisms of ozone pollution in Shandong Province—especially over the major port city of Qingdao—during summer 2014–2019. During this period, ozone pollution in Qingdao exceeded the tier II standard of the Chinese National Ambient Air Quality (GB 3095-2012) for 75 days. From the perspective of meteorology, the high-pressure ridge over Baikal Lake and to its northeast, which leads to a relatively low humidity and sufficient sunlight, is the most critical weather system inducing high-ozone events in Qingdao. In terms of emissions, biogenic emissions contribute to ozone enhancement close to 10 ppb in the west and north of Shandong Province. Numerical experiments show that the local impact of biogenic emissions on ozone production in Shandong Province is relatively small, whereas biogenic emissions on the southern flank of Shandong Province enhance ozone production and further transport northeastward, resulting in an increase in ozone concentrations over Shandong Province. For the port city of Qingdao, ship emissions increase ozone concentrations when sea breezes (easterlies) prevail over Qingdao, with the 95th percentile reaching 8.7 ppb. The findings in this study have important implications for future ozone pollution in Shandong Province, as well as the northern and coastal areas in China. Although the Clean Air Action Plan of 2013 has led to reductions in the majority of air pollutant emissions [trations , the tretrations . Surfacetrations ,8 and pltrations ,10; thusX ≡ NO + NO2) and volatile organic carbons (VOCs) are the primary precursors of ozone [3–NOx–VOCs relationship can be understood through Empirical Kinetic Modeling Approach (EKMA) diagrams that delineate the different governance regimes of either VOC or NOx limitation [3–NOx–VOC EKMA nonlinear relationship [Surface ozone is produced mainly through complex photochemical reactions. Nitrogen oxides  and severe ozone pollution, selected to reveal the mechanisms of large-scale systems influencing the high-ozone events and the effects of emissions on ozone concentrations—especially ship emissions. Qingdao, situated in the southeast of Shandong Peninsula, and partly bordered by the Yellow Sea, is a major coastal port city in China with a population of approximately 10 million. We first describe model configurations and datasets in http://www.pm25.in, last access: 2 February 2022), with the majority of sites located in urban areas, and were interpolated to 0.5° × 0.5° grids to ease the analysis. The ERA5 reanalysis dataset [https://cds.climate.copernicus.eu/#!/home; accessed on 2 February 2022) was used to investigate the meteorological influences on the ozone concentrations, such as two-meter air temperature, surface shortwave radiation, and relative humidity.Observations of maximum daily 8 h ozone concentration (MDA8) were downloaded from the China National Environmental Monitoring Centre , with horizontal resolutions of 36 km × 36 km and 34 vertical layers. The model domain covered the entirety of China , with thhttp://www.meicmodel.org; accessed on 2 February 2022) [This study uses the Community Multiscale Air Quality  model for atmospheric chemistry simulation. Aerosol Module Version 7 (AERO7) and Carbon Bond Version 6 (CB6)  represenry 2022) . Ship emry 2022) ,47. The ry 2022)  was usedry 2022) . We alsoThe ozone distribution in Shandong Province  in the summer of 2019, compared with only a total of 45 days during the summers of 2014–2018. The more severe ozone pollution in Qingdao in June and July 2019 than in previous years is consistent with that in the North China Plain [To elucidate the temporal characteristics of ozone pollution in Qingdao, the monthly variation of MDA8 ozone concentrations in Qingdao during summer 2014–2019 is presented in na Plain ,50.In this section, the meteorological variables from the ERA5 dataset are averaged over Shandong Province and Qingdao. Moreover, regional transport is also an essential factor affecting ozone concentrations. Although the wind speed at 10 m shows no significant relationship with MDA8 ozone concentrations in Shandong Province, zonal wind speed at 10 m is closely associated with MDA8 ozone concentrations in Shandong Province, especially in Qingdao, with a correlation coefficient of 0.42  in coastal areas . TherefoTo delve into the large-scale weather systems influencing ozone pollution in summer, a simple method was designed to identify high-ozone events objectively. A high-ozone event is detected when ozone concentrations in an area exceed 82 ppb  for at least four consecutive days. During the summer of 2014–2019, five high-ozone events were identified in Qingdao, occurring at 7 June–10 June 2017, 21 June–24 June 2018, 1 June–4 June 2019, 12 July–16 July 2019, and 19 August–22 August 2019. These all took place in the latter three years (2017–2019), with three during the summer of 2019, further highlighting the trend of increasing ozone in recent years.2, respectively, compared with the summer mean during 2014–2019, conducive to the occurrence of high-ozone events.Geopotential height at 500 hPa and ozone anomalies compared with the summer mean of 2014–2019 were composited for five high-ozone events in Qingdao to delineate the atmospheric circulations . A conspThe observed data were first used to evaluate the meteorological factors in WRF-CMAQ during summer 2014–2019, e.g., hourly air temperature at 2 m, wind direction at 10 m, and wind speed at 10 m . The perThe model evaluation was also conducted between observed and simulated MDA8 ozone concentrations in Shandong Province each year during summer 2014–2019 . The meaConsidering that isoprene is generally the main biogenic VOC contributing to ozone production ,60, we pTo unveil the effects of isoprene emissions—either in or outside Shandong Province—on ozone pollution in Shandong Province, the ozone episode (18–28 July 2017) with the largest contribution from isoprene emissions during 2014–2019—i.e., more than 10 ppb averaged in Shandong Province continuously for 11 days a—was selThe contribution of isoprene emissions in Shandong to MDA8 ozone concentrations therein was generally less than 1 ppb during this period b, due toTo reveal the effect of regional transport on the ozone concentrations contributed by biogenic emissions in Shandong Province, the hourly ozone concentrations during 09:00–16:00 local time due to isoprene emissions outside Shandong Province, as well as an 850 hPa wind vector, were composited during 18 July to 28 July 2017, and are presented in The distribution of MDA8 ozone concentrations contributed by ship emissions see  was analOverall, biogenic and ship emissions play important roles in ozone concentration enhancement under favorable weather conditions, paving the way for ozone control and prediction. Under a changing climate—in particular of the increase in the frequency and intensity of extreme weather events, i.e., heat waves and stagnation ,64—it isThis study investigated the underlying mechanisms affecting the surface ozone concentrations in Shandong Province, including the major port city of Qingdao, during summer 2014–2019, by utilizing observation, ERA5 reanalysis datasets, and WRF-CMAQ modeling. The summer ozone concentrations in Qingdao were the highest in June, consistent with but lower than those in other inland cities in Shandong Province. The MDA8 ozone concentrations over Qingdao during summer 2014–2019 were highest in 2019, with 30 days exceeding 82 ppb, in contrast to an average of 9 days during summers in 2014–2018.Surface shortwave radiation and relative humidity were most closely related to MDA8 ozone concentrations in Shandong Province. The high-pressure ridge appearing over Lake Baikal and to its northeast resulted in relatively low humidity and sufficient sunlight in Qingdao, critical for the occurrence of local high-ozone events.The ozone concentrations contributed by biogenic emissions were highest in July during 2014–2019—close to 10 ppb in the west and north of Shandong Province. Numerical experiments indicate that the local impact of biogenic emissions on ozone in Shandong Province is relatively small. In contrast, the biogenic emissions over the southern flank of Shandong Province trigger ozone enhancement and further transport northeastward, leading to an increase in ozone concentration in Shandong Province. Additionally, in Qingdao, the ozone concentrations due to ship emissions are associated with sea breeze. The summer average ozone concentrations from ship emissions were 2.4 ppb in Qingdao during 2014–2019, with the 95th percentile of 8.7 ppb.


R made of special concrete supported with bioactive materials collected from marine algal sources were studied. Three seasons , three coral reef localities and control models (SCE) without bioactive material and (NCE) made of normal cement were compared. The structure of the microbial pattern exhibited successive shifts from the natural environment to the ABRs supported with bioactive materials (ABAM). Cyanobacteria, Proteobacteria, and Planctomycetota were shown to be the most three dominant phyla. Their relative abundances pointedly increased on ABAM and SCE models compared to the environment. Amplicon Sequence Variant (ASV) Richness and Shannon index were obviously higher on ABAM models and showed significant positive relationship with that of macrobenthos than those on the controls and the natural reef (XR). Our results offer successful establishment of healthy microbial films on the ABR surfaces enhanced the restoration of macrobenthic community in the damaged coral reefs which better understands the ecological role of the ABRs.Many Artificial Reefs (ARs) have been used worldwide for marine habitat and coral reef restoration. However, the microbial community structure that colonize the ARs and their progressive development have been seldom investigated. In this study, the successive development of the microbial communities on environmentally friendly Artificial Biological Reef structures (ABRs) Different substances have been used to construct ARs, for example, cement, plastic, metal, rock, wood, fiberglass, and clay5.Over the past decade, many underwater artificial structures have been used worldwide to resist coral decline as well as repairing of marine habitats damaged by environmental disturbances including human impact and climate change5, in which four main phases are recognized: (1) instantly following installation, exterior acclimatizing takes place by absorption of organic and inorganic particles with the formation of a prefilm (seconds to minutes); (2) primary adherence and development of prokaryotic cells as well as lower eukaryotes on the substratum or to the primary formed film (minutes to hours); (3) germination of algal spores in addition to invertebrate larvae (days to weeks); and (4) establishment of a multi-community of advanced and variable species on the substrates (weeks to years)7. More attention has been drawn towards invertebrate and macroalgal communities colonizing ARs with little focus on the microbial community pattern although they are the very first developer of biofilms on Ars. However, they got disturbed by biological and nonbiological constraints. To our knowledge, only few studies have examined the microbial community composition and succession on ARs9. Thus, exploring the development of microbial communities over time enhances revealing the ecological part of ARs10. Microbial community established on a solid substratum rise in abundance over time at the primary phase of biofilm establishment13. This is in consistence with Abed et al. who reported a substantial reduction in the bacterial α-diversity over the 28 days of installing Ar models14.Artificial reefs are concerned with restoring the typical habitat by gradual settlement of periphyton  on their surfaces then succession stages follow15. Physical, biological and chemical factors govern the formation, development and succession of microbial biofilm16. Although attachment and development of microbial community structure have been proven to be shaped according to the substrate shape, color, roughness, material6, but also seems to rely more on geographical distances17 in addition to environmental biochemical parameters18. Microbiota is influenced by the AR materials to as far as the species abundance and richness levels20. Close tracing for the emergence of such microbial communities over time in coincidence with the recovery of benthic habitat associated with artificial substrates could be the puzzle solving key and the chain link to the coral reef ecosystem rehabilitation. In Weizhou Island, the biggest and youngest volcanic island in China sea, many ARs have been applied to understand why it could not benefit from the climate change and the Sea Surface Temperature (SST) rise according to the refuge theory to resist coral decline with a high latitude environment. However, the microbial community in coral reefs remains unexplored specially in correlation with the two types of impact and to the used artificial structures. In this study, microbial communities covered artificial biological reefs (ABRs)R for sexual marine habitat restoration  of affected sites were investigated in comparison to the natural coral ecosystem of three selected sites in Weizhou Island. Moreover, correlation between microbial and macrobenthic diversity succession on the ABRs is explored over three seasons. (ABRs) are made up of special concrete structures supported with specific bioactive materials of marine algal origin known to support marine habitat larval attraction including corals . The three selected sites in Weizhou Island were chosen according to the health status of the coral reef and according to live and dead coral coverage.In aquatic networks, microbial biofilms are the initial manufacturers that provide the biochemical needs and support the settlement and recruitment of eukaryotic larvae and algal sporesAfter filtering unqualified sequences, for the natural reef samples  collected from the three sites A, B, and C, a total of 40,491, 39,986, and 30,765 clean 16S tags were obtained in March, 30,769, 36,272, and 28,316 in July and 25,227, 31,466, and 33,803 in October respectively. Archaea showed a very low percentage (< 0.5%) of combined Bacteria and Archaea. We only focus on the bacteria in the following analysis. XR samples, yielded 497, 194, and 356 unique ASVs in March, 489, 37, and 372 in July and 73, 89, and 85 in October, representing five bacterial phyla (based on SILVA classification), with a maximum of 497 unique ASVs in sample As in March , site B with Mitochondria (94.78%), and site C with Pleurocapsa_PCC-7319 (30.10%). On the other hand, treatment models started to attract more diversity in the three sites. ABAM treatment showed variation in the dominant species among different sites, for example, site A exhibited significant abundance of 6%, site The variation in community pattern among different treatment samples using a heat map of the relative abundance of the 20 most abundant Class displayed average linkage among groups over the three seasons FI. The fi. In addition, LDA was used to detect variations in taxon pattern among the four samples . Within the SCE groups, the Shannon diversity value in B34 was also remarkably higher than in A20 and C55 in March, but highest in C53 in July and highest in A20 in October (p < 0.05). For the NCE, groups, the Shannon diversity value in A59 was clearly lower than B36 and C60 in the three seasons (p < 0.05). For the XR groups, the Shannon diversity value in Bs was slightly lower than As and Cs in March and continued to decrease significantly in July (p < 0.05) while jumped to the highest with slight increase than As and Cs in October  ordination was used to detect the difference in the microbial community patterns between the ABRs with different treatments and natural reef samples (XR) from the three selected localities over the three seasons Fig. A. The MaA PCA was performed according to the ASVs level to examine the correlation among the microbial community structures on different ABRs and in XR samples collected from the three selected localities over three seasons Fig. B. PCA ilThe co-occurrence networks of microbial community based on the strong and significant correlations were created to discover synergetic relations among samples from the different treatment models and the XR group Fig. . In totaThe positive co-occurrence and negative co-exclusion of microbial genera structure were diverse among ABAM treatment and the others. Negative interactions were higher in ABAM network (47.89%) than in NCE’s (45.10%) and SCE’s (26.47%), signifying higher co-exclusion among genera in ABAM groups. The network nodes of ABAM, SCE, NCE, and XR were similar at the phylum level, whereas Proteobacteria, and Cyanobacteria were the dominant phyla, accounting together for 72.73%, 70%, 65.21%, and 68.75% of the nodes in ABAM, SCE, NCE and XR, respectively.Polysiphonia macrocarpa, Ulva prolifera and Ectocarpus siliculosus were the most dominant species detected on the ABAM models in March, followed by Hydrolithon rupestre, Lithophyllum pustulatum and Porolithon onkodes in July while Gelidium austral and Lithophyllum pustulatum were found as dominant species in October (Supplementary Table Polysiphonia macrocarpa (in XR and ABAM) and Ceratodictyon intricatum (in XR and SCE). The dominant species also changed in XR: (Lobophora variegata and Ceratodictyon intricatum), , (Peyssonnelia novae hollandiae and Heterosiphonia pulchra) were the dominant species in March, July and October, respectively. Linear least-squares regression was performed between the α-diversity of the microbial communities and that of macrobenthos to examine the correlations between them , Ochrophyta (3 taxa), and Chlorophyta (2 taxa) 18, in addition to physical and chemical parameters which are known to affect the development of microbial biofilms on the surface of hard substrates, such as; geographic site, substrate positioning, and substrate unevenness16. In this study, we report that the Shannon diversity index and the average number of ASVs of the microbial community on the ABAM treatment were the highest followed by SCE compared to the NCE and the natural XR samples. Similar results were obtained with the Shannon index of macrobenthos indicating positive correlation between alpha diversity of microbial community and macrobenthos on the ABAM, and SCE models in contrast to NCE and XR samples which reflects toxicity of the normal cement used to make the control model NCE that leads to similar result to the XR with no restoration. Although there was relative abundance shift among the four types of samples, Cyanobacteria, Proteobacteria and Planctomycetota conserved their dominance on the natural reef which was partially in consistence with earlier studies21 who reported Bacteriodota and Proteobacteria as the most dominant phyla. Proteobacteria are developer colonizers22, as they represent the most metabolically miscellaneous bacterial phylum which undertakes a wide range of nutritional variability that might be a reason for its widespread. Here, Proteobacteria had slightly increased in abundance in ABAM and SCE models compared to the XR indicating improvement of a useful phylum abundance and it exhibited significant increase over time, indicating better effect of ABAM treatment in attracting functionally required phyla due to the supplement with bioactive materials. Also, Proteobacteria showed faster increase with ABAM and SCE models in site B compared to A and C, indicating that ABAM treatment worked best in site B. This could be due to water eutrophication caused by human impact at different geographical locations of Weizhou Island23, which led to the shift in the microbial community pattern and abundance in the XR samples as well as the corresponding ABRs models.Our results deliver essential information on microbial structure during the early placement of ABRs and prior to coral larval recruitment as well as two following seasons. Moreover, the macrobenthic community composition has been studied on the installed models in comparison to the natural reef in the three sites as well as in correlation with the developed microbial films over time. The epibenthic communities showed temporal succession on the ABAM models over time. The periphyton including microbes as well as higher algae and invertebrates successively colonized the available suitable substrate surfaces after deployment Fig. . Followi24, have exhibited unique dominance on all treatments with higher abundance in the ABAM after twelve weeks of deployment, indicating that introduction of any new substrate can attract better Cyanobacteria compared to the natural reef which has damaged substrate indicating human impact rather than environmental stress, with ABAM showing better attraction of Cyanobacteria because of added supplementary materials. Later, ABAM and SCE models showed faster decrease of Cyanobacteria in site B than in site C. We speculate that the Cyanobacteria either occupied the ABRs just after their installment or undertook Cyanobacterial blooming over different seasons and it showed decline in abundance over time when its role in integrating the first biofilm has ended and started to decline for better colonization with the next beneficial phyla at that specific time such as Proteobacteria as shown in the result. This hypothesis also goes well with the fact that ABAM treatment showed the best control on Cyanobacteria to act in this manner. This might also explain the fact that site B which is known for having the most deteriorating coral reef status had the least Cyanobacteria abundance in March compared to the rest of the sites. This could be interpreted that the stressful surrounding has led to less cyanobacteria to colonize the available substrates and hence, the consequence beneficial phyla. However, it’s difficult to confirm the effect of Cyanobacteria on the successful succession of microbial taxa because of lack of species identification. Moreover, the ABAM treatment had greater influence on attracting functionally important bacterial taxa for consequent marine habitat restoration.Cyanobacteria: a chief constituent of the biofouling structure, which can yield extracellular polymeric matters to help for better integrative biofilm progressVibrio started to appear significantly in all samples with the highest abundance in the XR and NCE samples. Consequently, diversity and abundance of the rest of the phyla have been greatly affected28. In contrast, later Vibrio was effectively controlled by ABAM treatment and SCE with consistent increase in diversity and abundance of the rest of the phyla. One of the important Genus is Rugeria which exhibited consistent abundance with ABAM and SCE compared to NCE and XR samples. This species is known to offer probiotic benefits to corals, such as B12 vitamin production and protection against some coral pathogens like Vibrio coralliilyticus25. This clearly indicates that ABAM models have attracted bacterial genera known for their defense against coral pathogens, and thus explains better tolerance of microbial biofilms on this model compared to the control models and the natural reef. In October, Vibrio continued to share the dominance with Propioneginium in the XR group while diminished in the rest of the samples. In most cases, ABAM treatment showed close results to SCE control treatment and that NCE treatment was closer to the XR result indicating high toxicity of the type of cement in the NCE treatment and explains the declined health in site B and A. Thus, ABAM has stable symbioses with beneficial bacteria. In contrast, the bacterial community exhibited flexibility in the XR samples with old biofilm and harmful microbial community. For example, potentially pathogenic Vibrio  showed high relative abundance in XR and NCE groups, of which existence may cause rise in gene expression linked to virulence27. Some research reported that the relative abundance of Vibrio as heterotrophic bacteria increases in coral reefs in response to high temperature, water contamination27, or due to coral diseases31. Our study does not only highlight the importance of abundance but also considering the persistence to determine functionally essential bacteria over time linked with ABR and coral reefs. Here, two bacterial phylotypes were highly ubiquitous and abundant among all community structures (Proteobacteria was highly persistent across site/season levels while Cyanobacteria was persistent across site only). PCA illustrated that all the samples could be separated based on the phylum abundance data set R supported with bioactive material for marine habitat larval attraction, associated with severely, moderately, and slightly damaged coral reef in three coral sites in Weizhou Island with different health status.Coral reef microbiome has multi essential functions in managing life of marine habitat; including larval settlement and metamorphosis, biofilm colonization, and pathogen domination under extreme environmental conditions, thus, controlling developed acclimatization of the whole coral reef ecosystems. We here introduce artificial structure, ABAM models supported with bioactive materials which have shown enhanced microbial biofilm preior to coral larval recruitment as well as following two seasons, including control over The study area is located 21 km south of Beihai city, China, that outspreads in Beibu Gulf. Three sites of coral reef, A, B, and C  were chosen using Global Positioning System (GPS)  was used to construct a conical like artificial biological reef (ABR) as shown in Fig. 32 with scuba diving utilities using a hammer, chisel, and gloves. Samples were kept in ice then stored at – 20 °C. The average of the replicates of each line transects represents the percentage values for each site.A total of thirty-six samples were collected over three seasons in 2021; Spring (end of March) just prior to coral larval spooning in April, Summer (July), and Autumn (October), twelve from each season; three from the sediment of natural reef of the three sites (XR group) and nine from the surface of the ABRs installed in the three sites; three from ABAM, three from SCE and three from NCE model structures Table . Samples33. Monthly average salinity at Weizhou Island ranges from 31.17 to 32.66 over 20 years34. The dissolved oxygen saturation is above 90 and no anoxia was spotted through the year35. Weizhou Island is characterized of rainfall, and ambient light. The acidification of the water ranges between 8.0 and 8.23. Ocean is clear, with transparency ranging from 3.0 to 10.0. Moreover, there is cyclonic movement all across the year33.The physical and chemical parameters indicating water quality in Weizhou Island have been collected from previous resources. The average annual SST in Weizhou Island is 24.62 °C. The mean annual air temperature is 22.6 °C; the mean annual precipitation is 1380.2 mm; mean wind speed is 4.8 m/sg. The upper layer was removed to a clean tube and the purification step was carried out using DNA Clean and Concentration Kit  following the manufacturer’s protocol: The primers 515F (5′-GTGCCAGCMGCCGCGG-3′)/907R (5′-CCGTCAATTCMTTTRAGT-3′)36 targeting the V4–V5 domain of prokaryotic SSU rDNA were used. The primers TAReuk454FWD1 (50-CCAGCASCYGCGGTAATTCC-30) and TAReukREV3 (50-ACTTTCGTTCTTGATYRA-30)37 targeting the V4–V5 domain of eukaryotic SSU rDNA were also used. Polymerase chain reaction was performed following the protocol of Ammon et al.38 with minor changes. The Ex Taq PCR-kit  was used according to protocol with primer concentration of 0.2 μM. The PCR reaction involved, denaturation step for 34 cycles at 95 °C for 30 s, 56 °C for 30 s, annealing at 72 °C for 1 min 30 s and extension at 72 °C for 5 min. Amplicons were purified then libraries were performed using the Ion Plus Fragment Library Kit 48 rxns . The 16 s rDNA and 18 s rDNA libraries were sequenced on an Illumina MiSeq platform  using a paired-end (2 × 250 bp) HiSeq Reagent Kit following manufacturer’s instructions in Beijing Genomics Institute . Generated sequences were assessed and analyzed using DADA2 (Divisive Amplicon Denoising Algorithm40 software package in R. The sequences were filtered and cleaned using the DADA2 workflow40 amplicon sequence variants (ASVs) determination. The ASVs were aligned to the SILVA rRNA database41; https://www.arb-silva.de/) for 16 s sequencing, and PR2 rRNA database42 (http://ssu-rrna.org/)) for 18 s sequencing. Illumina next-generation DNA sequences were deposited in the Sequencing Read Archive (SRA) of the National Centre for Biotechnology Information (NCBI) under BioProject accession PRJNA851196 ID: 851196, Biosample accessions SAMN29390395–SAMN29390466.DNA extraction was conducted using CTAB protocol and Zymo DNA Clean & Concentrator kit  following the manufacturer’s protocol. The samples were suspended in 600 μL of DNA lysis buffer and 10 μL proteinase K  at 55 °C for 48 h. To the suspension, 82.5 μL of 10% w/v CTAB  and 82.5 μL of 5 M NaCl, were added and incubated at 55 °C for 10 min. Next, Chloroform (600 μL) was added then vortexed and centrifuged for 10 min at 11,00043, Chao144, Pielou index45, Simpson diversity46, Shannon diversity47, rarefaction curves were analyzed using R48 using the package vegan49. Venny’s on-line website (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used for conducting the venn diagrams. The β-diversity analyses of bacterial Bray–Curtis dissimilarity was performed using the vegdist function in vegan package. To detect taxonomic variability among different treatments and XR samples, a least discriminant analysis (LDA) was conducted to recognize biomarkers (threshold > 4.0)50. The co-occurrence networks were performed by calculating the Spearman’s rank correlations with the psych package based on the relative abundance of genera, with |ρ| greater than 0.6 and a p value of less than 0.05, using Gephi (v. 0.9.2)51. The linear least-squares regression mode was used to identify the correlation between α-diversity of both macrobenthos and microbial community (ASV richness and Shannon index) and to assess the persistence of the relative abundance of dominant phyla over time.The α-diversity of bacterial community composition including the ASV richness, ACEABRR was registered for Chinese patency with application no. 202111290154.5.Supplementary Tables.


PurposeVitamin D receptor (VDR) has been proposed as a possible marker for fibromyalgia syndrome (FMS). The purpose of this study is to characterize the expression pattern of BsmI polymorphism (rs1544410) in the VDR gene in women with FMS and the genotype-phenotype association.MethodsA total of 105 FMS patients and 105 controls were included in this study. VDR gene BsmI polymorphism was assessed by polymerase chain reaction (PCR) and restriction fragment length polymerase (RFLP) method.ResultsThere was no significant difference in the frequency distribution of both genotypes and alleles for VDR gene BsmI polymorphism between FMS patients and controls (p>0.05). The frequencies of BB, Bb, and bb in the VDR gene BsmI polymorphism were 19%, 43%, and 37% in patients, while in controls were 22.9%, 55.2%, and 21.9%. However, we did not find any significant association between the clinical symptoms of this disease and VDR BsmI genotypes among FMS patients (p>0.05).ConclusionsThe relationship between the VDR gene BsmI polymorphism and FMS could not be determined in this study. However, further studies with a larger sample size may be required to show a relation between the VDR gene BsmI polymorphism and FMS. Fibromyalgia syndrome (FMS) is a widespread chronic musculoskeletal pain disorder characterized by symptoms like morning stiffness, fatigue, multiple tender points, sleep disorder, headache, low pain threshold, anxiety, and depression . The incThe action of vitamin D receptor (VDR) and the enzyme-mediated activity of 1-hydroxylase in many areas of the central nervous system, especially in the hypothalamus, may contribute to the central sensitization which is responsible for the development of chronic widespread pain in FMS. It has been proposed that VDR and the 1-hydroxylase may contribute to the triggering of chronic headaches and migraines . VitaminStudy populationThe Research Ethics Committee of the Era's Lucknow Medical College and Hospital (ELMCH), Era University, Lucknow, India approved this study. All the procedures including human subjects were performed in accordance with the ethical standards of ELMCH, Era University, Lucknow, India. A total number of 105 FMS patients and 105 controls were involved in this case-control study.Patients and controls selection criteriaAll the FMS patients and controls were female and came from the same ethnic group and geographical region. Patients were participants of the ELMCH, Lucknow, who attended the outpatient department (OPD) from the department of rheumatology. The study includes only those patients with FMS who will fulfill the American College of Rheumatology criteria 2016 . PatientData and sample collectionDuring the time of enrolment, the patients and controls were provided with questionnaires to be filled out, and the questionnaire inquired about the duration of symptoms, age, body mass index (BMI), Fibromyalgia Impact Questionnaire Revised (FIQR), pain intensity is measured by visual analog scales (VAS), weight loss, jaw pain, frequent awakening, family history, fatigue, headache, stiffness, feeling of fever, lack of energy, abdominal pain, restless leg, and irritable bowel syndrome. After signing the informed consent form, blood samples were collected from the patients and controls in tubes containing ethylene diamine tetra acetic acid (EDTA) and stored at 20°C for genetic analysis.VDR BsmI genotypingThe peripheral blood was taken in a blood collection tube from the FMS patients and controls after taking written consent. The salting out method was used for the DNA isolation from the blood samples. Extracted DNA was used for the amplification of the VDR BsmI gene polymorphism by polymerase chain reaction-restriction fragment length polymerase (PCR-RFLP) method. The primer details for the VDR BsmI gene polymorphism were given in Table PCR was performed in a 25µl reaction mixture of 150-200ng genomic DNA, 25pmol of each primer, and 2X master mix  per tube using a gradient MasterCycler . The PCR protocol is as follows: initial denaturation at 94°C for five minutes, followed by 30 cycles of denaturation at 94°C for 40 seconds, annealing at 62°C for 40 seconds, extension at 72°C for 40 seconds, final extension at 72°C for five min. After amplification, PCR products (825bp) were digested with BsmI  restriction endonuclease at 65°C for one hour. Digested products were run on agarose gel (2%) containing ethidium bromide and visualized by a gel documentation system . The absence of a BsmI restriction site was indicated as a B allele, while the presence of the BsmI site was indicated as a b allele that generates two or three fragments. The single uncut fragment of 825bp bands was indicated as BB homozygous. The bb homozygous generates two fragments of 650bp and 175bp, while the Bb heterozygous generates three fragments 825bp, 650bp, and 175bp. Statistical analysisFMS patients and controls were compared using statistical analysis performed with sufficient post hoc analysis using SPSS statics 28.0 software . The chi-square (χ2) test or Fisher's exact test were used to compare the genotyping data between FMS patients and controls. Odds ratio (OR), risk ratio (RR), and 95% confidence interval (CI) were used to assess the risk factors. χ2 test was used to evaluate the Hardy-Weinberg Equilibrium (HWE) for the genotype distribution of the patients and controls. The value was considered to be significant when p<0.05.Table Table Table The assumption is that environmental and genetic multi-factors play a significant role in FMS aetiology, as shown by numerous polymorphisms. According to several studies, genetic predispositions have a significant influence on the widespread development of chronic pain in FMS. FMS can occur in individuals with a genetic vulnerability due to environmental variables and/or the pathophysiology of neuroendocrine and autonomous abnormalities ,15-16. SVitamin D is necessary for calcium absorption. Vitamin D works by regulating gene expression when it binds to VDR. Potential genetic variations occur in specific genes after vitamin D exposure, which may modify bioavailability, transitions, and distribution in the pool for lipid accumulation, retention, and vitamin D action . VitaminTo the best of our knowledge, no previous studies have examined the association of the VDR gene BsmI polymorphism in North Indian women with FMS. There was no statistically significant difference between the genotypes and alleles frequency distribution of the VDR gene BsmI polymorphism in the FMS patients and controls. Our results are in agreement with other studies. Santos et al. observed that no significant differences in the distribution of the genotypes and alleles frequency of the VDR gene BsmI polymorphism among FMS patients and controls in Brazilian women (p=0.06 and p=0.07) . In addiOther authors found no association between the VDR gene BsmI polymorphism in postmenopausal women with osteoporosis  and obesThe limitation of this study was the evaluation of only the VDR BsmI gene polymorphism in FMS. The relation between the VDR BsmI gene polymorphism and FMS could be better understood in the future by examining BsmI polymorphism in a larger sample size. Therefore, more accurate results can be obtained between the VDR BsmI gene polymorphism and FMS.Our study is the first report of VDR BsmI genetic polymorphism in North Indian women with FMS. No significant results were observed between the VDR BsmI gene polymorphism and FMS. Thus, we could say that VDR BsmI gene polymorphism is not an important contributor for FMS in the North Indian ethnic group.