Results: research results have shown that 100% replacement of river sand with finely ground natural white sand, in concrete containing 20% of the mass as part of a multicomponent binder, fly ash and from 5 to 15% by weight of silica, contributes to the increase of its strength properties: the values of concrete compressive strength after 28 days were in the range from 118.5 to 128 MPa, tensile strength during bending and splitting, respectively, from 18.8 to 25.4 MPa and from 10.2 to 11.9 MPa, which is higher than the strength of concrete samples containing river sand. Conclusions: the achieved results have demonstrated the efficiency of using finely ground natural white sand as an alternative to river sand for producing high-strength concrete, thus helping to save the river sand resources in Vietnam. The use of fly ash and micro silicon, which are power and metallurgy wastes, as part of a multicomponent binder in order to partially replace cement reduces the carbon footprint in the production of binders and will also have a beneficial effect on environmental protection against industrial waste pollution.Introduction: the article discusses the effect of the complex of active mineral additives consisting of silica and fly ash, and a fine aggregate, including finely ground natural-white quartz sand for partial replacement of river sand, on the mechanical properties of high-strength concrete containing steel fiber. Materials and methods: high-strength concrete containing Dramix High-strength concrete is characterized by high compressive strength and is comparable to such high-strength natural rocks as basalt ,2,3. TheImportant characteristics of high-strength concretes are strength, durability and economy. High strength can be achieved by adding finely dispersed materials with pozzolanic properties to the concrete mixture , modern super- and hyper plasticizers, by the use of micro-reinforcing fibers , low water-to-binder ratio, increased cement content, as well by the longer mixing during concrete mix preparation and heat treatment of products and structures.High-strength fiber-reinforced concrete (HSFRC) can be viewed as the result of combining three concrete technologies: self-compacting concrete (SCC), fiber-reinforced concrete (FRC), and high-performance concrete (HPC) ,10. HighThe high strength of high-strength concrete is achieved by improving its production technology and the used materials (by adding ultra-thin pozzolans), as well as by a very low water–cement ratio, the use of highly effective superplasticizers, a high binder content, and an optimal volume of high-strength steel fibers. The use of steel fiber is aimed at preventing the growth and development of microcracks by reducing tensile stresses, which prevents their association into macrocracks ,14. The In the concrete mixture, the binder content is almost 40% of its total. At the same time, micro silica accounts for almost 25% of the binder mass, and its amount by mass can reach 30% ,18. The Quartz flour is another fine filler, which accounts for 8.4% of the total mass of the mixture. Quartz flour has an average particle diameter slightly smaller than that of Portland cement, which allows it to fill possible voids between sand grains, cement particles, and hydration products and thereby seal the structure of concrete cement stone, which increases its strength and reduces permeability. However, the use of finely ground quartz materials may not be required due to a significant proportion of non-hydrated Portland cement, which fills voids and forms a dense matrix of cement stone. Karine V and Maximilien et al.  found thFor high-strength concretes, the filler is an important component, as its content is almost 50% from the mass, and affects the concrete’s strength and cost. The grain size of the aggregates also has a general effect on the concrete’s compressive strength. The composition presented in 3 of superplasticizer to be suitable for producing concrete mix with 360 mm cone flow.The use of water-reducing superplasticizer (HRWR) is necessary to obtain high-strength concrete and its dosage can vary Gerlicher et al.  determinIt is of interest to study the possibility of obtaining concrete containing steel fiber and having high strength by using a developed complex of active mineral additives consisting of silica and fly ash, as well as a fine aggregate including finely ground natural white quartz sand, still little used, in order to partially replace the scarce river sand in Vietnam, and to investigate the mechanical properties of the resulting high-strength concrete. In addition, the use of fly ash and silica, which are energy and metallurgy wastes, as part of a multicomponent binder for the purpose of partial replacement of cement will help reduce the carbon footprint in the production of binders and will also have a beneficial effect on environmental protection from industrial waste pollution.High-performance concretes have a high particle packing density contributing to low porosity, high mechanical strength, and impermeability. The composition of high-performance concretes, in addition to cement, fine aggregates, and additives, also includes steel fiber and finely dispersed nanofillers, which, in terms of achieving high strength to compressive and tensile loads, replace coarse aggregates in their composition .The most widely used theoretical model for creating the maximum dense packing of particles in the design of UHPC is the model by Anderson and Andreasen . HoweverThe following raw materials were used in the work.3A content and 21.23% C4AF + C3A, the density of 3150 kg/m3, meeting the requirements of TCVN 7711 [The binder is the sulfate-resistant Portland cement PCsr40 manufactured by Luks Cement Ltd.  with 3 and 28-day compressive strength, respectively, 34 MPa and 50 MPa, the setting onset of 130 min and the setting end of 170 min, 2.49% CCVN 7711  and GOST®PP1–Sika Limited , with the particle size of <0.1 µm, the true density of 2150 kg/m3 according to TCVN 8827-2011 [Active mineral additives with pozzolanic reactivity and facilitating structure densification are: micro silica Sikacrete827-2011  and GOST827-2011 , as well827-2011  and GOST827-2011 . The che3 and the particle size modulus of 2.91; natural white sand with the average particle size of 0.1–1.8 mm compliant with the requirements of TCVN 7570-2006 [Fine aggregate-quartz sand from the Huong River, with the true density of 2650 kg/m570-2006 , TCVN 10570-2006 , and GOS570-2006 , and fin®ViscoCrete®151–Sika (Vietnam), with the density of 1.09 kg/L, according to TCVN 10302-2014 [Superplasticizer (SP) Sika302-2014  and GOST302-2014 .®3D 65/35 steel fiber with 0.55 mm diameter (D), 35 mm length (L), and L:D ratio ≈ 65 —the percentage of particles that can pass the sieve with smaller than D; iD—average particle size, mm; maxD—maximum particle size, mm; minD—minimum particle size, mm; q—the particle size distribution modulus.In this study, this modified model was used to design the specific matrix in accordance with Equation (1):RSS—residual sum of squares (at defined particle sizes); mixP—composed mixture; targetP—the target mix of the granulometric composition calculated by Equation (1); n is the number of points (between minD and maxD) used to calculate the deviation.Using Equation (1), it is possible to design different concrete compositions, taking different values of q, which determines the percentage ratio between small and large particles in the concrete mixture. Brouwers and Radix  suggesteR2, as shown in Equation (3), since this factor provides the correlation value between the gradation of the target curve and the represented concrete mix:The quality of the fit of the resulting cumulative particle size distribution curve is evaluated by using the determination factor However, the dry particle packing method does not account for the effects of water and HRWR. In practice, water and superplasticizer play an important role in particle packing and therefore affect the properties of UHPC .2.Packing method for wet particlesThe high friction of dry particles prevents their packing density increase . The preTherefore, when using the model of wet packing of particles, higher packing density will be achieved as compared to the dry packing model , since tw is the water density, ρα, ρβ, and ργ are the densities of the solid components of various binders; Rα, Rβ, and Rγ—volumetric ratios of the solid components of various binders.The high wet packing density improves the macro002Dmeso-micropore structure, resulting in the increase of the compressive strength of UHPC . The pacThe authors of  shows anThe developed compositions of high-strength concretes using industrial waste are presented in In order to obtain UHPC, it was necessary to follow the following sequence of steps in preparing the concrete mixture: (1) fine aggregates were mixed in a dry state for 2 min for preventing agglomeration, as well as evenly distributing fine particles; (2) thereafter, adding binders, including cement, fly ash, and micro silica, followed by 5 min of mixing; (3) steel fibers were then gradually added and stirred for another 3 min until these fibers have been completely dispersed; (4) then, continuing stirring for about 10 min, gradually adding the solution of superplasticizer previously mixed with water to the resulting mixture; (5) the workability of the prepared concrete mixture was determined by the flow of the cone, which should be in the range from 280 to 310 mm without delamination, and then filled with the concrete mixture of the form.The consistency of the concrete mixture was determined according to TCVN 12209-2018 standards  and GOSTThe test results are illustrated in We consider the change in compressive strength of the studied concretes with and without natural white quartz sand, obtained as the result of hardening for the concrete mixtures of twelve developed compositions. These compositions containing steel fiber , dependiUsing fly ash has reduced the compressive strength values as compared to the test composition (UC) at the early age of concrete hardening, which can be explained by the cement content decrease in the binder composition. It can be seen that the increase in the fly ash content is accompanied by the increase in the compressive strength of concrete samples at the longer age, namely, the compressive strength decrease at the age of 7 days ranged from 13 to 39%, while, in contrast, at the hardening age of 120 days, we observe the strength increasing by 5–18% as compared to the test concrete composition containing multicomponent binder from 20 to 40% from the mass fly ash to replace part of the sulfate-resistant Portland cement. This indicates the gradual increase in the strength of such concretes in longer hardening periods. This trend can be probably explained by the cumulative effect of cement hydration in combination with the pozzolanic reaction of the used fly ash, leading to a gradual increase in the content of low-basic calcium hydro silicates in the composition of the concrete cement stone ,55,56. F2):The product of the C-S-H pozzolanic reaction not only improves the adhesion between the cement stone and the surface of fine aggregate grains, but also results in compaction of the concrete structure. Thus, the pozzolanic reaction has the double effect: the increase in the compressive strength of concrete and the decrease in the total pore volume in its structure. In addition, micro silica particles < 0.1 µm can fill voids created by free water in the matrix.The rapid increase in compressive strength of concretes containing river sand was observed at the age from 3 to 28 days. At the same time, the increase of strength at 28-day age as compared to 3-day strength with the micro silica content of 7.5, 10, 12.5 and 15% from the mass amounted to 56.3%, 43%, 56.9%, and 56%, respectively. After 28 days, these concretes showed a slight increase in strength, which by 120 days reached values of 106, 108, 109, and 117 MPa, respectively. The highest compressive strength (137 MPa at the age of 120 days) was obtained with a micro silica content of 7.5% from the mass of the multicomponent binder. However, the decrease of the concrete compressive strength was observed with the larger increase of the micro silica content. The obtained results can be due to the fact that, with adding the larger amount of micro silica in place of sulfate-resistant cement, the part of it is formed without entering into a pozzolanic reaction with the formation of low-basic calcium hydro silicates strengthening the cement stone of concrete. In addition, their amount is reduced due to a decrease in the cement content in a multicomponent binder. Thus, the obtained experimental results showed that the optimal content of micro silica in the concrete mixture is 7.5% from the mass of the multicomponent binder, other things being equal.The axial tensile strength of the developed concretes using white and river sand as a fine aggregate was determined after 28 days of hardening of the samples . The resThe tensile strength in bending of 28-day age concrete samples was 9.5–29.2 MPa . When thWith the use of the local Vietnamese raw materials, high-strength fiber-reinforced concretes can be obtained, of the high potential for use in the different constructions. The conducted studies have discovered that the highest compressive strength, equal to 137 MPa at 120-day age, as well as the axial tension and tensile bending equal to 11.9 MPa and 29.2 MPa, respectively, at 28-day age, were shown by the concrete composition with natural white quartz sand, contained in the multicomponent binder composition based on sulfate-resistant Portland cement 7.5% from the mass microsilica combined with 20% from the mass fly ash from thermal power plants. Thus, it was determined that the optimal content of micro silica and low-calcium fly ash in a multicomponent binder is 7.5% and 20% from the mass, respectively.It has been established that using the natural white sand with grain size from 5 to 1800 μm increases the concrete strength characteristics as compared to river sand. Therefore, it is efficient to use white sand for producing concretes of high strength characteristics. The above use will also contribute to protecting the local river sand resources from depletion, which is relevant for Vietnam.The use of local waste from the power and metallurgy industries in the form of fly ash from thermal power plants and micro silica as a partial replacement for Portland cement in multicomponent binder compositions reduces the carbon footprint of the cementing components production and helps to protect the environment from industrial waste pollution. In addition, it is beneficial in terms of saving power resources and reducing the concrete cost.Based on the results of the conducted research, the following conclusions can be drawn:


MET-targeting antibody–drug conjugate that has demonstrated a good treatment response in patients with EGFR wild-type MET-overexpressing non-squamous non-small cell lung cancer. However, patients have been reported to acquire resistance to this drug, and the subsequent therapy has not been standardized. Here, we present a case of a 56-year-old woman diagnosed with KIF5B-MET fusion-positive non-small cell lung cancer who had a durable response to capmatinib after acquired resistance to telisotuzumab vedotin.Telisotuzumab vedotin is a  EGFR) wild-type MET-overexpressing non-squamous non-small cell lung cancer (NSCLC)  . HoweverEGFR, and the immunohistochemical staining for ALK and ROS1 was negative. Concurrent chemoradiotherapy followed by 12-month durvalumab consolidation was administered, and the tumor showed significant shrinkage. Eleven months after completion of durvalumab therapy, the patient experienced disease progression with enlargement of the left axillary lymph node. Sonography-guided biopsy revealed an adenocarcinoma. Next-generation sequencing (NGS) by the QIAact Lung All-in-One assay revealed KIF5B-MET fusion . Thus, capmatinib was administered, and further imaging showed a dramatic response after 3 months of therapy. The therapeutic response was determined based on the radiographic evidence according to Response Evaluation Criteria in Solid Tumors version 1.1 (However, 8 months later, the patient experienced disease progression again with new-onset left chest wall metastasis. Re-biopsy showed adenocarcinoma, and NGS using FoundationOneutations Table 1,MET fusion is a rare oncogenic driver mutation in NSCLC, which comprises only 0.5% of the total number of patients with lung cancer (MET gene partners have been described in lung adenocarcinomas, including KIF5B, HLA-DRB1, UBE2H, CD47, ATXN7L1, SPECC1L, and CAV1 (MET exon 14 skipping mutation, MET fusion induces ligand-independent activation of downstream signaling pathways, resulting in cellular proliferation, survival, migration, and angiogenesis (MET fusion might also result in overexpression of MET protein on the cell surface. In a study conducted by Gow et al., there were two NSCLC cases with the KIF5B-MET rearrangement, which is a fusion between exons 1-24 of KIF5B and exons 15-21 of MET. The loss of exon 14 in MET results in a lack of the juxtamembrane domain of the MET protein, leading to its overexpression during immunohistochemical staining, which is secondary to failure of ubiquitin-dependent protein degradation (KIF5B-MET rearrangement with a fusion between exons 1-24 of KIF5B and exons 15-21 of MET (KIF5B-MET rearrangement also contain the exon 1-15 of the KIF5B, which preserves the kinesin motor and coiled-coil domains that mediate homodimerization and subsequent activation of MET signaling pathway (KIF5B is also an active promoter, which had been reported to activate and enhance the downstream oncogenic pathway of ALK and RET (g cancer , 5. Thisg cancer . Severaland CAV1 . Similarogenesis . In addi pathway . The KIF and RET , 11.KIF5B-MET fusion, and no other oncogenic driver mutation was discovered, which implies that the tumor might be responsive to targeted therapy. In a in vivo study, the lung cancer xenograft model of KIF5B-MET fusion exhibited a good treatment response to crizotinib, a type Ia MET-TKI (de noo MET fusion (MET fusion could also be a resistance mechanism in patients with EGFR-mutant NSCLC who experience disease progression after treatment with EGFR-TKI. The combination of EGFR-TKIs and MET-TKIs can provide clinical benefits (MET-TKIs, including tepotinib and capmatinib, have demonstrated their effectiveness and have been approved to treat patients with MET exon 14 skipping mutation-positive NSCLC (in vitro study (MET fusion. In the present case report, our patient experienced significant tumor shrinkage after receiving capmatinib, which implies that capmatinib could be a potential salvage therapy for patients with NSCLC with MET fusion and acquired resistance to telisotuzumab vedotin.The therapeutic strategy after acquiring resistance to telisotuzumab vedotin remains to be standardized. In our case report, NGS of the re-biopsied tumor revealed  MET-TKI . SeveralT fusion , 12–15. benefits , 17. Recve NSCLC . Capmatiro study ; howeverMET fusion could potentially respond to capmatinib treatment. More importantly, it could be used as salvage therapy for patients with acquired resistance to telisotuzumab vedotin. Further prospective clinical trials are warranted to validate these results.In summary, our case report highlights that patients with The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by The Review Board and Ethics Committee of National Cheng Kung University Hospital. The patient provided their written informed consent to participate in this study.C-YL, S-HW, and P-LS had full access to data in this case report and takes responsibility for the integrity and accuracy of data analysis. Y-LC and C-LH contributed to the genomic data analysis. C-TL, S-YW, C-LH, DP, P-LS, and C-CL contributed to the scientific review and final approval of this manuscript. All authors contributed to the article and approved the submitted version.The present study was funded by grant no. MOST 110-2314-B-006-102, MOST 111-2314-B-006 -092-MY3 from the Ministry of Science and Technology, Taiwan, and grant no. NCKUH-11102019 from National Cheng Kung University Hospital, Taiwan.The patient involved in this case report gave his informed consent authorizing use and disclosure of his health information. We also thank the Molecular Medicine Core Laboratory, Clinical Medicine Research Center, National Cheng Kung University Hospital for technical support, experimental design, and data analysis with the GeneReader NGS system.DP is employed by Foundation Medicine Inc.The remaining 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.


Wolbachia endosymbionts. After the symbionts have been removed, the hosts must recover for some generations from the side effects of the antibiotics. However, most studies do not assess the direct and indirect longer‐term effects of antibiotics used to remove Wolbachia, which may question the exact contribution of this endosymbiont to the effects observed. Here, we used the fly Drosophila nigrosparsa treated or not with tetracycline for three generations followed by two generations of recovery to investigate the effects of this antibiotic on the fly locomotion, wing morphology, and the gut microbiome. We found that antibiotic treatment did not affect fly locomotion two generations after being treated with the antibiotic. In addition, gut‐microbiome restoration was tested as a more efficient solution to reduce the potential side effects of tetracycline on the microbiome. There was no significant difference in alpha diversity between gut restoration and other treatments, but the abundance of some bacterial taxa differed significantly between the gut‐restoration treatment and the control. We conclude that in D. nigrosparsa the recovery period of two generations after being treated with the antibiotic is sufficient for locomotion, and suggest a general assessment of direct and indirect effects of antibiotics after a particular recovery time.Antibiotics, such as tetracycline, have been frequently used to cure arthropods of  Drosophila nigrosparsa and differentiation of wing shape and differences in bacterial diversity between the control and gut‐restoration groups three generations after tetracycline treatment.We found no effect of tetracycline on locomotion in  The order of the larvae was chosen randomly. The locomotion of each larva was recorded for three min using a Sony XR155 Full HD video camera (Sony). The total crawling distance (mm) and mean speed (mm s2.1.5Drosophila Activity Monitor (DAM5M) device (Trikinetics). RING allows for differentiation between walking and jumping, and DAM5 allows for assessing the number of moves over long periods. For each experiment, 20 two‐week‐old female flies of each line were randomly selected, anesthetized with CO2 for sexing three days before the experiment, and were put into separate vials with malt food without any further treatment. For both methods, flies were put at room temperature (19°C) for one h before the start of the experiments. The experiments took place between 9:00 AM and 1:00 PM.Two methods were used for the adult locomotion experiments in Generation 6, the Rapid Iterative Negative Geotaxis (RING) assay , the flies were transferred individually into heptane‐cleaned vials  and clamped in random order in the RING apparatus. The fly‐containing vials were tapped quickly on the table, and the locomotion activities  of flies were video recorded for three min with a video camera . All fly lines were included in every run. The recorded videos were analyzed with ImageJ version 1.53c , each fly was transferred into a glass vial and placed in the DAM5M in random order. All fly lines were included in every run. The activities were measured every 20 s for an hour. The number of moves was detected automatically once a fly cross the infrared beam. The recorded data were then checked with DAMFileScan111X version 1.11 (Trikinetics). The statistical analyzes were the same as for the larvae.2.1.6http://www.sbmorphometrics.org/soft-utility.html). Thirteen landmarks were digitized manually using tpsDIG2w32 version 2.31 (http://www.sbmorphometrics.org/soft-dataacq.html) on every photo . The wing photos were converted to a tps file using tpsUtil32 version 1.79  were checked in Generation 6. We did not analyze the microbiome of the control flies in Generation 2 because we assumed that all bacterial communities would be stable over time as they were reared in the same controlled environment. Each fly was killed in liquid nitrogen, surface‐sterilized using 2.5% bleach for 2.5 min, and washed twice with sterile MilliQ‐water, each for one min .In larval locomotion, all treatments had similar crawling speed and distance Figure , and theχ2 = 10.30, p = .11; jump, χ2 = 4.24, p = .64).In the walk and jump activities (RING locomotion assays), flies of all treatments on average walked around three times and jumped 0.5 times in three min Figure , which rχ2 = 2.23, p = .90).In the move activity (DAM5M locomotion assays), the control moved the most, followed by gut‐restoration and antibiotic‐treated treatments. However, no significant difference among treatments was found .F1,4 = 0.07, p = .80; shape F22,88 = 0.13, p = 1.00), the antibiotic‐treated treatment , and the gut‐restoration treatment .No significant differences in size and shape were found between left and right wings of the flies of the control treatment  and in all pairwise comparisons of treatments ; between antibiotic‐treated and gut‐restoration treatments, R was 0.01 (p = .08); and between control and gut‐restoration treatments, R was less than 0.01 (p = .57).The significant difference in wing shape among treatments we found, however, was not clearly projected by CVA. CVA showed that the gut‐restoration treatment overlapped more with the antibiotic‐treated treatment on the first axis, and the antibiotic‐treated treatment overlapped more with the control on the second axis Figure . The ANO3.3After trimming, the forward and the reverse sequences were 280 and 220 bases long, respectively. A minimum merged read was 37,034 from the blank sample and a maximum of 390,048 reads from the −T3 sample of Generation 6. The cell and DNA mock communities revealed a minor extraction and sequencing error. The mock cell extraction deviated by a total of 5% and 1% from the relative abundance of the mock cell and mock DNA community standards, respectively Figure .p = .04) but not for other comparisons   Figure .R = 0.74) .Beta diversity using NMDS based on ASVs showed a separation between the antibiotic‐treated samples from Generation 2 and all the samples from Generation 6 (ANOSIM ) Figure . Yet, saLactobacillus (phylum Firmicutes) were the most dominant bacteria of the antibiotic‐treated treatment in Generation 2. After recovering, Acetobacter became the most dominant genus in the antibiotic‐treated treatment, whereas Lactobacillus was the second most abundant  had significantly higher abundances in Generation 2 (while being treated with the antibiotic) than in Generation 6 (after recovery).In Generation 6, several nt Table . In contp = .07), and taxa with significantly different abundances between the gut‐restoration treatment and the control decreased from twelve to only eight; however, still, no significant difference between antibiotic‐treated and gut‐restoration treatments arose , the restoration method could be considered successful as there were no significant differences in alpha diversity among treatments. In any case, some taxa still differ significantly between the gut‐restoration treatment and the control also after potential removal of that sample.We found significant differences in wing shape among treatments  but no significant effect of tetracycline on larval and adult locomotion of Wolbachia‐infected flies had higher locomotion activities than antibiotic‐treated flies  and Acetobacter (Proteobacteria) of the antibiotic‐treated treatment was significantly different between Generations 2 and 6. Lactobacillus and Acetobacter had the highest relative abundance during and after the antibiotic treatment, respectively. There was no genus with the same abundance across treatments as another one. Thus, taxa not influenced by antibiotics or antibiotics plus restoration were potentially resistant to the antibiotic, but not as competitive as Acetobacter. A decrease in Proteobacteria and an increase in Firmicutes during antibiotic treatment were observed in earthworms (Chao et al., Lactobacillus species, during tetracycline treatment, is that Firmicutes bacteria have higher resistance to tetracycline than other bacteria because they have a high number of tetracycline‐resistance genes (Berglund et al., Laodelphax striatellus (Zhang et al., Lactobacillus and the increase of Acetobacter in generation 6 might be due to competition between them (Wong et al., The abundance of several bacterial taxa such as The significant differences in the abundances of some bacterial taxa between the control and the antibiotic‐treated treatment in Generation 6 mean that two generations after the last antibiotic treatment might not be enough for the gut microbiome to recover, and the differences between the control and the gut‐restoration treatment suggest that the gut microbiota of the gut‐restoration samples possibly was not fully restored Table . NeverthD. nigrosparsa after two generations of recovery. Assessing the effect of a tetracycline treatment lasting more than three generations would be a good test of the potential effects of antibiotics on wing morphology. In any case, our study illustrates the importance of assessing both direct and indirect effects of any antibiotic after a particular recovery time, before or while assessing the effect of Wolbachia infection.In summary, we found a significant difference in wing shape among treatments, and there were no significant differences in neither larval nor adult locomotion. There were some differences in alpha diversity and abundances of the gut microbiome among treatments. We cannot make a clear statement that the difference in wing morphology we found was an effect of differences in the microbiome as we lack firm evidence, but we can conclude that there was no effect of tetracycline on locomotion activities of Simon O. Weiland: Data curation‐Equal, Formal analysis‐Equal, Writing—original draft‐Lead, Writing—review & editing‐Equal. Matsapume Detcharoen: Data curation‐Equal, Formal analysis‐Equal, Writing—original draft‐Equal, Writing—review & editing‐Equal. Birgit C. Schlick‐Steiner: Conceptualization‐Equal, Formal analysis‐Equal, Funding acquisition‐Equal, Project administration‐Equal, Supervision‐Equal, Writing—review & editing‐Equal. Florian M. Steiner: Conceptualization‐Equal, Formal analysis‐Equal, Funding acquisition‐Equal, Project administration‐Equal, Supervision‐Equal, Writing—review & editing‐Equal.None declared.None required.


The basic tests performed included tests of the compressive strength of concrete, and of the split and flexural tensile strength for different dosing amounts. Within the structural tests of reinforced concrete beams without shear reinforcement, two variants of spans with different degrees of reinforcement were implemented. Herein, the test results are evaluated graphically with a detailed analysis of the positive effect of fibers, and we also provide general recommendations for the structural uses of the fibers used and the design of fiber-reinforced concrete structures. Among the important results of this experimental program was the observation of a significant increase (of the order of tens) of the percentage of the split tensile strength and an increase of the overall load-bearing capacity of the reinforced concrete beams without shear reinforcement. Among the important aspects of our findings is the fact that a fine-grained concrete mixture was used, which increased resistance to pressure water seepage, and therefore, the effect of shrinkage can be influenced by the method of production and the treatment of the concrete used. We also provide detailed figures of the microstructure.The current findings on concrete with fibers show that research has focused primarily on individual aspects, especially in terms of mechanical properties and structural uses. However, no broader view of the problems solved has been provided. In this study, we present a conceptual overview of a new, comprehensive experimental program for the assessment of fiber-reinforced concrete, which includes the analysis of microstructural and structural elements, as well as specific features such as shrinkage and resistance to pressurized water. The proposed experimental program included several variants of schemes for the dosing of fibers into concrete, using steel fibers that were short and straight. Fiber dosing was performed up to 110 kg/m As part of sustainable development, in technical areas, including construction, people strive to reduce costs or demands in the production of a particular product, while improving its useful properties. One of the options within concrete structures is fiber-reinforced concrete ,2,3,4 orSeveral recommendations, design codes, and standards are available for fiber-reinforced concrete, which provide basic information about this area of knowledge, for example, from the International Federation for Structural Concrete (FIB) ,16, the The research concept presented here is shown in The greater advantage—also related to its disadvantages—of fiber-reinforced concrete is that it allows the individual design of concrete compositions and the dosing of fibers, which makes it difficult to generalize knowledge and limits the possibility of structural design use. It should also be noted that there are many variants of fibers. Selected variants and possible divisions of fiber types for fiber-reinforced concrete are shown in 3 [From the technological point of view regarding the production of concrete and fiber-reinforced concrete, it is necessary to ensure the homogeneity of a structure, and the resulting properties are also significantly affected by the concrete composition , especia3 ,27,28,293 ,31,32,333  and the 3 ,35,36,373 , test st3 ,39,40, b3 , industr3 , and bea3 ,44. Impo3 ,35, and 3 . Adequat3 ,47,48,49However, the current findings in the literature show that researchers and research tasks have focused primarily on individual aspects of fiber-reinforced concrete, especially in terms of its mechanical properties or structural uses. No broader view of the solved problem has been provided, and the partial results of individual studies cannot be easily connected based on previous research, considering the number of variants of fiber-reinforced concrete and the various individual composition designs presented. For example, Katzer  deals wiThe motivation for the present study was, therefore, to provide a comprehensive experimental program for the assessment of fiber-reinforced concrete, including the analysis of microstructural and structural elements, as well as specific features such as shrinkage and resistance to pressurized water.3 to 110 kg/m3 in up to 6 test series. The fiber parameters are presented in The research area of this study was focused on several goals, including basic mechanical properties, bending tests, structural tests, and specialized tests. Specialized tests included the study of the microstructure using optical and electron microscopy, shrinkage tests, and tests of resistance to pressurized water. A concrete matrix based on fine-grained concrete ,44 was cThis section deals with the evaluation of the basic mechanical properties of fiber-reinforced concrete. The mechanical properties selected for study were the compressive strength, split tensile strength, and modulus of elasticity. A cube of nominal size 150 mm × 150 mm × 150 mm was chosen for compressive strength  and spliEach series had three or six samples. The samples were tested after 28 days. The samples were stored in a water bath. Details on the number of samples and the test method are given in Sample photographs from the tests of basic mechanical properties are shown in 3. From the evaluation of the results of the bulk density testing, the effect of the dosing of fibers on the bulk density was clearly evident, gradually increasing from 2205 to 2303 kg/m3. The negative difference in the increase of bulk density was observed only for dosings of 90 and 110 kg/m3, and the difference was minimal. In the case of compressive strength, the positive effect of the fibers was evident.The results of the basic tests are summarized in 3, and the compressive strength increased up to 64.01 MPa from the reference value of 57.1 MPa for plain concrete. However, for higher fiber dosing, the compressive strength began to decrease slightly, but still remained greater than the reference value of plain concrete. The decrease of compressive strength was probably also related to a decrease of bulk density, which results in an increase of porosity. High fiber dosing is characterized by significantly more difficult processing in concrete production.However, the increase of strength was only observed for dosing amounts up to 75 kg/mThe basic tests also included the determination of the static and dynamic moduli of elasticity, which were tested using cylinders. The reference value for plain concrete was determined with regard to the capacity of the laboratory and the small influence of fibers on the moduli. The resulting average static modulus of elasticity was 30.8 GPa, with a select variation coefficient of 3.6%, and dynamic modulus of elasticity 43.3 GPa, with a select variation coefficient of 1.6%.Another important type of tests are bending tests, which make it possible to determine the tensile strength in bending. Load-displacement diagrams are an important output of these tests. As part of the bending tests, three-point tests were performed with a notch of 25 mm and a span of 500 mm . The res3. Three samples (tests) were performed for each dose. The three-point bending test results are shown in 3, the residual strength was relatively small. For fiber dosages of 60 and 75 kg/m3, increases in the residual tensile strength were clearly visible. In the case of a fiber dosage of 110 kg/m3, the effect of tensile strengthening after the formation of the initial crack was noticeable. The final residual strength values observed for deformations of 8 mm were in a relatively small range for all tested specimens. Test samples with a fiber dosage of 60 kg/m3 show higher peak strength than the other samples, but the next section of the load-displacement curve displayed the expected residual tensile strength, between the curves of 40 and 75 kg/m3, as can be assumed. The higher peak load values observed for a fiber dosage of 60 kg/m3 could be caused by the material variability of fiber-reinforced concrete, and it would be appropriate to perform more tests. However, our overall assessment of the course of the curves has a representative character. The addition of fibers always increased the peak value of the load, and the influence of dosage was particularly visible on the residual tensile strengths, i.e., on the downward (softening) curve of the load diagram.Bending tests of fiber-reinforced concrete were performed at fibers dosages of 40, 60, 75, and 110 kg/mFor the results of the tensile tests, it was possible to determine the regression dependence of strength, depending on the dosing of the fibers. A graphical representation of these results is shown in x is the dosage of fibers (kg/m3), and the overall reliability is very high and reaches values greater than 0.996. In the case of bending tensile strength, the resulting regressive analysis is formulated asx is the dosage of fibers (kg/m3), when the reliability is good and reaches a value of around 0.7432.The influence of fibers on the mechanical properties of fiber-reinforced concrete was also analyzed by assessing functional dependence. In the case of split tensile strength, it is possible to formulate the resulting regressive analysis as follows:The study of the microstructure is one of the important sources of information on concrete materials. Particularly in the case of quasi-brittle materials, one can study the microstructure to obtain information about the character of the fracture surface and details about the bonds at the microstructural level. Two approaches were therefore chosen for this area, the first of which was focused on the fracture surface after destructive testing. The partial goal was to focus on inequalities for different components of the material composition of concrete. A digital optical microscope with an extremely large depth of field was used for the measurements, which also allowed the visualization of graphical images.The first selected area is shown in Another part of the study of the microstructure focused on a more detailed depiction of the connection between the concrete and the steel fibers. An SEM (Scanning Electron Microscope), which is an electron microscope that uses a moving electron beam for imaging, was used for this investigation. The specific area between the concrete matrix and the fiber, with a magnification 140×, is shown in 3 was already technologically inefficient and uneconomical. With regard to the limited number of samples and finding the largest possible difference, a dosage of 90 kg/m3 was chosen for the specialized test. The concrete matrix without fibers that was designed could be typically used for repairs or for the water management of structures with increased durability. The depth of penetration with pressurized water was tested on three cube samples made of plain concrete and three fiber-reinforced concrete cube samples with a fiber dosage of 90 kg/m3 and an edge length of 150 mm.Another part of this study focused on finding the effect of fibers on the pressurized water resistance  of concrSchleibinger Testing Systems [3. In both cases, volume changes were observed in three mixtures in measuring troughs measuring 60 × 100 × 1000 mm (with a height of 60 mm) for 42 days, with the mixtures in the two troughs being wrapped in a protective foil throughout the maturation process, and a mixture in one gutter matured in the air  troughs showed a smaller increase of extensibility in the first days, but at the end of the test period, the shrinkage values were incomparably higher than those for the mixtures in the gutters provided with the protective foil. It was also true that the fiber-reinforced concrete samples achieved lower shrinkage values compared to the plain concrete samples. In An evaluation of the results presented in 3.Applications of fiber-reinforced concrete can be found, for example, in reinforced concrete beams without shear reinforcement, where its use can be very advantageous from a technological point of view . For thi3 was. The average maximum load bearing capacity was 108.0 kN for the beams with dosage.A selected image of a small beam specimen after the experiment is shown in 2 = 96.18%. Thus, we can assume a very high dependence of the maximum load-bearing capacity on the dosing of MasterFiber 482 [A regression analysis was performed, and the agreement reached Riber 482  fibers.maxP,600, in the testing machine on the dosing of fibers in the three-point bending test for small beams has the following form:x is the dosing of fibers (kg/m3). The second variant of the bending test was for beams with nominal dimensions of 190 × 100 × 1150 mm (height 190 mm) and a span of 900 mm. As in the previous test, B500B reinforced bars with a diameter of 10 mm, two pieces in number, and a reinforcement cover of 20 mm were chosen. With regard to the dimensions and capacity of the laboratory, tests were performed for fiber dosages of 60 kg/m3 and 90 kg/m3.The function of the dependence of the achieved load-bearing capacity, The mechanism of failure was very similar in both cases. A shear crack formed in the beam. A selected beam after testing is shown in maxP,900, in the testing machine on the dosing of fibers in the three-point bending test for beams has the following form:x is the dosing of fibers (kg/m3). The results of the graph in 3 was approximately 13 kN higher than that of a beam with a dosage of 60 kg/m3, which represents an increase of load-bearing capacity of approximately 17.5%. Based on the results of both bending tests, it can be concluded that the presence of steel fibers in the reinforced concrete structural elements contributes favorably to its load-bearing capacity.The function of the dependence of the achieved load-bearing capacity, This study deals with the field of fiber-reinforced concrete. The introduction contains information about fiber-reinforced concrete, including the most important testing and research methods. The main motivation for this research was to provide a comprehensive view of the research area of fiber-reinforced concrete, ranging from its microstructure to its use in structural elements. The basic part of the manuscript was focused on determining the mechanical properties of fiber-reinforced concrete and monitoring the effect of fiber dosing on these properties, including regression analysis. The tests included a plain concrete mix as a reference. Based on the compressive strength results, it can be stated that with an increasing proportion of fibers in the fiber concrete mixture, the compressive strength also increases, but suitable processing of the concrete mixture must be ensured.3 up to a value of 4.18 MPa. The very positive effect of the fibers was documented by three-point bending tests, and residual tensile strength after peak loading was evident.The split tensile strength increased from 2.99 to 5.88 MPa, i.e., almost twofold. The increase of split tensile strength was significantly pronounced already for the dosage of 40 kg/mThe bulk density of the concrete sample must also increase. However, a steady increase of bulk density was not achieved for all dosages. With regard to the primary use of steel fibers in concrete, the results of split tensile and bending strength tests are more relevant.One of the important parts of this study was the study of the microstructure, which verified that the fracture surface showed relatively small differences. In addition, elemental composition analysis was also performed for selected areas. We have provided detailed images of the matrix between the concrete and the steel fiber. The results of the analysis were in agreement with the expected assumptions, displaying the expected mechanical properties and resistance.Among the important results of this experimental program, we verified the effect of the use of fibers on volume changes in concrete with respect to different environments (foil/air). The fibers had a significant positive effect on the elimination of volume changes. Based on the measured depths of penetration with pressurized water, we found that the presence of fibers in the concrete mix can adversely contribute to its watertightness, which may result in a reduction in its durability. However, the resulting water penetration values were minimal and a high resistance of concrete against pressurized water was observed. Through implementing similar studies of the surface and structure, it can be stated that the increased depth of leakage could probably be eliminated in a more suitable way through a final surface treatment.3 were selected for the testing of structural elements. The structural elements were reinforced concrete beams without shear reinforcement, which can present a typical suitable use of fiber-reinforced concrete. Based on the results, it can be stated that increasing the content of steel fibers from 60 to 90 kg/m3 increased the load-bearing capacity by approximately 17.5%. However, in comparison with reinforced concrete beams made of plain concrete, the overall load-bearing capacity was increased by almost twofold.After the basic tests of mechanical properties, dosages of 60 and 90 kg/m3, it is necessary to realize that the workability of the concrete mixture deteriorates significantly. It is also important to monitor the even distribution of the fibers very carefully. The samples can also have a more problematic surface treatment, which greatly influences the results regarding the resistance to penetration with pressurized water. There can also be problems with the compaction of the mixture itself.In the case of dosing steel fibers above 90 kg/mThe research program implemented here included experiments and results assessing both microstructural and structural elements, as well as specific tests on features such as shrinkage and resistance to pressurized water. The results and information obtained through the research program can be described in the following partial conclusions.The positive effect of fibers on the tensile strength of fine-grained concrete with aggregates up to 4 mm was verified. With higher dosages of fibers, the tensile strength was also greater, both in the split tensile tests and in the bending tests. The deformation capacity of concrete was increased significantly.In the bending test, the effect of higher dosing on the post-peak course of the load-displacement diagram was clearly visible.3 are very difficult to process.For the practical application and dosing of fibers in concrete, the production technology is limiting, and dosages above 90 kg/mTypically, higher fiber dosing can adversely affect compressive strength, which can also result in a change in the microstructure, a pore volume increase, or a slight increase or decrease of bulk density.The advantages of using fiber-reinforced concrete include the fact that it contributes to reducing the impact of the shrinkage of concrete, as has been experimentally verified.The results of the microstructure analysis were in agreement with the expected assumptions regarding structural bonds. The analyzed composition corresponded to the assumptions, exhibiting the expected mechanical properties and resistance. The results of the experimental program provide a new perspective on the detailed microstructure of concrete and fiber-reinforced concrete.In the case of the requirement of resistance to pressurized water in fiber-reinforced concrete, it is necessary to very carefully check the processing technology and the final surface treatment used. The use of fibers in concrete can affect the resistance to pressurized water locally.The very positive effect of the use of fibers was verified by experiments of reinforced concrete beams without shear reinforcement, with tests performed for different spans and cross-sections. The experiments reported here are suitable for numerical modeling, and the authors will focus on this area in further research.


Lobaria pulmonaria has more than 800 associated bacterial species, a microbiome which has been hypothesized to increase the lichen’s fitness. We artificially exposed samples of L. pulmonaria to antibiotics and a stepwise temperature increase to determine the relative effects of antibiotic treatments vs. temperature on the mycobiont and photobiont gene expression and the viability and on the community structure of the lichen-associated bacteria. We found that the mycobiont and photobiont highly reacted to different antibiotics, independently of temperature exposure. We did not find major differences in bacterial community composition or alpha diversity between antibiotic treatments and controls. For these reasons, the upregulation of stress-related genes in antibiotic-treated samples could be caused by genotoxicity in L. pulmonaria and its photobiont caused by exposure to antibiotics, and the observed stress responses are reactions of the symbiotic partners to reduce damage to their cells. Our study is of great interest for the community of researchers studying symbiotic organisms as it represents one of the first steps to understanding gene expression in an endangered lichen in response to exposure to toxic environments, along with dynamics in its associated bacterial communities.Antibiotics are primarily found in the environment due to human activity, which has been reported to influence the structure of biotic communities and the ecological functions of soil and water ecosystems. Nonetheless, their effects in other terrestrial ecosystems have not been well studied. As a result of oxidative stress in organisms exposed to high levels of antibiotics, genotoxicity can lead to DNA damage and, potentially, cell death. In addition, in symbiotic organisms, removal of the associated microbiome by antibiotic treatment has been observed to have a big impact on the host, e.g., corals. The lung lichen  Penicillium chrysogenum, from which the first antibiotic was discovered, penicillin. Even though antibiotics are also naturally produced in ecosystems [Antibiotics are instrumental in medicine and cattle farming. Several organisms can produce antibiotics as a strategy to control population sizes of harmful microbes in their environment, such as the mold fungus osystems , the antosystems ,3. It haosystems ,5. NonetRaphidocelis subcapitata  [Damage associated with exposure to high levels of antibiotics has been reported in several organisms, such as muscle and liver damage in zebra finch , trout ,8 and rapleales) ,16. Asobara tabida, the elimination of their symbiont bacteria Wolbachia using antibiotic treatment influenced their reproduction efficiency, as female wasps were completely incapable of producing viable oocytes [Coccotrypesdactyliperda, as females treated with antibiotics were incapable of laying eggs [Corals previously treated with antibiotics suffer tissue loss and a substantial decrease in photosynthetic efficiency because they lack their symbiotic microbiome . In the  oocytes . This efing eggs . Lobaria pulmonaria (L.) Hoffm. , has more than 800 associated bacterial species and its bacterial microbiome has been hypothesized to be a third partner in the lichen symbiosis [Lichens are traditionally described as self-sustaining symbioses of fungi and algae/cyanobacteria which develop a symbiotic phenotype known as the lichen thallus. The photosynthetic partners, the photobionts, fix carbohydrates (and some fix atmospheric nitrogen) and the fungal partners provide shelter to their photobionts between their peripheral layers, which optimizes photosynthetic functioning . In the ymbiosis . L. pulmonaria responds to sudden temperature increases from 5 to 25 °C by upregulating and differentially expressing some heat shock genes [L. pulmonaria and its green-algal primary photobiont, Symbiochloris reticulata. They found that both symbionts upregulated heat shock genes when there was an unexpected increase in temperature. However, whether other environmental factors such as exposure to antibiotics influence gene expression of the lichen, and if there is an interaction between responses to antibiotic treatments and to temperature increases, remain to be investigated. The tolerance of environmental stress, for example tolerance of exposure to antibiotics in the environment, allows individuals and populations to persevere in changing environments ,36,37,38ck genes . In addick genes  studied L. pulmonaria by both their genotoxicity properties and because they could change the lichen’s associated bacterial communities, as has been previously found in other organisms with a symbiotic microbiome. For those reasons, we analyzed the mycobiont and photobiont transcriptomes of L. pulmonaria samples which had been exposed to antibiotic and temperature treatments to better understand their possible impact on gene expression of the lichens and their associated bacteria. Our main objectives were (1) to identify differential gene expression of lichen samples treated with different antibiotics vs. controls; (2) to analyze differential gene expression of lichens exposed to antibiotics and thermal increases to test the hypothesis that control samples exhibited less severe stress responses at high temperatures than antibiotic-treated samples; and (3) to investigate changes in microbiome abundance before and after antibiotic treatment. To our knowledge, this is the first study to assess the effects of lichen exposure to antibiotic treatments and possible consequences on symbiotic microbiome composition and to quantify gene expression differences of different symbionts. We hypothesized antibiotic treatments could represent a stress factor for Lobaria pulmonaria is an epiphytic species which is rare or locally extinct in most regions of Central Europe, except in some Alpine regions [L. pulmonaria associates with a green-algal primary photobiont, a species belonging to the Trebouxiaceae, i.e., Symbiochloris reticulata (Tschermak-Woess) Skaloud, Friedl, A.Beck and Dal Grande [Nostoc sp.  [L. pulmonaria, with Proteobacteria being the predominant phylum and an especially high abundance of Alphaproteobacteria [L. pulmonaria and S. reticulata reference genomes and transcriptomes have been sequenced and the data have been released by the DOE-JGI Joint Genome Institute. The lung lichen  regions . The mycl Grande  and a mitocales) . There ibacteria ,27,45,46L. pulmonaria is locally abundant. We collected parts of individuals of L. pulmonaria with a large size . The samples were randomly collected from different trees which were at least 2 m apart. Lichen lobes were placed in Petri dishes on filter paper which were kept with a lid on and acclimated in constant light (30–50 μmole m−2 s−1) at 4 °C in the laboratory for 3 weeks. Samples were placed randomly and were sprayed with deionized water frequently. During May 2019, we visited a locality at Rauher Weg  in Hinterstein Valley in the Alps in southern Germany, where Bacterial cells in lichen thalli were visualized using fluorescence in situ hybridization (FISH) following the protocol of Cardinale et al.  with hybBacterial cells in the stained samples were investigated using confocal laser scanning microscopy (CLSM) on a Zeiss LSM 880 (Zeiss) equipped with an argon ion laser and a helium neon laser for excitation of Atto633 (633 nm) and SytoOrange81 (561 nm). Bacterial cells were observed with a C-Apochromat 63×/1.20 W Korr M27 water immersion objective. Zeiss software Zen Black Edition 2.3 SP1 FP1  was used for imaging. −2 s−1) at 4 °C, we split each collected lichen thallus into four  lobes and applied different treatments to each as follows. For ten days, we sprayed the antibiotic treatment once every two days, alternating between sterile deionized H2O and antibiotics (control: same volume of sterile deionized H2O only) in a surface-sterilized fume hood, and the samples were exposed to a daily hydration–dehydration cycle in the 4 °C cold chamber and to constant light. Each of the four lobes originating from a single thallus received a different treatment: antibiotic against (i) Gram-negatives and Gram-positives (Ampicillin); (ii) Gram-negative bacteria (Ciprofloxacin); (iii) mix of Ampicillin and Ciprofloxacin; (iv) sterile deionized H2O (control). The antibiotic treatments were performed in a fume hood as follows: antibiotics were sprayed onto lichen lobes residing in Petri dishes inside a plastic cover with an opening that allowed sample manipulation. After each antibiotic spray treatment, the samples were immediately put back in the cold room at 4 °C. Antibiotics were used at concentrations routinely utilized in lichen culture media (50 µg/mL), and approximately 500 µL was sprayed onto each lichen lobe each time. We repeated the antibiotic treatment three times during the ten-day period (2) from different parts of one lobe (sampling at 4 °C). The samples were frozen in liquid nitrogen and stored until processing at −80 °C. Three samples were taken from each lichen to account for intrathalline variability. Then, we transferred the lichen lobes into a growth chamber at 25 °C and we incubated them there for two hours. After this incubation period, an additional three samples were taken from the lichen lobes from a previously untouched area of the samples as detailed above and the material was flash-frozen and stored as before. The temperatures were chosen based on the recorded temperature spectrum at the sampling sites where L. pulmonaria is locally common and known stress conditions . The ex2O, with a NanoDrop ND-1000 UV/Vis-Spectrophotometer  and a Qubit 2.0 fluorometer employing the RNA HS assay kit . The RNA concentrations varied between 15 and 200 ng/µL. Then, we used DNase 1 digestion  in our RNA samples to eliminate any remaining genomic DNA. All digested RNA samples were adjusted to a concentration of 10 ng/µL.RNA extraction was performed according to the manufacturer’s protocol with the RNeasy Plant Mini Kit . We measured the RNA concentration and quality, after the extracted RNA had been eluted in RNase-free HRNA concentration and quality were checked using a high-sensitivity RNA chip on an Agilent Bioanalyzer. RNA sequencing was executed applying an adapted version of the prime-seq protocol , with soL. pulmonaria mycobiont reference genome , as well as to the reference genome of the green-algal photobiont, S. reticulata . The assembled fungal and algal genomes contained 91.0 and 90.5% of the core eukaryotic genes, respectively.We followed the analysis outline used by Chavarria-Pizarro et al. . First, L. pulmonaria reference genome GTF file [L. pulmonaria and S. reticulata using the same parameters used by 41. We used the ‘tximport’ function from the tximport R-package [Secondly, we used Samtools to convert quality-controlled bam files produced by zUMIs to fastq files using ‘bam2fq’ . The topGTF file . Then, wGTF file  to gener-package  to impor-package . Then, w-package ,58 .We performed principal component analysis (PCA) in R version 3.6.3 using normalized counts (vst). The Adonis test (a randomization/Monte Carlo permutation test) was utilized to test for significant differences in gene expression variation between samples and individuals exposed to different temperatures and different antibiotic treatments. Differential expression analysis was conducted using the DESeq2 package in R. Then, we used the Wald test to compare differentially expressed transcripts between two groups using the same parameters as Chavarria-Pizarro et al. . We carrp ≤ 0.05) for each of the two explanatory variables for the mycobiont and the green-algal photobiont separately . The WGCNA code is included in the R script (see above).Weighted Gene Co-Expression Network Analysis (WGCNA)  was perfLobaria pulmonaria reference genome hosted at DOE-JGI, https://mycocosm.jgi.doe.gov/LobpulSc1/LobpulSc1.home.html, (accessed on 16 August 2021) were used to match differentially expressed (DE) transcripts with protein and GO-term information . We searched for homology of L. pulmonaria mycobiont transcript sequences to protein sequences available for several whole-genome-sequenced species of fungi. For the S. reticulata photobiont, we searched for homology of transcript sequences to protein sequences available for algae . When to et al.  against o et al. ). A locuticulata . The GO We also used the demultiplexed, filtered and cleaned sequences produced by zUMIs software  . In S. reticulata, we obtained 11,326 sequences after filtering low-count transcripts. The sequencing resulted in 3.5 to 9 million reads per sample, with an average of 6 million. The sequence reads resulted in 4.0 to 10.5 million reads per sample, with an average of 7.5 M reads per sample in p ≥ 0.05). Correspondingly, in the photobiont, there was no clear grouping of samples by the type of antibiotic treatment visible in the PCA (p ≥ 0.05), nor between high and low temperatures (p ≥ 0.05). Finally, we found no detectable grouping of individual thalli treated with the different types of antibiotics or the water controls  (p ≥ 0.05) (p ≥ 0.05) (p ≥ 0.05)  , nor tem ≥ 0.05) . Consequ ≥ 0.05)  nor by tA total of 235 differentially expressed transcripts were found in the mycobiont when comparing samples exposed to antibiotic treatments with controls. We found that overall, 110 transcripts were downregulated, and 125 transcripts were upregulated. A total of 49 transcripts had functional information available and could be used to derive functions. In the samples exposed to antibiotic treatments, the upregulated expressed transcripts were identified as Transcription Factors (TFs), oxidation enzymes, protein refolding proteins, DNA repair proteins, ABC multidrug transporters and efflux pumps, and genes encoding stress-activated proteins such as Hog 1 and NST1, NST2, NST3 .https://datadryad.org/stash/share/6ObxvBWNeaKcQ0Fu3Wxse1GN3am0RNwRebaAGlyjIWU, accessed on 15 May 2022). For 24 of the differentially expressed mycobiont transcripts, protein sequence information was available  and “brown” (270) . We found five gene modules positively associated with antibiotic treatments, i.e., “blue” (950) , “pink” (220) , “midnight-blue” (650) , “green” (200) , and “turquoise” (250) . Unfortunately, we did not find GO terms for most of the genes, just for some genes in the modules “yellow” and “turquoise”. According to the functional enrichment analysis, some biological processes that the L. pulmonaria mycobiont downregulated in response to antibiotic treatment in the “yellow” module were carbohydrate metabolic process (GO:0005975) and phosphotransferase activity (GO:0016868). In the “turquoise” module, the process that was upregulated in response to antibiotic treatment was the activity of some transmembrane transporters . In the WGCNA, all 14,885 mycobiont transcripts were assigned into 25 modules, designated by color, with each module containing highly correlated genes A–C. Thes2 = −0.51, p < 0.05), “midnight-blue” (650) , “green” (200) , and “grey” (192)  , the oxidation process  and mitosis   (R < 0.05) . Accordi0051443) . https://datadryad.org/stash/share/6ObxvBWNeaKcQ0Fu3Wxse1GN3am0RNwRebaAGlyjIWU, accessed on 15 May 2022). Our whole-transcriptome level results for the mycobiont were consistent with the results from the pairwise differential expression analysis provided by DESeq2, as there was a higher number of differentially expressed mycobiont genes due to antibiotic treatments than due to the temperature change. All DE transcripts that were found in the DESeq2 analysis were also found in the gene modules associated with antibiotic treatments in the WGCNA  and “turquoise” (400 genes) , were negatively associated with antibiotic treatments . The majority of bacterial OTUs belonged to the phyla Proteobacteria (37%), Cyanobacteria (22%), Chlamydia (11%), Firmicutes (11%), and Actinobacteria (11%) . The mosThe alpha diversity estimators Chao1, ACE, and InvSimpson showed some differences between the temperatures, especially between samples treated with Cipro and Mix . NonetheL. pulmonaria and its green-algal primary photobiont, S. reticulata, when they were treated with antibiotics and exposed to an unanticipated temperature increase. Our study has four major conclusions: (1) L. pulmonaria and its photobiont S. reticulata upregulated some stress-related genes and multidrug transporter efflux pumps when they were treated with antibiotics. (2) The mycobiont and green-algal photobiont had similar responses to the antibiotic treatments. (3) Temperature increases had a lower impact on the mycobiont and photobiont gene expression responses compared with antibiotic treatments. (4) Diversity estimates of bacterial communities did not differ, neither between antibiotic nor temperature treatments. We examined the response of Porites astreoides revealed that gene expression patterns during coral bleaching respond to loss of their symbiont rather than heat stress per se [Peltigera membranacea, along with the downregulation of gene expression of the lectin gene (lec2) at higher temperatures, there was a drop of cyanobacterial DNA repair gene expression [Vibrio shiloi infections and bleaching [Nitocra spinipes, as it caused alterations in their associated microbiome [Organisms in a symbiotic relationship with the microbiome decrease their resistance to abiotic and biotic stress factors when they have been manipulated with antibiotics ,70,71. As per se . In addipression . Similarpression . In contpression ,75,76. Icrobiome . L. pulmonaria and its photobiont S. reticulata upregulated genes associated with stress responses such as TFs, oxidation enzymes and stress-activated protein kinases, which are usually upregulated when organisms are under stress [L. pulmonaria also upregulated genes encoding Hog1 and MAPK, which are common stress response signal pathways that have been found to activate in another fungi under stress conditions [2H2 and the oxidation enzymes cytochrome P450 and GMC have been found to be upregulated when fungi are exposed to stress conditions such as heat, superoxide and osmotic pressure in previous studies [As discussed above, we assumed that antibiotic treatments could lead to lichen genotoxicity and could reduce vitality or cause shifts in species composition of lichen-associated bacterial communities. Our results show no differentiation in the bacterial communities between antibiotic treatments and controls. Nonetheless, r stress ,77,78,79nditions ,80,81,82 studies ,36,41. We found that several transcripts upregulated in the mycobiont and photobiont were efflux pumps. Multidrug resistance (MDR) efflux pump proteins can provide resistance to multiple antibiotics in bacterial cells ,84. ATP-L. pulmonaria and its green-algal photobiont could be due to cell genotoxicity caused by exposure to the antibiotics, and by upregulating these genes, the organisms are trying to reduce antibiotic levels and avoid or repair damage to their cells. Our experimental antibiotic treatments exposed the lichen samples to oxidative stress or genotoxicity, which has been previously reported for several organisms treated with antibiotics ,12,13,14L. pulmonaria mycobiont and photobiont is due to experimental changes in the bacterial communities. However, as we predicted, the antibiotic treatments stressed the lichen, because it has been hypothesized that by diverse functions, e.g., production of specific metabolites, bacteria increase the fitness of lichens. However, the bacterial communities of the samples treated with antibiotics had similar alpha diversity and community composition. This coincided with our microscopic pictures where we were able to find bacteria even after the antibiotic treatment  and stress-induced proteins help repair or remove heat-induced cellular damage by preventing the aggregation of denatured proteins [L. pulmonaria and its photobiont could be under long-term stress when we made the RNA extractions, as they had been exposed for 10 days to antibiotic treatments, and for that reason we may not have been able to detect the exact moment when the organisms overexpressed Hsps. Future research can consider the time frame to characterize in detail the acclimation response of L. pulmonaria and its photobiont to antibiotic treatments [Previous studies have found that ectively ,41. Surpproteins ,105,106,proteins . Elevateproteins ,108,109.L. pulmonaria than in its photobiont. Therefore, it seems as if antibiotic and temperature treatments affected the mycobiont more than the photobiont at the whole-transcriptome level, or maybe the photobiont has a slower response to temperature increases. An interesting finding of the WGCNA of the mycobiont and photobiont is that transcripts related to metabolic processes, catalytic activity and genes related to DNA replication were downregulated and those related to transmembrane transporter activity were upregulated, which coincides with our results of the pairwise comparisons and with past studies which have found that L. pulmonaria shows high levels of transcriptome plasticity to deal with unexpected environmental changes [Temperature and antibiotic treatments played a greater role in the response of the mycobiont of  changes ,41. Four main response pathways allow organisms to cope with stress: antioxidation, osmoregulation, repair of damaged DNA and refolding or degradation of misfolded proteins . These aL. pulmonaria carries out several functions such as stress responses and resistance to toxic abiotic factors [L. pulmonaria, reflected in its gene expression patterns, as the treatments would reduce vitality or cause shifts in species composition of lichen-associated bacterial communities which provide important functions to the lichen. Our results did not find differences between communities exposed to antibiotic treatments vs. controls, which may be attributed to a lack of selectivity in our treatments, as bacteria could be preadapted to deal with high levels of antibiotics produced by the lichen or by microbes in the surrounding environment. Some secondary compounds produced by lichens protect them against chemical and biological negative effects; some of them have antibacterial activity [L. pulmonaria possesses antibacterial activity against both Gram-positive and Gram-negative bacteria [L. pulmonaria are resilient to acute perturbations as they must adapt to deal with antibiotic metabolites produced by their host. In addition, we cannot exclude that we were not able to detect the exact moment when the communities changed or the bacterial population decreased, as the samples had been exposed for 10 days to the antibiotic treatments when we first sampled them. It has been documented that the symbiotic microbiome in  factors ,111,112.Escherichia coli cultures became tolerant to ampicillin by acquiring mutations that extended their lag phase, when populations were subjected to constant exposure to that antibiotic [L. pulmonaria samples treated with different antibiotics. They found that treatments with antibiotics or with deionized water decreased the activity of the corresponding bacteria, which coincides with our microscope pictures (L. pulmonaria were decreasing their active growth as a strategy to increase their tolerance to antibiotic exposure during our experiment. Antibiotic resistance mechanisms in bacteria include mutations that could decrease the binding of the drug to its target and increase expression of efflux pumps, which could diminish active bacterial growth, as bactericidal antibiotics require bacterial activity for the bacteria to be killed . Escheritibiotic ,114. Krapictures . It is pL. pulmonaria. As our experiment did not lead to major differences in community composition or alpha diversity between antibiotic treatments and controls, upregulation of stress-related genes in antibiotic-treated samples could be caused by genotoxicity that exposure to antibiotics could have caused, and that the stress responses associated with antibiotic treatments are a response of the symbiotic partners to reduce damage to their cells. The lack of differences in bacterial communities between antibiotic treatments and controls could be due to the communities’ resilience to acute perturbations, as bacteria associated with L. pulmonaria must adapt to deal with the antibiotic metabolites produced by their host. In addition, it could be that our experimental timing did not allow us to detect the exact moment when the communities changed or decreased. We have demonstrated here that antibiotic treatment has crucial effects on differential gene expression in the mycobiont and photobiont of the lichen Moreover, we did not find any heat shock genes upregulated with elevated temperature, which could confirm the high stress that our antibiotic treatment posed on the lichens, as organisms under chronic stress do not sustain elevated Hsp expression as it is associated with high energy cost. Furthermore, future studies can try to separate the costs and benefits of lichen gene expression using a time series of the response to antibiotic and temperature treatments. Our research is relevant for researchers studying stress responses in symbiotic organisms as it provides insights on gene expression of multiple mutualistic symbionts. It also provides a first baseline to understand gene expression in an endangered lichen in response to exposure to toxic environments, along with dynamics in its associated bacterial communities.