In the study, experiments were performed on two eco-efficient self-compacting concrete mixes of reduced binder content containing supplementary cementitious materials. The behaviour of the eco-efficient self-compacting concrete (SCC) mixture was examined to determine whether it is suitable for multilayer casting. It is recommended that the SCC should be poured in an uninterrupted manner. However, it is not uncommon that contractors are forced to take breaks as a result of delivery delays. Casting the elements in multiple poorly prepared layers may cause the creation of cold joints between them. Two technological variants of the multilayer casting of eco-efficient SCC on beam elements were analysed: pouring the mixture from a minor height on the previously placed layer and placing the subsequent layer on the mechanically disturbed surface of the underlying material. Different delay times were used: 15, 30, 45 and 60 min between the execution of two layers of eco-efficient SCC. The load-bearing capacity of the joint was determined using a splitting tensile strength test on cubic elements. It was observed that, regardless of the mixture and casting variant, the interlayer bond strength decreased as the delay time increased. This effect was less pronounced when the first layer was mechanically disturbed. It was also demonstrated that concrete with reduced binder content is characterized by a lower drop in bond strength between successive layers. Finally, it is noted that the current recommendations and normative guidelines for the multilayer casting of self-compacting concrete should be specified with regard to the time delay allowed for the execution of the next layer in the absence of interference with the previously placed layer. Lack of clarity in this respect may result in the creation of a cold joint and hence a reduction in the load-bearing capacity between layers. The largest contribution to the negative influence of concrete lies primarily in the significant environmental imprint of Portland cement and the substantial amount of concrete production overall. The related air and water pollution contributes to global warming as well as acidification and eutrophication . In receIn the case of large-scale construction works such as high walls and considerable foundations, contractors often use multilayer casting, which involves the application of technological delays between successive layers of SCC. Such delays are usually caused by disruptions in the supply of the concrete mix. A higher viscosity layer can form on the surface of the already cast SCC mix, which, when not properly combined with new concrete, can create not only unaesthetic lift lines and discolorations, but most importantly reduce the bond strength of the joint ,4,5, whiHowever, each standard specifies different methods that do not have a clear timeframe regarding when they should be used. The ACI standard  states tGiven the guidelines for multilayer casting of SCC elements, an essential issue in self-compacting mixtures is their thixotropy. This time-dependent phenomenon is responsible for the continuous loss of apparent viscosity when there is shear and stiffening of the mixture during its rest ,12. The A considerable number of studies with new generation concretes ,5,16,17 Given the fact that it is hardly ever possible to carry out continuous concrete placing works and there is increasing consumption of concrete of lower environmental impact, including eco-efficient SCC, the effect of introducing breaks into the concrete layer placing procedure ought to be analysed thoroughly in line with state-of-the-art placement technologies. Furthermore, the significant environmental footprint of concrete raises the issue of the implementation of eco-efficient concrete, which should also be examined in terms of multilayer casting. This study looks into the effect of multilayer casting technology on the load-bearing capacity of a joint between layers of eco-efficient SCC based on the testing of two different mixtures.3 which, in the light of the ICI Rheocenter SCC classification ; F—peak load, [N]; L—the length of the sample contact line, [mm]; d—declared cross-sectional dimension, [mm].The load-bearing capacity of the joint was computed according to the procedure used to specify the concrete splitting tensile strength using the formula presented in Equation (1):500, thus classifying the mixtures to both VS1 and VS2 classes, depending on the rest time. In the case of the second batch, in order to maintain similar flow properties, an additional amount of superplasticizer was added during resting time. Moreover, the eco-efficient SCC mixtures stayed stable and showed no signs of segregation nor bleeding during the testing time. Comparing both types of eco-efficient concrete, the Green-SCC was characterized by a higher viscosity and lower slump–flow than Eco-SCC, which resulted in a lower L-box ratio that still was in a normative passing ability range. The L-box ratio could also be affected by a contribution of coarse aggregate in a mixture; as Green-SCC had a higher volume of coarse aggregate than Eco-SCC, the L-box ratio was expected to be lower for Green-SCC [The results of the fresh mix properties are presented in reen-SCC .The results of the compressive strength tests performed on the reference samples (cast in a single layer) with their standard deviation (SD) are demonstrated in The parameter describing the dynamics of a decrease in load-bearing capacity of a joint with respect to one minute is also presented in 2 = 0.966) with a clear negative slope were noted for Variant I. In the case of Variant II, the drop was relatively minor and the coefficient of determination (R2 = 0.81) did not indicate a clear linear relationship. The same relationship was observed for elements made with layers of Green-SCC. In the case of both variants of placement technology, the tendency of the reduction in load-bearing capacity with the delay time increase was noted. A more pronounced effect of surface preparation was observed for the more viscous Green-SCC. Comparing the load-bearing capacity ratios for 60 min delay time in both types of eco-efficient SCC, the difference between the values found for Variant II and Variant I was approximately equivalent to 14% and 22% for Eco-SCC and Green-SCC, respectively.The relation between the average load-bearing capacity ratio and the interval time for each variant of surface preparation, with the corresponding linear trendlines, are presented in In an attempt to explain the phenomena responsible for the decrease in the load-bearing capacity of the joint between two eco-efficient SCC layers, the failure surface condition after splitting tensile strength tests was analysed. In both types of eco-efficient SCC, the failure mechanisms of each variant of surface preparation were alike. The typical failure surface condition after splitting tensile strength investigation for the reference specimen and two variants of surface preparation for a delay time of 30 min between layers of Eco-SCC is presented in In the case of the reference samples, the failure plane passed through the contact zone of the binder matrix and the aggregate. This is a commonly observed failure mechanism for concretes with low and medium strength properties. This results from a different microstructure of the hydrated matrix in the direct proximity of the aggregate grains as compared to the other areas. In the vicinity of the aggregate grains, the matrix is generally more diluted and thus more porous. Microcracks in the matrix–aggregate interface are therefore the primary reason for the failure of the bond strength .In Variant I of surface preparation, the zone of sufficient layer mixing was situated in the middle part of the specimen cross-section where the fresh mix had impacted the incompletely solidified layer from a low height. In this area, the second layer mix penetrated the first layer forming a high-quality joint. The observed failure plane of the joint was similar to the reference samples. The dimension of the proper mixing zone depended on the delay time. The longer the time between laying the first layer and the subsequent one, the smaller the zone of proper mixing was. In the other parts of the sample, the second layer moved across freely over the first layer that had partially stiffened. The shear stress was not sufficient to reverse the process of structural build-up at rest. As a result, there was no mixing of the first and second layers. The failure in these zones was mainly caused by the debonding of the interlayer joint as it passed along the surface of the structural build-up. Hence, the joint was structurally comparable to a joint between hardened and new concrete. The research revealed that the larger the area of the undisturbed surface of the mix laid earlier, the lower the load-bearing capacity of a joint between two layers .In the case of Variant II of the surface preparation, the occurrence of the proper mixing of both layers was observed and the failure mechanism was similar to the reference samples made without interruption in concreting. Only at the edges of the specimens were there minor areas of failure between the second layer and the first layer, where structural build-up was noted. Variant II of the surface preparation caused adequate plasticisation of the previously laid mixture, thus facilitating good mixing with a fresh mixture.The results of the research allowed us to propose guidelines for casting eco-efficient SCC in layers so as to limit the effect of delays between layers on the mechanical strength of the beamlike structures, which assume the process of restoring a plastic state of the mixture that was previously made by mechanical surface disturbance (Variant II of placement technology). In this variant, no significant reduction in the load-bearing capacity of a joint between two layers of both Eco-SCC and Green-SCC was noted in the analysed delay time range. The effective performance of Variant II was a result of a uniform and limited interference of the mechanical disturbance across the whole joint surface. Analysis of the failure mechanism of specimens made in this variant showed a similar mechanism to monolithic samples. Moreover, in the case of this method, it was possible to eliminate traces and discolorations on the concrete surface (lift lines) at the interface between layers. However, it ought to be kept in mind that this sort of interference may trigger unwanted segregation, hence it should be performed with a great amount of control and following previous schedules.Variant I of placement technology resulted in a greater decrease in the load-bearing capacity of a joint between two layers of eco-efficient SCCs. The inspection of the failure surface condition revealed that in this variant the satisfactory mixing of successive layers is limited as a result of a partial structural build-up in the rest of the first layer mixture and insufficient impact-induced plasticisation. As the intact surface area of the of the base layer increases, the load-bearing capacity of a joint between the two layers decreases. For this variant, a clear linear dependence of the decrease in load-bearing capacity of a joint with delay time was recorded in both types of eco-efficient SCC. Similar observations were reported by Megid and Khayat ,4, that 3 [The authors drew analogous conclusions in their previous publication based on self-compacting concrete with a binder content of 450 kg/m3 . That miFurthermore, the failure mechanism under splitting tensile in eco-efficient SCCs was found to be more comparable to the behaviour of normal concrete rather than self-compacting concrete. The reason for this is the low powder content in the mixture and the more porous cement matrix in the aggregate area, which consequently cause failure along the interface between the matrix and the aggregate.It should be taken into account that the occurrence of a partially reversible structural build-up at rest may be of importance for the layer-to-layer bond strength when placing concretes according to the international guidelines ,10,31 alIn general, a decrease in the load-bearing capacity of a joint between two layers of eco-efficient SCC was observed with an increase in the duration of the delay time. The most substantial reduction was found for the technological variant connected with the free-flowing of the mixture over the previously laid layer. For delay times of 15, 30, 45, 60 min in the case of Eco-SCC, the average decrease in bond in relation to the reference sample was 8.5%, 12.9%, 16.1% and 24.3%, respectively. In the case of Green-SCC, for which delay times of 30 and 60 min were investigated, the reduction in relation to reference samples were 18.9% and 28.7%, respectively. In the technology variant connected with mechanical disturbance of the previously placed layer, a slight decrease, ranging between 2.1% and 10.5%, was obtained for Eco-SCC, and between 4.2% and 6.7% for Green-SCC.The current recommendations and normative guidelines for multilayer casting of self-compacting concrete appear to be insufficiently detailed regarding the time of delay between layers if there is no interference with the previously placed batch. On the basis of this research, in the case of concreting with intervals longer than 15 min between layers, it is recommended that a fresh layer should follow superficial plasticisation of the underlying layer to improve the bond at their interface.Due to its reduced binder content, eco-efficient SCC concrete is characterised by reduced structural build-up at rest, which contributes to a smaller decrease in the load-bearing capacity of a joint between two layers compared to traditional SCC concrete. In general, eco-efficient SCC would be more convenient in the multilayer casting technology than traditional SCCs that are rich in powder content.From the experimental studies conducted, a set of conclusions and recommendations can be drawn:


In order to be sustainable, the concrete must meet processing, mechanical and durability properties while taking cost aspects into account. The paper presents (i) the “micro-filler/eco-filler concept” for achieving a clinker reduced, optimised binder and (ii) a performance-based approach to put sustainable “Eco-concrete” into practice. Clinker is substituted by locally available inert fillers by at least two different particle size fractions and supplementary cementitious materials. The method is based on particle packing optimisation, reduction in water demand and optimisation of the mix ratio of the binder blend, which allows the performance requirements to be met. The new Eco-concretes deliver the desired performance in terms of processability, strength and durability  while lowering the environmental impact in comparison to standard concrete. One of the new mixes was used for a small animal passage tunnel. The direct comparison of the developed Eco-concrete and standard concrete showed a 24% reduction in CO2, while achieving satisfactory workability, stripping strength and durability performance.In times of climate change, the reduction in embodied greenhouse gas emissions is a premise for sustainable concrete infrastructure. As Portland cement clinker is mainly responsible for the high CO It would help to achieve short-term CO2 reduction targets in 5 to 10 years. This requires either the provision of new, low-clinker composite cements or the mixing of existing cements with a high portion of supplementary cementitious materials (SCMs) and fillers to produce low-carbon concretes on an application-specific basis.A maximum reduction in Portland Cement clinker in cementitious binders, cement and concrete is one major step on the pathway to the decarbonization of concrete production by 2050 . This isconcrete ,4. In co2-reduction in construction practice already today.In the last few decades, the use of Portland composite cements  is increasing, e.g., in Germany and Austria ,6. The aTo ensure an adequate concrete performance, the pros and cons of different SCMs and fillers have to be considered. Latent hydraulically active and pozzolanic SCMs help to produce a dense microstructure during hydration and therefore will increase the resistance of concrete against chloride and chemical attack ,13,14. ITo link workability, early strength (for stripping formwork), durability and environmental performance, a method, according to Juhart et al. , for desMF/dEF/OPC ≤ 0.33 with an optimum reached at a ratio ⋍0.1. Practically MFs have a d50 < 3 µm in any case, significantly smaller than OPC. The physical filler effect increases the packing density of the mix and helps to accelerate hydraulic reactions inter alia by its nucleation effect and higher specific surface area (SSA) [t ratio  but the same or an even higher w/bh ratio  compared to pure PC pastes. In general, their portion of clinker is reduced and thus their w/c ratio (water/clinker ratio) increases.The performance-based mix design follows a stepwise approach: (i) suitable source materials selection, (ii) binder design with paste experiments by the micro-filler/eco-filler concept including tests to determine the compatibility of binders and additives and (iii) final development  of the concrete mix as well as its performance proof in terms of workability, strength and durability. The general approach of optimizing binder composition is illustrated in ea (SSA) ,23. In tBy means of the presented concept, optimal combinations of OPC and SCMs and finally—by applying the above steps (iii) and (iv)—optimal concrete mixes are found that are tailored to the requirements of the desired application.The concept presented was verified in practice by means of a prototype building, an animal passage tunnel under a railway line in Austria. All steps of realization were carried out, from concrete development, approval testing in a project-specific special admission, the dosage and way of adding of micro and eco-fillers in a ready-mix concrete plant and the delivery as well as concreting the prototype.For direct comparison of the performance one half of the animal passage was manufactured with a well proven normal concrete as reference (Ref-C) and the other half with the Eco-concrete, as it will be described in the next sections. Both concretes should fulfil all requirements of concrete strength class C 25/30 and exposure classes XC4/XW1/XD2/XF3 according to ÖN B 4710-1   test methods and the interpretation of the test results has to be conducted with care, as the test methods were not developed and evaluated for such Eco-concretes in detail. For the building owner, implementation of performance-based, COMixes were designed in steps: (i) characterizing and selecting appropriate powder materials  considering fineness, clinker content or reactivity and ecological impact; (ii) designing eco-efficient binders or pastes by optimizing binder blends (combinations of OPC/EF/MF), water-binder ratios and SP-compatibility; and (iii) up-scaling the most promising blends to concrete and, finally, manufacturing them. In the last step, all project-specific relevant concrete properties and, in particular, the durability characteristics were tested and, if necessary, optimized by adapting the mix design.3, 1 wt.% MgCO3) as well as 4 different micro-fillers were used. One of the micro-fillers is a dolomite powder  and the others are finely processed limestone powders of the same supplier. The latter products have a high content of CaCO3 (97–98 wt.%) and are essentially distinguished in their fineness according to The project-related standard normal concrete used as reference (Ref-C) contained a cement CEM II/A-L 42.5 N according to ÖN EN 197-1 . For theTo determine the clinker content of the binder blends, as discussed later, the minimum clinker content of 95 wt.% of CEM I and 80 wt.% of CEM II/A-L in ÖN EN 197-1  was take50 value given in A surfactant-based air entraining agent (AEA) and 4 different PCE-based superplasticizers depending on the binder and their compatibility were used. The powder materials were characterized including the determination of (i) dry particle density according to ÖN EN ISO 1183-1 , (ii) Bl3, 1% MgCO3) in 3 fractions  according to ÖNORM EN 12620 [Carbonate aggregates (95–97 wt.% CaCOEN 12620  were useEN 12620 ).The PSD analysis in 2 emissions of concrete production. The considered data for the environmental indicators GWP and PEt of the used concrete constituents are shown in 50 values of 2.8 µm for MF and 8.5 µm for EF, neglecting even greater fineness), resulting in GWP and PEt values of MF and EF according to 3 fresh concrete was calculated in accordance with the values for GWP and PEt of the source materials and their fresh concrete quantities—the results can be found in Beside the physical requirements, the ecological impact is an important selection criterion in times of climate change with the main aim of reducing the CO2-eq., while achieving equivalent workability and strength to a standard binder as reference. In our case, the reference was the water–binder mix of the project´s usually desired normal concrete, i.e., Ref-paste with 88 wt.% CEM II/A-L 42.5 N and 12 wt.% GGBFS-mix. As a starting Eco-mix, a basic Eco-paste of 55% CEM I and 45% GGBFS was defined, which, from experience, is very workable and ecological, but develops strength more slowly than pure OPC [t value of 0.50 and on the other hand at constant w/bh value of 0.45 , while, in contrast, w/bh is the water/hydraulically active binder ratio, where all hydraulically active materials (bh is OPC and GGBFS) are accounted for by 100% .Based on the combined filler or “micro-filler/eco-filler” concept , optimumpure OPC . To incr of 0.45 . In the The cement paste variants  were mixt of 0.50 in h. At 15% of MF-LS-UF the early strength value of Ref-paste can be reached (8.3 N/mm2). The coarser MF and EF do not have this potential, but keep the early strength from 3% to 15% substitution of GGBFS nearly at a constant level. The reasons for the different behavior are manifold and overlapping each other. Increasing packing density and specific surface area of the blend would increase strength, as they accelerate hydration. On the other hand, increasing w/bh by greater GGBFS substitution-rates by inert LSP results in lower strength.The paste experiments at const. w/bh = 0.45 = const. That means water content (and w/bt) is reduced with greater GGBFS substitution rates by inert LSP. For 15 wt.% of MF-LS-UF a doubling of the 24 h strength, but with negative influence on workability is achieved. However, an increased usage of MF-LS-D also results in increasing early strength while at the same time improving the workability. With an MF-LS-D content of 15%, an increase in early strength is achieved that is above the Ref-paste strength (14.2 N/mm2 > 13.3 N/mm2) while far exceeding its flowability (280 mm > 161 mm).For further optimization, the workability and early strength had to be adjusted with increasing MF content. For the first purpose, either suitable SP or an MF with plasticizing effect, i.e., MF-LS-D, could be used. For the latter purpose, w/b ratios can be reduced. Note that, in our case, no (fluid) SP was added in all the paste experiments, as this measure was reserved for further concrete development. In the final evaluation of the results and based on the experiences of the former research ,38, it wCompatibility tests according to ÖN EN 1015-3  were con2 technologies [From the paste experiments the two most promising mixes were chosen to develop clinker-reduced, sustainable concrete: Firstly, the blend “UF Eco”, with 55 wt.% CEM I 52.5 R, 40 wt.% GGBFS and 5 wt.% of MF-LS-UF of the binder. It is characterized by a high content of hydraulic and latent-hydraulic binder (OPC + GGBFS) and—in order to increase packing density and early strength—a small amount of MF-LS-UF. Secondly, the blend “D Eco”, where the binder is composed of 55 wt.% CEM I 52.5 R, 30 wt.% GGBFS, 10 wt.% EF-LS and 5 wt.% of plasticizing MF-LS-D. The second approach would be very economical and would use GGBFS sparingly. GGBFS will become increasingly scarce in the future, when iron and steel production will produce less GGBFS because of new low-COnologies .c  of at least 300 kg/m3 and a water/binder (w/bc) value of maximum 0.53 is prescribed for the concrete type of the standard concrete C 25/30 XC4/XW1/XD2/XF3. Clinker was substituted by GGBFS and LSPs and water was reduced. Due to the reduced water dosage at constant total binder content  of Eco-mixes, their paste volume decreased compared to the reference. The latter is defined as the volume of water, SP, air voids, binder and fines of the aggregates < 125 µm particle diameter. The w/bt value of Eco-concretes was lowered in order to increase the early strength and to ensure sufficient durability potential, in particular, sufficient carbonation resistance despite clinker reduction. As a consequence, the w/bh ratio remained constant (UF Eco) or increased slightly (+6% for D Eco) compared to the reference. For further comparison, the rough proportion of clinker and GGBFS in the total binder is given in The significant parameters of the mix composition are given in 3 each of Ref-C, D-Eco and UF-Eco were mixed in a ready-mixed concrete plant in a double-shaft batch mixer DKX (BHS-Sonthofen) and transferred in quick succession to a truck mixer. For evaluation of the concrete properties, fresh concrete samples were taken from the truck mixer still on the site of the concrete manufacturer and tested. In addition, concrete specimens were produced and transferred into the laboratory for the strength and durability tests described below. In order to ensure that the two Eco-concretes demonstrably comply with requirements of the concrete C 25/30 F52 XC4/XW1/XD2/XF3 specified for the project, the following strength, workability and durability tests were conducted.Two concrete mixtures of 1.5 mFor consistency evaluation flow-table tests according to ÖN EN 12350-5  were carDurability checks include water penetration, freeze–thaw resistance, carbonation resistance and chloride diffusion.The water penetration was tested on three prisms (200/200/120 mm) according to ÖN EN 12390-8  with spe3). After demolding, the prisms were first stored in plastic sheets until the 7th day and from the 7th to the 28th day underwater. The sonic travel time before and after exposing 3 prisms of each mix to 56 freeze–thaw cycles was measured. Each cycle lasted 12 h and went through temperatures of +20 °C/−20 °C and back to +20 °C in a climatic chamber. The change of the sonic travel time after 56 cycles of each mix compared to that of a normatively produced “zero” concrete mix according to ONR 23303 [The freeze–thaw resistance was tested according to ONR 23303  based onNR 23303  was eval2-content of 3%. To determine the carbonation depth at different exposure times of 0, 7, 28 and 70 days, slices were split off the prism (thickness of 7–8 mm) and phenolphthalein indicator solution was sprayed onto the fracture surfaces. The remaining pieces were each time returned to the climate chamber storage. 30–75 min after spraying, the carbonation depth was measured at 5 points at each of the 4 sides, with an accuracy of 0.5 mm. Outlier at pores and aggregates were not considered.The carbonation resistance test was carried out according to ÖN EN 12390-12 , using tk at different exposure time passing through the measured value at time t = 0 as fixed point. In x-axis, so that the linear slope of the regression line corresponds to the accelerated carbonation rate KAC (in mm/√days). The conversion from the measured accelerated carbonation rate to a theoretical natural carbonation rate KNAC is carried out by Equation (1) and the conversion parameters 2 solution before they were put into boxes filled with a 3% NaCl solution for 90 days. For the determination of the chloride diffusion coefficient, powder samples were obtained with a profile grinding machine by dry grinding of 9 individual layers starting from the exposed side to depth (0–25 mm). The chloride content of each layer was measured by potentiometric titration with silver nitrate according to ÖN EN 14629 [− concentration values in various depths were used to calculate the diffusion coefficient (Dnss) and the Cl− surface concentration (CS) according to Equation (2) and − profiles and fitted Cl− diffusion curves are shown in The chloride penetration test was performed according to ÖN EN 12390-11  using a EN 14629 . The obt2 at an age of 28 d according to ÖN B 4710-1 [Consistency, air content and 28 d strength are the properties by which concrete is typically assigned to classes by limiting values . They caB 4710-1  was reacThe water penetration depth threshold of 50 mm (according to ÖN B 4710-1 ) is satiThe freeze–thaw resistance of all concretes is very close. The sonic travel time change of all variants is within the required range of differing not more than ±2.5% from the standardized zero concrete after 56 freeze–thaw cycles (according to ÖN B 4710-1 ).NAC of 4.5 mm/√year, which is undercut by all the concretes  than the Dnss of Ref-C (9.7 × 10−12 m2/s)  see .3 of fresh concrete. Due to the clinker reduction and the use of materials with a low CO2 rating, the GWP value could be reduced by 24% in both Eco variants. The PEt value could also be reduced by 17% and 18%, respectively, through the adjustments. SP had to be added a bit more to Eco-mixes than to the reference, which had a corresponding effect on the environmental impact of the eco-concretes due to its high GWP and PEt values, in total still very advantageous. With each m3 of concrete installed, more than 50 kg of CO2 could be saved, which corresponds to 2500 kg of CO2 for a quantity of 50 m3 installed.The “ecological impact” was calculated according to the values in The blended binders developed consist of 55 wt.% clinker and 45 wt.% of a mix of GGBFS and one or two LSP. Thus, they are equivalent to (future) CEM II/C cement composition according to EN 197-5 ,23, whilThe performance of the concretes was compared and evaluated in its entirety in order to be able to select the most suitable variant. For this purpose, the individual performance indicators see  for the t value. The main reason for the good workability is the appropriate choice of type and dosage of SP. However, D Eco surpasses the limits after 90 min clearly, which means, SP dosage could be reduced a bit, especially regarding the “consistency keeping” part of the SP-mix.2 emissions. Moreover, if strength is related to clinker content, eco-concretes show a remarkably higher strength per wt. of clinker or higher clinker efficiency than standard concrete.It is also noteworthy that both Eco-concrete variants show a higher 2d strength than the standard normal concrete despite their low clinker content and high proportion of GGBFS. This is due to the above-mentioned effect of the fine or micro-fine limestone powders, which accelerate cement hydration. The goal of being able to strip formwork of eco-concretes at the same early stage as standard normal concrete was achieved. However, at 28 d and especially at the “high” age of 90 d, “overstrength” is achieved. In particular, the mix with the highest proportion of GGBFS (UF Eco) shows the highest strength . This meIn terms of durability properties , all con2 emissions, the application of performance-based design is essential. The principle of “equivalent concrete (durability) performance” of Eco-concrete to standard concrete with traditional composition  were made of CO2-reduced concrete and the other half of standard concrete  in the planned construction sections, which were foundations, walls and the ceiling. In addition, two mock-up-walls see  made of With respect to workability, D Eco was equivalent to Ref-C. Only a somewhat faster stiffening at the execution temperatures of 27–30 °C on site compared to 20 °C in lab was observed. It was also noticed that Eco-concrete had a slightly higher viscosity than standard concrete. This corresponds to the lower water content and higher packing density of the fresh Eco-concrete compared to standard concrete.Hydration heat development was monitored by wireless sensors embedded in the mock-up walls as well as in some cube specimen that were used for strength evaluation. The results see  show no The desired fair-faced concrete quality was achieved for both variants, see 2 reduction of 24% compared to standard concrete. Further on, Eco-concrete was successfully applied in an infrastructure construction project. The practical implementation showed that the outlined, performance-based design concept for clinker-reduced concrete allowed to fulfil application-specific requirements as early formwork stripping and high durability. The production, processing, installation and stripping of the eco-concrete was to a large extent successful in the same way as that of the standard normal concrete. However, it was observed that the eco-concrete was somewhat more viscous and stiffened more quickly in the slightly warmer environment during execution compared to laboratory conditions.In this study, Eco-concrete was designed by the MF/EF concept with the aim of optimizing its performance in respect to functionality, durability and environmental impact as the top optimization goal. Two Eco-concrete variants passed the performance tests with regard to the normative limits and proved to be at least equivalent to the standard normal concrete. At the same time the concept allowed a CO2 reduction. Rather, limit values for performance requirements should be specified and met, while reducing the environmental impact and/or improving the service lifetime should be the top optimization goals. As concrete optimization is a multi-criteria decision, final selection of the concrete to be used is a question of weighting of individual criteria in an overall (owner’s) decision.We conclude that the principle of “equivalent concrete performance” of actual standards is not suitable for optimizing concrete in terms of COIn a systematic monitoring of the structure, characteristic values of the damage progress  are to be recorded over several years and a model for service life prognosis will be derived from this.


CBS gene, c.969G>A, p.(Trp323Ter); c.982G>A, p.(Asp328Asn); and the Qatari founder variant c.1006C>T, p.(Arg336Cys). This is the first study to review the screening of newborns in Kuwait for classic homocystinuria, starting with the detection of elevated blood methionine and providing a follow-up strategy for positive results, including plasma total homocysteine and amino acid analyses. Further, we have demonstrated an increase in the specificity of the current newborn screening test for classic homocystinuria by including the methionine to phenylalanine ratio along with the elevated methionine blood levels in first-tier testing. Here, we provide evidence that the newborn screening in Kuwait has led to the early detection of classic homocystinuria cases and enabled the affected individuals to lead active and productive lives.Kuwait is a small Arabian Gulf country with a high rate of consanguinity and where a national newborn screening program was expanded in October 2014 to include a wide range of endocrine and metabolic disorders. A retrospective study conducted between January 2015 and December 2020 revealed a total of 304,086 newborns have been screened in Kuwait. Six newborns were diagnosed with classic homocystinuria with an incidence of 1:50,000, which is not as high as in Qatar but higher than the global incidence. Molecular testing for five of them has revealed three previously reported pathogenic variants in the  Kuwait is a small country situated in the northwestern part of the Arabian Gulf with a total area of 17,818 square kilometers . There aIn October 2014, the Kuwait Ministry of Health started a publicly funded expanded newborn screening program (NBS) meeting the highest international standards to screen for a wide range of metabolic and endocrine disorders, including a total of 22 disorders  via testClassical homocystinuria (HCU) OMIM 236200), is an inborn error of methionine (Met) and homocysteine (Hcy) metabolism with a global incidence of ~1:260,000 , and the200, is aThe clinical manifestations of untreated HCU include cognitive impairment, behavioral problems, ocular abnormalities , connective tissue involvement , and thromboembolism . Due to The management of HCU resides mainly in lowering Hcy level to near normal levels via Met restricted diet, betaine, folic acid, and pyridoxine supplementation. However, poor compliance to diet and medications as well as the considerable fraction of pyridoxine nonresponsive individuals noted in the Qatari population , has ledThis paper is the first to review the current practice of the national NBS for HCU in the State of Kuwait as well as the follow-up strategy for positive screens since the expansion of the NBS program in October 2014.A retrospective analysis of the data registry for the NBS over the 6-year period between January 2015 and December 2020 in Kuwait was performed after obtaining consent from the NBS program and KMGC. These data included newborns delivered at both private and public hospitals all over Kuwait according to the percentage of coverage shown in The first DBS samples were collected on Whatman 903 filter papers within 48–72 h of life but could be accepted up to one month after birth. For any DBS collected before 48 h of age, a second DBS would be repeated within 7 days. All DBS were sent to the NBS laboratory at the KMGC for analysis. Samples showing high Met levels were typically repeated three times from different punches from the same DBS filter paper to ensure that the DBS was homogenous with adequate quality for accurate quantitative analysis. The mean value of the three readings of Met was reported as positive initial screen if it exceeded the cutoff value. Positive initial screens were followed by a second DBS for amino acid analysis in addition to the plasma total homocysteine (tHcy) analysis at Al-Sabah Hospital Biochemistry Laboratory and Ibn Sina Hospital Biochemistry Laboratory respectively, the main laboratories that provide diagnostic testing for metabolic disorders, as recommended in NBS ACT Sheets and Confirmatory Algorithms for Met by the American College of Medical Genetics to confirm or exclude the diagnosis of HCU  Figure . If the The method used to measure Met in the first DBS is semi-quantitative determination via tandem mass spectrometry (MS/MS) without a butylation step ,19. ElecAn API 3200 triple quadrupole tandem mass spectrometer (AB-SCIEX) and liquid chromatography-tandem mass spectrometry (LC–MS/MS) were used to analyze amino acids with the butylation step, as previously described with minor modifications .High-performance liquid chromatography (HPLC)  was used to analyze plasma amino acids as previously described with minor modifications .Plasma total homocysteine was measured by competitive immunoassay as previously described ,24.Phe was analyzed in DBS similarly to Met using MS/MS. We have measured the Met/Phe ratio for all NBS samples  for the period 2015–2020, including the six confirmed HCU cases. We have then compared the Met/Phe ratio to Met levels as a primary marker for HCU and determined its specificity as a screening test. The Met/Phe ratios ranged from 0.2 to 3.4 with an average of 0.3 and standard deviation of 0.09. The Met/Phe cutoff of 0.75 corresponds to the 99th percentile of the controls. All statistical analyses were performed using SPSS22.CBS gene due to their association with HCU. Variants were further prioritized if they were rare in gnomAD database  https://gnomad.broadinstitute.org/ (accessed on 27 May 2021) or if they were previously reported to be associated with HCU in the literature.All biochemically confirmed HCU cases, except for the first individual , underweThe Kuwait NBS data registry included 338,379 samples for 304,086 screened neonates  born in Kuwait between January 2015 and December 2020 . The numCBS gene: c.969G>A, p.(Trp323Ter); c.1006C>T, p.(Arg336Cys); and c.982G>A, p.(Asp328Asn)  for the period between 2015 and 2017, which was decreased to 44 μmol/L (mean+ 5SD) thereafter. If we apply the cutoff of Met > 44 μmol/L (mean + 5SD) for the period 2015 until 2020, the total number of newborns with exclusive hypermethioninemia is 512. A total of six confirmed cases of HCU based on elevated plasma Met and tHcy have been identified since the year 2015 through the NBS program. These six cases  were from six different families, four of whom were Kuwaitis, one was Saudi Arabian, and the last individual was Egyptian . This hap328Asn) . Using a Met/Phe cutoff of 0.75 in addition to Met levels (with a cutoff of 44 μmol/L), led to a percentage of 0.05 (174 cases) of suspected HCU  in DBS followed by collecting another blood specimen for biochemical confirmatory tests for positive cases, which includes analyzing blood for tHcy and amino acid chromatography. The positive rate based on the first Met DBS screening was about 1:846 (0.12%)  . Based oAs demonstrated by Al-Dewik et al. and Yamada et al., individuals with late diagnoses of HCU have a poorer outcome, mainly in terms of social and intellectual outcomes compared to individuals diagnosed based on NBS and treated early in infancy, emphasizing the importance of early diagnosis of treatment via NBS ,41. HoweThe incidence of HCU in Kuwait in this study is shown to be 1:50,000, relatively similar to the incidence reported in Saudi Arabia . With thIn Kuwait, the biochemical confirmation of HCU is typically followed by a confirmatory molecular testing for the CBS gene. Molecular testing of affected newborns as well as late diagnosed individuals with HCU has revealed the same founder missense variant reported in Qatar, c.1006C>T, p.(Arg336Cys) , while tOur experience of NBS for HCU in Kuwait is successful for several reasons: 1. The high consanguinity rate in the population and thus the high incidence of IEM; 2. The small size of the country that allowed centralization of NBS laboratory screening service and facilitated the close coordination between different hospitals including private sectors; 3. NBS and early treatment not only reduce the morbidity and mortality typically associated with IEM but also allow for carrier screening of at-risk family members via molecular testing, enabling a sort of prevention strategy, providing better counselling options for the families, and reducing the future incidence of the disease.In summary, our study is the first to review the experience of the NBS program for HCU in Kuwait. We demonstrated that our national NBS for HCU is highly effective, the screened positive cases are successfully followed up, and the affected infants are treated within an average of 9 days of their lives. We report the incidence of HCU in Kuwait to be 1 in 50,000 since the expansion of the NBS program in October 2014, emphasizing its effectiveness and importance. Further, we recommend adding Met/Phe ratio to blood Met level as a first-tier and tHcy as a second-tier strategy in our national HCU screening to further increase the specificity of NBS testing.


Background/objectives: Brazil, Russia, India China and South Africa have prioritized cooperation regarding health, including malnutrition. Anaemia in children, adolescents and women of reproductive age has been on the increase in these countries, placing a huge strain on healthcare systems. This study aimed to map the scientific perspective and research publications on anaemia in children and adolescents in the BRICS countries. Methods: Bibliometric analyses were employed to map scientific publications related to anaemia in children and adolescents in BRICS countries using VOSviewer software. Research documents from 1990 to 2020 were imported from PubMed. Descriptive statistics was used to analyse trends in research publications, authorship and keywords over the 30-year period. Findings: BRICS countries accounted for 15% of all publications on the subject within the last three decades. While India had the highest number of publications, China had the author with the highest number of research publications and co-authorship links. Of all article types, India had the highest number of letters, while China and South Africa published the highest number of RCT/clinical trial and review articles, respectively. Conclusion: The review of all scientific studies on anaemia in BRICS nations for the past 30 years revealed gaps in research collaborations on anaemia between authors in BRICS nations. However, collaborative research projects may contribute to building a shared base of evidence, innovations, data and methodologies for a more comprehensive understanding of the risks and vulnerabilities of child and adolescent anaemia. This will aid in the development and evaluation of interventions and policies to alleviate anaemia and nutrient deficiencies. Anaemia is a major public health concern, affecting young children, adolescents and women of child-bearing age. According to the World Health Organization (WHO), an estimated 42% of children under the age of five years and 40% of pregnant women globally are anaemic . AnaemiaSuch nutrition and health related research advancements are developing among Brazil, Russia, India, China and South Africa, commonly known as the BRICS nations, who may not yet have formal political alliance but do have prioritized cooperation regarding health. A recent study found that despite this health cooperation, India and South Africa had the poorest healthcare system performance, with the authors identifying strong correlations between mortality and health expenditure, funding, and human resources deficiencies, as well as magnifying the activities to reduce unhealthy lifestyles in BRICS countries as prioritization points to improve health system outcomes . With reIron-deficiency anaemia is directly linked to malnutrition and poverty. Children are likely to become stunted or wasted due to poverty and inability to consume well-balanced diets; women are unable to access iron and folic tablets during pregnancy; and poor sanitation, malaria occurrences and worm infestations also result in high occurrences of anaemia . Thus, tBibliometric analysis is a methodology applied to quantitatively measure scientific publications to determine the research productivity of journals ,15. In bChild and adolescent nutrition remain an important public health concern globally. As previously mentioned, young children and adolescents are at greater risk of anaemia due to the increased iron and other micronutrient demand, required for growth and protection against susceptibility to infections ,3. A sciWe conducted bibliometric analysis on research conducted by BRICS  member countries on Anaemia in children and adolescents between January 1990 and December 2020. We retrieved published documents from PubMed by using new and varied search terms ((“anaemia” [All Fields] OR “anemia” [MeSH Terms] OR “anemia” [All Fields] OR “anaemias” [All Fields] OR “anemias” [All Fields]) AND (“child” [MeSH Terms] OR “child” [All Fields] OR “children” [All Fields] OR “child s” [All Fields] OR “childrens” [All Fields] OR “childrens” [All Fields] OR “childs” [All Fields]) AND (“adolescences” [All Fields] OR “adolescency” [All Fields] OR “adolescent” [MeSH Terms] OR “adolescent” [All Fields] OR “adolescence” [All Fields] OR “adolescents” [All Fields] OR “adolescents” [All Fields]) AND [Brazil OR Russia OR India OR China OR South Africa]). These terms were built based on previous publications about anaemia in children and adolescents. PubMed was our database of choice as it allows for basic and advanced search options. In our search, we included all publications including books, journal articles, letters and clinical trials to capture all information on the subject. Where articles have authors from two or more member countries, we designated such articles to the country of the first author. Our search yielded 15,345 articles for publications across the world from which 10,000 were imported, 13,055 articles  from non-BRICS countries and 2298 publications from BRICS countries. To ensure quality assurance, articles were double checked to eliminate repetitive articles.The PubMed outputs were imported, from which we generated an excel spreadsheet for each BRICS country. From this, bar charts and graphs were produced to describe trends in publications, authors with highest publication and keywords that were most used in each country and collectively. The same was also undertaken for BRICS countries combined against non-BRICS countries to assess trends in publications. Additional analysis was performed using VOSviewer version 1.6.15 to visualise publications on the subject by BRICS countries. VOSviewer was selected because it is easily accessible, free, and user-friendly.In the results below, we analysed the number of publications, author’s keywords, article types and research organizations for each BRICS country and collectively. From Interestingly, the number of publications from South Africa has generally remained the same in the first two decades until 2011. Meanwhile, the number of publications from Brazil has significantly increased over the years, with an increase of over 200% between 2008 and 2014. Comparatively, Brazil and South Africa recorded a spike in the number of publications compared to India and China in the year 2017. The top 10 authors in each country, with at least two published documents overall within the three decades, were analysed. For each of the authors, the total co-authorship link with other authors was calculated. Authors with the greatest total strength were selected and were ranked before considering the number of publications. The results are shown in the figures below. It should be noted that the total number of publications per author is the sum of the number of publications depicted as blue and co-authorship links in brown. From the figures, the authors with the highest publications are Bonfim Carmen with 18 publications among Brazilian authors, Zamran Ari with four publications among Russian authors, Chandra, Jagdish with 22 publications among Indian authors, Fu Rong with 34 publications among Chinese authors, and Wonkam Ambroise with nine publications among South African authors. Interestingly, it could be seen that the number of publications is not directly proportional to the number of co-authorship links. For example, although Wonkam Ambriose from South Africa had nine publications, there were 11 co-authorship links, compared to Rabie Helena with only three publications but as many as 21 co-authorship links. The same dynamics are seen among Indian authors where Chandra Jagdish had 22 publications and 25 co-authorships links compared to George Biju who had 11 publications and 41 co-authorships links. In addition, consider Cipolotti Rosanna from Brazil with eight publications without a co-authorship link; however, Silva Gisele Sampaio, with six publications, had a total of 11 co-authorships links. From this, it could be deduced that there is a poor collaboration among some authors and that authors with higher publications tend to collaborate with wide range of other authors. In addition, we analysed research collaborations between authors from these five countries, as displayed in From the layout algorithm displayed in the figure below, it appears there is weaker or no collaboration between authors across the BRICS countries compared to within a country. The minimum number of occurrences of an author’s keyword is five; for Russia it is one. For each of the author’s keywords, the total strength of the co-occurrence link with other keywords was calculated. The top five authors’ keywords with corresponding total link strength were selected and tabulated as represented in the graphs below .Article types were determined from PubMed manually for all countries. For each BRICS country, each article type was sourced and counted from the larger search results, tabulated, and plotted as shown below.For each organisation from BRICS, the highest number of published documents and the corresponding total strength of the co-authorship links with other organizations were calculated. The organisations with the highest number of publications were ranked first and those with the greatest total link strength were selected, as shown in the graphs below. Documents with authors > 25 were excluded. The minimum number of documents of an organisation in a country was set to two. For BRICS combined, minimum number of an organisation set to one to allow all organisation. For each organisation, the total strength of the co-authorship links with other organizations was calculated. The organisations with the greatest total link strength were selected. In terms of collaborations among these top five organisations, which are depicted here in terms of total link strength, no collaboration was found between and within the BRICS countries when set to a minimum of five documents per organisation, as seen in the graphs above. This means that no two BRICS organisations have collaborated on five or more publications within the past 3 decades. However, collaborations existed between and within BRICS countries if the minimum number of documents per organisation was set to four or less. The majority of these collaborations were between Chinese and Indian organisations.A network analysis of the collaboration between a total of 5812 BRICS organisations which published within the period of 3 decades was conducted. From this number, 1000 were selected and analysed. To allow for fair representation of all organizations, the minimum number of documents per organization was set to one for the VOSviewer software analysis. From the figure above, it could be seen that there are generally poor collaborations between BRICS organizations and weak interconnections in terms of the subject matter and publications during the period. This study used bibliometric data to report on published research on anaemia in children and adolescents in BRICS countries between 1990 and 2020. Overall, 2298 studies from BRICS countries were found, contributing to 15% of the total studies on the subject worldwide. The results were presented according to (i) the number of publications over time, (ii) most published authors, (iii) keywords, (iv) article types, and (v) research organizations, for each BRICS country and collectively.The overview bibliometric analysis conducted on BRICS countries indicated that there has been a rapid increase in the number of publications, with the highest in India and the least in Russia, since 2000. After 2006, there was a significant rise in the number of publications from China, Brazil and South Africa. Notably, China’s rate of publications grew after 2010, as described by Xie and Freeman 2019) [ [19], wiFurthermore, our analysis found that between 1990 and 2012, there was no change in the publication rate in Russia. However, this finding cannot be taken at face value when taking into account the fact that the Russian population has always been reporting a significant lower anaemia prevalence as compared to populations from other countries with a similar socio-demographic index . SeveralThe study also touched upon the evolution of anemia research in BRICS countries based on keywords and most published authors from the publications. Certainly, published research on these topics contributes significantly to the knowledge of the scientific community and leads to better understanding of the research areas that most require further contributions; however, it could be seen that the number of publications is not directly proportional to the number of co-authorship links, which was also observed in other bibliometric analysis studies ; as illuThe most used keywords varied by member country. The co-occurrence link between keywords was generally low. Keywords relating to sickle cell disease tended to cluster separately from the other clusters of keywords. Furthermore, sickle cell disease and sickle cell anaemia were among the top keywords in studies from Brazil.The majority of studies were journal articles followed by randomized control trials or clinical trials. While China and India far exceeded the other member countries in their absolute numbers of journal articles, the relative percentage of journal articles published within each country were similar. South Africa’s studies comprised a higher proportion of reviews than the other countries. Russia, despite having the lowest absolute number of RCTs and clinical trial studies, had a higher proportion of these studies compared to the other countries.The top five research organizations with respect to the number of publications comprised two organizations from India, followed by two from China and one from South Africa. The Chinese and Indian organizations with the most publications generally had fewer collaborations with other organizations in their respective countries, while within-country collaborations were more prevalent among the top ranking South African, Brazilian and Russian organizations. The findings from this bibliometric analysis provide evidence that China and India have dominated in terms of numbers of publications over the past three decades. Furthermore, the inter-country collaborations have mostly involved organizations from these two countries. However, there was minimal collaboration involving organizations from the other BRICS countries. There are, therefore, opportunities for increased collaboration between BRICS countries on anaemia research in children and adolescents. This study included documents published only in English, indexed and retrieved from PubMed. It is likely that the conclusion from this analysis may not reflect the actual publication trends as affected by BRICS member countries, organisations and authors whose publications were in languages other than English and have not been indexed or appeared in PubMed.VOSviewer warning: PubMed data on organisations may not have been harmonized. Organization names may not have a consistent format. Other observation was that publications by organisations and authors outside BRICS countries where the published documents included study site and data from BRICS member countries were difficult to eliminate from the PubMed search result/output.In this study, a comprehensive bibliometric analysis has been undertaken on research conducted by BRICS  member countries on Anaemia in children and adolescents between January 1990 and October 2020. Published research and review articles were retrieved from PubMed using new and varied search words. A significant amount of interesting and revealing findings were gleaned from this study. It could be seen that there is a general increase in the number of publications over the years. While South Africa published the highest number of review articles (25 out of 87), the highest number of journal articles and letters were published by China and India, respectively. China published the highest number of RCT/Clinical Trials (80 out of 197) and Russia the least (9 out of 197). It is also of note that no two BRICS organisations have collaborated on five or more publications within the past three decades.The BRICS countries have shared health priorities, including the alleviation of all forms of malnutrition such as undernourishment and micronutrient deficiencies. The ninth BRICS Health Ministers meeting held in Brazil in 2019 reaffirmed the importance of BRICS countries investing and collaborating in research and development activities in various spheres of healthcare and wellbeing . Researc


Spodoptera exigua is a worldwide pest afflicting edible vegetables and has developed varying levels of resistance to insecticides. Methoxyfenozide (MET), an ecdysteroid agonist, is effective against lepidopteran pests such as S. exigua. However, the mechanism of MET to S. exigua remains unclear. In this study, we analyzed the expression patterns of genes related to the ecdysone signaling pathway in transcriptome data treated with sublethal doses of MET and analyzed how expression levels of key genes affect the toxicity of MET on S. exigua. Our results demonstrated that 2639 genes were up-regulated and 2512 genes were down-regulated in S. exigua treated with LC30 of MET. Of these, 15 genes were involved in the ecdysone signaling pathway. qPCR results demonstrated that ecdysone receptor A (EcRA) expression levels significantly increased in S. exigua when treated with different doses of MET, and that the RNAi-mediated silencing of EcRA significantly increased mortality to 55.43% at 72 h when L3 S. exigua larvae were exposed to MET at the LC30 dose. Additionally, knocking down EcRA suppressed the most genes expressed in the ecdysone signaling pathway. The combination of MET and dsEcRA affected the expression of E74 and enhanced the expression of TREA. These results demonstrate that the adverse effects of sublethal MET disturb the ecdysone signaling pathway in S. exigua, and EcRA is closely related to MET toxic effect. This study increases our collective understanding of the mechanisms of MET in insect pests. Spodoptera exigua treated with LC30 of Methoxyfenozide, all of which were involved in the ecdysone signaling pathway.Five genes were up-regulated and 10 genes were down-regulated in EcRA was up-regulated as treatments of Methoxyfenozide on S. exigua increased.The expression of EcRA expression by RNAi dramatically increases the sensitivity of S. exigua to Methoxyfenozide.Knockdown of EcRA expression affects the expression of the ecdysone signaling pathway genes in Methoxyfenozide-treated S. exigua. Knockdown of Spodoptera exigua  is a polyphagous pest. It attacks important cultivated and industrial crops, including cotton and sugar beets, resulting in significant losses [S. exigua is important for agricultural production, and insecticides can be used for this purpose [S. exigua [t losses . Therefo purpose ,3,4. Tra. exigua ,3,4. How. exigua . Integra. exigua . Compare. exigua .EcR, USP, and several other ecdysone-responsive genes, including the ecdysone-induced protein 74EF (E74), nuclear hormone receptor HR3 (HR3), hormone receptor 4 (HR4), hormone receptor FTZ-F1 (FTZ-F1), hormone receptor HR38 (HR38), and chitin biosynthesis pathway genes [Trehalose 6-phosphate (TPS), Trehalase (TRE), Hexokinase (HK), Glucose-6-phosphate isomerase (G6PI), Glutamine fructose-6-phosphate aminotransferase (GFAT), Phosphoacetyl Glucosamine Mutase (PGM), N-acetyltransferase UDP-N-acetylglucosamine Pyrophosphorylase (UAP), and Chitin Synthase (CHS), all of which have been found in several insect species [IGRs contain Juvenile Hormone agonists (JH), ecdysteroid agonists (20E), and Chitin Biosynthesis Inhibitors (CSI). 20E is a molting hormone and combines with the ecdysone receptor (EcR) and ultraspiracle (USP), turning into a compound . This coay genes ,10,11,12 species ,14,15,16Helicoverpa armigera that were fed Escherichia coli expressing amiRNA-HaEcR exhibited developmental defects and high rates of mortality [EcR influenced developmental duration, pupae formation, adult eclosion, and the chitin content of S. exigua [EcR in physiological processes, the relationship between EcR and insecticides has been reported. In Conopomorpha sinensis, EcR expression was changed by chlorbenzuron [S. exigua with tebufenozide or RH-5849 demonstrated that ecdysone agonists binding with EcR [EcR expression was enhanced, and subsequently impaired fecundity [S. exigua treated with MET [S. exigua.In Lepidoptera, the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis and larval growth are involved in two ways, mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR) pathways ,17,18. Eortality . The sup. exigua . To elucbenzuron . Treatinwith EcR . After tecundity . MET is ecundity . Therefowith MET ,25. HoweS. exigua fed with MET were sequenced using an Illumina TruseqTM RNA Sample Prep Kit. We determined that the ecdysone signaling pathway genes associated with S. exigua fed with MET. Using quantitative real-time PCR (qPCR), identified the gene expression change and used RNAi to assess the role that EcRA involve in increasing S. exigua sensitivity to MET. We also assessed the expression patterns of ecdysone signaling pathway gene changes in MET and RNAi treated larvae. These results reveal different doses of MET affects gene expression on ecdysone signaling pathway, exhibiting that knocking down EcRA impacts mortality of MET and the expression of ecdysone signaling pathway genes of S. exigu. This study attempts to determine the interaction of EcRA and MET on S. exigua, and further explore the action mechanisms of MET.In this study, the transcriptome of S. exigua larvae in MET and control groups. Quality control was performed on the original sequencing data to obtain high-quality data and conduct statistical analyses  and 49% (MET).The total number of transcripts and UniGenes were 118,834 and 93,874. After filtering, the average error rate located with 0.02~0.03%. The results show that >98% of each sample had a quality score of Q20, and >95% of each sample had a quality score of Q30. The GC content of the S. exigua was determined by comparing its transcriptomes in response to MET. Compared with the control group, 2639 genes were up-regulated and 2512 genes were down-regulated  in egulated a. Accordegulated b. By come of MET . KEGG ene of MET c. There e of MET . In qPCRreliable .10, LC30, and LC50) of MET were used to examine the dose-dependent effects in ecdysone signaling pathway gene expression in S. exigua. EcRA, USP, and HR3 were positively up-regulated and E74, HR4, FTZ-F1, and HR38 were down-regulated after the larvae were treated with different doses of MET. Notably, the up-regulation of EcRA increased to 10.85-fold under LC10 treatment, 14.02-fold under LC30 treatment, and 14.81-fold under LC50 treatment . Knock down of EcRA in L3 larvae resulted in increased mortality. Approximately 55.43% of MET-dsEcRA treated larvae at the LC30 dose of MET  is a polyphagous pest. It attacks high value cultivated and industrial crops, including cotton and sugar beets [S. exigua treated with MET [S. exigua. In this study, we analyzed S. exigua after it was treated with MET to assess the role of genes involved in the toxicity of MET. The result of the transcriptome sequencing, DEGs, qPCR, RNAi, and bioassay analysis exhibited that sublethal doses of MET could adversely affect the ecdysone signaling pathway, and knock down of EcRA increased the mortality of MET to S. exigua. ar beets . Therefowith MET ,25. HoweSpodoptera frugiperda provided several genes for defensive mechanisms [S. exigua using transcriptome sequencing. This lays a foundation for studying the relationship between insecticides and ecdysone signaling pathway genes. EcR and SfUSP-2 were significantly induced by the hormone agonist MET [PxTre increased in flubendiamide [HK, G6PI2, GFAT, PAGM1, and UAP genes decreased significantly after the injection of validamycin [ChsA dramatically decreased in response to chlorantraniliprole [S. exigua [H. armigera 20E-related genes [Leptinotarsa decemlineata, as halofenozide can activate detoxification, and the ability of agonists to regulate the expression of the 20E-response genes [EcRA was significantly regulated in three different sublethal doses. Interestingly, Plutella xylostella was treated with LC25 and LC50 of fufenozide for 48 h, and PxEcR-B expression was downregulated [EcR expression trends in the two species of Lepidoptera differed following treatment with the same kind of insecticides. Hence, we identified the EcRA gene as the primary research objective after treating S. exigua with MET. However, little is known about the interaction relationship between EcRA and MET.Transcriptome sequencing is used to identify the relationship between insecticides, pests, and key genes involved in pest control. Analysis of the transcriptome sequencing of Lufenuron against chanisms . We founnist MET . The expndiamide . The expidamycin , while miliprole . MET aff. exigua . Previoued genes . Our resse genes . It has se genes ; therefoegulated . EcR expEcRA was an important gene in the ecdysone signaling pathway, and knock down of EcRA in S. exigua increased the sensitivity to MET. Similarly, miR-189942 suppressed PxEcR-B expression and influenced the tolerance of P. xylostella to fufenozide [EcRA significantly decreased after dsEcRA injection compared with the control. Moreover, we applied gene silencing followed bioassay to evaluate the role of EcRA in the MET toxicity to S. exigua. After the EcRA was knocked down, the results of the bioassay showed that mortality of S. exigua was increased when exposed to MET. It indicates that EcRA involves in the detoxification of MET. Overall, these findings indicate that EcRA plays an important role in regulating the toxicity of MET, but that crosstalk between the MET and dsEcRA treatments for the genes in the ecdysone signaling pathway requires further study.fenozide . In thisEcRA influenced the expression of genes in the ecdysone signaling pathway. Similarly, the mRNA level of 20E signaling genes was significantly changed after the knockdown of EcR [E74 differed from previous research. There are two possible reasons for this. First, Leptinotarsa decemlineata and Henosepilachna vigintioctopunctata belong to the order Coleoptera, while S. exigua belongs to the order Lepidoptera, and the structure and composition could differ among the three pests. Second, MET affects the expression of E74. After the injection of dsEcRcom, the chitin contents in the cuticle significantly decreased [G6PI, UAP, and CHSA significantly changed in S. exigua treated with MET-dsEcRA. The expression levels of SeTre-1, SeG6PI, SeUAP, and SeCHSA were significantly reduced when dsEcRcom was injected at the larval stage of S. exigua [In this study, knockdown of n of EcR ,36,37,38ecreased . In our . exigua . S. exigua treated with different doses of MET, and that knocking down EcRA affects mortality of MET and the ecdysone signaling pathway. These results suggest that EcRA, as an important gene in ecdysone signaling pathway, plays an important role of totoxic process of MET to the target pest. Therefore, our findings offer a deep insight into interaction of EcRA and MET, and the action mechanisms of MET.Overall, our study reveals the gene expression of the ecdysone signaling pathway in The insecticide used in this study was Methoxyfenozide (MET) (97% purity), which was provided by Anyang Quanfeng Biotechnology Co., Ltd. . All compounds were technical grade materials, and stock solutions of 100 mg/L of technical grade materials were prepared using acetone as the solvent .S. exigua larvae were collected from the cotton fields of Anyang, Henan, China, and had been raised for at least 30 generations in a greenhouse before the bioassay [w/v) honey solution.bioassay . The lar10 = 7.00 ng/cm2, LC30 = 21.00 ng/cm2, LC50 = 38.20 ng/cm2) was added to every orifice, and 100 μL of DEPC water was used as a control group [S. exigua were not changed.The MET was dissolved in acetone and diluted with DEPC water to the desired concentration. L3 larvae were starved 4 h and placed into 24 orifice tissue culture plates, containing the artificial diet, via the drug–membrane method. A total of 100 μL different sublethal doses of MET  was isolated from the cuticle tissues using TRIzol Reagent  according to the manufacturer’s instructions. Total RNA was stored at −80 °C.After 72 h of MET induction, samples of the L4 larvae were taken from all treatments. Larvae were fixed and dissected, after which the cuticles were collected with tweezers. The cuticles were cleaned and placed in a 0.7% saline solution. The filter paper absorbed the residual saline from the cuticle. Biological samples were performed in triplicate, with each comprising one L4 larva cuticle that was released in a 1.5 mL tube. Total RNA (LC30 and DEPC water), obtained as described above, were isolated using Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Total RNA was sent to MAJORBIO CLOUD for cDNA library construction and transcriptome sequencing. The library was constructed using an Illumina TruseqTM RNA sample prep kit and the software was used to assemble all of the clean data from scratch. The reads were decomposed, after which we built the k-mer (default k = 25), selected the seed k-mer, and extended both sides to form a contig. Overlapping contigs were clustered to form components, and each component became a set of possible representations of mutable shear isoforms or homologous genes. Each component had a corresponding DE Bruijn Graph. The DE Bruijn Graph of each component was simplified to output the full-length transcript of the variable-shear subtype, and the transcript corresponding to the homologous gene was combed to obtain the splicing result file. All UniGenes and transcripts obtained by transcriptome assembly were compared with six databases , with results providing statistics on the annotation and KEGG of each database.Approximately 1 μg of total RNA  method reduces the influence of differences in gene length and sequencing volume on the calculated gene expression, which can be directly used to compare differences in gene expression between different samples. Raw counts were statistically analyzed using the DESeq2 software to obtain the UniGenes and Transcripts, and compare differences in expression between the groups based on certain screening conditions. S. exigua, we compiled all the known genes involved in this pathway based on reports on S. exigua and other insects and sequenced them based on their functional annotation in the transcriptome database [To identify the ecdysone signaling pathway genes in database ,40,41. P10, LC30, LC50, and DEPC water), prepared according to the above section, were selected to synthesize cDNA. The cDNA was synthesized from total RNA using Evo M-MLV Reverse Transcriptase Kit . The cDNA synthesized mixture (10 μL) contained 500 ng total RNA, 1 μL gDNA Clean Reagent, 2 μL 5× gDNA Clean Buffer, and RNase Free water to 10 μL. The reaction conditions were 42 °C for 2 min, and 4 °C for 10 min. The mix of step one contained 1 μL Evo M-MLV RTase Enzyme Mix, 5 μL 5× RTase Reaction Buffer Mix I, 1 μL RT Primer MIX, and 4 μL RNase Free water to 20 μL. The cDNA was synthesized following these cycling conditions: 15 min at 37 °C, 5 s at 85 °C, and 10 min at 4 °C. All cDNA was stored at −20 °C. To investigate sublethal doses of MET induction, approximately 500 ng of total RNA  and a 467 bp fragment of the green fluorescent protein gene  were amplified using specific primers [RNA was amplified using primers with the T7 RiboMAXTM Express RNAi system. The resulting dsRNA was dissolved in DEPC water and quantified at 260 nm using a spectrophotometer Nanodrop2000C . The diluted dsRNA was used immediately or stored in 10 μL aliquot at −80 °C.The interference primers were subjected to biosynthesis according to the T7 RNA polymerase promoter . The DNA primers . PCR proRNA was injected into L3 larva (molt < 3 h) using a microsyringe . After the injection, the L3 larvae were placed into 24 orifice tissue culture plates containing the artificial diet via the drug–membrane method; 100 μL of MET (LC30 = 21.00 ng/cm2) was added to each orifice. The control was injected with dsGFP. All treatments contained MET-dsGFP and MET-dsEcRA. Every treatment was replicated five times with 120 larvae, for a total of 240 treated larvae. After the successful knockdown of EcRA, the mortality of S. exigua larvae to MET was calculated after 72 h.The one μg dsWe collected the surviving L3 larvae following treatment at 12 h, 24 h, and 48 h, and the L4 larvae following treatment at 72 h. The larval cuticles were dissected, and total RNA extracted. The cDNA was synthesized from total RNA and stored at −20 °C.EcRA, survival L4 larvae treated with MET-dsGFP or MET-dsEcRA, were collected in In order to analyze the expression of ecdysone signaling pathway genes regulated by MET-dsEcRA, USP, E74, HR3, HR4, FTZ-F1, HR38, TPS, TREA, HK, G6PI, GFAT, PGM, UAP, and CHSA were analyzed after the larvae were treated with sublethal doses of MET and MET-dsRNA via qPCR. The interference efficiency of dsEcRA was performed by qPCR from 12 h to 72 h. The qPCR program was set to the following cycling conditions: 30 s at 95 °C, 40 cycles of 5 s at 95 °C, 10 s at 60 °C, and 25 s at 72 °C. The reference genes were the elongation factor (SeEF) and Glyceraldehyde-3-phosphate dehydrogenase (SeGAPDH) [−ΔΔCT). The expression of SeGAPDH) . All samt-test, or one-way ANOVA, followed by a least significant difference test for mean comparison. The bioassay data were analyzed using IBM SPSS Statistics 20 . The primers were designed in Beacon Designer 7.0 software  and the pictures were composite using GraphPad Prism 7.0 .All the results in the experiment were presented as the mean ± standard error (SE). Significant differences were determined by Student’s


Vibrio alginolyticus were loaded into mesoporous silica nanoparticles (MSN) to compose the vaccine delivery system. Hydroxypropyl methylcellulose phthalate (HP55) was coated to provide protection of immunogen. The morphology, loading capacity, acid-base triggered release were characterized and the toxicity of nanoparticle vaccine was determined in vitro. Further, the vaccine immune effects were evaluated in large yellow croaker via oral administration. In vitro studies confirmed that the antigen could be stable in enzymes-rich artificial gastric fluid and released under artificial intestinal fluid environment. In vitro cytotoxicity assessment demonstrated the vaccines within 120 μg/ml have good biocompatibility for large yellow croaker kidney cells. Our data confirmed that the nanoparticle vaccine in vivo could elicit innate and adaptive immune response, and provide good protection against Vibrio alginolyticus challenge. The MSN delivery system prepared may be a potential candidate carrier for fish vaccine via oral administration feeding. Further, we provide theoretical basis for developing convenient, high-performance, and cost-efficient vaccine against infectious diseases in aquaculture.The development of effective vaccines and delivery systems in aquaculture is a long-term challenge for controlling emerging and reemerging infections. Cost-efficient and advanced nanoparticle vaccines are of tremendous applicability in prevention of infectious diseases of fish. In this study, dihydrolipoamide dehydrogenase (DLDH) antigens of  Larimichthys crocea) is the largest mariculture fish in China and in 2017, the annual production of large yellow croaker in marine fish has reached 177,640 tons  (PMMMA-PLGA) that was capable of effectively targeting surface immunogenic protein (SIP) to the rear intestine, eliciting a robust immunity in tilapia  served as coatings for the nanoparticle, providing protection for MSN-DLDH nanoparticles. After assembly, the properties of the nanoparticle vaccine were further characterized and evaluated by in vitro and in vivo experiments. In vitro studies confirmed that the nanoparticles were pH-controlled release of antigens and non-toxic to the kidney cells of large yellow croaker. Further, the vaccine was administered orally by feeding to large yellow croaker. Our study demonstrated that the cytokines of IFNγ, IL-1β, IL-2, IL-4, and IL-13 in difference immune tissues  were significantly up-regulated at different time via oral administration. And the levels of serum antibody against large yellow croaker steadily increased. Our study suggested that the vaccine is capable of eliciting both innate and adaptive immune response and providing protection for large yellow croaker, with the relative percent survival (RPS) of 76.92%. This study provides a theoretical basis for the future development and clinical application of economical oral vaccines based on the MSN delivery system.We previously reported an oral vaccine with an intelligent carrier, poly [(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]-poly . N, N, N-trimethylhexadecan-1-aminium 4-methylbenzenesulfonate (CTATos), horseradish peroxidase (HRP) conjugated goat anti-mouse IgG, and o-phenylenediamine (OPD) were purchased from Sigma-Aldrich . Triethanolamine (TEAH3), tetraethyl orthosilicate (TEOS), polyvinyl alcohol (PVA) and triethyl citrate were purchased from Aladdin . CellTiter-Lumi™ plus luminescent cell viability assay kit was purchased from Beyotime . TRIzol™ Reagent was purchased from Thermo Fisher Scientific . Primescript™ RT Reagent kit with gDNA Eraser (RR047A) and TB Green™ Premix Ex Taq™ II kit (RR820A) were purchased from TaKaRa .The experimental large yellow croaker  was purchased from Ningde Fufa Fisheries Co., Ltd. . The kidney cell of large yellow croaker (YCK), DLDH gene (GenBank: MK281384) was amplified by PCR with a hexa-histidine TEV protease digestion site added to the 5′ end and then subcloned into the pET-28a vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) for expression. After culturing for 3 h in LB, IPTG (BBI Life Science) was added to the media at a final concentration of 0.3 mM for induction of the target protein at 16°C for 12 h. The cells were collected by centrifugation at 6,720 g for 10 min, cells were broken by an ultrasonic cell disruptor in buffer containing 50 mM Tris pH 8.0, 300 mM NaCl, 0.1% Tween-20 and 5% glycerol. Cell fragments were removed by centrifugation at 98,900 g, and supernatant was purified by nickel-nitrilotriacetic acid (Ni2+-NTA) chromatography column . The His tag was removed by TEV protease digestion and dialysis at 4°C overnight, and the overexpressed DLDH proteins were harvested by Ni2+-NTA column for the second time. The purified protein was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Total DLDH proteins were concentrated to approximately 5 mg/ml with a 30 kDa cut off ultracentrifugal filter device .The Colwellia psychrerythraea 34H  was used as a template for modeling, and the three-dimensional structure diagram was performed using PyMOL software  and at the ratio of 1:1.28 (mMSN/mDLDH), and then gently rotated at 7°C overnight, and the protein was loaded into the MSN, resulting in MSN-DLDH. The loading degree of the protein in MSN-DLDH was calculated by the following formula: DLDH load ratio (%) = DLDHload/(DLDHload + MSNmass) × 100%. In order to protect the antigen from destruction by strong acid environment in the stomach, HP55 enteric coatings were prepared by the double emulsion method . The primary emulsion (W1/O) was obtained after ultrasonic emulsification at 475 W for 2 min. The primary emulsion was transferred to the external aqueous phase W2 (containing 0.1% PVA), and the complex emulsion (W1/O/W2) was obtained after ultrasonic emulsification at 160 W for 3 min. Then, the mixture of CH2CH2OH and CH3COCH3  was added slowly under stirring. MSN-DLDH@HP55 vaccine was obtained by centrifugation at 17300 g for 10 min after stirring at room temperature until the organic solvent evaporated. Subsequently, the vaccine was attached to the feed, adhering to detailed method as follows: The commercial feed of large yellow croaker was mixed with the binder, and then the vaccine suspension was sprayed on the feed evenly. Finally, the feed was soaked in the chitosan solution  for 3 s to form coatings and quickly transferred to distilled water for 5 s, and then dried using vacuum drying technique.MSN preparation was performed according to previously described protocols with minor modification . CTATos n method . The MSN2O, and the droplets of sample suspension were air-dried on the copper grid. The CHNS elemental analysis was performed using an Elemental Analyzer . The particle potential of the sample was analyzed by laser particle size and zeta potential analyzer . The samples were dissolved in ddH2O (1 mg/ml) and measured immediately at 25°C for five times, 3 s/round. The loading capacity of the material was analyzed by thermal gravimetric analyzer . The sample was heated in nitrogen atmosphere, ranging from 35°C to 900°C, at 10°C/min heating rate. The specific surface area and porosity of the sample were analyzed by chemisorption-mass spectrometry . The specific surface area was calculated by the Brunauer-Emmett-Teller (BET) equation; the pore size distribution was calculated using the Barrett-Joyner-Halenda (BJH) model; and the pore volume was determined by the amount of adsorption at relative pressure (P/P0) of 0.97.The morphology of sample was analyzed by field emission transmission electron microscope  at an operating voltage of 200 kV. The sample was dispersed evenly in ddHThe acid-base release performance of MSN-DLDH@HP55 was analyzed by artificial gastric fluid at pH 1.2 and artificial intestinal fluid at pH 7.5. The MSN-DLDH@HP55 vaccine suspension was centrifuged, and the supernatant was discarded. Then, the pellet was resuspended in artificial gastric and intestinal fluid, respectively. Both groups were incubated in a shaker at 30°C for 6 h at 260 rpm. The control MSN-DLDH was incubated in artificial intestinal fluid under the same condition. In order to determine whether the vaccine could release the antigen normally after treatment in an acidic environment, the vaccine was incubated in artificial gastric fluid at pH 1.2 for 3 h and then transferred to artificial intestinal fluid at pH 7.5 for 3 h. The DLDH concentration in the supernatant was measured by ultramicro ultraviolet visible spectrophotometer  every 30 min for each group.in vitro were assessed by CellTiter-Lumi™ plus luminescent cell viability assay kit . YCK cells were grown in 96-well plates (104/well) in M199 media supplemented with 10% fetal bovine serum for 24 h at 27°C. Then YCK cells were incubated with different concentrations  of DLDH proteins and MSN-DLDH@HP55 for 24 h, respectively. To each well, 100 μl CellTiter-Lumi™ plus solution was added and wells were shaken at room temperature for 2 min to lyse the cells. Next the plates were incubated at 25°C for 10 min. The luminescence of each well was measured by microplate reader , and cell viability was evaluated according to the following equation: Cell viability (%) = (Sample/Control) × 100%.The cytotoxicity of DLDH proteins and MSN-DLDH@HP55 Vibrio alginolyticus. The healthy fish were randomly divided into four groups (50 fish per group), each group had three biological repeats. The nanovaccine group and DLDH group were orally administered the MSN-DLDH@HP55 vaccine feed and DLDH vaccine feed, respectively, two times at 1 week interval, while the challenge group was given commercial feed all the time. Each time, the nanovaccine group was given 2 μg of the vaccine per gram of large yellow croaker each time of immunization. One week after the second stimulation, the challenge group, nanovaccine group and DLDH group were challenged with 10-fold median lethal dose (1.1 × 107 CFU/ml) of Vibrio alginolyticus in large yellow croaker, while the immunized fish was intraperitoneal inoculated with 0.2 ml PBS as control. The relative percentage survival (RPS) of the large yellow croaker was recorded for consecutive 14 days and the RPS was calculated according to the following formula (The RPS assay was performed to demonstrate the immune protection effects against  formula :5F4 against large yellow croaker (1:500), and the third antibody was goat anti-mouse IgG conjugated to HRP . The optical density (OD) value of each well was determined by microplate spectrophotometer . The OD value was transformed to percent of inhibition (PI) of the antibody in serum, determined with the following equation: PI (%) = (1-OD490 of sample serum/OD490 of control serum) ×100%. When the absolute value of the PI value was no less than 18.4%, the sample was determined to be positive. All tests were performed in triplicate.Three fish were randomly selected from each group for serum collection at day 0, 7, 14, and 21. Immune response was assessed by antibody serum levels determined by blocking ELISA . A 96-wevia reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using TB GreenTM Premix Ex Taq™ II kit (RR820A). β-actin was used as an internal control with β-actin forward and reverse primers. The relative gene expression of each group was compared with that of day 0 according to the formula: Relative expression = 2−ΔΔCt , energy dispersive X-ray spectroscopy (EDX), dynamic light scattering, thermo gravimetric analysis (TGA) and nitrogen adsorption-desorption analysis. The results of TEM showed that MSN had a circular divergent pore structure with a diameter size of 71.39 ± 8.00 nm Table S1Analysis of acid-base release performance of the vaccine is shown in In vitro cytotoxicity analysis results of DLDH and MSN-DLDH@HP55 are shown in Vibrio alginolyticus, and the DLDH control group was 26.67%, while that in the nanovaccine group was 80%  and down-regulated at 21 days. The highest expression level of IFNγ was at 14 days, while the IL-1β was at 28 days. The expression of IL-2 in the head kidney of immunized group was up-regulated at 7 and 14 days after immunization , with the highest expression level at 7 days, while it was down-regulated at 21 and 28 days. The IL-4 and IL-13 in the head kidney of immunized group were up-regulated at 7 and 14 days , down-regulated at 21 days, and up-regulated at 28 days . The highest expression level of IL-4 was at 7 days, and the IL-13 was at 28 days.via oral administration has proven to be widely applicable, efficient in time and cost, and greatly applicable to the safety management of fish of various sizes and stages  in Vibrio. It was the common protective antigen with cross-protection effect for the pathogen of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi  can be found in the article/The animal study was reviewed and approved by The Research Ethics Committee of Institute of Biotechnology, Fujian Academy of Agricultural Sciences.YW: conceptualization, funding acquisition, project administration, writing—review and editing. WZ: methodology, software, validation, formal analysis, investigation, writing—original draft. CZ: conceptualization, methodology, project administration, formal analysis, writing—review and editing. FX and FC: methodology. XL: formal analysis, writing—review and editing. HG: methodology, writing—review and editing, funding acquisition, resources. All authors contributed to the article and approved the submitted version.This work was supported by the National Thousand Talents Program of China, the STS Project of Chinese Academy of Sciences and Fujian Province (2016T3041), the Key Project of Fujian Province (2017N0031), the Fujian Province Natural Science Foundation of China (2019J01280), Special Funds of the Central Government Guiding Local Science and Technology Development (2020L3008), National Key R&D Program of China (2019YFD0900102), Fujian Provincial Public Research Institute of Fundamental Research (2019R1026-4).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.