The experimental results have shown that the average diameter and particle size distribution of silicone particles depend on the chemical structure and architecture of the compatibilizers. One copolymer has been identified as the best compatibilizer, allowing a lower mean diameter and particle size distribution in addition to the best mechanical properties of the final network (less brittle character). This study has consequently evidenced the possibility of creating in situ silicone capsules inside an epoxy network by adding tailored compatibilizers to epoxy/silicone formulations.The objective of this study was to prepare crosslinked epoxy networks containing liquid silicone particles in order to improve their mechanical properties and obtain less brittle materials. Different copolymers were used as compatibilizers. These copolymers vary in their chemical composition and structure. All of the copolymers contain hydrophobic (PDMS sequences) and hydrophilic groups. The effect of their chemical structure and architecture on the morphology of the dispersed phase, and on the final physico-chemical and flexural characteristics of epoxy/silicone blends, was explored. The morphology of crosslinked formulations was studied by scanning electron microscopy (SEM), and the thermal characteristics (glass transition temperature, T Epoxy resins form a class of widely used high-performance thermosetting polymers, due to their excellent mechanical properties, moisture and chemical resistances, heat and dimensional stabilities, very good electrical insulation, and very good adhesion to different substrates ,2,3. Theg). One of the first approaches has been to add reactive liquid elastomers such as carboxyl-terminated butadiene-acrylonitrile copolymer (CTBN rubber) to the uncured epoxy formulations and to cure these reactive systems [Several strategies have been developed to consistently increase the epoxy toughness without a significant lowering of the overall mechanical performance or the glass transition temperature  and a shell able to ensure strong bonding to the resin matrix. Since this strategy generates similar biphasic final morphologies as in the case of reactive liquid rubbers, it was assumed that the mechanisms described for rubber-toughening are also valid here ,17,18,19A very recent strategy to epoxy-toughening suggests using self-organizing thermoplastic copolymers that are miscible with the uncured epoxy matrix, and conduct phase-separation during epoxy curing, generating different nanostructured elastomeric domains inside the 3D epoxy network. The major advantage of this approach consists of the much lower viscosity of the epoxy formulation, making it easier to process ,21,22,232 [Other recent exploratory strategies for epoxy-toughening propose using nanoparticles such as silica ,25,26, g2 ,29, and 2 ,31,32,332 ,34,35,362 ,37,38 of2 ,24,39.In addition to the interest in improving epoxy toughness and impact resistance, some certain new challenges have appeared in recent years, based on the great potential of epoxy resins to produce innovative advanced functional materials exhibiting self-healing, self-cleaning, shape memory, or other functional properties such as transparent-to-opaque transitions ,42,43,44In the present work, we investigated the possibility of preparing 3D epoxy networks containing liquid silicone particles and tailoring their biphasic morphology by choosing an appropriate compatibilizer . Using a−1. Consequently, the exact EEW and 1H NMR data [The equivalent epoxy weight (EEW) of a DGEBA resin represents very important data for developing formulations with a stoichiometric epoxy/hardener ratio. For the present work, the DGEBA supplier’s data only indicated a batch with NMR data ,48, know1H NMR spectrum, as follows [pR is the ratio between the proton signals associated with the aromatic rings of DGEBA repetitive units, (a+bI) and the proton signals corresponding to the terminal epoxy units (g+h+eI), and tR corresponds to the same ratio in the case of the DGEBA oligomer (when n = 0).On the other hand,  follows ,48:DP¯n=1H NMR spectrum of DGEBA used in this study.−1, well below the 700 g·mol−1 indicated by the supplier. Consequently, for all formulations, the experimental EEW value was taken for the DGEBA/IPD ratio.The results show that the DGEBA resin used in this study has a The results of the miscibility tests are presented in The last column from Preliminary DSC measurements have shown that the neat DGEBA/IPD epoxy system reached full crosslinking after one hour of curing at 150 °C see . Based oThe main chemical reactions for DGEBA + IPD resins are presented in −1) offered additional heat for epoxy and diamine to regain thermodynamic mobility and continue the crosslinking reactions. The post-curing stage for one hour at 150 °C was intended to fully crosslink the epoxy network.During the curing of all of the epoxy systems, the formation of the first crosslinks induced an increase in the matrix viscosity and glass temperature transition. The curing phase at a slow temperature ramp (2.5 °C·min−1. The onset of cure (Tonset), the exothermic peak temperature (Tpeak), and the heat of cure ΔHcure of the epoxy formulations were determined from the DSC curves.The curing behavior of epoxy reactive systems (with and without silicone) was observed by non-isothermal DSC, from room temperature to 210 °C, at 10 °C·minonset or the temperature of the maximum curing rate, Tpeak. For instance, The presence of liquid inclusions of 5 to 10 wt% of chemically inert silicone did not change the large exothermic profile due to the curing of the epoxy matrix see . When cocure with the addition of silicone that corresponds to the decrease in epoxy matrix concentration. Similar results have been obtained for the formulations containing the other compatibilizers.The only difference concerns the decrease in ΔHThe morphological analysis of cured systems was performed by SEM. Cured neat epoxy resins presented a homogeneous transparent aspect. At the end of the curing procedure, all epoxy/silicone formulations containing PDMS and the compatibilizers presented biphasic morphologies with well-dispersed spherical particles inside a continuous rigid epoxy network. The SEM images show that all of the compatibilizers assured a good epoxy/silicone compatibilization, especially for 5 wt% silicone see . All invAt equal concentrations , the most efficient compatibilizers for epoxy/silicone resins based on DGEBA are coPo1 and 3, which gave fine morphologies with narrow particle size distributions. Both copolymers present linear hydrophilic polyether chains which are OH terminated.g values obtained by DSC and DMA, which are not unusual since these two methods are not describing the same physical phenomena from the sample, no clear dependence could be detected depending on the particle sizes of the silicone inclusions or the effectiveness of the different compatibilizers.crosslink) of epoxy/silicone blends. According to the theory of rubber elasticity, the storage modulus G’ from the rubbery plateau  is directly proportional to the crosslink density of cured resins but can also be influenced by the presence of PDMS inclusions [However, DMA data allowed further evaluation of the effect of silicone and compatibilizers on the thermomechanical characteristics and crosslink density , this study suggests that the most appropriate compatibilizers for reaching this goal are coPo1 and coPo3.Since coPo1 and coPo3 exhibit the best compatibilizing capacities, their mechanical properties will be compared. An increase in the flexural strength (at break) is observed for the blend containing coPo1, for both PDMS contents, contrary to the blend containing coPo3 for which a decrease in the flexural strength can be noticed. For the other formulations, no significant trend is observed, proving that the mechanical properties depend not only on the dispersion quality but also on other parameters such as the chemical architecture  or the chemical composition . However, some of them prove to be more effective. In particular, coPo1 and coPo3 induce smaller particle sizes and a narrower distribution, for both of the PDMS concentrations studied. These two linear copolymers with polyether/silicone molar ratios close to one and OH terminal groups are consequently the most efficient as compatibilizers. The ether and hydroxyl groups allow a good compatibility with DGEBA chains, which also possess OH and ether groups, while their linear PDMS sequences allow a good compatibility with the PDMS dispersed phase.The experimental results show that all copolymers allow a stable dispersion of liquid silicone inclusions inside the epoxy network , which proves that both PDMS and copolymers macromolecules are well included in the network, avoiding any migration at the surface. This property is important for coating or bonding epoxy surfaces.The study of the flexural properties, in particular the deformation at break, indicates an improvement (less brittle character) for all of the copolymers, which was the objective of this study. Nevertheless, coPo1 makes it possible to obtain the most interesting properties, with a higher deformation at break.w = 28,000 g·mol−1) was purchased from Alfa Aesar, Illkirch, France. The different linear di-functional or multi-pendant silicone polyethers used as compatibilizers  was purchased from Huntsman Advanced Materials, Europe, Basel, Switzerland. The curing agent, isophorone diamine (IPD), was purchased from Sigma-Aldrich, Saint-Quentin-Falavier, France. Trimethylsiloxy-terminated polydimethylsiloxane  and mixed for another 10 min at 33 °C. Thermosetting formulations were poured into different silicone molds to obtain rectangular specimens for physico-chemical and mechanical characterizations. The formulations were pre-cured in an oven at 33 °C for 60 min, then heated with a temperature ramp up to 150 °C at 2.5 °C·min1H NMR analyses were performed with a Bruker 300 UltraShield spectrometer, Bruker, Marne La Vallée, France, at 300 MHz, driven by the XWIN-NMR V3.5 software. DGEBA epoxy resin was dissolved in deuterated chloroform at a concentration of 10 wt%.Miscibility tests of the different compatibilizers with either DGEBA or silicone were performed at room temperature and 33 °C (in a heating bath), to determine the best pre-mixing procedure for optimally incorporating all the components of the epoxy formulations. Equal quantities of silicone–polyether copolymers and either DGEBA or PDMS were vigorously mixed during 10 min, and the visual aspect of the mixture was recorded for five hours.Morphological analysis of epoxy formulations was performed by scanning electron microscopy (SEM). The morphology of fractured specimens of cured resins was also investigated by SEM, by means of a JEOL JCM 6000, Jeol, Croissy-sur-Seine, France. The fractured specimens of cured resins were previously immersed in butyl acetate (a good solvent for PDMS) for 24 h, at room temperature, to remove the silicone contamination from the surface and potentially from a few microns in depth. After removal from butyl acetate, the samples were dried and analyzed by SEM. The particle size distribution and average diameter value of inclusions were determined by image analysis, after verifying the homogeneity of the morphological patterns over 3 samples for each specimen.g, and to study the influence of silicone inclusions on the curing kinetics of epoxy materials. The DSC analyses were carried out with a TA Instruments’ DSC-Q200, TA Instruments, Guyancourt, France under a nitrogen atmosphere, using hermetically sealed aluminum pans.DSC was used to investigate the curing of epoxy formulations, to determine the T−1. In the case of cured samples, the first heating scan was used to verify whether the curing was complete or not, and the second to determine the Tg of the cured samples. The first heating scan applied on uncured epoxy formulations indicated the onset temperature of curing, and the exothermic peak temperature. The area under the exothermic peak due to the crosslinking reaction gave, by integration, the heat of cure, ΔHcure (J/g). The second heating scan was used as previously, to determine the Tg of in situ cured formulations.A first heating scan was applied on uncured and cured samples, from 25 to 200 °C, followed by cooling down to 25 °C, and a second heating scan from 25 to 200 °C; all scans were performed at constant rate of 10 °C·min−1. The viscoelastic moduli, storage (G′) and loss modulus (G″), and mechanical loss tangent (tan δ) were obtained for a frequency of 1 Hz. The glass-transition temperature values were assumed as the temperatures at the maximum tan δ peak.Dynamic–mechanical analyses were performed on a modular Anton Parr MCR 302 rheometer, Anton Paar, Les Ulis, France. Cured rectangular specimens (40 mm × 12 mm × 2.3 mm) were mounted in single cantilever bending clamps and submitted to a temperature scan from 25 to 180 °C, with a heating rate of 2 °C·minThe flexural properties of cured epoxy formulations were measured using a Testometric M500-30AT universal testing machine, Testometric, Rochdale, UK. Three-point bending tests were performed on rectangular specimens (142 mm × 12 mm × 2.3 mm). The measurements were performed with a crosshead speed of 0.5 mm/min, at room temperature. Seven specimens were tested for each epoxy formulation.Contact angle measurements were performed to evaluate the water wettability of cured epoxy resins with and without silicone in the formulation. The experiments were carried out with a Krüss DSA100 Drop Shape Analyzer, Krüss, Villebon sur Yvette, France using the sessile drop method. Water droplets of 0.2 µL were deposited on epoxy surfaces (both upper and lower surfaces of rectangular specimens), with a 0.53 mm diameter syringe, at a 500 µL/min flow rate. At least 10 measurements were conducted for each surface.This study has evidenced the possibility of creating in situ liquid silicone capsules inside an epoxy network and to control their size and distribution by adding compatibilizers to epoxy/silicone formulations.The experimental results have shown that the average diameter and particle size distribution of silicone capsules depend on the chemical structure and architecture of the silicone-compatibilizers. The copolymers coPo1 and coPo3 were the most efficient as compatibilizers. CoPo1 appears to be the best choice to decrease the brittle character of epoxy networks while improving the hydrophilicity of the cured sample. The next step will be to study aging by following the evolution of properties over time and under aggressive conditions .


Purpose: To study the accuracy of non-invasive chromosomal screening (NICS) results, in normal chromosomes and chromosomal rearrangement groups and to investigate whether using trophoblast cell biopsy along with NICS, to choose embryos for transfer can improve the clinical outcomes of assisted pregnancy.Methods: We retrospectively analyzed 101 couples who underwent preimplantation genetic testing at our center from January 2019 to June 2021 and collected 492 blastocysts for trophocyte (TE) biopsy. D3-5 blastocyst culture fluid and blastocyst cavity fluid were collected for the NICS. Amongst them, 278 blastocysts (58 couples) and 214 blastocysts (43 couples) were included in the normal chromosomes and chromosomal rearrangement groups, respectively. Couples undergoing embryo transfer were divided into group A, in which both the NICS and TE biopsy results were euploid (52 embryos), and group B, in which the TE biopsy results were euploid and the NICS results were aneuploid (33 embryos).Results: In the normal karyotype group, concordance for embryo ploidy was 78.1%, sensitivity was 94.9%, specificity was 51.4%, the positive predictive value (PPV) was 75.7%, and the negative predictive value (NPV) was 86.4%. In the chromosomal rearrangement group, concordance for embryo ploidy was 73.1%, sensitivity was 93.3%, specificity was 53.3%, the PPV was 66.3%, and the NPV was 89%. In euploid TE/euploid NICS group, 52 embryos were transferred; the clinical pregnancy rate was 71.2%, miscarriage rate was 5.4%, and ongoing pregnancy rate was 67.3%. In euploid TE/aneuploid NICS group, 33 embryos were transferred; the clinic pregnancy rate was 54.5%, miscarriage rate was 5.6%, and ongoingpregnancy rate was 51.5%. The clinical pregnancy and ongoing pregnancy rates were higher in the TE and NICS euploid group.Conclusion: NICS was similarly effective in assessing both normal and abnormal populations. Identification of euploidy and aneuploidy alone may lead to the wastage of embryos due to high false positives. More suitable reporting methods for NICS and countermeasures for a high number of false positives in NICS are needed. In summary, our results suggest that combining biopsy and NICS results could improve the outcomes of assisted pregnancy. With the increase in work pressure and competition in modern society, the average childbearing age of couples is increasing; there is also a decline in fertility rates and an increase in infertility due to various reasons, such as chromosomal abnormalities of embryos which is mainly caused by age-dependent chromosome segregation errors during meiosis I . Therefoin vitro fertilization, due to its high accuracy and non-invasiveness  and spent culture medium . In 2013siveness . Previousiveness . Moreovesiveness . Moreovesiveness . The onesiveness . In addisiveness , determiFor embryo screening, both the trophoblast cell biopsy and NICS methods have their advantages and disadvantages. We wanted to understand whether combining the two methods, to select embryos for transfer, improves the clinical outcome, as there are few such reports.Therefore, in this study, we compared the consistency, sensitivity, specificity, PPV, and NPV of NICS in normal chromosome and chromosomal rearrangement groups. This is the first time that such a large sample size of different populations has been used to evaluate the performance of NICS. Furthermore, based on the results of TE biopsies and NICS, patients undergoing an embryo transfer were divided into two groups: euploid TE/euploid NICS group in which both the TE biopsy and NICS results suggested euploidy, and euploid TE/aneuploid NICS group in which the TE biopsy results suggested euploidy and the NICS results suggested aneuploidy. A series of clinical results from the two groups were compared in order to determine whether TE biopsies combined with NICS could improve clinical outcomes.We performed a retrospective analysis of 101 couples who received PGT in our center from January 2019 to June 2021; 492 blastocysts were collected for TE biopsy. Meanwhile, D3-5 SCM and BF were collected for NICS. Among them, 278 blastocysts (58 couples) were included in the normal chromosome group and 214 blastocysts (43 couples) were included in the chromosomal rearrangement group. This study was approved by the Ethics Committee of the Peking University of Shenzhen Hospital ([2018] Issue no [014]) and was performed in accordance with the principles of the Declaration of Helsinki (1964) and its later amendments. Informed consent was obtained from all the patients included in this study.2, 5% O2). After the D5-D6 balstocyst was completely formed, artificial collapse of the blastocoel was induced by applying a laser pulse (300 μs), using the ZILOS-tkTM laser system , at the junction of TE cells and the location of the juction was far from the inner cell mass. After 5 min of treatment, all the cluture media with the released blastocyst fluid were collected with a drawn glass straw and placed into a PCR tube containing 5 µL of cell lysis buffer . The shriveled blastocyst was put into the biopsy operation dish and biopsied under an inverted microscope. The fixed needle was used to hold the cell mass inside the blastocyst at 9 o’clock, and a small hole was punctured, in the trophoblast cell junction, at three points opposite to the ICM. The biopsy needle entered the blastocyst at the perforated position and absorbed 3–6 trophectoderm ectoderm cells. The biopsied cells were completely separated from the blastocyst by a laser pulse (300 μs), using the ZILOS-tkTM laser system , while being pulled. The biopsied blastocyst was removed under a stereomicroscope, and the biopsied trophoblast ectoderm cells were cleaned several times in phosphate-buffered saline. The egg stripping pipette (135 µm inner diameter) was used to move the biopsied trophoblast ectoderm cells into the PCR tube containing 5 µL of cell lysis buffer.In our study, we carefully removed and washed the cumulus-corona colliculus complex before intracytoplasmic sperm injection (ICSI). The embryos derived from ICSI were cultured to cleavage stage (D3). If there were any remaining granulosa cells, we removed the ramaining granulosa cells completely with a glass pipe. Then, each embryos was rinsed and transferred into an 20 µL droplet of blastocyst media, in the BD353001 petri dish  to equilibration overnight. On the afternoon of D4, the embryos cultured in the collective culture were repeatedly blown, washed, and placed successively into the previously prepared blastocyst culture medium. Each embryo was placed into an individual drop of fresh blastocyst culture medium, which was then cultured in a three-gas incubator  technique and library generation as described previously. The amplification products were sequenced on the Illumina HiSeq 2,500 platform  with approximately two million sequencing reads per sample. The read numbers were counted along the whole genome with a bin size of 1 Mb and normalized based on GC content and a reference dataset. A copy number gain from two to three copies results in a 50% increase in read counts, whereas a copy number loss from two copies to one copy results in a 50% decrease in read counts.The standard for mosaicism in NICS  and TE-PFirst, the NICS results were compared to the TE biopsy results in both groups. The consistency, sensitivity, specificity, PPV, and NPV were calculated. The parameters between the two groups were statistically analyzed . Thereaft-tests and Mann–Whitney tests were used for parametric and non-parametric data, respectively. The chi-square or Fisher exact tests were used for the categorical variables of each group. All analyses were done using the SPSS version 25.0 . Multiple logistic regression analyses were conducted to compare the outcomes of group A and group B after controlling for p < 0.10 and co-variables considered clinically influential, including female age, morphology , and cycle type of embryo transfer.Descriptive data for the continuous variables are presented as the mean and standard deviation, and categorical data as numbers and percentages. Student’s There were 278 blastocysts in the normal karyotype group; amongst these 9 blastocysts were contaminated with the parent source, 3 blastocysts were contaminated by exogenous sources , 21 blastocysts DNA samples from SCM and BF failed to amplify and could not be detected by NICS, and 3 blastocysts failed to amplify and could not be detected by TE biopsy. Finally, 242 blastocysts were used for consistent comparison . In the p > 0.05) in the performance characteristics between the chromosomal rearrangement and normal karyotype groups.There were 214 blastocysts in the chromosomal rearrangement group; amongst these 8 blastocysts were contaminated with the parent source, 4 blastocysts were contaminated by exogenous sources, 9 blastocysts DNA samples from SCM and BF failed to amplify and could not be detected by NICS, and 1 blastocyst failed to amplify and could not be detected by TE biopsy. Finally, 192 were used for consistent comparison . In the p > 0.05) in the performance characteristics between the chromosomal rearrangement and normal karyotype groups.We further evaluated the embryos with different NICS CNV results. According the classification method of previous research , The CNVIn all, 85 TE euploid embryos were transferred, which were further divided into two groups: euploid TE/euploid NICS (52 embryos) and euploid TE/aneuploid NICS (33 embryos) groups . There wIn recent years, more and more scholars have studied the effectiveness of NICS. Although NICS has the advantage of being a relatively simple and non-invasive sampling process compared to TE biopsy, contamination from the mother source and the low content of DNA in the culture medium can easily lead to the failure of amplification and affect the results. Wei Qiang Liu et al., observed that the DNA dectiction rate in SCM was slightly higher than that in BF samples , a findiMaternal contamination is a common problem among discordant results. It is known that contamination by maternal DNA often leads to female bias in the sex ratio. Our research found that 17 samples had gender inconsistency, the TE sex chromosome was XY and the BCM was XX, which may be due to maternal contamination. Our maternal contamination rate (17 of 492) is lower than previously reported: 2 out of 27 , and higversus whole embryos and NICS, mostly because of mosaics (In previous studies, the consistency rate of NICS was low . Current mosaics . What’s  mosaics . This se mosaics ; this maBoth TE biopsy and NICS have their limitations, that affect the accuracy of the two methods. These may affect the outcomes of assisted pregnancy, such as reduced pregnancy rate, increased abortion rate, etc., Rubio et al. reported that the ongoing pregnancy rates of euploidy embryos screened by TE biopsy and NICS were higher than that of NICS aneuploidy embryos after embryo transplantation, but there was no statistical difference between the two groups . In our In summary, our results suggest that NICS was similarly effective in assessing both euploid and aneuploid chromosomes in embryos. Therefore, combining the TE biopsy and NICS results may improve the outcomes of assisted pregnancy; however, this needs to be studied further.


However, a direct measurement of cellular mitochondrial function provides valuable information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium . As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC . Previoue fibers ,3,4,5. I NBTdfz) . Accordimax was performed in transverse sections of skeletal muscle fibers cut at 6 µm thickness by cryo-sectioning, thereby controlling pathlength for light absorbance [Previously, SDHsorbance ,6,7,8,9.sorbance .max varies across skeletal muscle fiber types and changes with activity, age, and disease conditions [max across skeletal muscle fibers corresponded to differences in mitochondrial volume density [max and mitochondrial volume density in individual hASM cells, which has not been previously explored.Previously, we found that SDHnditions ,20,21,22 density ,8,9. Suc density ,24. Thusmax in individual hASM cells based on the progressive precipitation of NBTdfz. In the present study, the relationship between SDHmax and mitochondrial volume density was examined. In addition, measurements of SDHmax were compared to respirometry-based OCR measurements. Finally, the rigor and reproducibility of SDHmax measurements across hASM cells were assessed both within and across patients. The goal of the present study was to validate a novel microscopic optical sectioning technique to measure SDHdfz (change in OD) with time  and was significantly lower compared to the change in OD in the presence of succinate ) to obtain a 3D image of the NBTdfz precipitate distributed throughout the hASM cell. The sampling depth was restricted to 5 µm in the z-axis with a fixed optical slice thickness of 0.5 µm  averaging ~6% across intact hASM cells and ~4% across permeabilized cells .To simultaneously measure SDHASM cell A,B. SDHml volume C. The mil volume . The relrelation D. Acrossmax measurements, we tested the use of CellLight™ Mitochondria-GFP for the simultaneous measurement of SDHmax in individual hASM cells. To validate that the pattern of CellLight, labeling was restricted to mitochondria; hASM cells were loaded with both the CellLight™ Mitochondria-GFP and MitoTracker® Red FM . In separate experiments, SDHSM cells F. The rerelation G. Acrossmax in intact hASM cells . In addition, the maximum OCR measurements obtained were normalized to mean mitochondrial volume density (MVD), measured from independent determinations of mitochondrial volume  in individual hASM cells. The method used in this study was similar to that previously used to measure SDHmax in skeletal muscle fibers [max in hASM cells involved the control of pathlength for light absorbance by confocal-based optical slicing rather than cryo-sectioning , which is critical in the quantification of the SDH reaction rate. The main findings of the present study were: (1) the SDH reaction rates in both intact and saponin-permeabilized hASM cells were linear across an 8 min period; (2) the optimal succinate concentration for measuring SDHmax was ~10 mM in permeabilized hASM cells compared to ~80 mM for intact hASM cells; (3) at optimal succinate concentrations, the SDHmax values for intact and permeabilized hASM cells from the same patient were comparable; (4) SDHmax varies with mitochondrial volume density across hASM cells; and (5) when SDHmax was compared to OCR measurements normalized for mitochondrial volume density, there was a strong correlation (R2 = 0.96). We conclude that measurements of SDHmax provide a reliable estimate of the maximum respiratory capacity of individual hASM cells.In the present study, we validate the use of a quantitative histochemical technique to measure the maximum velocity of the SDH reaction . The reaction is performed using mPMS as an exogenous electron carrier and azide to inhibit cytochrome oxidase (Complex IV), thereby reducing the non-specific reduction of NBT. The optimal concentration of the various compounds used in the assay has been tested in previous studies and has been consistently shown to give reliable results in various testing models [The technique for measuring SDHk et al. , followek et al. . The quag models ,21,22,25max, the Beer–Lambert equation requires control of the pathlength for light absorbance in determining the accumulation of the reaction product . In measuring SDHmax in skeletal muscle fibers, the pathlength for light absorbance was controlled by cryo-sectioning the fibers at a defined thickness [max measurements regardless of the number of optical slices sampled or the thickness of the optical slices. The similar measurements across optical slices indicate that NBTdfz is distributed within the cytosol rather than within the mitochondria and the change in OD is proportional to the thickness of the optical slices.In the quantitative histochemical method for measuring SDHhickness ,8,9,22. hickness ,26,27. Hmax was achieved at a succinate concentration of 80 mM in the reaction media. However, the exogenous succinate concentration in intact hASM cells does not necessarily reflect the available succinate for the SDH reaction. Therefore, we compared the optimal succinate concentration for SDHmax in intact hASM cells to that in saponin-permeabilized cells, where the cell membrane diffusion barrier was removed. Since saponin can also permeabilize the mitochondrial membrane, we carefully titrated the concentration of saponin and confirmed that the mitochondrial membrane potential was not affected. In permeabilized hASM cells, SDHmax was achieved at a succinate concentration of 10 mM. Importantly, at optimal succinate concentrations, the SDHmax values measured in intact and permeabilized hASM cells were comparable. This indicated that succinate transport across the cell membrane is necessary, and most likely achieved by a Na+-dependent dicarboxylic acid transporter [max measurements further indicated that an adequate amount of succinate is being transported into intact cells to achieve SDHmax. Furthermore, in both intact and permeabilized hASM cells, we confirmed that the SDH reaction is linear over an 8 min period. The temporal linearity of the SDH reaction is similar to that found in skeletal muscle fibers [max in hASM cells in each patient. Based on the sample size used, we observed that the variability of SDHmax measurements across hASM cells within a patient was less than that observed across patients.In the quantitative histochemical technique, it is essential to use an optimal concentration of succinate to avoid substrate limiting the reaction. In intact hASM cells, we found that SDHnsporter ,29,30,31e fibers ,9,22. ThIn the present study, we used 3D confocal imaging to identify mitochondria and measure mitochondrial volume density within hASM cells across patients. We observed significant variability in mitochondrial volume density across hASM cells from a single patient implying the presence of inherent differences in mitochondrial volume between individual hASM cells and patients. In previous studies, we compared the use of 3D confocal imaging to determine mitochondrial volume to that obtained by electron microscopy (EM) and found the mitochondrial volume densities determined by the two methods to be comparable . Howevermax values compared to less active type IIx/IIb fibers. Imposing reduced activity on diaphragm muscle fibers reduces mitochondrial volume density and SDHmax of type I and IIa fibers . These previous results indicate that mitochondria are highly dynamic organelles undergoing cycles of fission/fragmentation and fusion as well as mitochondrial biogenesis to meet energy demands [In previous studies, we observed that mitochondrial volume density is greater in hASM during various inflammatory conditions, which reflects an increase in energy demand within hASM cells ,33,34,35 demands ,37,38,39max to the mitochondrial volume provides important information on the relationship between mitochondrial structure and function. SDHmax normalized for mitochondrial volume density is much higher in type I and IIa diaphragm muscle fibers compared to type IIx/IIb fibers, thus reflecting a higher maximum respiratory capacity per mitochondrion in these fibers (18). Unfortunately, simultaneous measurements of SDHmax and mitochondrial imaging by MitoTracker labeling are not possible in hASM cells since we found that MitoTracker decreased SDHmax. Thus, MitoTracker labeling appears to impact mitochondrial function, perhaps because mitochondrial labeling depends initially on the proton gradient followed by binding to thiol groups in the mitochondrial matrix. By comparison, CellLight, a BacMam expression vector encoding GFP fused to the leader sequence of E1α pyruvate dehydrogenase [max measurements. Additionally, the relationship between SDHmax and mitochondrial volume density determined by CellLight labeling was much stronger than that observed using MitoTracker. Normalizing SDHrogenase , labels rogenase ,42. Whenmax within patients. However, when OCR measurements were normalized to mitochondrial volume density there was a strong correlation to the average SDHmax across cells. Studies have also provided evidence that NBTdfz is specific for measuring SDH activity and thus effective for determining mitochondrial oxidative activity [max are comparable to those in lysate-based biochemical measurements [max provides measurements of mitochondrial respiratory capacity within individual cells with lower variability.In the present study, maximum OCR measurements normalized to cell count did not correlate to the average SDHactivity ,45,46,47urements ,49,50,51max measurement is a reliable technique, we acknowledge the presence of certain limitations associated with the assay. We show evidence that the measurement of SDHmax reflects the maximum respiratory capacity of mitochondria in hASM cells but does not imply that SDHmax is equivalent to OCR measurements. Unlike respirometry-based OCR measurements, SDHmax does not inform us how much of the respiratory capacity measured is used to meet the cellular energy demands, ATP-linked respiration, and proton leak, among others.While our results confirm that SDHThe study was reviewed by Institutional Review Board at Mayo Clinic (IRB #16-009655) and considered to be exempt for the following reasons. Patient consent was obtained during pre-surgical evaluation  prior to surgery in non-threatening environments and patients remained anonymous. No one on the team was involved in patient selection or in the surgery itself. In no cases were patients contacted by any of the investigators involved in this proposal. Patient history was provided at the time of tissue acquisition for the recording of sex, demographics, pulmonary disease status, pulmonary function testing, imaging, co-morbidities, and medications, but no patient identifiers were stored. Accordingly, samples were numbered, but patient identifiers were not attached to the samples, preventing retrospective patient identification from samples alone.2—95% air) in phenol red-free DMEM/F-12 medium  supplemented with 10% fetal bovine serum  and 100 U/mL of penicillin/streptomycin  in sterile culture dishes. Isolated hASM cells from 1 to 3 subcultures were used in all experiments. Before experiments, the cells were serum deprived for 48 h by changing the growth medium to DMEM/F-12 medium lacking FBS.Bronchial tissue samples were obtained from anonymized female and male patients undergoing lung resection surgeries. The selected patients had no history of asthma or other chronic lung diseases. After pathological assessment, normal regions were identified, and third to sixth-generation bronchi were isolated. The smooth muscle layer was dissected under a microscope, and hASM cells were dissociated by enzymatic digestion using papain and collagenase with ovomucoid/albumin separation as per the manufacturer’s instructions  as previously described ,53. hASMBefore conducting any experiments, the phenotype of hASM cells was confirmed by immunocytochemical analysis of α-smooth muscle actin (α-SMA) expression as previously described ,54,55,56Dissociated cells were imaged to distinguish α-SMA expressing hASM cells and the percentage of hASM cells was determined as a fraction of total dissociated cells (determined from DAPI) . Consistdfz [For the quantitative histochemical procedure, the Nikon Eclipse A1 laser scanning confocal microscope with a ×60/1.4 NA oil-immersion objective at 12-bit resolution into a 1024 × 1024-pixel array using the transmitted light channel was used for image acquisition. An interference filter with a peak emission wavelength of 570 nm was placed in the light path to limit the spectral range of the light source to the maximum absorption wavelength of NBTThe measured gray level (GL) of the microscope was calibrated to known OD units using a photographic density stepwedge tablet  (0.04–2.max in intact cells was compared to that of saponin-permeabilized cells (see below for details).Following calibration, the quantitative histochemical procedure for measuring the maximum velocity of the SDH reaction was performed in hASM cells. This technique has been previously described in detail in single skeletal muscle fibers ,20,21,25dfz due to the reduction of NBT is used as the reaction indicator. Experiments were performed at room temperature (22 ± 1 °C), with similar parameters maintained across preparations. hASM cells were delineated as the ROI while the nucleus was excluded, and NBTdfz precipitation was measured as the progressive change in OD as the SDH reaction proceeded. The change in average OD within the selected ROI was measured every 15 s across a 10 min period. The SDHmax was then determined using the Beer–Lambert equation (below):dfz accumulation, k is the molar extinction coefficient for NBTdfz , and l is the pathlength for light absorbance . The SDHmax is expressed as millimoles of fumarate per liter of cell per min.Intact and permeabilized hASM cells from the same patients were exposed to solutions containing either no succinate (without substrate) or a succinate-containing solution. Both solutions contained (in mM): 1.5 NBT (reaction indicator), 5 Ethylenediaminetetraacetic acid (EDTA), 1 mPMS, and 0.75 azide in 0.1 M phosphate buffer (pH: 7.4). In the quantitative histochemical procedure, the progressive precipitation of NBTmax was previously determined in skeletal muscle fibers [max in intact and saponin-permeabilized hASM cells was compared. Prior to performing the SDH assay, we assessed saponin-induced permeabilization of the cell membrane by the uptake of trypan blue in hASM cells. We also assessed any potential disruption of the mitochondrial membrane by using TMRM to monitor the mitochondrial membrane potential during saponin titration. For permeabilization of cells, isolated hASM cells were first incubated in serum-free medium containing 25 µg/mL of saponin for ~1 min at room temperature to allow permeabilization. The media was aspirated, and permeabilized hASM cells were then incubated with different succinate concentrations , and the rate of the SDH reaction was measured as described above. For comparison, intact hASM cells from the same patient were incubated with the same succinate concentrations  and the rate of the SDH reaction was measured as described above.The maximum substrate (succinate) concentration required for measuring SDHe fibers ,3 but nomax measurements, two sampling parameters were used to obtain 3D confocal images during the SDH assay. First, a step size of 0.5 µm or 1.0 µm between optical slices were obtained while controlling the aperture setting to provide a 0.5 µm optical slice thickness  were measured within each cell as described above. To reduce time of acquisition and assess the impact of 3D confocal sampling parameters on SDHhickness A–C. Secohickness A–C. In bmax measurements in hASM cells, 20–30 hASM cells were analyzed in each of the 6 bronchial samples (patients). In all assays, multiple hASM cells (identified by size and shape) were visualized within a single microscopic field. Due to the large size of hASM cells, the borders of some cells overlapped, and we analyzed only those hASM cells whose borders were not overlapping. Typically, this selection process resulted in analyzing 3 to 4 hASM cells per field. Based on an a priori power analysis of variance in SDH® Red FM  or 200 nM MitoTracker® Green FM  and 1 µg/mL Hoechst 33342 Solution , to label the nucleus, in serum-free media pre-warmed to 37 °C, for 15 min followed by extensive washing with 1X Hanks’ Balanced Salt Solution (HBSS). hASM cells were plated at a density of ~15,000 cells/well into 8-well Ibidi μ-slide plates  and incubated to allow for cell adherence and subsequently serum deprived. The cells were loaded with 200 nM MitoTrackerhASM cells were plated at a density of ~15,000 cells/well into 8-well Ibidi μ-slide plates  and incubated to allow for cell adherence. Adhered hASM cells were transduced with CellLight™ Mitochondria-GFP, BacMam 2.0  to label the mitochondria as per the manufacturer’s instructions. This is a baculovirus system with a fusion construct of the leader sequence of E1 alpha pyruvate dehydrogenase and emGFP, providing accurate and specific targeting to cellular mitochondria. hASM cells were incubated in growth media containing the CellLight reagent for 24 h at 37 °C. Subsequently, the hASM cells were washed with 1X PBS and serum deprived for 48 h for further experimentation. Although the efficiency of transduction was relatively low (~60%), GFP protein could be observed in the mitochondria.Mitochondria in hASM cells (distinguished by their size and shape) were imaged using the same confocal microscope system used for SDH measurements as described above ,32,34,58https://imagej.nih.gov/ij/). Using the mitochondrial analyzer plugin on ImageJ, the Z-stacks were then processed for background correction and ridge filter detection [The 3D images obtained were deconvolved by using the automatic deconvolution algorithm on NIS-Elements analysis software (Version 5.20.02)  ,23,34,59etection ,60,61,62etection ,63. Mitoetection ,9,23,59.2 sensor, was hydrated using Seahorse XF Calibrant Solution for 24 h prior to assaying. Titration of mitochondrial inhibitors and FCCP (Seahorse XF Cell Mito Stress Test kit) were performed across patients to determine the optimal concentrations that were subsequently used to test mitochondrial respiration. The optimal concentration of the inhibitors and FCCP in hASM cells remained consistent across patients and were as follows: 1 µM oligomycin (ATP uncoupler), 1.25 µM FCCP (accelerates electron transport chain), and 1 µM antimycin A (Complex III inhibitor) with 1 µM rotenone (Complex I inhibitor) [2. Since the total area of the well covered by the adherent cells was 27.5 mm2, the total number of adherent cells in each well was estimated by extrapolating the counts obtained from the single center image by multiplying them by a factor of ~3.71 (ratio of the area of the well to the area of the image obtained). hASM cells from 6 patients were used for OCR measurement (n = 4 wells/patient).hASM cells were plated into the Seahorse XF24 cell culture microplates  as recommended by the manufacturer for optimal adherence and growth of mammalian cells. hASM cells were then incubated to allow for cell adherence and formation of a confluent monolayer, and subsequently, serum-deprived for 48 h. Following serum deprivation, oxygen consumption rate (OCR) was measured by the mitochondrial stress test assay performed using the Seahorse XFe24 Bioanalyzer  as per the manufacturer’s instructions and as described previously . Prior thibitor) ,67,68,69hibitor) ,70. The max and mitochondrial volume density measurements, each dot represents data obtained from a single hASM cell and each color represents a single patient . For OCR measurements, each dot represents data obtained from one well (4 wells/patient) and each color represents a single patient . For measurements of SDHmax with varying number of optical slices, substrate dependence (in intact and permeabilized cells), and mitochondrial volume density (CellLight and MitoTracker), each dot represents a single cell from one patient. The number of cells per patient used was determined by a power analysis of primary outcome measures. The expected effect size was calculated with an a priori biologically relevant difference of 20% and equal variance, with sample size estimated using d = 1.4, α = 0.05, and β = 0.8. A Shapiro–Wilk test was used to confirm a normal distribution in the data. Statistical analysis was conducted using Prism 9 , and a paired t-test was used to compare groups. The linearity of the SDHmax assay was assessed using the simple linear regression model. The linear relationship between SDHmax, OCR, and mitochondrial volume density was assessed with Pearson’s correlation. Statistical significance (*) was concluded if p < 0.05. All data are represented as mean ± SEM or mean ± SD.For each of the experiments, hASM cells were dissociated from bronchial tissue samples obtained from both female and male patients. Sex is an important biological variable, but the study was not powered to detect sex differences. The patient inclusion criteria were based on the phenotype of dissociated cells as confirmed by immunoreactivity to α-SMA. If less than 90% of the dissociated cells were not immunoreactive for α-SMA, the samples were discarded. Three out of nine patient samples were excluded based on these criteria, resulting in the inclusion of six patients. Differences across patients were considered a random variable in the statistical design. Sample size (n) represents the number of hASM cells or biological replicates analyzed per patient and the n for each experiment are provided in the figure legends. For SDHmax in hASM cells is reliable and reproducible. The SDHmax measurements were not comparable to respirometry-based OCR measurements, which is considered the “gold standard” for the measurement of mitochondrial function. However, we provide evidence that respirometry-based OCR measurements are more susceptible to variability than SDHmax measurements. We conclude that the quantitative histochemical technique to measure SDHmax can be used in other cell populations. Future studies targeting the SDH complex may unravel further information on the mechanism and specificity of the SDH assay.In conclusion, we present evidence that the quantitative histochemical technique for measuring SDH


The impact of human sewage on environmental and food contamination constitutes an important safety issue. Indeed, human sewage reflects the microbiome of the local population, and a variety of human viruses can be detected in wastewater samples. Being able to describe the diversity of viruses present in sewage will provide information on the health of the surrounding population health and will help to prevent further transmission. Metagenomic developments, allowing the description of all the different genomes present in a sample, are very promising tools for virome analysis. However, looking for human enteric viruses with short RNA genomes which are present at low concentrations is challenging. In this study we demonstrate the benefits of performing technical replicates to improve viral identification by increasing contig length, and the set-up of quality criteria to increase confidence in results. Our approach was able to effectively identify some virus sequences and successfully describe the viral diversity. The method yielded full genomes either for norovirus, enterovirus and rotavirus, even if, for these segmented genomes, combining genes remain a difficult issue. Developing reliable viromic methods is important as wastewater sample analysis provides an important tool to prevent further virus transmission by raising alerts in case of viral outbreaks or emergence. Contaminated food and water are a major pathway for the transmission of infectious diseases, this poses a global health issue, with viruses being one of the most frequently reported infectious agents. Considering the host specificity of most human enteric viruses, despite rare exception such as for hepatitis E virus, this contamination mainly occurs through contact with human sewage that have not been treated properly. People infected with enteric viruses, pathogenic or non-pathogenic, that are derived from the human gut, excreted in the stools and vomitus. Early evidence of the importance of sewage contamination with human viruses emerged in the late 1930’s, raising the question of possible transmission of poliovirus through contaminated water . Since tUp to now, the major restriction of sewage analysis is that only previously described viruses are searched for and therefore detected. In 1995, a review described studies of sewage analysis up to the development of molecular biology and the possibility to detect a large range of viruses, even though many of these do not grow or grow poorly in cell culture . Since tMetagenomic developments propose a new step for the analysis of environmental samples. Next-generation sequencing (NGS) or high-throughput deep sequencing allow the massive, parallel sequencing of DNA making possible, which in theory can describe all micro-organisms present in one sample. Such NGS method has been shown to be very promising for sewage analysis as it described the diversity of virus strains circulating in the population . In an eHere the previously developed method was applied for the analysis of archived wastewater samples with the aim to evaluate the benefits of performing technical replicates to improve viral identification, with a focus on virus that may cause food contamination.Twelve archival samples collected from six wastewater sewage treatment plants (WWTP), all located in northwestern France within 200 km of each other, were selected . All WWTAll samples but one (2006-D) were 24-h composite raw sewage samples, and had been stored at −20 ± 1°C in 1-liter aliquots before the metagenomics study (storage length varying from 14 to 2 years).The 12 samples were concentrated from 40 ml raw sewage by adding 10 ml of 50% polyethylene glycol , and then 10 ml of 50% of PEG 6,000 were added. After overnight incubation at 4°C under gentle agitation, the mixture was centrifuged for 1.5 h at 13,500 × g, the pellet was suspended in 2 ml of 0.05 M glycine buffer (pH 9).All 12 samples were analyzed under four technical replicates . The 48 The re-suspended pellets were filtrated using a cascade of 5, 1.2, and 0.45 μM filter pores  to eliminate cells and the filtrate treated with 2000 units of OmniCleave Endonuclease  for 1 h at 37°C to eliminate free DNA and RNA. Nucleic acids (NA) were extracted by adding 10 ml of a guanidium-based lysis buffer (bioMerieux) and 140 μl of paramagnetic beads  using 5 μl of extracted NA per well  in duplicate on a Mx3000P QPCR System . Standard curves based on double strand DNA  corresponding to nucleotides 4,484 to 5,668 of the GI.1 Norwalk virus (Genbank M87661) or nucleotides 4,217 to 5,355 of the GII.4 Houston virus (Genbank EU310927), were included in each run. Concentrations range from 8.1 and 3.4 RNA copies/μl to 8.1 × 104 and 3.4 × 104 RNA copies/μl for norovirus GI.1 and GII.4, respectively.Norovirus quantification was performed for the two main genogroups I and II (GI and GII) using primers and probes targeting the conserved region at the beginning of ORF2 . The rRTTwo negative amplification controls  were included in each amplification series and filter tips and dedicated rooms were used to prevent false-positives. Standard curves included in each run were analyzed and retained for NA quantification if amplification efficiencies were comprised between 85 and 110%. For the screening, concentrations were calculated per sample by adding GI concentrations to GII. When samples were prepared for metagenomics, concentrations obtained for each replicate (4 per sample), were calculated separately, then GI and GII concentrations were added and the four values obtained were used to calculate the geometric mean concentration (GMC) per sample.Libraries were prepared by series of eight. All the 48 NA extracts were transcribed into cDNA using the Superscript II  and random hexamers (New England Biolabs (NEB), USA). Completion of this step was checked by PCR quantification for norovirus GI and GII as described above, except that enzyme mix was replaced by Platinum taq (ThermoFisher Scientific) and the RT step was removed from the thermal profile.After double strand DNA synthesis using second strand reaction buffer from NEBNext Ultra RNA Library prep, physical fragmentation of the DNA was performed using sonication  and libraries were prepared using the kit KAPA Hyper Prep (Roche), according to the manufacturer’s instructions. Viral sequences were enriched using VirCapSeq-Vert probe panel Roche; . A negatDe novo assembly was then performed using metaSPAdes with kmer length 21, 33, 55, 77, 99 (nt database (download 2022-05-08) with an e-value of 10−5, and nr database (download 2021-02-06) using diamond with an evalue of 10−3. When both approaches gave results, the BLASTn match was kept. To evaluate the coverage of each contig, post-process reads were mapped using Bowtie2  and L the length of the considered contig  were calculated using the number of reads per family and the total number of reads after trimming and deduplication. The heatmap was done using Graphpad prism v 9.0.0 .Genotyping of viral sequences was performed using several web-based tools, and only contigs represented more than 100 reads were considered. For norovirus, sapovirus or hepatitis A & E virus sequences, the identification was done using the respective online Typing Tools and for rotavirus the Rotac tool . Other cPhylogenetic analysis were performed by aligned sequences using Muscle or ClustalW2, followed by IQ-TREE algorithm to choose the best fit model according to the BIC (Bayesian Information Criterion) and to construct phylogenetic trees with a boostrap of 1,000.Samples were collected from sewage treatment plants from relatively small communities over a period of 10 years . Eight sFor metagenomics, the 12 samples were treated and extracted 4 times each .After the nucleic acid extractions of the four replicates from each of the 12 samples, 29 were positive for norovirus GI and 40 for norovirus GII. After cDNA synthesis, 22 extracts were positives for norovirus GI and 45 for norovirus GII (data not shown). One replicate of sample 2009-P did not give a GI or GII Ct value and none of the reads were identified as norovirus. Norovirus concentrations, expressed as GI + GII, obtained after cDNA synthesis were similar or half-a log higher compared to concentrations obtained for the RNA . All fouAmong the 818 million of reads obtained after the Illumina sequencing, 44 libraries gave comparable read numbers ranging from 1,565,594 to 85,453,164 reads, and four libraries yielded a low number of reads  presented a large variability between replicates when considering raw reads, clean reads or viral reads, while some others  presented a similar number of reads for all replicates . The coeTo further decide which approach allows better viral identification, the length of contigs obtained for three genera of enteric viruses , were compared considering contigs obtained for each separate replicate (blue) and with merged replicates orange; . For mamTherefore, only the merged-replicate datasets were used for subsequent analyses.Astroviridae, Caliciviridae, Hepeviridae, Picobirnaviridae, Picornaviridae, and Reoviridae , the proportions of ‘other viruses’  ranged from 17 to 63% (Hepeviridae family were identified only in two samples (2014-K and 2016-B).An initial taxonomic identification was conducted directly on the clean reads of merged replicates. Among the 76,076,257 viral reads, the large majority of reads were identified as sequences related to viral families infecting humans such as oviridae . Reads bal reads ,B. A lar7 to 63% . Reads bAfter assembly, contigs were identified using different tools (see method section) and 1,375 contigs were identified as belonging to nine selected viral families comprising known human pathogens : seven s4 reads, complete genomes were assembled. For kobuvirus, all the different samples provided a low number of reads and contigs obtained were all under 1 kb.Long contigs or complete genomes were obtained for different viral genera with a clear relationship to read numbers . HoweverCaliciviridae family, when considering contigs longer than 1 kb obtained with more than 100 reads, 32 contigs were identified as norovirus and three as sapovirus. A large range of norovirus read numbers per sample was observed from 264 reads (sample 2009-P) to 1,309,920 reads  for which none of the cDNA replicates were positive for norovirus GI by PCR provided no norovirus GI reads, while some GII reads were identified. Similarly, no norovirus reads were obtained from the replicate of sample 2009-P that did not provided any Ct value. Norovirus sequences were genotyped using the Typing tool and the Noronet database, for the ORF1 and ORF2 regions separately . OverallHepeviridae family, four contigs were identified as orthohepevirus from samples collected in site B and K  of the contigs, were identified as mamastrovirus 1 (HAstV-1), using the genome detective web tool. This genus was represented in all samples. Feline astrovirus 2 (FAstV-2) was the second most identified genus (17%) with 22 contigs. Other contigs were identified as mamastrovirus 2 (HAstV-2) 1 contig, mamastrovirus 5 (HAstV5) 1 contig and mamastrovirus 9 (HAstV-9) 3 contigs. Twenty one contigs were close to reference sequences that were not been approved as a species by the ICTV, but belong to the mamastrovirus genus: 16 contigs of Human astrovirus MLB2 (HAstV-MLB2), 4 contigs of the Culex Bastrovirus-like virus and 1 contig of Canine astrovirus (CaAstV-1). Twelve long contigs (more than 6 kb) were found in all samples except sample 2009-P.For the Among the complete genomes obtained in the different samples, six clustered to HAstV-6, and six to HAstV-1. Phylogenetic analysis of the complete RdRP protein sequence confirmed identification of the complete HAstV genomes compared to previously available sequences in GenBank .Picobirnaviridae reads were identified in all samples  are really helpful. However, assembling the 11 rotavirus genes from a sewage sample, remain a delicate challenge, performed on previously described constellations obtained in Human or animal stool samples . It is iThe aim of this study was to evaluate the relevance of technical replicates and quality criteria such as a minimum of reads or contigs length to identify human pathogenic virus sequence with confidence. We chose samples from diverse WWTP and different seasons to ensure that our protocols could be applied to diverse wastewater samples. As some samples were kept frozen for more than 10 years and could be degraded, we did not expect to raise conclusion in terms of sequence diversity or the presence of specific strains. Yet, we included samples linked to hepatitis outbreaks to assess the possibility to detect the causing agents retrospectively. Our approach was found efficient to identify some sequences and successfully described the viral diversity, yielding full genomes either for norovirus, enterovirus or even rotavirus, even if, for these segmented genomes, combining genes remains a difficult issue.Metagenomics allowing the description of all genomes present in a sample applied to wastewater samples that received all different viruses replicating in the gut of the local population constitute a promising approach for epidemiological surveillance. Our results show that performing biological replicates and merging the obtained data allow to obtain longer contigs and a reliable strain identification, an important issue for public health surveillance. Indeed, confidence in result interpretation is strengthened by quality criteria in terms of read numbers and contig length . ImportaThe sequencing data presented in this study are deposited in the NCBI repository. Accession numbers can be found in JS, MD, and FG contributed to the conception, design of the study, contributed to data interpretation, and manuscript preparation. JS performed experiments. JS and AB performed the bio-informatics analysis. All authors contributed to the article and approved the submitted version.This study was supported by the COMPARE and VEO European projects (H2020 grant agreement N°643476 and H2020 SC1-2019-874735) and by support from the Direction Générale de l’Alimentation (DGAl) through the annual convention.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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This study aimed to develop a rapid and efficient method for measuring the growth of entomopathogenic fungi, which are used as environmentally friendly alternatives to chemical insecticides. The traditional methods used to measure fungal growth are time-consuming and limited in their ability to capture important aspects of growth. In this research, we implemented an indirect measure using a microplate reader, which uses the optical density of small-volume cultures to estimate fungal growth. We directly related changes in optical density to the amount of fungal biomass, and compared the results of this method with traditional measurements on solid agar plates. We found that the microspectrophotometric approach provided accurate and reliable measurements. Our findings revealed differences in growth rates and biomass production among different species and isolates of the fungi. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi, and has practical applications in assessing their virulence and pathogenic potential. It can contribute to our understanding of how these fungi grow during infection, and aid in the development of effective biological control strategies against insect pests.Metarhizium to validate spectrophotometric growth measurements, and investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures by extracting, drying, and weighing mycelial mats. From the relationship established between OD and biomass, we generated standard curves for predicting biomass based on the OD values. The OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium species. The logistic growth phase, as captured by the OD measurements, could be accurately assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense, this technique was demonstrated to be an effective, reproducible, and simple method for rapidly measuring filamentous fungal growth with high precision. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence their growth.Quantifying the growth of entomopathogenic fungi is crucial for understanding their virulence and pathogenic potential. Traditional methods for determining growth, such as biomass determination or colony growth area, are time-consuming and quantitatively and spatially limited in scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical density (OD) changes were directly correlated with dry weight of samples for six isolates from three species of the genus  Entomopathogenic fungi are important natural regulators of insect populations, and are widely used as environmentally friendly biological control alternatives to synthetic chemical insecticides ,2,3,4. IMeasurement of the radial expansion of fungal colonies on solid media is a widely-used direct method to quantify growth. The method is straightforward, and allows for measurement of other phenotypic traits such as spore production and colony morphology  ,8,21. HoIn addition to this, the solid media environment may not be biologically appropriate for some fungi based on their specific ecologies. In entomopathogenic fungi, in vivo growth progresses primarily through the insect hemocoel, which is a submerged liquid environment . The phyMetarhizium spp. This correlation allows for the construction of isolate-specific standard curves, enabling the quantification of biomass based on OD measurements.In situ spectrophotometry provides a viable alternative to these methods by directly correlating optical density (OD) values with an increase in fungal biomass. It is generally understood that growing fungus changes the OD of liquid cultures, because turbidity directly correlates with unit population size, which serves as the basis for the traditional spectrophotometric analysis of fungal growth . In thisMetarhizium were compared to investigate variations among species and isolates. This was assessed in addition to the effect on the relationship between dry weight and OD. Six fungal isolates of the genus Metarhizium were used to produce standard curves of OD by dry weight: M. brunneum KVL 16_36 , M. brunneum KVL 12_30 [M. acridum KVL 18_06 (ARSEF 6421), M. acridum KVL 04_55 (ARSEF 7486), M. guizhouense KVL 19_24 (ARSEF 977), and M guizhouense KVL 19_28 (ARSEF 3611). The acronym ARSEF refers to the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) collection of Entomopathogenic Fungal cultures . The acronym KVL refers to the entomopathogenic fungus culture collection maintained at the Section for Organismal Biology, Department of Plant and Environmental Sciences, University of Copenhagen. Metarhizium (Metschnikoff) Sorokin  was selected as the focus of our study, due to its multifaceted importance in both evolutionary ecology and practical applications in pest management [The growth of two isolates within each of three different species of VL 12_30 , M. acri6 ARSEF 621, M. acv/v) Tween® 80  via agitation with a Drigalski spatula from sporulating colonies, and the resulting suspension was centrifuged; the supernatant was removed, and the colonies were rinsed twice with 0.1% (v/v) Tween® 80 to remove all fragments of mycelia. Conidial suspensions were prepared at a concentration of 2 × 106 conidia per mL by dilution using 0.1% (v/v) Tween® 80. The concentration of the stock suspension was determined by counting conidia from 1000× serially diluted stock suspension in a Fuchs Rosenthall hemocytometer (×400 magnification). To verify germination, 100 µL of 100× diluted stock suspension was spread with a Drigalski spatula on an agar plate of SDAY/4 and incubated for 24 h at 23 °C. Four microscope coverslips (22 mm × 22 mm) were then placed over the culture surface, and 100 conidia were counted under each coverslip. Conidia were considered to have germinated with the germ tube that was at least as long as the width of the conidium, and conidial germination after 24 h was verified as >98% in all cases before being used in further assays.The fungal cultures were grown on quarter-strength Sabouraud dextrose agar + yeast media , 10 g/L dextrose , 2.5 g/L yeast extract (Neogen Corp.), 15 g/L agar  No. 1, Neogen Corp.))) in Petri dishes  at 23 °C, and conidia were harvested after 14 days. The conidia were collected in 0.1%  by inoculating 100 µL of 2 × 106 conidial suspension into 100 µL Sabouraud dextrose + yeast media . The OD of each well was measured at 405 nm in a SynergyTM HT MultiDetection Microplate Reader  with Gen5 software Version 2.00.18 every 10 min for 96 h at 24 °C, without removal of the microplate from the plate reader. The wavelength of 405 nm was previously described as being fit for this purpose [To determine whether measurement by spectrophotometry in liquid microcultures can accurately capture the growth curve of an entomopathogenic fungus, a growth curve of TM HT MultiDetection microplate reader with Gen5 software. At each time point, one microplate per isolate was collected after OD measurement and stored at −20 °C. The OD values of all of the microplates were also measured after conidia settled (15 min post-inoculation) to establish the baseline reading OD of each culture. This baseline reading was subtracted from subsequent measurements to determine changes in OD for the construction of standard curves.To establish a correlation between OD and fungal biomass, OD values were measured, and the mycelial mats were subsequently extracted and weighed for the six isolates at four time points  during the linear growth phase. The period of logistic growth was determined from the continuous growth curve generated, as detailed in g) at 4000 rpm for three minutes to force fungal material to the bottom of the wells, and the remaining media supernatant was removed using a pipette. The wells were then filled with 200 µL of 99% ethanol, and mycelia were scraped from the bottom of the well with a pipette tip to re-suspend the fungal material. The entire content of the well was then transferred to a pre-weighed aluminum weigh boat using a cut pipette tip. The process was repeated three times, refilling each well with 200 µL of ethanol and scraping to ensure complete removal of residual mycelial matter and rinsing of the pipette tip. Thus, a total of 600 µL of ethanol was utilized to thoroughly wash each well. Complete extraction of mycelial material from the wells was confirmed through microscopic examination of the microplates. If any residual mycelial material was observed, the extraction process was repeated until the wells were free of any remaining matter. For each isolate, ten replicate wells containing resultant mycelial suspensions were pooled into pre-weighed aluminum boats, resulting in a total of six pooled dry weight measurements per microplate with an approximate volume of 6 mL. The pooled samples were subsequently dried in an oven for 72 h at 60 °C within a heat-resistant, lidded box. Finally, the dried samples were weighed on a precision scale .To quantify the changes in biomass of the microcultures, mycelial mats were extracted for the determination of dry weight. Thawed microplates were centrifuged in an Eppendorf Centrifuge 5810R  was measured using both microspectrophotometry and radial expansion methods. These experiments were conducted under the same conditions at 23 °C in SDY/4 and SDAY/4 media, respectively. For the microspectrophotmetric analysis, media and conidial suspensions were prepared as described in To evaluate how the microspectrophotometric method compared to traditional growth assays performed on solid media, the growth of two isolates  of SDAY/4 were each inoculated with five cultures (n = 20). The inoculation was performed using 5 µL of a conidial suspension with a concentration of 2.4 × 10Metarhizium fungus  could be captured within a short time period (96 h) using microspectrophotometry (M. guizhouense (M. acridum (M. brunneum (2 = 0.93–0.95) (M. guizhouense isolates (KVL 19_28 and KVL 19_24) exhibited distinct slope estimates compared to most other isolates, as well as each other  and KVL 16_36 (p < 0.05), with a smaller dry weight estimation compared to these two isolates  , but wit93–0.95) . In the 93–0.95) . Notablych other . The sloisolates .M. guizhouense) grew more slowly, and had a lower overall biomass than all other isolates  over 56 h, from 20 to 80 h after inoculation (M. acridum) isolate had a faster growth rate, growing more overall compared to KVL 19_28 (M. guizhouense), whether that was on the solid agar medium  values correlated strongly with biomass, and it was demonstrated that it is possible to produce a standard curve for the quantification of dry weight from OD by extracting, drying, and weighing the microcultures of mycelia on a precision scale .In addition, we showed that change in the OD accurately represents biomass accumulation. This is not always the case in measurements of radial expansion, as the expansion of fungal colonies on solid media can often be equal in area, while having significantly different densities of mycelial growth . Fungal Metarhizium, M. acridum and M. guizhouense. Our findings demonstrated that the microspectrophotometric analysis yielded similar end point results and linear growth rates compared to the radial growth assay (In this study, we compared the use of microspectrophotometry with radial expansion analysis to assess the growth dynamics of two species of th assay A–C. Howeth assay . The finth assay .M. acridum and M. guizhouense through finer measurement intervals during critical growth phases, particularly the early linear phase, which could not be discerned through radial expansion measurements performed over several days (M. acridum KVL 04_55 isolate generally exhibited more growth over time compared to other isolates (M. guizhouense KVL 19_28. This observation provides important insights into the growth dynamics and pathogenic potential during early infection processes for this particular isolate [Importantly, our study revealed a different relationship between ral days . This deisolates . However isolate .Metarhizium. Significantly differing slope estimates and total biomass production were observed not only between species, but also among isolates, indicating variations in pathogenicity potential within this in vitro setting (Additionally, our findings have practical implications regarding the pathogenic potential of the different isolates and species measured. By comparing the growth rates and total biomass produced between isolates, our findings shed light on the potential speed of host invasion across different species of  setting . This hiMetarhizium isolates could be measured within a few days, whereas radial growth on agar plates typically takes 10–14 days.Previous studies demonstrated the use of microspectrophotometry in monitoring the growth of filamentous fungi, but were unable to determine the dry weight of individual microcultures, and therefore relied on comparisons of indirect metrics to extrapolate the relationship between OD and fungal dry weight ,13, or uThe need to produce entomopathogenic fungi for biocontrol at massive scales makes it important to be able to investigate the effects of different media and nutrients on entomopathogenic fungal growth, and identify optimal growth parameters . In addiBeauveria spp., Hirsutella spp., Cordyceps spp., and Lecanicillium spp. [In conclusion, this technique allows for the rapid generation of growth curves of entomopathogenic fungi at a fine timescale with many replicates, and in a medium that is more ecologically relevant to entomopathogenic fungi than what typical solid media bioassays provide. Furthermore, this approach has the potential for application to other species of filamentous entomopathogenic fungi under investigation, such as ium spp. . The met


Shinkaia crosnieri and Munidopsis verrilli are both dominant species in cold-seep ecosystems, and they have different distributions and feeding behaviors. These species may have evolved to have distinct epibiotic microbiota. Here, we compared the epibiotic bacterial communities on the M. verrilli carapace (MVcarapace), S. crosnieri carapace (SCcarapace), and S. crosnieri ventral plumose setae (SCsetae). The epibiotic bacteria on SCsetae were dense and diverse and had a multi-layer configuration, while those on MVcarapace and SCcarapace were sparse and had a monolayer configuration. Chemoautotrophic bacteria had the highest relative abundance in all epibiotic bacterial communities. The relative abundance of amplicon sequence variant 3 , which is associated with sulfide oxidation, was significantly higher in SCsetae than MVcarapace and SCcarapace. Thiotrichales species seemed to be specifically enriched on SCsetae, potentially due to the synthetic substrate supply, adhesion preference, and host behaviors. We hypothesize that the S. crosnieri episymbionts use chemical fluxes near cold seeps more efficiently, thereby supporting the host’s nutrient strategies, resulting in a different distribution of the two species of squat lobster.The relationships between epibiotic bacteria on deep-sea hosts and host lifestyle factors are of particular interest in the field of deep-sea chemoautotrophic environmental adaptations. The squat lobsters  Munidopsis andamanica  to use as food, and it was adapted to environments with high concentrations of sulfides  was first recorded in hydrothermal vents in the Okinawa Trough, mainly dwelling in chemosynthetic ecosystems in the west Pacific Ocean  has been widely reported in various deep-sea ecosystems around the world, such as the San Clemente Basin (S. crosnieri (SCsetae), which harbors dense epibiotic bacterial communities, are the main basis for distinguishing between the two squat lobsters  and hydrothermal vents in the Okinawa Trough  to visualize the morphology of the epibiotic bacteria, quantitative real-time PCR to determine the difference in the absolute density of the bacteria, and high-throughput sequencing of 16S rRNA gene to explore the differences in the composition of the epibiotic bacterial communities.Shinkaia crosnieri was caught around the cold seep, where conspicuous methane bubbles can be observed. Munidopsis verrilli was mainly caught at peripheral mussel beds around 10 m away from the cold seep. Specimens were collected using a carousel suction sampler on the remotely operated vehicle (ROV) Faxian on the research vessel “Kexue.” They were transported in a homothermic macrofauna carrier which maintained a temperature similar to that at the cold seep, at around 4°C.Squat lobsters were collected from Site F in the South China Sea  during a cruise in May, 2021. S. crosnieri or M. verrilli via morphological classification, according to the descriptions given by carapace and SCcarapace) and the plumose setae of S. crosnieri (SCsetae) were rinsed with sterilized seawater and then immediately dissected with scissors on board. Samples for molecular analysis were snap-frozen in liquid nitrogen and stored at −80°C until DNA extraction. Cold seep interface water associated with the community of S. crosnieri (designated environment [ENV]), which was used to investigate the bacterioplankton, was filtered using an in-situ microbe sampler with a 0.22-μm filter membrane and stored at −80°C.All animals were identified as The animal samples for SEM were preserved in glutaraldehyde, underwent replacement of glutaraldehyde after 24 h, and were then stored at 4°C. In the laboratory, the samples were dehydrated in a graded ethanol series, underwent critical-point drying, were coated with gold , and were then observed using SEM .M. verrilli, three carapace samples of S. crosnieri, three setae samples of S. crosnieri, and one environmental water sample for bacterioplankton. We used a DNeasy PowerWater Kit  to extract total DNA from these samples, following the manufacturer’s instructions. The DNA was then diluted to 1 ng/μl for PCR amplification. The PCR amplification was performed using the primer pair 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′), which target the hypervariable regions between V3 and V4 of the prokaryotic 16S rRNA gene. All PCRs were performed with 10 μl Phusion® HighFidelity PCR Master Mix , 1 μM forward primer, 1 μM reverse primer, and 1 μL template DNA. The thermal cycling program involved initial denaturation at 98°C for 1 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, and a final elongation at 72°C for 5 min. Electrophoresis was then performed using 2% agarose gel. Sequencing libraries were generated using an NEBNext® Ultra™ II DNA Library Prep Kit and were quantified by Qubit and qPCR. After that, NovaSeq 6000 (Illumina) was used for on-machine sequencing.For 16S amplicon sequencing, we obtained ten different samples. These included three carapace samples of ® Premix Ex Taq™ (TaKaRa), 1 μl template DNA, 1 μM forward primer, and 1 μM reverse primer (2 to 108 copies/μl) obtained from E. coli genomic DNA by using the bacterial 16S rRNA gene-specific primers 27F (5′-AGAGTT TGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′)  in triplicate. 331F (5′-TCCTACGGGAGGCAGCAGT-3′) and 797R (5′-GGACTACCAGGGTATCTAATCCTGTT-3′) primers were used and each reaction (20 μL) contained 1× SYBRe primer . The staACTT-3′) . The theBioinformatic analyses were performed using QIIME 2 (v2022.2) . Raw seqS. crosnieri was covered with numerous plumose setae, whereas the ventral parts of M. verrilli was much smoother with only nolittle setae. The SEM images showed that the percentage of surfaces covered by biofilm was significantly higher for SCsetae . However, the differences between the bacterioplankton (in the single ENV sample) and the epibiotic bacteria were insignificant.The alpha diversity of the epibiotic bacterial and bacterioplankton communities were compared. The Shannon index varied from 5.13 to 8.68, and differed significantly among the three groups of epibiotic bacteria. The Shannon index was highest for MVMethylococcales (10.25 to 36.75%), Campylobacterales (4.32 to 34.32%), and Thiotrichales (6.28 to 51.29%) were all in the top three orders in each community, although their ranks varied. In the SCsetae epibiotic bacterial communities, Thiotrichales accounted for the highest relative abundance (42.25%). In contrast, in the MVcarapace and SCcarapace epibiotic bacterial communities, the relative abundance of Thiotrichales  was lower than that of Methylococcales  or Campylobacterales . Furthermore, the ranking of the top three orders was similar between the epibiotic bacterial communities of MVcarapace and SCcarapace. The bacterioplankton community in the ENV sample wasdominated by Alteromonadales (15.99%), Campylobacterales (15.03%), and Methylococcales (14.77%). Notably, Alteromonadales was only exclusively dominated the bacterioplankton community, and the proportion of Thiotrichales was much lower , Thiotrichales mainly involved ASV3 , and Methylococcales mainly involved ASV2 (genus Marine_Methylotrophic_Group_2). ANOSIM revealed significant differences in the epibiotic bacterial communities within each of the three groups (p < 0.01). The SCsetae epibiotic bacterial community was highly separated from the others based on PCoA and heatmap clustering , associated with sulfide oxidation, in SCsetae seemed to be a major source of variation among the groups  associated with sulfur oxidation, was much higher in SCsetae than MVcarapace and SCcarapace, which might indicate an enrichment of specific sulfur-oxidizing bacteria in SCsetae.To further explore the compositional differences at the ASV level, PCoA, heatmap analysis, and bacterial co-occurrence network analysis were used . The domustering ,B. Accore groups . There w results . The bac results , which mThiotrichales, Methylococcales, and Campylobacterales; M. verrilli and the first comparative study of the epibiotic bacterial communities on Munidopsidae species in cold seeps.Cold seeps harbor thriving communities of unique organisms, supported mainly by chemosynthetic microbes  could be scraped by the host to use as food  accounted for a high proportion of the epibiotic bacterial communities (>60%), while the proportions of these orders were less in the bacterioplankton community (<40%). These differences indicated that the epibiotic bacteria were not random recruited from the bacterioplankton.To identify the enriched taxa, we performed various analyses at the ASV level. Studies have shown that mutualistic host–microbe relationships likely exist for verrilli . Additioverrilli . TherefoThiotrichales) among the epibiotic bacterial communities is striking. Thiotrichales, which are often involved in primary production in chemosynthetic communities  was strikingly higher for SCsetae than MVcarapace and SCcarapace, which might indicate that sulfide oxidation was quite important in the formation of epibiotic bacterial communities on S. crosnieri setae.Notably, a recent study found that chemoautotrophic bacteria belonging to Gammaproteobacteria  could coevolve with their hosts, whereas Campylobacteria  had a weak preference for hosts  in SCsetae and explains the high relative abundance, across all samples, of ASV1 , which is also a sulfur-oxidizing bacteria like ASV3 .The difference in the relative abundance of ASV3  of the host can indirectly deepen the symbiotic relationship to increase the productivity of its episymbionts and the adaptation of the host, such as the yeti crab Kiwa puravida  on the setae to support host survival.mpaction . The chempaction . The mulpuravida . Researcts setae . At Sitets setae . Higher M. verrilli, although the relative abundance of chemoautotrophic bacteria was high, their body surfaces are lacking dense setae, and endogenous water flow and dancing behavior have not been reported or observed. These chemoautotrophic bacteria could not form as dense community as S. crosnieri. Previous studies indicated that M. verrilli tended to be opportunists and had broader feeding targets . We hypothesize that the presence of setae shaped the structure of the epibiotic bacterial communities and that the numerous chemosynthetic epibiotic bacteria tightly adhering to SCsetae form an ideal biofilm, in contrast to the relatively sparse and simple epibiotic bacterial communities on carapaces, improving metabolic efficiency and the overall primary productivity.As for  targets . This misetae group and the higher relative abundance of ASV3 , we suggest that the morphology of S. crosnieri might allow the epibiotic bacterial community to form a bushy jungle in the plumose setae, which improves primary production and provides S. crosnieri with sufficient nutrition by selectively obtaining sulfur-oxidizing episymbionts from the environment. Consistently, S. crosnieri clusters around the vents in order to use the high chemical fluxes for chemosynthetic reactions. In contrast, M. verrilli has a relatively low dependence on epibiotic bacterial communities for its nutrient supply, so they are more dispersed. Our research showed that the different distributions of S. crosnieri and M. verrilli relate to the differences in their epibiotic bacterial communities, involving differential adaptation mechanisms around cold seeps.In light of the unique composition of the SCThe data presented in the study are deposited in the NCBI repository, accession number PRJNA947936 and PRJNA954131.The studies involving animals were reviewed and approved by the Science and Technology Ethics Committee of the Institute of Oceanology Chinese Academy of Sciences.WF performed the statistical analysis and wrote the first draft of the manuscript. MW and CL contributed to conception and design of the study. ZX, DD, MH, ZZ, HZ, and LF contributed to sampling. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China, grant/award number: 42076091 and 41906124, the NSFC Innovative Group Grant (No. 42221005), the National Key R&D Program of China (2022YFC2804003), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA22050303 and XDB42020401).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.