The Arrhenius constitutive model was built for the corresponding conditions, and the model’s accuracy was verified through error analysis. Additionally, hot processing maps were constructed to analyze the processing zone of the steel under different hot deformation conditions. Finally, the microstructure of the processing zones was observed and verified using the electron backscattered diffraction (EBSD). The results indicate that the interaction of work hardening and dynamic softening influences the hot deformation behavior of 26CrMo7S steel. The Arrhenius constitutive equation with a value of the correlation coefficient (r = 0.99523) accurately predicts the flow behavior of 26CrMo7S steel under different strains. The optimal processing zone obtained with the hot processing maps is within a deformation range of 1010~1190 °C and a strain rate range of 0.01~10−1.5 s−1, and the obtained microstructure is in good agreement with the analysis results.The hot deformation behavior and flow stress characteristics of experimental 26CrMo7S steel were analyzed using a thermal simulator under a range of conditions, including a strain rate range of 0.01~10 s With the decrease in traditional oil resources, unconventional oil fields like shale and tight oil are being explored. Deeper oil wells mean increased temperature and pressure, leading to a more adverse operating environment for oil well pipes, resulting in the need for steel with higher safety and reliability standards ,2,3. The−1. The research found that fully recrystallized structures corresponded to a high-energy dissipation zone. Zhang et al. [−1, using the Gleeble-1500D thermal simulation test machine. they were able to develop stress–strain curves, a complex flow stress constitutive equation, and hot processing maps. Furthermore, they determined the optimal hot deformation parameters within the temperature range of 800~850 °C and strain rate range of 0.01~1 s−1. These studies showed that the establishment of a constitutive equation and the use of hot processing maps can accurately predict deformation mechanisms and prevent processing instability of material during hot deformation. In addition, the constitutive and hot processing maps are beneficial to identify the appropriate hot deformation parameters of material and provide valuable guidance for safe process and gaining the suitable performance of material during the hot deformation. Although numerous studies concern the constitutive models of various alloys merged with hot processing maps, there is a lack of systematic research on 26CrMo7S steel, and the microstructure and final properties of 26CrMo7S steel are affected by hot deformation conditions. Therefore, it is necessary to study the hot deformation parameters of 26CrMo7S steel and derive a corresponding constitutive model and hot processing maps, facilitating the optimization of hot deformation parameters for this particular steel.The study of hot processing maps helps control microstructure and ensure optimal performance by avoiding generating defects caused by inappropriate hot deformation parameters. In recent years, experts have studied different alloys to create models and maps for hot processing. Zhou et al.  tested tg et al.  studied g et al.  performeIn this paper, a high-temperature simulation compression test was conducted on the 26CrMo7S steel sample. The true stress–strain curve was plotted from the experimental data, and then the Arrhenius constitutive equation was modeled. According to the curve and the model, the distribution of stress, strain, and temperature fields, as well as the relevant flow properties, were investigated under different hot deformation situations. The relationship between flow stress and the deformation process was analyzed quantitatively, and hot processing maps were created to examine the cooperative deformation and microstructural characteristics of 26CrMo7S steel. These results provide a mathematical model for predicting flow stress under different hot deformation conditions and a theoretical basis for obtaining suitable properties of experimental steel through the building of hot processing maps.The experimental material is 26CrMo7S steel, and its chemical composition is shown in The experimental procedure and the hot compression test carried out on a Gleeble-3800 thermal simulation testing machine are presented following the schematic map in −1 to 10 s−1. But with the deformation temperature increased from 850 °C to 1250 °C, the flow stress value gradually decreased.The true stress–strain curves of 26CrMo7S steel are depicted in −1 and 0.1 s−1, the flow stress curve exhibited a dynamic recovery type at deformation temperatures of 850 °C and 950 °C. When the deformation temperature was 1050 °C or above, the flow stress curve exhibited dynamic recrystallization, the same result as Chen et al. [When the strain rate was 0.01 sn et al. , and then et al.  suggeste−1, at a deformation temperature of 850~1050 °C, the flow stress curve increased with increasing strain, displaying dynamic recovery [−1, the flow stress curve exhibited a dynamic recovery type at deformation temperatures ranging from 850 °C to 1250 °C. As the strain increased, the flow stress peaked and demonstrated a tendency towards flattening or continuous rise.When the strain was 1 srecovery ,26, whilThe constitutive equation can determine the relationship among the deformation parameters, such as flow stress, temperature, and strain rate. The strain rate was controlled by the hot activation process, which occurred during hot deformation. Flow stress is essential data for high-temperature flow characteristics, while deformation temperature and strain rate greatly influence it.Based on the true stress–strain curve, the relationship between flow stress and deformation parameters was often expressed using the classical Arrhenius constitutive model.The Arrhenius constitutive model proposed by Sellars and Tegart is widely acknowledged for its ability to demonstrate the correlation among flow stress, temperature, and strain rate . The ArrThe Arrhenius constitutive equation can be expressed in three equations following the different stress levels. The constitutive equation of 26CrMo7S steel is based on the following three forms, respectively .When When T is the absolute temperature, and Q is the activation energy of deformation, All Z parameter.Then, using the definition of the hyperbolic sine function to solve Equation (6), the flow stress The natural logarithm of Equations (2)–(4) was taken respectively, and the obtained expression is shown in Equations (8)–(10).Equation (10) is deformed to obtain Equation (11).The data points of the peak stress shown in Based on Equations (8) and (9), the relationships of n = 5.957176.Similarly, Q = 452,648 kJ/mol, and ln A is calculated to be 38.98675.The substitution of the flow stress value under peak conditions into Equation (11) enabled the linear fitting of the scatters of The Q value was substituted to obtain the parameter Equation (13).In Equation (1), the calculated hot deformation activation energy Z parameters under peak conditions, as shown in Equation (14).In Equation (7), substituting the calculated parameters can obtain the stress equation of 26CrMo7S steel expressed in r can express the linear strength between the predicted and experimental values, and r can be computed as shown in Equation (15).Error analysis of the model prediction and experimental values can further describe the accuracy of the above constitutive equations. The correlation coefficient The deviation between the predicted and experimental values can be reflected by the absolute error , and the experimental data were substituted into Equations (15)–(17). The corresponding the correlation coefficient and absolute error were obtained, as illustrated in It can be seen from Based on the Dynamic Material Model (DMM) theory, the material dissipates as a power dissipator during hot deformation. The absorbed energy is mainly dissipated in two forms: one is the deformation viscoplastic heat generated at high temperature and high strain rate, which is dissipated through heat; the other one is the microstructure evolution, which is dissipated through dynamic recovery, dynamic recrystallization, hole formation, and phase transformation. σ), strain (ε), and strain rate .m is the strain rate sensitivity coefficient. Also, they are decisive for the allocation of K is the material constant, and Deriving from m = 1, the material exhibits the ideal linear dissipation process, as shown in According to the dynamic material models, when m ≤ 1, the material does not exhibit energy dissipation behavior. When the m-value is between 0 and 1, it indicates that a nonlinear dissipation process occurs, as shown in η to represent the microscopic deformation mechanism in the hot deformation process. The η value is the ratio between the Jmax, as shown in Equation (21).η is the power dissipation factor, J is the dissipative covariate in the nonlinear dissipation process, and maxJ is the ideal linear dissipative covariate.Prasad et al. ,33,34 inJ could be expressed as Equation (22).(1) Prasad et al.  proposedm = 0, m = 1, η, the dimensionless parameter, can be expressed with Equation (23).Thus, according to Equation (22), when The flow instability criterion is shown in Equation (24).(2) The Murty criterion believed that only tionship . Then, GWhen The corresponding flow instability criterion is shown in Equation (27).Various criteria, such as the Malas and Gegel stabilization zone and Semiatin instability criteria, are useful in predicting the behavior of specific materials ,37. Howeη values. The flow instability values are computed through the flow instability criterion. The hot processing safety zone region is the area with high power dissipation. For example, when the strain was 0.1 , as shown in The high-power dissipation occurred mostly in areas with high temperatures and low strain rates because it is more beneficial for dislocation to migrate at such temperatures. A strain of 0.7 resulted in peak Different regions in the processing maps indicated that some specific microstructural evolutions occurred in the samples treated by corresponding processing parameters. To better understand the microscopic evolution characteristics of the samples, various areas from the hot processing maps of strain 0.7 were selected for EBSD testing. The EBSD results of experimental steel under different thermal conditions are shown in −1, which was dissipation low-value region η = 0.09. It can be obviously seen that there were recrystallization and grain growth phenomena in −1, which belonged to the flow instability region of medium temperature and high strain rate, and η = 0.15. It can be seen that the original grain of the material was significantly elongated, and the dynamic recrystallization (DRX) grew at the original grain boundary. This phenomenon can lead to uneven structure, making mechanical instability during hot deformation more likely. −1, the power dissipation coefficient η hit 0.49; the corresponding EBSD images are shown in When the temperature reached 1150 °C, and the strain rate was 0.01 sBased on the recrystallization distribution maps and IPF maps, it can be observed that new dynamically recrystallized grains occur mainly at triple junctions and original boundaries marked respectively in black boxes and circles, as shown in Ultimately, these findings above emphasized that hot processing maps of the 26CrMo7S steel were crucial in forecasting the microstructure of hot deformation.(1)Experimental steel has negative temperature sensitivity and positive strain rate sensitivity, and the peak stress decreases with increasing temperature or decreasing strain rate, which is the result of the interaction of dynamic softening and work hardening.(2)The developed Arrhenius constitutive model has high accuracy in predicting the peak stress of the 26CrMo7S steel. The hot deformation activation energy is 452.648 kJ/mol. The predicted value is strongly linearly correlated with the experimental value, and the correlation coefficient reaches 0.99523.(3)−1.5 s−1, where the peak efficiency η exceeds 0.4, and more fine grains occurred, leaving only a few initially deformed grains.The suitable domain of hot processing found from the hot processing maps is at a temperature of approximately 1010~1190 °C and a strain rate of 0.01~10The hot deformation behaviors and microstructure of 26CrMo7S steel were investigated in order to understand the hot deformation mechanism and hot processing parameters. The major conclusions are as follows:


Under field conditions, the recycled virus inoculum produced maize yield comparable to that of commercial insecticides but similar to that of the control. This study has highlighted the potential of recycled virus inoculum from larvae treated with a commercial product. This approach offers affordable means of controlling FAW since farmers only need to purchase the commercial product once and can use recycled virus inoculum from treated larvae for repeat applications.Maize is the staple food in Sub-Saharan Africa and a source of livelihood for millions of smallholder farmers. Constraints in production, which include pest damage, lead to loss of production and hunger. Since its arrival in Kenya in 2016, fall armyworm (FAW) has caused huge destruction. Chemical control is the preferred choice by farmers despite its negative effects. Baculoviruses offer a sustainable alternative to pesticides. However, their cost and special storage requirements make them unattractive to smallholder farmers, especially where repeat applications are required. The potential to use recycled virus inoculum from FAW larvae treated with a commercial baculovirus product has not been documented. This study evaluated the efficacy of recycled virus inoculum from larvae treated with Littovir, a commercial product, under laboratory and field conditions. Under laboratory conditions, the recycled virus inoculum caused varying mortality in different FAW instars, with the highest mortality recorded in the 1st–3rd instars was caused by Littovir followed by recycled virus inoculum from 100 FAW larvae (36%). Under field conditions, even though recycled virus inoculum did not offer adequate protection against FAW damage, the maize yield was comparable to that of commercial insecticide-treated plots and similar to that of control plots. This study has shown the potential use of recycled virus inoculum from infected larvae for the management of FAW. This would offer the farmers a sustainable and affordable option for the management of FAW as it would require the farmers to purchase the commercial baculovirus once and collect larvae from treated plots for repeat applications.Fall armyworm (FAW) is a major pest of maize and causes huge losses. Chemical pesticides are the commonly used control strategy among farmers. The efficacy of baculoviruses against FAW has been proven; however, farmers may not be able to afford the products. The use of farmer-produced baculovirus mixtures could provide an opportunity for a nature-based solution for FAW at a low cost. This study evaluated the potential of recycled virus inoculum from FAW larvae treated with a commercial baculovirus (Littovir) for the management of FAW under laboratory and field conditions. In the laboratory, the virus from 25, 50, 75 and 100 FAW larvae caused variable mortality among FAW instars. The highest mortality (45%) among 1 Zea mays L.) is the main staple food crop in sub-Saharan Africa (SSA) and is mainly grown by smallholder farmers [Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is one of the most destructive pests [https://www.pcpb.go.ke/crops/ (accessed on 10 June 2022). Despite their popularity, pesticides have negative and undesirable effects, including human health effects, food safety concerns emanating from pesticide residues, and development of resistance by the pest, and they also affect beneficial non-target organisms [S. frugiperda multiple nucleopolyhedrovirus (SfMNPV), has been demonstrated, and some commercial products are available in various countries, including Kenya [Maize  on the treated leaf surface. The midgut of the larvae is alkaline and dissolves the OBs, releasing the virions that bind to, and infect, the epithelial cells of the midgut after crossing the peritrophic membrane (PM) . The infA limitation of baculoviruses is that they do not cause instantaneous death of the pest. Mortality is observed days after applications; however, this is not a major challenge in maize because the crop can withstand moderate defoliation without compromising yield . StudiesSpodoptera littoralis nucleopolyhedrovirus (SpliNPV) as the active ingredient.Therefore, this study evaluated the efficacy of recycled virus inoculum from infected FAW larvae sprayed with a commercial baculovirus product, to test an approach that would reduce the cost of FAW control by smallholder farmers . In thisThe experiment was carried out at Kenya Agricultural and Livestock Research Organization (KALRO) Muguga, Kiambu County, between August 2020 and October 2021. The station lies at an altitude of 1675 m above sea level, longitude 36.6579649 E and latitude −1.2551409 S. The laboratory experiments were carried out between August 2020 and February 2021, while the field experiments were carried out between March and October 2021. Under field conditions, treatment applications were applied between May and June 2021. During the experimental period, the mean daily temperature was 21 °C, and the mean annual precipitation was 136.6 mm.Eight maize seeds, variety H514, per pot were planted in pots measuring 17 cm (diameter) and 17 cm (depth) and placed in the open field to allow germination, after which they were thinned to 4 seedlings per pot. After 7 days post-germination, the seedlings were transferred to a greenhouse to avoid infestation by FAW and other pests. They were regularly watered and used to feed FAW larvae.st to 3rd instars and soft forceps to pick larger instars (4th–6th), into boxes (22 cm (length) and 15 cm (width)) lined with a paper towel to absorb moisture from the maize leaves. The boxes had tops with fine nets to allow for air circulation. The net apertures were small enough to prevent larvae from escaping. The FAW larvae were fed with cultivated maize leaves of variety H514. The maize leaves were harvested and cleaned with water; then, they were air-dried for 10 min to remove excess moisture before being fed to the larvae. The larvae were fed every 2 days. The mean temperature in the FAW rearing room was 28 °C, and RH was 80%. A thermostatic heater was used to maintain favorable temperatures for FAW growth and development since the night temperatures could drop to as low as 8 °C.The initial FAW colony was established by collecting FAW larvae from infested maize plants, in the open field at KALRO Muguga, and then transferred to the laboratory for rearing. FAW larvae were transferred, using a soft camel brush to pick 1(a)Initial inoculum treatment with baculovirus (Littovir)11 OB/L and left for 10 min. Maize seedlings were prepared by cutting from the base and cleaning with tap water. Excess water was removed using paper towels. They were then immersed in a basin containing 5 × 1011 OB/L Littovir suspension for 5 min. Subsequently, they were air-dried for 10 min to remove excess moisture and transferred into the aerated boxes. Twenty (20) FAW larvae (1st–6th instars) were then transferred into the containers and kept at room temperature. The different larval instars were evaluated separately. Maize seedlings were replaced regularly to avoid cannibalism among the larvae. Mortality was recorded daily for 7 days, and dead larvae were collected and stored in a fridge at 4 °C for later use. This treatment had 4 replications to obtain enough infected larvae to use in the bioassays. In the laboratory experiment, FAW instars were separated into two groups, i.e., 1st–3rd instars and 4th–6th instars , before being subjected to treatment. Each group contained 30–40 larvae. The initial inoculum was prepared by suspending 5 × 10(b)Virus from FAW larvae treated with baculovirus (Littovir)When preparing recycled virus inoculum from FAW larvae, the larvae were first placed in the freezer at 4 °C for 20 min to immobilize them. The larvae were then transferred into a glass vial and crushed, using a pestle, into a paste. The dead larvae were crushed and then suspended in 10 mL of tap water to form a suspension.st–6th instars) were used in each treatment, and each treatment was replicated 3 times.There were 6 treatments, as follows: (1) Littovir, (2) untreated control, (3) recycled virus inoculum from 25 larvae, (4) recycled virus inoculum from 50 larvae, (5) recycled virus inoculum from 75 larvae and (6) recycled virus inoculum from 100 larvae. Twenty (20) FAW larvae  was applied at the recommended rate of 8 mL/20 L of water or 150 mL/Ha in 500 L of water, while Escort (Emamectin benzoate 19 g/L) was applied at the recommended rate of 25 mL/20 L of water or 500 mL/ha in 400 L of water. The pesticides were thoroughly mixed with tap water prior to application and applied using separate knapsack sprayers to avoid contamination.The Muguga area has an average temperature of 16 °C with daily temperatures rarely exceeding 28 °C or falling below 8 °C . Muguga is near the equator; therefore, there are minimal day-length variations. The Muguga area has gently sloping hills and well-drained clay-loam soils. The fertile soils in the area are originally from lava and are generally very deep .Maize variety H614, acquired from local agrodealers, was planted in plots measuring 10 × 10 m. Two seeds per hole were planted and allowed to germinate. The intra-plant and inter-row spacing was 30 cm by 75 cm, respectively. After germination, thinning was performed to leave 1 seedling per hole. The total plant population was 333 plants per plot. All the agronomic practices were carried out safe for the application of pesticides. The experiment was carried out between April and October 2021. The treatment application was performed between May and June 2021.Following the laboratory experimental results where recycled virus inoculum from 25, 50 and 75 larvae caused low mortalities, they were dropped from the field experiment, and recycled virus inocula from 100, 150, 200 and 250 FAW larvae were used instead. There were 8 treatments, as follows: (1) Littovir, (2) untreated control, (3) Engeo insecticide, (4) Escort insecticide, (5) recycled virus inoculum from 100 FAW larvae, (6) recycled virus inoculum from 150 FAW larvae, (7) recycled virus inoculum from 200 FAW larvae and (8) recycled virus inoculum from 250 FAW larvae. The experimental design was a completely randomized block design. Each treatment was replicated 5 times; hence, there were 40 experimental plots. Treatment applications were applied weekly for 5 weeks.Immediately after maize germination, FAW pheromone traps ) were installed at the rate of 4 traps per hectare to monitor FAW infestation. Twenty (20) maize plants per treatment were randomly selected from each experimental plot. The plants were thoroughly examined on the leaves and whorls for the presence of FAW larvae and eggs. The larvae found on the plants were counted and recorded. Sampling was carried out weekly and began 4 weeks after germination. Leaf damage assessment was carried out once before treatment application and thereafter once every week by assessing twenty (20) random maize plants from each experimental plot. The plants were thoroughly examined on the leaves and whorls for FAW damage. The damage was scored using the following damage scale: 1 = no damage to any ears; 2 = tip (<3 cm) damage to 1–3 ears; 3 = tip damage to 4–7 ears; 4 = tip damage to 7 or more ears and damage to 1–3 kernels below ear tips on 1 to 3 ears; 5 = tip damage to 7 or more ears and damage to 1–3 kernels of 4 to 6 ears; 6 = ear tip damage to 7–10 ears and damage to 1–4 kernels below tips of 7 to 10 ears; 7 = ear tip damage to 7–10 ears and damage to 4–6 kernels destroyed on 7–8 ears; 8 = ear tip damage to all ears and 4–6 kernels destroyed on 7–8 ears; 9 = ear tip damage to all ears and 5 or more kernels destroyed below tips of 9–10 ears . Samplin−1.Maize grain yield was assessed at the end of the experiment. Maize was harvested from all the experimental plots and dried to remove excess moisture. The maize cobs were then threshed using a thresher, cleaned and weighed. The yield data were then recorded. The grain yield was computed in kilograms per hectare and extrapolated to tons ha−1. Data were analyzed using R software version 4.1.2.The FAW infestation, damage assessment scores and maize grain yield were first log-transformed before the data were subjected to analysis of variance (ANOVA) and means were separated using Tukey HSD. Grain yield was expressed in kilograms per hectare and was extrapolated to tons hast–3rd instars varied significantly among the treatments . Littovir caused the highest mortality at 44.79%, while the recycled virus inoculum from 25 FAW larvae caused the lowest mortality at 8.3% , F = 11.7, d.f = 4, 18; p < 0.001.Mortality of the 1 at 8.3% . Howeverth–6th instars was also significantly different across the different treatments . A similar trend where Littovir caused the highest mortality while virus inoculum caused the lowest mortality was observed  (p < 0.001). Among the treatments, the synthetic pesticides Escort and Engeo were more effective in protecting maize against damage by FAW compared to the baculovirus (Littovir) and the recycled virus inocula from 100, 150, 200 and 250 FAW larvae  . The damW larvae . There wW larvae . p = 0.02)  . Escort st–3rd instars compared to the 4th–6th instars even though the mortality was below 50% in all the treatments. This variation may have resulted from the fact that the young instars have not developed defense mechanisms and hence are more susceptible to virus infection. Under field conditions, inducing infections of FAW resulting in dead caterpillars is an important source of inocula for the occurrence and maintenance of epizootics [Management of FAW relies mostly on the use of synthetic chemical pesticides which are associated with health and environmental risks, and high cost, especially for resource-poor smallholder farmers. It is therefore important to develop affordable and sustainable strategies for the management of FAW. This study evaluated the potential of using recycled virus inoculum from larvae treated with a commercial baculovirus (Littovir) under laboratory and field conditions. In the laboratory, both the Littovir and recycled virus inoculum, from FAW larvae, induced mortality, with the highest mortality induced by Littovir followed by recycled virus inoculum from 100 FAW larvae. The induced mortality was higher in the 1izootics ,16. Epizizootics .S. littoralis, not S. frugiperda, and this could explain the low levels of mortality even under optimum laboratory conditions. Fall armyworm damage to maize crops has been shown to be influenced by cropping systems and agricultural practices and varies between monocrops and intercrops, with monocrops having more damage [In the field, FAW damage and yield were evaluated for eight treatments. Among the treatments, Escort (insecticide) produced the best protection against FAW damage. Littovir performed the same as Engeo (insecticide), although there was no significant difference between Littovir, the recycled virus inoculum and the untreated control. Increasing the number of treated larvae from which the virus inoculum was prepared, and used as a treatment, did not offer any additional advantages. In fact, there was no significant difference in terms of crop protection from damage to maize crops between the 100 and 250 larvae treatments. This is advantageous because farmers will spend less time collecting 100 larvae compared to 250 larvae. The baculovirus used in this experiment (Littovir (SpliNPV)) is adapted to infect e damage ,25. The e damage ,27. The e damage ,25,28. I−1. In this study, only the treatment with Escort produced a yield that was significantly different from that of the untreated control. The yield in all the other treatments was similar to that of the untreated control. This implies that the maize damage level influenced the grain yield. This study agrees with a previous study that showed that defoliation by FAW larvae on maize minimally influences grain yield even though the damage is detectable [Maize grain yield was evaluated at the end of the season and expressed as tons hatectable . The Esctectable . Previoutectable ,31,32,33Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is highly effective in the management of FAW and is a preferred choice for biological control [The lack of significant differences in the maize grain yield between Engeo (insecticide) and the baculovirus (Littovir) as well as the recycled virus inoculum from FAW larvae treated with Littovir demonstrates the potential of the baculovirus and the recycled virus inoculum as a sustainable FAW management strategy. This could offer resource-poor smallholder farmers a sustainable and affordable FAW management option as it negates the need for repeat applications of expensive insecticides. Previous studies have demonstrated that  control ,34,35,36However, farmers should appreciate that the virus is slow-acting and does not have a knockdown effect like chemical pesticides. Hence, early application of the virus would be recommended. S. frugiperda even when applied at very high rates (250 larvae). The idea of recycling was demonstrated with SpliNPV but might work considerably better with SfMNPV. The possibility that farmers could apply baculovirus once and collect larvae and recycle the virus for repeat or subsequent applications could save farmers money. The recycled virus inoculum from FAW larvae produced maize grain yield comparable to those of insecticide-treated plots, and this suggests that this approach would offer farmers benefits that include human safety, environmental protection and enhanced biological control of FAW since the virus is highly specific and does not affect non-target organisms and thus is compatible with integrated pest management strategy.This study has established that Littovir recycled from dead larvae provided low mortality of It is recommended that further studies be undertaken using natural isolates of SfMNPV in Kenya to find a more pathogenic virus. There is also a need to determine how long the farmers should keep the FAW larvae after collecting them from the field or the virus inoculum before applying a repeat application. Further studies should also be carried out to evaluate the effect of ecological parameters on the efficacy of recycled virus inocula from dead FAW larvae under different agro-ecological zones.


To evaluate the magnitude, prevalence, and trend of the financial relationship between Japanese head and neck surgeons and pharmaceutical companies between 2016 and 2019.Cross‐sectional analysis.Japan.This study evaluated personal payments concerning lecturing, consulting, and writing paid by 92 major pharmaceutical companies to all Japanese head and neck surgeons board‐certified by the Japan Society for Head and Neck Surgery between 2016 and 2019. The payments were descriptively analyzed and payment trend were assessed using population‐averaged generalized estimating equations. Further, the payments to board executive board members with specialist certification were also evaluated separately.p < .001) and the executive board specialists without a voting right . The payments per specialist and prevalence of specialists with payments annually increased by 11.4%  and 7.3% , respectively.Of all 443 board‐certified head and neck surgeons in Japan, 365 (82.4%) received an average of $6443 , while median payments were $2002 (interquartile ranges [IQR] $792‐$4802). Executive board specialists with a voting right received much higher personal payments  than the non‐executive specialists (median $1926, IQR $765‒$4134, There were increasingly widespread and growing financial relationships with pharmaceutical companies among head and neck surgeons in Japan, alongside of introduction of novel drugs. The leading head and neck surgeons received much higher personal payments from pharmaceutical companies, and no sufficient regulation was implemented by the society in Japan. The concern for the influence of physicians' financial conflicts of interest (COIs) with pharmaceutical companies on healthcare jeopardized trust in healthcare and evoked motivation demanding greater transparency in the relationships worldwide.Among several specialties, we speculated head and neck surgeons (HNSs) have increasing financial ties with pharmaceutical companies. One of the main diseases in the specialty is head and neck cancers including oral cavity cancer, pharyngeal cancer, and thyroid cancer. While surgery and radiotherapy are the key treatment for head and neck cancers, chemotherapy has advanced dramatically over the past 2 decades.HNSs' financial relationships with pharmaceutical companies in the United States (US) were well‐described in studies conducted on otolaryngologist‐head and neck surgeons since the launch of Open Payments Database.This study is a cross‐sectional analysis evaluating the financial relationships between all board‐certified HNS specialists and pharmaceutical companies in Japan. All HNSs who were certified by the Japan Society of Head and Neck Surgery (JSHNS) were included in this study. The JSHNS, the sole and largest professional medical society for head and neck surgery in Japan, trains and certifies HNSs who have abundant skills and knowledge in head and neck surgery and can provide multidisciplinary treatment of head and neck cancers under the name of “Head and Neck Cancer Specialist.”https://www.jshns.org/modules/list_specialist/index.php) on February 10, 2022. At the time of our data extraction, the name list of board‐certified HNSs was last updated on January 20, 2022. The name list of executive board members of the JSHNS between 2020 and 2021 were also collected from the JSHNS webpage (https://www.jshns.org/modules/about/index.php?content_id=6) and the affiliation and position of the board‐certified HNSs were manually collected online to evaluate the strength of financial relationships between pharmaceutical companies and HNSs with a leading role.HNSs' names and affiliation were extracted from the official JSHNS webpage  was performed, using the panel data of the personal payments in each specialist. As the payment distribution was highly skewed  received at least 1 payment from 55 (60.0%) pharmaceutical companies between 2016 and 2019. Total payment amounts and number of instances were $2,351,621 and 3348 instances over the 4 years. The median was $2002 (interquartile range [IQR] $792‐$4802) in payments; 4.0 (IQR 2.0‐9.0) in payment instances; and 3.0 (IQR 2.0‐5.0) in number of pharmaceutical companies per specialist , while the proportion of HNSs receiving payments did not reach statistical significance . 52 HNSs (11.7%) continuously received more than $1000 for the 4 years, and among them 31 (59.6%) were university professors  worked at universities or university hospital  and the executive board HNSs without a voting right .We further investigated all 31 executive board members in 2021. Of 31 members, 18 (58.1%) were the board‐certified HNSs. All 18 executive board members with specialist certification accepted personal payments from pharmaceutical companies between 2016 and 2019 (Table p < .001) (Table p < .001) per year was observed. Limiting payments from 50 (90.9%) companies with 4‐years data, the payments per HNS and fraction of HNSs with payments also annually increased by 11.4%  and 7.3% , respectively. 20.3% to 29.1% of HNSs received more than $1000 per year and a total of 45.4% (201) of HNSs received more than $1000 per year at least 1 year.The median annual payments per specialist increased from $817 (IQR $511‐$2248) in 2016 to $1027 (IQR $520‐$2284) in 2019, with an average annual change of 12.4%  Table . The nump < .001) and prevalence of HNSs with payments . However, there were no increasing trend of payment values and prevalence of HNSs with payments among the executive board HNSs with a voting right.In a subgroup analysis on the HNSs with executive board memberships, this increasing trend of payment values and prevalence of were observed among the HNSs without executive board memberships in both of payment values  of total payments, respectively. The largest payments were made by Ono Pharmaceutical , followed by Merck Biopharma , and Taiho Pharmaceutical . Taiho Pharmaceutical distributed personal payments to the largest number of 165 HNSs, totaling 37.2% of all HNSs, followed by Merck Biopharma , Eisai . Payment trends and payment categories were described in Figures r(50) = 0.53; p < .001), as well as number of HNSs with payments (r(50) = 0.54, p < .001) in the Spearman's correlation.There were moderate positive correlations between number of new or additional indications for head and neck cancers between 2011 and 2021 and the 4‐years total payments to the board‐certified HNSs  in the pediatric hematologists/oncologists,The payment analysis on the company level has provided plausible reason for the increasing trends. We found positive associations between number of novel approved drugs and magnitude of personal payments to the HNSs, as well as the number of HNSs distributed payments. Payments from top 5 companies occupied for 60% of total payments, and among them, 4 companies had novel drugs for head and neck cancers. Among the 5 companies, Ono Pharmaceutical and Bristol Myers Squibb increasingly made personal payments to the HNSs mainly for lecturing since 2017. Ono Pharmaceutical paid 4.1 times higher payments in 2017, when Nivolumab was approved for head and neck cancer in Japan, compared to those in 2016 . Also, Bristol Myers Squibb made 13.7 times and surprisingly 27.2 times higher payments in 2017  and 2018  than those in 2016 ($3780), respectively.Despite these increasing trend of payments as well as novel drugs for head and neck cancer, the JSHNS has not made any regulation for financial COI among the board‐certified HNSs. Accumulating evidence indicates financial COI influences physicians' clinical practiceWe also found that the leading HNSs in Japanese head and neck surgery such as the JSHNS executive board members received more substantial payments and they develop and implement regulations and statements for the HNSs endorsed by the JSHNS. Financial COI among the JSHNS executive board members were declared by each executive member, according to the JSHNS policy, but the COI information was not publicly disclosed due to the privacy of members. Financial independency from any entities and integrity to the patients are the most essential bases of all professional medical societies.This study included several limitations. First, there would be underestimated payments to the HNSs from nonmember companies of JPMA. However, as the member companies accounted for 80.8% of total pharmaceutical sales in Japan in 2018,In conclusion, the majority of the Japanese board‐certified HNSs were financially tied with pharmaceutical companies manufacturing novel drugs between 2016 and 2019. These financial ties became increasingly prevalent and strong in overall HNSs. Additionally, the HNSs in leading roles received much higher personal payments from pharmaceutical companies than those in other developed countries, and no sufficient regulation was implemented by the professional medical society in Japan.Anju Murayama, data collection, study concept and design, resources, statistical analysis, drafting of the manuscript, reviewing of the manuscript, and study supervision; Haruki Shigeta, data collection, study concept and design, and drafting of the manuscript; Sae Kamamoto, data collection, study concept and design, and drafting of the manuscript; Erika Yamashita, data collection, reviewing of the manuscript; Hiroaki Saito, study concept and design, statistical analysis, drafting of the manuscript, and reviewing of the manuscript; Toyoaki Sawano, study concept and design, and reviewing of the manuscript; Divya Bhandari, study concept and design, and critically reviewing of the manuscript; Sunil Shrestha, study concept and design, and critically reviewing of the manuscript; Eiji Kusumi, study concept and design, and critically reviewing of the manuscript; Tetsuya Tanimoto, study concept and design, drafting of the manuscript, and study supervision; Akihiko Ozaki, study concept and design, data analysis, drafting of the manuscript, and study supervision; all authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.Dr Saito received personal fees from TAIHO Pharmaceutical Co., Ltd. outside the scope of the submitted work. Dr Kusumi received personal fees from Otsuka Pharmaceutical outside the scope of the submitted work. Drs Ozaki and Tanimoto received personal fees from Medical Network Systems, a dispensing pharmacy, outside the scope of the submitted work. Dr Tanimoto also received personal fees from Bionics Co., Ltd., a medical device company, outside the scope of the submitted work. Regarding nonfinancial conflicts of interest among the study authors, all are engaged in ongoing research examining financial and nonfinancial conflicts of interest among healthcare professionals and pharmaceutical companies in Japan. Individually, Anju Murayama, Hiroaki Saito, Toyoaki Sawano, Tetsuya Tanimoto, and Akihiko Ozaki have contributed to several published studies addressing conflicts of interest and quality of evidence among clinical practice guideline authors in Japan and the United States. The other authors have no example conflicts of interest to disclose.This study was funded in part by the Medical Governance Research Institute. This non‐profit enterprise receives donations from a dispensing pharmacy, namely Ain Pharmacies, Inc., other organizations, and private individuals.This study received support from the Tansa (formerly known as the Waseda Chronicle), an independent non‐profit news organization dedicated to investigative journalism. None of the entities providing financial support for this study contributed to the design, execution, data analyses, or interpretation of study findings and the drafting of this manuscript.Supplemental Material 1. Anonymized raw payment dataset.Click here for additional data file.Supplemental Material 2. Distribution of payment values per specialist.Click here for additional data file.Supplemental Material 3. Payment concentration.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries’ decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries ( Tissue engineering is an emerging field in biotechnology that develops innovative solutions to repair, regenerate, or replace injured tissues and organs . Great aFemale reproductive tissues have singularities that distinguish them from the other tissues. They are highly responsive to sex hormones, which cause morphophysiological alterations, highlighting great periodic plasticity ,9. This The ovary has a unique microarchitecture that contributes directly to ovarian follicle maturation and maintenance, which provides a suitable microenvironment for oocyte survival ,12. OvarStudies have attempted to develop biomimetic ovarian tissues capable of supporting in vitro ovarian follicle culture or that were transplantable, reestablishing ovarian functions in infertility models ,25,26,27To overcome these limitations, it was proposed to use decellularized scaffolds, which present native tissue 3D structure and ovarian ECM components, which are essential for folliculogenesis, follicular maturation, and oocyte survival ,32,33,34Ovarian decellularization has been reported for murine, bovine, swine, and human ovaries ,37,38. HAn alternative to this method is the perfusion of solvents through the vasculature of the whole organ to diffuse the solutions throughout the tissue in a more controlled way that preserves the structures ,41. The Therefore, this study aimed to establish a new and efficient protocol for the entire porcine ovaries decellularization through detergents perfusion and ultrasonication application to generate an acellular scaffold with the three-dimensional microstructure and ECM components preserved that allows cell culture and survival.n = 5 per group) were collected from prepubertal sows at approximately 6 months of age. Immediately after the collection, the samples were stored on ice and transported to the laboratory. Adjacent ligaments and uterine remnants were removed, and ovarian major vessels were dissected. This research was conducted in compliance with the institutional ethics committee regulations of the University of São Paulo (CEUAx protocol no. 8756210222). The methodological pathway is summarized in Samples were obtained from the slaughterhouse of the Faculty of Animal Science and Food Engineering of the University of São Paulo, Pirassununga campus, Brazil. The ovaries (2O) and 2 h of sodium phosphate buffer (PBS) 1x at a rate of 1 mL/min; (2) decellularization—sodium dodecyl phosphate (SDS) 0.5% for 48 h and Triton X-100 1% for 5 h at a rate of 1 mL/min; (3) final washing—the ovaries follicles were ruptured with hypodermic needles to facilitate cell debris washing, then perfused for 24 h with deionized water and 24 h with sodium phosphate buffer (PBS) 1x at a rate of 3 mL/min while they underwent an ultrasonic bath for 15 min. After that, the ovarian follicles were ruptured with injection needles to facilitate the cell debris washing, and then deionized water was perfused at a rate of 3 mL/min in the ultrasonic bath , 3 cycles of 5 min. Ovarian scaffolds were stored in PBS 1x at 4 °C for further analysis.The ovaries were cannulated through the ovarian artery and perfused with the assistance of an ORCA Bioreactor™  perfusion bomb. The perfusion sequence took place in three stages: (1) initial washing—2 h of deionized water  and microsectioned using a cryostat . The slides were stained by DAPI solution  at room temperature without light for 10 min and then washed with PBS 1x for subsequent analysis using fluorescent microscopy .Histological evaluation was used to verify the decellularization protocol’s efficiency. Native and decellularized ovarian fragments from cortical and medullary regions were fixed in 4% buffered paraformaldehyde for 48 h, dehydrated using increasing concentrations of alcohol , diaphanized in xylol, and embedded in paraffin. Sections of 5 µm  were stained with hematoxylin and eosin (HE) to evaluate nuclei presence and ECM general condition; Masson’s trichrome for total collagen content detection; Picrosirius red to distinguish different stages of collagen maturation; Alcian blue (pH = 2.5) to evaluate GAGs general content; and Weigert’s fuchsin-resorcin to evidence elastic fibers. Slides were photographed and analyzed using a light microscope .®) and then sputter coated with gold . Finally, the samples were photographed under a scanning electron microscope .Ovarian samples were washed four times with deionized water in the ultrasonic bath, then in Karnovsky solution  for 48 h and dehydrated in increasing alcohol concentrations for 5 min each. After that, the fragments were dried in a supercritical point device  in PBS was used for non-specific protein interaction blockage. The primary antibodies used were: anti-collagen I , anti-collagen III , anti-fibronectin , anti-laminin subunit α2 , anti-elastin , anti-hyaluronic acid , and the secondary antibodies were IgG anti-mouse/anti-rabbit . The incubation occurred overnight in a wet chamber at 4 °C. The reaction was detected by Dako Advance HRP  and developed with DAB , according to the manufacturer’s instructions. Slides were photographed and analyzed using a light microscope .® DNA Mini Kit  was used to extract the genetic material from native and decellularized samples according to the manufacturer’s specifications. The fragments were digested overnight at 56 °C by the action of Proteinase K stabilized by the kit’s lysis buffer. The fragments were purified and analyzed by spectrophotometry at 260 nm .For DNA quantification, the QIAampn = 5) were sectioned into three regions: cortical, corticalmedullary (intermediate), and medullary regions. This compartmentalization was conducted to evaluate possible differences in the ECM molecular profile of each region. The spectrum of each ovary region, between 4000 and 400 cm−1, is an average of three measurements with 128 scans and 4 cm−1 of spectral resolution. All measurements were performed at room temperature, and the spectra were vector normalized using OPUS software 8.7 SP2.Fourier transform infrared spectroscopy (FTIR) was used to evaluate the ECM molecular composition of native and decellularized samples. The analysis was performed in a Bruker Vertex 70v FTIR spectrometer  with an attenuated total reflectance (ATR) accessory. The ovary samples  were sectioned into three regions as mentioned. The Raman spectra were obtained using a Senterra Confocal Raman microscope  equipped with an objective lens (20× magnification) focusing the excitation laser  on the sample. The spectrum of each ovary region, from 1750 to 400 cmAfter decellularization and washing, the whole-ovary scaffolds were perfused for 10 min with 70% alcohol, then the scaffolds were fragmented and immersed in progressive and regressive alcohol concentrations  for 5 min each in the laminar flow hood. Finalizing the alcohol cycle, the fragments were washed five times with PBS 1x with 2% antibiotics  to remove the alcohol. Then, the scaffolds were exposed to UV light for 5 min and posteriorly sealed for analysis. To evaluate the scaffold sterility, the fragments were immersed in culture medium and placed into the cell culture incubator for 72 h in α-MEM medium  supplemented with 10% bovine serum .4 3T3 cells were seeded on scaffold fragments immersed in supplemented α-MEM medium for 7 days at the same conditions previously described to evaluate cell adhesion and viability. The seeded scaffolds were collected and processed for histological analysis, DAPI staining, and SEM analysis.After the sterilization process, 5.0 × 10p < 0.05. Data were presented as the mean ± SD. PCA analysis was performed on FTIR and Raman data. A one-way ANOVA was performed to compare the means of quantitative FTIR-ATR and Raman quantitative data. Statistical significance was considered when p < 0.05. A Turkey post hoc test was applied to compare native and decellularized samples. The data were analyzed with GraphPad Prism 7.0 .Shapiro—Wilk normality tests were conducted to confirm the normal distribution of the data. DNA content was analyzed by Mann-Whitney test. Statistical significance was considered at 2O, PBS, SDS, Triton X-100, and final washing), there was a progressive whitening of the ovary until the final stage  content was demonstrated by Alcian blue staining, which stains the regions containing these components in bluish tones M,P. The The elastic fibers were highlighted by Weigert’s fuchsin-resorcin staining, which stains the elastic components in dark tones Q–T. The Immunohistochemical analyses were performed to confirm the presence of the main ECM components and if they remained after the decellularization process . Type I After histological and immunohistochemical analysis, an ultrastructural analysis of native A,C,E,G a2 stretches and may exhibit overlaps with the amide A band. Amides A and B can be found in the spectral range of 3440 to 3300 cm−1 and 2990 to 2830 cm−1, respectively [−1 is characteristic of amide I, mainly attributed to carbonyl C=O stretching along the protein chain [−1 and 1240 cm−1, respectively. These are associated with NH and CH2 molecules bending, as well as C–N bonds stretching [−1 can be related to C–O–C and C–OH vibrations [Both FTIR-ATR and Raman spectroscopy were used to evaluate physico-chemical alterations in the ECM from native and decellularized samples. These vibrational spectroscopies performed together promote a better understanding of the chemical characteristics of the samples since each one provides a different response of radiation–matter interaction, complementing each other. For a preliminary evaluation, the ovaries were sectioned into three portions: cortical, corticomedullary, and medullary regions. This classification was made to verify if there was a spectroscopic distinction between the regions regarding the ECM composition. The statistical method used to analyze the found spectra was principal component analysis (PCA) for both sample groups. The FTIR-ATR spectra of native and decellularized samples A exhibitectively ,48. The in chain ,49. Amidretching . Furtherretching ,48. FinaPrincipal components analysis (PCA) explored differences in the ovary regions spectra. −1 (CH2 deformation) [−1 was calculated as shown in Amide regions can be associated with organs with high collagen content, with amide I serving as a spectral marker for protein secondary structure. Amide II can be related to protein hydration but also can indicate collagen self-assembly, which consists of a spontaneous aggregation of several scales of collagen molecules to form staggered longitudinal matrices ,51. The rmation) . C–O–C armation) ,52. Base−1, the amide III band between 1288 and 1218 cm−1 [2 and CH3 molecules [−1. S=O symmetrical stretching at 1062 cm−1 can be attributed to GAG molecules and the ring stretching vibration mode of phenylalanine at 1003 cm−1 [−1 can be associated with the presence of C–C stretches present in the proline and hydroxyproline ring residues [−1 [In addition to FTIR results, Raman spectroscopy analysis was also applied to evaluate ECM physico-chemical alterations in both native and decellularized tissues. Raman spectra before and after the decellularization process A exhibit218 cm−1 ,55,56,57003 cm−1 ,59,60. Tresidues . The eladues [−1 . Raman bOnce the ECM composition of the scaffolds was characterized, an assay was conducted to evaluate the scaffolds’ cytocompatibility. To evaluate cell interaction with the scaffolds, 3T3 fibroblasts were seeded and cultured for 7 days in order to evaluate if the cells were able to survive, anchor, and migrate through the scaffold . Using HDecellularized ovarian scaffolds have already been described as promising platforms for cell culture and excellent sources of biological matrixes for hydrogel production, which may be combined with other biomaterials and applied for in vitro oocyte maturation and maintenance ,63,64,65Pre-pubertal porcine ovaries were chosen for this study because, as these animals were not into the reproductive cycle yet, the samples would be less variable and demonstrate similar structural and physiological properties as it is known that hormonal stimulation promotes drastic changes in ovarian tissue, and therefore in the ECM ,73,74,75A new decellularization protocol was presented to generate a biological acellular scaffold of the whole porcine ovary based on the perfusion of 0.5% SDS and 1% Triton X-100 detergents and ultrasonication. The detergents perfusion through the vascular system with a controlled flow rate allows the solutions to penetrate easily into the organ parenchyma, infusing into regions of difficult access with little contact surface . This teOnly three studies proposed decellularization methods for larger ovarian tissue portions or for the whole organ ,38,78. HPennarosa et al. ,39 were Different from the currently used methods for ovarian decellularization, the described protocol included ultrasonication, a physical method that provided additional stimulation to assist in cell rupture and debris removal without degradation of the ECM ,80. SimiThe acquisition and maintenance of three-dimensional structures of several organs and tissues have been one of the main tissue-engineering goals ,85. SeveRegarding the ovary, a compartmentalized organ whose portions have distinct and defined compositions and functions, the structure is essential for follicular maturation and maintenance . SeveralHistological and immunohistochemical findings suggest that ovarian ECM components were preserved in comparison to the native tissue. However, instead of performing image quantification that may contain errors, be biased, or not represent the entire tissue scenario , and to A suitable biomaterial for biotechnological applications can support and maintain viable cells and allow cellular adhesion, migration, and proliferation . DecelluIn this study, we established an efficient and reproducible protocol for porcine, whole ovary decellularization combining perfusion and ultrasonication. This protocol resulted in a whole-organ scaffold with the microarchitecture preserved, which indicates potential application in reproductive tissue engineering. The generated scaffolds were acellular, with the ECM components preserved, and non-cytotoxic, which allowed cell viability and attachment. The technique produced a viable and suitable biomaterial to be applied as a platform for ovarian microenvironment reconstruction and to support cell culture and application in in vitro reproduction biotechnologies.


The availability of water is an essential factor in food production and, therefore, for food security, since even with moderate water deficits, plants reduce their production significantly. In a context of climate change, in which water scarcity is becoming increasingly evident, food security can be seriously compromised. In this work, the use of plant-growth-promoting rhizobacteria (PGPR) strains is proposed as a biotechnological tool to improve the physiological conditions of tomato plants under conditions of moderate water stress with the aim of trying not to lose productive capacity under these conditions. To validate it, the effect of PGPR on photosynthesis, oxidative state, and innate defensive response were determined in plants growing under moderate water stress. The results obtained show that PGPR can alter the physiology of plants in different ways, improving their resistance under moderate water stress conditions, maintaining their productive capacity. The use of this technology in agricultural production could reduce water consumption without reducing food production, which would have a strong social impact at all levels.Pseudomonas sp.  inoculated in tomato plants subjected to moderate water stress  were studied. Photosynthesis efficiency, photosynthetic pigments, compatible osmolytes, reactive oxygen species (ROS) scavenging enzymes activities, oxidative stress level and expression of genes related to abscisic acid synthesis , proline synthesis (Pyrroline-5-carboxylate synthase P5CS gene), and plasma membrane ATPase (PM ATPase gene) were measured. Photosynthetic efficiency was compromised by PEG, but bacterial-inoculated plants reversed the effects: while N5.12 increased carbon fixation (37.5%) maintaining transpiration, N21.24 increased both (14.2% and 31%), negatively affecting stomatal closure, despite the enhanced expression of NCDE1 and plasma membrane ATPase genes, evidencing the activation of different adaptive mechanisms. Among all parameters evaluated, photosynthetic pigments and antioxidant enzymes guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) responded differently to both strains. N 5.12 increased photosynthetic pigments , proline (33%), glycine betaine (4.3%), and phenolic compounds (21.5%) to a greater extent, thereby decreasing oxidative stress . Both bacteria have highly beneficial effects on tomato plants subjected to moderate water stress, improving their physiological state. The use of these bacteria in agricultural production systems could reduce the amount of water for agricultural irrigation without having a negative impact on food production.Physiological, metabolic, and genetic changes produced by two plant growth promoting rhizobacteria (PGPR)  Water is a crucial resource for agriculture, representing 72% of the total volume of fresh water consumed. Severe water stress is considered when, on average, more than 40% of the available water supply is used each year and where the demand for water is higher than the amount available during a certain period of time. Spain is one of the 44 countries highly affected by water stress .In Spain, the agricultural sector accounts for around 80% of the total water consumption. Irrigation is a fundamental element of our agri-food system. In Spain, it represented 22.9% of the cultivated area in 2021 , contributing to 50% of the final crop production. Therefore, irrigation is of paramount importance to keeping Spain as the leading exporter of fruit and vegetables in the EU, and to be among the three leading exporters in the world, along with China and the United States.To date, the water scarcity problem has been addressed almost exclusively by improving irrigation systems. However, at no time has it been contemplated to reduce water consumption because, as indicated above, this has negative consequences on crop production.1O2), hydroxyl radicals (OH−), superoxide anion (O2−), and hydrogen peroxide (H2O2), which cause lipid peroxidation of cell membranes, protein denaturation, and nucleic acid destruction /(ε × FW), where FW is the plant fresh weight and ε the molar extinction coefficient (155 mM−1/cm). Results were expressed as μmol/g FW.Malondialdehyde (MDA) content was determined as described by Hu et al. . Brieflyw/v). After sonication for 10 min and centrifugation , total phenolic compounds and flavanols were determined on the supernatant. Quantitative analysis of total phenolic compounds (TPC) was performed with Folin–Ciocalteau reagent  [2CO3 solution was prepared. After incubating at room temperature for 30 min, a UV-Visible spectrophotometer (Biomate 5) was used to measure absorbance at 760 nm. A calibration curve was constructed with gallic acid. Results were expressed as mg gallic acid equivalent (GAE) per mg of fresh weight (FW).A methanolic extract was prepared with leaf powder  and 80% MO, USA)  with modPrior to RNA extraction, samples were ground to a fine powder with liquid nitrogen. Total RNA was isolated from each replicate with PureLink RNA Micro Kit , DNAase treatment included. RNA purity was confirmed using NanodropTM. A retrotranscription followed by RT-qPCR was performed.−∆∆Ct method [P5CS , NCED1 , and PM ATPase 1 (plasma membrane ATPase 1) genes were studied. Gene primers used are shown in The retrotranscription was performed using iScript tm cDNA Synthesis Kit . All retrotranscriptions were carried out using a GeneAmp PCR System 2700 : 5 min 25 °C, 30 min 42 °C, 5 min 85 °C, and hold at 4 °C. Amplification was carried out with a MiniOpticon Real Time PCR System (Bio-Rad): 3 min at 95 °C and then 39 cycles consisting of 15 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, followed by melting curve to check results. To describe the expression obtained in the analysis, cycle threshold (Ct) was used. Standard curves were calculated for each gene, and the efficiency values ranged between 90 and 110%. Results for gene expression were expressed as differential expression by the 2t-test was performed to check the statistical differences between control and PEG treatments. One-way ANOVA with replicates was used to check the statistical differences among treatments with PEG  in all data obtained. Prior to ANOVA analysis, homoscedasticity and normality of the variance were checked with Statgraphics Plus 5.1 for Windows, meeting requirements for analysis. When significant differences appeared (p < 0.05), a Fisher test was used [Student’s was used . PrincipNet photosynthesis (Pn), transpiration (E), and water use efficiency are shown in Photosynthetic efficiency measured through the chlorophyll fluorescence emitted by photosystem II is shown in Photosynthetic pigments are shown in Compatible solutes were measured in roots and leaves separately . In all Enzymatic activities related to oxidative stress are shown in 2O2 and MDA) are shown in 2O2 concentration  [w/v) [A critical point in drought experiments is the medium used and the way to achieve stress. Osmolovoskaya et al.  reviews w/v)  or even v) [w/v)  in the gv) [w/v) . TherefoOne of the primary plant responses to dehydration is stomata closure, which is intended to prevent transpiration-related water loss and is essential for the success of the drought avoidance strategy ,38. SincThese effects found on photosynthesis and chlorophyll concentration have been described by other authors working with other bacterial genera and plants, which suggests that it may be a common mechanism of action in plants subjected to water stress and elicited with PGPRs. ,44. RegaThe fluorescence emission data from photosystem II indicate a significant increase in Fv/Fm in the inoculated plants and an increase in energy dissipation (NPQ) in the presence of PEG regardless of the presence or absence of the bacterium. On the other hand, plants inoculated with N 5.12 have significantly higher carotenoid values (carotenes + xanthophylls) than the other treatments. The relationship between the different types of xanthophylls and the NPQ is well-known . Given tPseudomonas inoculated in Arabidopsis seedlings subjected to water stress improved their resistance by increasing the concentration of proline, glycine betaine, and chlorophyll concentration. The concentration of proline in the leaves of the plants inoculated with N5.12 is related to a significantly higher expression of the P5CS gene in these plants  in the leaf (The production of reactive oxygen species (ROS) under stress conditions is one of the first consequences derived from such situations. Its elimination is vital to avoid oxidative stress . PEG-trethe leaf c,d. The the leaf , so othethe leaf f which, the leaf ; in any NCDE1 gene expression , which could reinforce the maintenance of the open stomata, since these pumps play an important role in maintaining the turgor of the guard cells [ABA is a hormone that plays a major role in stomatal closure in situations of water stress and, in general, in different abiotic stress situation. The PEG-treated plants decreased carbon fixation and transpiration significantly with respect to the control, indicating a clear effect in stomatal closure to prevent water loss. pression  in PEG ceceptors ,63. It ird cells , as wellrd cells .As evidenced in the PCA , PEG conThe scarcity of water in agricultural systems is going to become one of the most important problems in the coming years in food production. Research in this field is shown to be key if we want to produce food for a growing population. In this work, we have addressed the issue through the use of PGPRs as a biotechnological tool capable of eliciting the innate systems of plants to resist water stress conditions without losing productive capacity. The bacteria used in this work have shown that their use can be very effective. Both have been able to improve photosynthetic efficiency under moderate water stress conditions, one of them (N 5.12) increasing the concentration of photosynthetic pigments, compatible solutes, and phenols, which led to less oxidative stress (measured by the MDA marker). Both strains modify the physiology of plants in different ways, making them adapt to stress conditions. The use of this type of bacteria could become a viable and economical alternative in agricultural systems exposed to water stress, reducing production losses that occur in these situations. Experiments are currently being carried out under real growing conditions to determine if the observed effects hold up under real crop conditions.


MAGI1 gene, and with type, hereby differentiating heavy from light horses on ECA5 within the POU2F1 gene. Both genes were previously described to affect growth, muscling and fatty deposits in sheep, cattle and pigs. Furthermore, we pin-pointed another suggestive QTL on ECA21, near the PTGER4 gene, associated with human ankylosing spondylitis, for shape differences in the back and pelvis (roach back vs sway back). Further differences in the shape of the back and abdomen were suggestively associated with the RYR1 gene, involved in core muscle weakness in humans. Therefore, we demonstrated that horse shape space data enhance the genomic investigations of horse conformation.Conformation traits are important selection criteria in equine breeding, as they describe the exterior aspects of the horse . However, the genetic architecture of conformation is not well understood, as data of these traits mainly consist of subjective evaluation scores. Here, we performed genome-wide association studies on two-dimensional shape data of Lipizzan horses. Based on this data, we identified significant quantitative trait loci (QTL) associated with cresty neck on equine chromosome (ECA)16 within the  Some traits, such as the concave (dished) head shape in the Arabian horse, are breed-specific and essentially aesthetic. Other traits are relevant for health (limb angles) or influence performance (slope of the shoulder or croup)4. While some of these traits, such as height length and angle measurements, can be objectively quantified, those describing shapes, proportions or having an aesthetic component are usually scored by experts of a particular breed. Conformation scores have been shown to be subjective and unreliable in many breeds, and are therefore difficult to be included in genetic analyses8. To date, only one recent genome-wide association analysis (GWAS) has identified a quantitative trait locus (QTL) for the conformation of the croup in the Icelandic horse based on subjective scoring data9. Advances in data imagery through the field of geometric morphometrics have led to the development of the horse shape space model, tracing the contour and certain anatomical landmarks on standardized photographs of horses10. Shape space data has been shown to be more heritable and reliable than traditional scores for the shoulder joint and fetlock joint angles of the front and hind limb in Franches–Montagnes (FM) horses11. Furthermore, a GWAS of joint angles from the horse shape space model applied to FM and Lipizzan horses revealed novel QTL for poll angle and elbow joint angle12. To date, two-dimensional (2D) geometric morphometric data have been already used to explore the genetic architecture of morphological differences in mollusks13, sorghum14, or house flies15, while three-dimensional (3D) data has been applied to study facial traits in humans (summarized in16), cats17 and dogs18.Conformation refers to all exterior, visible traits of the overall morphology of an animal . Horses have been selected based on conformation traits for centuries, resulting in diverse breeds with specific use 19. Currently, Lipizzan horses of a lighter type are associated with higher Arabian admixture proportions and have a more concave head and more salient withers20. Consequently, there is a relatively large phenotypic variation in the Lipizzan horse, which makes it an ideal model to explore conformation differences in the horse in a GWAS, while avoiding population stratification present when comparing different breeds.Our aim was to explore the genetic architecture of conformation in horses. For this purpose, we used 2D shape data from the Lipizzan horse breed. The Lipizzan breed is considered a baroque riding horse, traced back to a few known Italian and Spanish founder animals born in the eighteenth century. Between 1776 and 1945, Arabian horses were also introgressed to “ameliorate” its type, affecting the conformation of the head, neck, withers and extremitiesHere, we report QTL for shape-derived conformation traits representing a gradient from a heavy to light type, from a roach to sway back, as well as for the shape of neck. These findings may have implications for the selection of Lipizzan horses, and potentially, the domesticated horse in general.20. The eleventh PC, explaining 1.32% of the variance, described a gradient between a horse with a larger neck, torso and a more sloping croup compared to a horse with a slimer torso and horizontal croup.The first twelve principal components (PCs) accounting for more than 1% of the shape variance (246 landmarks) were visualised in Supplementary Fig. For the shapes of the body parts (torso and neck), we applied the same principle and selected the second and the fifth PC accounting for 21.38% and 3.51% of the torso and neck shape variance, respectively.p = 3.25 × 10–07), within the membrane-associated guanylate kinase, WW and PDZ containing 1 (MAGI1) gene  was located at 26,328,965 near the Prostaglandin E Receptor 4 (PTGER4) gene . Horses homozygous for the reference allele (C–C genotypes) tended to have a rounder croup, a hyperkyphotic back (roach back) and a tight abdomen  was located at 3,879,741 in the POU Class 2 Homeobox 1  gene . The horses with an A-A genotype  were of a lighter type, with less neck and hindquarter musculature and a more horizontal croup, while G-G horses were heavier built, with a sloping, muscular croup , near the Transglutaminase 3 (TGM3) gene   at 9,664,363, embedded in the Ryanodine receptor 1 (RYR1) gene   of the five shape-derived conformation traits ranged from 0.09 ± 0.12 (PC11) to 0.63 ± 0.14 . The aforementioned QTL, including surrounding SNPs ± 2.5 Mb, explained 33% up to 99% of h2SNP, while the standard errors were smaller.The genome-wide heritability  and PC2 (poll flexion–extension) were not as heritable as PC3 describing the width of the neck in the FM horse11. Therefore, it is important to notice, that posture effects might account for the majority of shape variance and need to be considered in the selection of PC associated with conformation of the horse.The first two PCs, accounting for more than 50% of the shape variation, were consistent with previous results applying the horse shape space model on either LipizzanMAGI1 gene was significantly associated with the shape and width of the neck. This gene has not been described previously in equine studies, while in sheep, MAGI1 was associated with average daily gain and Kleiber ratio (average daily gain/end weight0.75) in 6- to 9-month-old sheep22. Furthermore, this gene was reported in a selection signature analysis differentiating two sheep breeds, the Sarda and Sardinian Ancestral Black, the latter having a much smaller body size23. In the horse, a cresty neck is the consequence of localised fat deposits in the dorsal neck region delineated by the nuchal ligament25 and is highly prevalent in Baroque breeds such as Pura Raza Español , Lusitano, Morgan or Paso Fino horses26. Cresty neck is an undesirable trait in the PRE, and horses with high cresty neck scores are greatly penalised during breeding shows26. Furthermore, there are some health concerns associated with cresty neck, as it has been associated with insulin dysregulation, one of the risk factors for equine metabolic syndrome, especially when measuring the neck circumference near the base of the neck28. Interestingly, MAGI1 variants have previously been associated with glucose response29 and insulin resistance31 in human studies. We suggest that the identified QTL in the MAGI1 gene might be associated with cresty neck in Lipizzan horses by affecting insulin regulation. Further studies including additional data from other Baroque breeds including horses with or without cresty neck, in combination with metabolic data such as blood insulin and glucose levels, are required to confirm this QTL.Only a single SNP within the POU2F1 gene, a transcription factor expressed in many different tissues32. In the Brazilian Nellore cattle, a QTL containing the POU2F1 gene explained 41% of genetic variance in conformation and 29% in muscling scores33 and was also identified as one of the major transcription factors regulating growth and fatty deposits in Iberian × Landrace pigs34. In humans, certain Pou2f1 isoforms were either heavily up- or downregulated by repulsive guidance molecule a (RGMa), important in skeletal cell differentiation, cell fusion and hypertrophy35. POU2F1 variants were also associated with human Type 2 Diabetes Mellitus37, and it is common that adults affected by Type 2 Diabetes lose skeletal muscle mass38. Therefore, the identified QTL is likely associated with both muscle mass and fatty deposits.The QTL associated with type is located within the PTGER4 gene. In humans, this gene has previously been associated with the severity of ankylosing spondylitis 40. Main clinical signs include lower back pain, spinal stiffness and loss of mobility42. Hyperkyphosis (roach back) is particularly common in young men43. The hip and shoulders may also become affected45, with an observed shift between movements of the shoulder girdle and the pelvis46. Although we are comparing phenotypes between bipedal humans and quadrupedal horses, the shape variance explained by PC6 was still consistent with some of the aforementioned symptoms. The higher extreme showed a horse shape with a roach back and sloping croup, as opposed to the lowest extreme of a sway backed horse (hyperlordosis) with a horizontal croup, although we have no information on actual clinical signs in this dataset of horse photographs. Follow-up studies should include radiographs of genotyped horses with extreme roach back or sway back, as well as an objective movement analysis of homozygous horses from both genotype groups to determine whether the conformation would affect the gait quality of the horse, as stride length and over-tracking distance are highly dependent on the movement of the pelvis at walk, and to a lesser extent, at trot48. Previous genomic research investigated juvenile onset lordosis in the American Saddlebred horse identifying a large haplotype on ECA20 potentially involving the TRERF1, TAF8 and C6orf132 genes49. The causal variant could not be identified yet, which the authors attributed to the phenotyping method and the complexity of the trait50. The new QTL on ECA21 may be one of the contributing genetic factors influencing the severity of lordosis. However, this should be confirmed by multi-breed studies to better understand whether this QTL is only present in Lipizzan horses. There have been no genetic studies on roach back in the horse to date, although it is also an undesirable trait in horse breeding, similarly to sway back.The scores from PC6, representing the shape variation between a sway and a roach back, were suggestively associated with a QTL near the RYR1. Ryanodine receptor 1 gene mutations have been associated with malignant hyperthermia in many mammals including pigs51, dogs52 and horses53, but also with myopathies such as central core disease (CCD), Multiminicore Disease (MmD), Centronuclear myopathy (CNM) and axial hereditary myopathy56. Symptoms vary, but commonly include proximal muscle weakness, scoliosis and hip girdle weakness. In the case of the axial hereditary myopathy, pronounced lumbar hyperlordosis is frequent56. Joint hypermobility may lead to hip dislocations, although for MmD cases the spine is very rigid55. In the horse, the gravitational pull of the viscera will over time extend the back towards lordosis, especially in the case of pregnant mares. This effect can be counteracted by well-developed ventral musculature, which can flex the back57. The extreme phenotype of the equine torso indicates a weakness of the abdominal muscles, which may cause the back to sag towards the ground.The shape of the torso was suggestively associated with a QTL containing PTGER4 could influence the shape of the spine due to osseous fusion by increasing spinal rigidity, RYR1 could weaken the abdominal musculature, causing the spine to bend as a consequence. These results underline the complex phenotype and genetic architecture of conformation traits.For the shape of the back and abdomen, we have discovered two genes with different actions, but similar phenotypes. While TGM3 gene. TGM3 is involved in skin and hair physiology. Mutations in this gene were associated with uncombable hair syndrome in humans58 and wavy hair (“wellhaarig” phenotype) in mice59. TGM3 was identified as one of the cis-regulatory elements involved in the adaptation of the Yakutian horse to the Arctic environment, probably by increasing the thickness of the coat60. It is unclear how this gene might affect the shape of the neck and withers. It is possible that if the structure of the mane hair is more rigid, the outline of the neck would be overestimated during the digitising process. TGM3 was also associated with some forms of head and neck cancers, which would suggest a regulatory function in this anatomical region61.The scores from PC5 of the neck landmarks were significantly associated with a single SNP near the 2SNP of the five shape-derived morphological traits were low (< 0.10) to high (> 0.40) and exceeded high standard errors, most likely due to the small sample size. Interestingly, the redefined QTL accounted for 33% up to 99% of h2SNP. The scores from PC3 associated with the neck width variation had the highest h2SNP. This was consistent with findings from a study of the shape space in FM horses, where the PC describing the neck width also had a high heritability, albeit pedigree-based11.The h12). Based on this method it becomes feasible to derive objective conformation traits of hundreds or thousands of horses independently from breeding judges, which simultaneously improves the data quality and quantity for genetic analyses. Furthermore, shape-derived conformation traits can be pooled across breeds and therefore support, multi-breed comparisons. However, there are currently two major limitations: the posture of the horses when analysing a single photograph, and the association of the PCs with conformation, which remains subjective. Excluding the effect of posture, by standardising the position of the head and limbs during photo taking, or by using the mean of several shapes of the same horse photographed in different postures, should result in PCs associated with differences in conformation.This study has revealed several novel QTL associated with conformation in the Lipizzan horse. However, the sample size was small, and the applied SNP data did not include whole-genome sequence information. The horse shape space model is an efficient high-throughput phenotyping method to infer objective information on different aspects of equine conformation , born between 1987 and 2013; median of 2005). Samples were collected in the studs of Piber (Austria), Đakovo (Croatia), Topol’čianky (Slovakia) and Szilvasvárad (Hungary) between 2014 and 2017, following national rules and regulations. All horses were genotyped on the commercial Axiom™ Equine genotyping Array containing 670,795 evenly distributed single nucleotide polymorphisms (SNP)11. The photographs were digitised with tpsDig263 following the outline horse model described in Druml et al.10: 18 anatomical and 15 somatometric landmarks combined with eight outlines , for a total of 246 landmarks , where each score on a PC corresponded to one specific horse, and therefore to a specific shape pattern. The PCs explaining more than 1% of the variance for the outline horse model were considered for GWAS. From the 246 landmarks of the full horse model, we also extracted landmark configurations for the neck and torso  was influenced by age and posture5. Therefore, we corrected for age in our statistical analyses and excluded PCs affected by posture.The reproducibility of the horse shape space model has been extensively studied elsewhere: The digitisation process of single horse photographs was highly repeatable (same image and digitiser) and reproducible p < 0.0001 using PLINK66, 361,411 genome-wide SNPs were available for analysis. Genome-wide association studies (GWAS) were performed using a polygenic model (polygenic_hgml) in the R-package GenABEL67 with the R version 3.4.168, taking into account sex  and age by categories  as covariates in addition to the genome-derived kinship matrix. The age categories were previously defined by Druml et al.21 for the Lipizzan horse. The significance of each SNP was extracted using mmscore.After removing SNPs not located on the autosomes, with a minor allele frequency below 5%, a genotyping rate below 90% or departing from Hardy–Weinberg equilibrium (HWE) at p < 10–5, and the significance threshold as the effective number of independent loci (pind) by pruning the 361,411 SNPs for linkage disequilibrium (LD). Using a 50-kb sliding window size, a 5-kb window step size and an r2 exclusion threshold of 0.5, resulting in an LD-independent p-value threshold of pind < 4.98 × 10–7. We also evaluated the quantile–quantile (Q–Q) plots for the inflation of small p-values, hinting at false positive association signals. The effect of the SNP on the phenotype was evaluated graphically with boxplots to visualize differences in phenotypic values. We investigated which genes were located near significant SNPs using the NCBI Genome Data Viewer, based on the EquCab 3.0 reference genome assembly69. Genes functions in horses, humans and other livestock species were investigated in current published peer-reviewed literature (last research: 03.05.2022).The results were visualised with Manhattan plots, with a suggestive significance threshold at 2SNP) using the GCTA software70 by including all available SNPs simultaneously into a genetic relationship matrix (grm), followed by a restricted maximum likelihood estimation (REML) with the grm including the aforementioned covariates age categories and sex as predictors and each conformation trait as the response variable71. To estimate the proportion of the heritability explained by the SNP with the highest-log10 p-value, we defined a QTL window of ± 2.5 Mb surrounding the most significant SNP in order to establish an informative grm72. Thereafter, we estimated the heritability of the 5-Mb QTL (h2QTL) with the covariates age categories and sex, and calculated the proportion of the heritability explained by the 5-Mb QTL compared to all genome-wide SNPs (h2QTL/h2SNP).For each reported QTL, we estimated the genome-wide heritability (hThis study was discussed and approved by the institutional Commission for Ethics and Animal Welfare, University of Veterinary Medicine, Vienna, protocol number: ETK-06/05/2015, in accordance with GSP guidelines, national legislation and ARRIVE guidelines.Supplementary Figures.


Epioblasma brevidens) is an endangered freshwater mussel endemic to the Tennessee and Cumberland River drainages, major tributaries of the Ohio River of the eastern United States. We conducted mask and snorkel surveys in May and June of 2021 and 2022 to locate, observe, photograph, and video female E. brevidens to document their unique mantle lures at sites in the Clinch River in Tennessee and Virginia. The mantle lure is morphologically specialized mantle tissue that mimics prey items of the host fish. The mantle lure of E. brevidens appears to mimic four distinct characteristics of the reproductive anatomy of the underside  of a gravid female crayfish, to include: (1) the external apertures of the oviducts located on the base of the third pair of walking legs, (2) crayfish larvae still encased in the egg membrane, (3) pleopods or claws, and (4) postembryonic eggs. Surprisingly, we observed males of E. brevidens displaying mantle lures that were anatomically complex and closely resembled the female mantle lure. The male lure similarly mimics oviducts, eggs, and pleopods but is diminutive  to those same structures in females. We describe for the first time the mantle lure morphology and mimicry of E. brevidens, showing its close resemblance to the reproductive anatomy of a gravid female crayfish, and a novel form of mimicry in males. To our knowledge, mantle lure displays in males have not been previously documented in freshwater mussels.The Cumberlandian Combshell  of a gravid female crayfish, to include: (1) the external apertures of the oviducts located on the base of the third pair of walking legs, (2) crayfish larvae still encased in the egg membrane, (3) pleopods or claws, and (4) postembryonic eggs. Surprisingly, we observed males of E. brevidens displaying mantle lures that were anatomically complex and closely resembled the female mantle lure.We describe for the first time the mantle lure morphology and mimicry of  Listed by the U.S. Fish and Wildlife Service (USFWS) as an endangered species in 1997 under the U.S. Endangered Species Act (1973), E. brevidens only has one remaining stronghold, a large population of >50,000 individuals in the Clinch River in Tennessee (TN) and Virginia (VA)  to host fishes. Females of E. brevidens are long‐term winter brooders, eggs are fertilized in late summer and early fall and the embryos then mature into glochidia in the outer gills of females from early fall through winter for release to host fishes the following spring  depending on water temperature and then excyst to begin life on the river bottom  packets composed of an outer layer or inner core of undeveloped “structural” eggs wherein hundreds to thousands of glochidia are encased or embedded  and partners in the Powell River at Fletcher Ford, Virginia, where two separate capture events were observed. The first observation was made by Isabel Boyce  on September 14 of a captured Tangerine darter (Percina aurantiaca), and the second observation was made by Sarah Colletti (VDWR) on September 15 of a captured Gilt darter (Percina evides), where both fish were caught by the head and gill operculum by female E. brevidens  infesting and releasing a host fish, the Tangerine darter (Percina aurantiaca). Video was taken by John Hartley (Virginia Department of Conservation and Recreation) at Fletcher Ford on September 14, 2022 in the Powell River, Lee County, Virginia. Video is three seconds long and the time lapse is 5 frames per second and is shown at 1× speed but it can be slowed to 0.25× speed using the Playback speed option menu.Video of a female Cumberlandian combshell (Epioblasma brevidens) capturing and infesting a host fish, the Gilt darter (Percina evides). Video was taken by Brittany Bajo‐Walker (Virginia Department of Wildlife Resources) at Fletcher Ford on September 15, 2022 in the Powell River, Lee County, Virginia. Video is 4 min and 34 s long and the time lapse is 30 frames per second and is shown at normal 1× speed.Video of a female Cumberlandian combshell  a third species. Typically, mimicry involves three species, the mimic, the model, and the dupe or what is referred to as the species that receives the signal ; however, in some mimicry systems, two out of three or all three of the mimic, model, and dupe can be the same species. By deceiving the dupe, the mimic improves its individual fitness, gaining a selective advantage by evading predation, preying on the dupe, or in the case of freshwater mussels, facilitating reproduction. While no universally accepted definition of mimicry exists, Font  recentlyon Table . Thus, t2E. brevidens displaying their mantle lures at four sites in the Clinch River: (1) Bennett Island, Russell County, VA , (2) Wallens Bend, Hancock County, TN , (3) Kyles Ford, Hancock County, TN , and (4) Frost Ford, Hancock County, TN . These sites harbor highly diverse mussel assemblages of >30 species that co‐occur with E. brevidens  displaying and moving mantle lure. Video is of the same female mussel shown in Figure Video of a female Cumberlandian combshell , rockbass (Ambloplites rupestris), catfish , freshwater drum (Aplodinotus grunniens), sculpins (Cottus spp.), and many other fish species. Like these and other fish species, consumption of crayfish, especially their eggs and larvae, would be nutritious for logperch as well. Hence, we believe based on our field observations, photographs, and video of displaying females and males, E. brevidens in the Clinch River likely is mimicking crayfish as the primary model species, rather than fish eggs as proposed by Barnhart et al.  ; methodology (supporting); resources (supporting); validation (supporting); visualization (supporting); writing – review and editing . Timothy Lane: Investigation (supporting); methodology (supporting); resources (supporting); writing – review and editing . Jess W. Jones: Conceptualization (lead); investigation (lead); methodology (lead); resources (lead); supervision (lead); validation ; visualization ; writing – original draft (lead); writing – review and editing .The authors declare no conflict of interest.