Chrysobalanus icaco) is an ecologically significant native species to Southern Florida. Application of precision agriculture technologies such as optical sensors reduces the cost of over-fertilization and nutrient runoff. The aim of this work was to establish a base line sensor value for fertilizer treatment in cocoplum by monitoring chlorophyll content using the Soil Plant Analytical Development (SPAD), atLEAF, and Normalized Difference Vegetation Index (NDVI) sensors. Initial slow-released fertilizer treatment 8N-3P-9K was used at 15 g (control), 15 g , 15 g , 30 g , 30 g , and 45 g . Evaluations were conducted at 0 (base reading), 30, 60, 90, 120, 150, and 180 days after treatment. Growth parameters, optical non-destructive chlorophyll meters, leaf and soil total nitrogen and total carbon, and total nitrogen of leachate were analyzed. The results demonstrated that the treatment using 30 g slow-released fertilizer (8N-3P-9K) supplemented twice with 15 g in November and March after the first fertilization in October provided the least contamination through runoff while still providing adequate nutrients for plant growth compared to higher fertilizer concentrations. These results demonstrate that the highest treatment of nitrogen can cause considerable losses of N, causing extra costs to producers and environmental damage due to the flow of nutrients. Thus, techniques that help in N monitoring to avoid the excessive use of nitrogen fertilization are necessary. This study can serve as a basis for future research and for nurseries and farms, since it demonstrated from the monitoring of the chlorophyll content by optical sensors and by foliar and substrate analysis that lower treatments of nitrogen fertilization are sufficient to provide nutrients suitable for the growth of cocoplum plants.Cocoplum ( The total crop value of floriculture production in the United States topped $4.80 billion in 2020 with Florida representing 24% of the wholesale value for operations. Within the state of Florida, foliage was by far the largest produced category, evaluated to be $520 million in 2020 with garden plants representing the second highest at $245 million .Chrysobalanus icaco, colloquially known as cocoplum or paradise plum, is a woody shrub native to South Florida. Cocoplum is comprised of two distinct ecotypes, inland, and coastal, differentiated by growing conditions and growth habits. Cocoplum is both ecologically and economically significant to South Florida [ Florida .Nitrogen (N) is one of the most influential macronutrients and is crucial in plant development especially in landscaping plants, being a limiting element of production. Due to this characteristic, it is intensively used in productive crops, aiming to get the crop to reach its maximum potential . GeneralGood agricultural practices respond mainly to the need of protecting biodiversity, genetic resources and landscape, soil, and water resources, as well as the provision of public goods by farmers. Biodiversity conservation is inextricably linked to agricultural activity . PrecisiThe use of visible (VIR) and infrared (NIR) imaging provides information on crop health and growth stage. During the photosynthesis process, chlorophyll molecules of plants absorb blue and red light and reflect green light. On the other hand, infrared light penetrates the inner part of leaves reflecting infrared energy. Since leaf spectral reflectance changes with plants growing, affection of diseases and pest infections, employing these images allows adequate monitoring of crops . TechnolTM, a canopy-wide reflectance sensor.Several sensors have been designed to measure either the reflectance or the absorbance of green color present in the leaves. The greenness of the leaves represents the amount of chlorophyll found in the chloroplasts, which can be used as an indirect indicator for the photosynthetic processes of the plant to determine plant health and vigor. Growers can use this to monitor plant N levels using sensor readings to determine the nitrogen status of the potted plants . These sThe aim of this study is to establish a base line sensor value for fertilizer treatment in cocoplum, a woody shrub native to South Florida, by monitoring chlorophyll content using SPAD and atLEAF sensors, and Normalized Difference Vegetation Index (NDVI), by a precision horticulture point of view. At the end of the treatments for six months, growth parameters, total nitrogen in the soil and in the leaf, and a leachate sample were also carried out in order to measure the nutrient runoff to find the best management practices among fertilizer treatment. The results of this study are extremely important and can serve as a basis for future research and for nurses and farms for best management practices among fertilizer treatment for cocoplum.The p ≤ 0.05). The atLEAF values (66.22) and leaves number (215.09) were significantly (p ≤ 0.05) higher with using 45 g supplemented with +15 g  compared to 15 g (control) with 61.97 and 182.91, respectfully , and NDVI did not differ significantly for the interaction between fertilization rate and evaluation period represented by days after fertilization (DAF). Therefore, these factors were evaluated separately. Fertilizer treatments were not significantly different in plant height and NDVI. However, atLEAF values and number of leaves were significantly different (ectfully .p ≤ 0.05) higher, compared to 0, 30, and 60 DAF , respectively. The highest plant height was recorded at 180 DAF with 55.73 cm. These results show the plant growth and the increase in the relative chlorophyll content (atLEAF) and NDVI over the months, during six months of evaluation, showing the normal growth behavior of the plants (The highest leaf number (227) was recorded after 90 DAF which was significantly (e plants .p ≤ 0.05) higher (0.87) in 120 DAF compared to 0, 30, 60, 90, 150, and 180 DAF , respectively. Also, 150 DAF provided a higher value (0.84) for NDVI than 0, 30, and 180 DAF , respectively. Finally, 60 DAF provided a higher (0.81) value for the same feature compared to 0 and 30 DAF , respectively (Relative chlorophyll content (atLEAF) was significantly higher at 90 DAF (66.64) compared to 0, 30, 60, and 120 DAF , respectively. NDVI values were significantly (ectively .p ≤ 0.05) between fertilization rate and days after fertilization for relative chlorophyll content (SPAD). An increase in relative chlorophyll content (SPAD) (67.32) was observed in the treatment 30 g  at 90 days after fertilization (p ≤ 0.05).There was significant interaction (lization  by Tukeyp ≤ 0.05) between fertilization rate and days after fertilization for total nitrogen and total carbon of leaf samples. An increase in total nitrogen (2.51) was observed using 30 g  at 150 days after fertilization, and an increase in total carbon (48.27) was observed in the 15 g  at 150 days after fertilization (p ≤ 0.05).There was significant interaction (lization  by Tukeyp ≤ 0.05) interaction between fertilization rate and days after fertilization, contributing to an increase in total nitrogen and total carbon of soil samples in 180 days after fertilization. An increase in total nitrogen (1.67) was observed using 30 g  at 180 days after fertilization, while using 30 g  provided an increase (38.29) in total carbon at 180 days after fertilization (There was a significant (lization . The trep ≤ 0.05) interaction between fertilization rate and days after fertilization for salt, electric conductivity, pH, and total nitrogen. For leachate samples at days 60, 90, 150, and 180 DAF, the treatment 45 g  provided a higher value for salt , and EC  compared to 15 g (control)  and , respectively. On day 30 DAF, all treatments provided higher values for salt  and EC  compared to control treatment  (There was a significant (ctively) .Lowest total nitrogen (8.244 ppm) in leachate samples was recorded at 30 DAF using 30 g  compared to other treatments. At 60 and 150 DAF the treatment 45 g  provided a higher value of total nitrogen  in leachate samples compared to other treatments except the treatment 15 g  at 60 DAF (115.250 ppm), and treatment 15 g  at 150 DAF (124.470 ppm) . At 180 These results demonstrate that the highest treatment of nitrogen can cause considerable losses of N, causing extra costs to producers and environmental damage due to the flow of nutrients. Thus, techniques that help in N monitoring to avoid the excessive use of nitrogen fertilization are necessary.This study can serve as a basis for future research and for nurseries and farms, since it demonstrated from the monitoring of the chlorophyll content by optical sensors and by foliar and substrate analysis that lower treatments of nitrogen fertilization are sufficient to provide nutrients suitable for the growth of cocoplum plants . In addip ≤ 0.05) correlated at 90 DAF. Also, NDVI values were significantly (p ≤ 0.05) correlated with NL at 150 DAF. There were not significant correlations observed for any parameters at 30 and 180 DAF  between NDVI and number of leaves at 60 DAF. Also, negative significant and high correlations  between NDVI and atLEAF, and total carbon and number of leaves, respectively, at 120 DAF, were observed .TM are appropriate devices to estimate fertilizer need of the potted plants.In a study conducted by Freidenreich et al. , applicaNitrogen is an essential element for plant growth and development. It is a major component of chlorophyll in plant leaves. Several sensors have been designed to measure either the reflectance or the absorbance of the green color present in the leaves. The greenness of the leaves represents the amount of chlorophyll found in the chloroplasts, which can be used as an indirect indicator for the photosynthetic processes of the plant to determine plant health and vigor . In factChrysanthemum. In another study, Dunn et al. [Salvia, and Swearengin et al. [In this study, the highest fertilizer concentration showed a higher number of leaves when compared to the control; also, the values of relative chlorophyll content (atLEAF) increased with higher fertilizer concentrations, showing the relationship between plant growth and N status. Similar results to this study were reported; Khoddamzadeh and Dunn  observedn et al.  reportedn et al.  observedChlorophyll is the most important pigment of the leaf and one of the most important of the plant since it is through it that plants manage to capture sunlight and use it as an energy source. By means of sensors it is possible to estimate the amount of chlorophyll in the leaf, and thus be able to evaluate the deficiency of nitrogen in the plant, indicating the necessity of nitrogen fertilizer .Gaillardia, Khoddamzadeh and Dunn [Chrysanthemum, Dunn et al. [Salvia, and Swearengin et al. [Justicia brandegeana.The highest value for the relative chlorophyll content (SPAD) was provided by treatment 30 g  at 90 DAF 67.32). Other authors observed SPAD readings increased with increasing N content, such as Dunn et al. [. Other aand Dunn  in Chrysn et al.  in Salvin et al.  observedLeaf tissue N analysis refers to the measurement of total N content in leaf blades of the most recently fully expanded leaves. It is a long-established method for monitoring crop N status ,34. AlthKhoddamzadeh and Dunn  reportedCarbon-nitrogen metabolism is the most basic and important nutrient metabolism of plants, and its dynamic changes in the plant directly affect the absorption, transformation of mineral nutrition, formation of protein, and so on ,37. TherThe correlation analysis demonstrated the association between SPAD and atLEAF sensors at 90 DAF; both sensors can be used to monitor the fertilizer status of the potted cocoplums. Another important observation was the association between NDVI and number of leaves at 150 DAF; this correlation demonstrates that the normalized difference vegetation index is positively related to the number of leaves; that is, when there is a linear increase in one parameter, the same thing occurs for the other parameter. Significant negative correlations were also observed between NDVI and number of leaves at 60 DAF, for NDVI and atLEAF, and between total carbon and number of leaves at 120 DAF. For negative correlations, as there is a linear increase in one parameter, the other parameter decreases.®) was used at 15 g (control), 15 g , 15 g , 30 g , 30 g , and 45 g , applied on the surface of each pot, and well water was then used during irrigations. The treatments T1, T2, T3, T4, and T5 were supplemented with 15 g after the first fertilizing in October (Cocoplum plants (1 year old) were purchased from Santa Barbara Nursery  in September 2021. The plants were grown at the FIU Organic Garden shade house located at Florida International University, in Miami, Florida. In October 2021, the initial slow-released fertilizer treatment 8N-3P-9K , and at day 0 (day before fertilization—DBF) as base reading .The performed analyses were:Five plants per treatment were evaluated monthly to the number of leaves (unit) by a counter, and plant height (cm) by a tape measure. Two branches of each plant, one larger and one smaller, were marked and measured, and the average of these two branches represented the plant height.TM Normalized Difference Vegetation Index (NDVI) sensor . During measurements, the NDVI , an atLEAF chlorophyll meter , and a GreenSeekerthe NDVI a sensor the NDVI b,c, measThis analysis was performed from individual containers to determine nutrient runoff rates. Each plant was irrigated until a saturated state was reached. Once containers reached the saturation point, a tray was placed underneath, serving as a collection reservoir. The plants were further irrigated with 350 mL of water, allowing the collection of 50 mL leachate. Samples were stored in 50 mL conical tubes that were immediately refrigerated at 4 °C until laboratory analysis in the CAChE Nutrient Analysis Core Facility at Florida International University; the test was performed for total nitrogen (ppm). The electric conductivity (EC), and salt of each leachate sample was measured in situ.Five plants per treatment were used for leaf samples and they were collected monthly. For the substrate samples, five plants per treatment were used and they were collected at the beginning and at the end of the experiment. The leaf and substrate samples were dried at 70 °C for 48 h, ground, and then analyzed for the total nitrogen (%) and total carbon (%). These analyses were performed at the CAChE Nutrient Analysis Core Facility at Florida International University.p ≤ 0.05) using the SISVAR statistical program [www.graphpad.com, accessed on 27 July 2022).The experiment was established in a completely randomized design with six treatments that were replicated five times with single pot replications (one plant in each pot), totaling 30 plants. Data were subjected to analysis of variance (ANOVA), and the means were compared by Tukey’s test  supplemented twice with 15 g in November and March after the first fertilization in October provided the least contamination through runoff while still providing adequate nutrients for plant growth compared to higher fertilizer concentrations. The results of this study could serve as a guideline for nursery producers and landscape personnel as a fast and non-destructive tool for sustainable fertilizer management practices within the ornamental plant industry.


Equine Gastric Ulcer Syndrome (EGUS) is a highly prevalent disorder in horses, which can be classified, based on the localization of the lesions, as Equine Squamous Gastric Disease (ESGD) or Equine Glandular Gastric Disease (EGGD). Although EGUS is recognized as a common cause of poor performance in racehorses, objective investigations about its relation with athletic capacity are lacking. Therefore, the present retrospective study aims to evaluate the associations between EGUS severity and some fitness parameters measured during an incremental treadmill test in Standardbred racehorses in training.p<0.05.With this aim, data from 87 Standard bred racehorses which underwent a complete diagnostic evaluation for poor performance was reviewed. During gastroscopic examination, a 0-4 score was assigned to ESGD, while EGGD was evaluated for absence/presence; a total EGUS score was obtained by adding 1 point to ESGD score in horses showing concomitant EGGD. Fitness parameters obtained during incremental treadmill test included speed at a heart rate of 200 bpm (V200), speed and heart rate at a blood lactate of 4 mmol/L , peak lactate, lactate and heart rate at 30 minutes post-exercise, maximum speed, minimum pH and maximum hematocrit. The associations between fitness parameters and EGUS and ESGD scores were evaluated by Spearman correlation, while Mann-Whitney test was used to compare them between horses with or without EGGD. Statistical significance was set at p = 0.0025) and minimum pH (p = 0.0469); ESGD grade was inversely correlated with V200 (p = 0.0025) and VLa4 (p = 0.0363). Although a trend was observed, no significant differences in V200 were observed between horses with or without EGGD (p = 0.073); horses with EGGD reached a lower minimum pH (p = 0.0087).EGUS grade was inversely correlated with V200 (These results show a negative association between aerobic capacity and EGUS, in particular ESGD. Although different hypotheses have been proposed, including abdominal pain and decreased appetite due to lactate accumulation, the underlying mechanisms are still unknown, and it is not clear whether EGUS represents a cause or a consequence of an early lactate accumulation and post-exercise acidosis. Equine Gastric Ulcer Syndrome (EGUS) is a common disease in horses, defined as the presence of erosive and ulcerative lesions in the terminal esophagus, proximal squamous stomach, distal glandular stomach, and proximal duodenum , 2. BaseClinical signs of EGUS are usually subtle and non-specific –28. AmonIn equine medicine, studies objectively investigating physiological responses to incremental treadmill exercise tests in horses with naturally occurring EGUS are lacking. Therefore, the present retrospective study aims to evaluate the potential impact of naturally occurring ESGD and EGGD on horses' athletic capacity, by measuring some fitness parameters during a standardized incremental treadmill test in poorly performing Standardbred racehorses.The clinical records of Standardbred racehorses referred for poor performance evaluation to the Equine Sports Medicine Unit of the Veterinary Teaching Hospital of the University of Milan  between 2002 and 2021 were retrospectively reviewed. As all the procedures were performed on clinical patients for diagnostic purposes and included informed owner consent for the use of clinical data, ethical review and approval were waived. All horses were in full training upon admission and underwent the following diagnostic protocol:Day 1: collection of history, clinical examination and lameness evaluation, laboratory analyses, electrocardiogram at rest, upper airway endoscopy at rest, acclimation on treadmill (one or two sessions);Day 2: incremental treadmill metabolic test, including plasma lactate analysis, creatine-kinase (CK) measurement at 6 h after exercise and Holter registration;Day 3: dynamic upper airway endoscopy on high-speed treadmill and tracheobronchoscopy performed 30 min after exercise for the evaluation of exercise-induced pulmonary hemorrhage (EIPH);Day 4: lower airway endoscopy, bronchoalveolar lavage fluid (BALf) collection and cytological examination;Day 5: gastroscopic examination.Horses showing signs of systemic illness, lameness, clinically significant cardiac arrhythmias or valvular regurgitation, dynamic upper airway obstructions (DUAO) or rhabdomyolysis were excluded from the study, since these disorders may influence athletic performance. As lower airway inflammation is highly prevalent among racehorses, its presence was not an exclusion criterium: however, BALf cytological findings were taken into consideration during statistical analysis as a possible bias.On Day 1 the horses were conditioned to the high-speed treadmill  with one or two training sessions. On Day 2, the incremental treadmill metabolic test was performed: the belt was inclined with a 3° slope and horses were warmed-up with 4-min walk at 1.5 m/s and 3-min trot at 6 m/s. The warm-up was followed by 1-min phases, during which the speed was increased by 1 m/s, until the horse was no longer able to maintain the treadmill speed; at the end of the test, the horses were walked for 30 min with a 0° slope to cool down .n = 56) and hematocrit (n = 44), by means of a blood gas analyzer   and reagents supplied by the manufacturer . In somell, USA) .® Click Holter, Trento, Italy) ; moreover, an ECG was obtained continuously before, during and after exercise, by means of a Holter recorder  . To excl, Italy) .The fitness parameters obtained from the treadmill test were collected on an electronic sheet , and included:VLa4: speed at a plasma lactate concentration of 4 mmol/L;HRLa4: heart rate at a plasma lactate concentration of 4 mmol/L;V200: speed at a heart rate of 200 bpm;Lac 30: plasma lactate concentrations at 30 min during the cool down;HR 30: heart rate at 30 min during the cool down;Lac max: maximum plasma lactate concentration reached during treadmill test or cool down;V max: maximum speed reached during the test until fatigue;pH min: minimum pH reached during the test;Ht max: maximum hematocrit reached during the test.The values of VLa4 and HRLa4 were calculated by means of a specific software (Lactate-E 1.0), which provides precise lactate threshold markers by inverse prediction .A complete evaluation of the upper and lower airways was included as part of the diagnostic protocol for poor performance. On Day 1, the anatomy and function of the upper airways were evaluated through an endoscopic examination at rest: horses were restrained in stocks without pharmacological restraint, a flexible videoendoscope  was passed through the left nasal passage, and the upper tract of the respiratory tract was visualized. Laryngeal function was assessed during spontaneous breathing and after the stimulation of movements by inducing swallowing, performing nasal occlusion, and during the “slap test” . Horses On Day 3, a high-speed treadmill endoscopy (HSTE) was performed: after 4-min walk and 5-min trot warm up with a 5% slope, treadmill was temporarily stopped, a videoendoscope  was passed into the nasopharynx of the horse and held in position with straps. Then, the treadmill was rapidly accelerated up to maximal speed until the horse's fatigue. The endoscopic images were visualized in real-time on a monitor and digitally recorded to allow slow-motion analysis . Horses g for 5 min. The slides were air dried, stained with May-Grünwald Giemsa and Perl's Prussian blue, and observed under a light microscope at 400× and 1,000× for 400-cell leukocyte differential count and calculation of a simplified total hemosiderin score , as previously described, and a BALf sample was collected for cytological examination: briefly, at the level of the carena, 60 mL of a 0.5% lidocaine hydrochloride solution were sprayed to inhibit the cough reflex, and the endoscope was passed into the bronchial tree until it was wedged within a segmental bronchus; here, 300 mL sterile saline 0.9% was instilled, and the fluid immediately aspirated. The collected BALf was stored in sterile EDTA tubes and processed within 90 min. A few drops of pooled BALf were cytocentrifugated  at 26 in score .margo plicatus and the pylorus, the stomach was insufflated with air and the mucosa was rinsed of adherent food material and mucus with water  on fitness parameters, horses were divided into MEA and non-MEA groups; horses were included in the MEA group when presenting a percentage of BALf neutrophils >10% and/or eosinophils >5%, and/or metachromatic cells >5% .Data are presented as mean ± standard deviation (SD) if normally distributed and as median and interquartile range (IQR) if not normally distributed. Statistical significance was set at Eighty-seven Standardbred racehorses met the inclusion criteria. The study population consisted of 10 geldings, 43 stallions and 34 mares, aged from 2 to 8 years , and weighing from 373 to 527 kg (mean 445.7 ± 32.39 kg). The fitness parameters obtained by the incremental treadmill metabolic test and the results of BALf cytology are displayed in margo plicatus, and the pyloric antrum were visualized and evaluated in all horses. In the study population, ESGD had a prevalence of 98.85% (95% CI: 93.77–99.94%). In particular, no horses showed grade 1 ESGD, 8.05% of horses showed grade 2 ESGD, 21.84% grade 3, and 68.96% grade 4 . The presence of EGGD was observed in 46 horses , while no lesions of the glandular mucosa were detected in 41 horses (47.13%). All horses with EGGD were concomitantly affected by ESGD.The squamous and glandular mucosae, the p = 0.0457, r = 0.21), V max  and Ht max ; moreover, it was inversely correlated with BALf eosinophils count . Sex and weight were not associated with any fitness parameters, or BALf cytology results.No association between ESGD grade or presence/absence of EGGD and age, sex, nor weight was observed. Age was positively correlated with the values of VLa4 , HRLa4 , and Ht max . The BALf lymphocyte percentage was positively correlated with the values of VLa4 , HRLa4 , and V max ; conversely, it was inversely correlated with the value of Lac 30 . The BALf neutrophil count was inversely correlated with the values of VLa4 , HRLa4 , V max , and positively correlated with Lac 30  and Lac max . The percentages of BALf eosinophils and mast cells and the total hemosiderin score were not associated with any fitness parameter. When evaluating MEA and non-MEA groups, horses in the MEA group showed lower values of VLa4 (p = 0.0341), while no other fitness parameter differed between groups.No relationship was detected between BALf cytological results and ESGD grade, or presence/absence of EGGD. Moreover, no association was observed between presence/absence of MEA and ESGD grade, or presence/absence of EGGD. The count of BALf macrophages was inversely correlated with the values of VLa4  and VLa4 . The presence of EGGD was associated with lower values of pH min (p = 0.012).The ESGD grade was inversely correlated with V200  for diagnostic purposes, while no procedures were performed for research aims. Moreover, all the procedures were performed according to relevant guidelines as a part of standard diagnostic protocols. Written informed consent was obtained from the owners for the participation of their animals in this study.CL, LS, and FF contributed to conceptualization and design of the study. CL visualized, defined the methodology of the study, and performed formal analysis. CL, LS, BC, GS, EZ, and FF contributed to investigation. CL wrote the first draft of the manuscript, under the supervision of LS and FF. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fisheries are forecasted to shrink in the tropics due to climate change. In Vietnam, fisheries are a pro-poor economic sector and essential nutrition source; however, welfares of producers and consumers in the climate change context are not well understood. While most studies focus on the gains or losses of total products and revenues, this paper pays additional attention to the changes in surpluses of market players in the long run. A combination of the production function, demand and supply functions, and partial equilibrium analysis is employed to measure the production and welfare impacts based on time series data from 1976 to 2018 and a Vietnam household living standards survey in 2018. The results show that relative to the present, catch yield is likely to reduce 35%-45% by mid-century and 45%-80% by the end of the century. Consumers may lose their surplus of 7-9 billion USD  by 2035 and 10-18 billion USD by 2065 due to supply reduction, while producers may gain additional profit of 3.5-4.5 billion USD by 2035 and 5-9 billion USD by 2065 owing to a price increase. The research findings suggest that Vietnam could impose measures to limit capture effort, as set out in the Law of Fisheries 2017, without harming fisher welfare. The expansion of aquaculture could reduce the gap between supply and demand of wild fish to mitigate consumer welfare loss; however, this impact is still ambiguous. Vietnam is a coastal country on the west bank of the East Sea, with a coastline of 3,444 km stretching from North to South and more than 3,000 large and small islands . There aClimate change impacts are witnessed in various aspects of human life and living organisms around the globe, in which fisheries are widely recognized to be affected –7. ClimaSeveral studies have quantified the impacts of climate change on fisheries, revealing different magnitudes in different places and species. The catch potential of Indian mackerel for the period 2020-2100 under RCP6.0 will decline from 2035 (+14%) to 2100 (−44%) . The preCheung, Lam  forecastFisheries are a pro-poor economic sector and important nutrition source in Vietnam . This seReviews of the literature reveal that most studies related to impacts of climate change focus on the gains or losses of fish stocks, yields and revenues, while the welfare of fishery producers and consumers under climate change is not well understood. When the supply falls short of demand, the price will increase, leading to the uncertain fluctuation in welfares of market players. The study aims to identify the welfare changes via a partial equilibrium analysis and supporting methods. It is the first attempt to quantify the losses, or gains, if it is the case, of wild fish consumers, producers, and both due to climate change in Vietnam.There are several approaches to analyze the various aspects of climate change impacts, such as vulnerability , 33–36, There is ample literature depicting the impacts of climate change on aqua species , 17, 24,st century [As shown in  century . Its man century , 49; cha century ; more in century , which l century , 52; inc century .Marine fishes and other aquatic species are poikilotherms whose internal temperature varies considerably. They are susceptible to variation in environmental temperature and migrate to water areas with their preferred warmth , 54–56. Fishing costs may increase due to incremental investment in vessels and gears to adapt to changes in fish stocks, species composition, and distribution . ChangesOn the demand side, total wild fish consumption depends on several factors such as population size, income, preferences, and the availability of substitutes, especially aquacultural products. The relative fishery price is likely to go up when the potential catch goes down in the long run.Profit will alter following changes in catch yields, costs, and prices. The welfare benefits of consumers, producers, and society will be reallocated.Following the flow of climate change impact on fisheries, this paper tests and measures (1) the capture reduction due to climate change, and (2) the potential changes in producer and consumer welfares.This study employs a set of several methods. Impact on catch yields is forecasted using a production function and the climate change scenarios for Vietnam. Then, a partial equilibrium analysis measures welfare changes. The price elasticity of demand is identified via a Marshallian demand function. The price elasticity of supply is inherited from the IMPACT model of IFPRI . DiffereThe production function is widely used in literature to describe the relationship between outputs and inputs in agriculture and fisheries , 65–68. βi and εt are coefficients and white noise, respectively.The production function to evaluate impacts of climate change on capture fisheries in Vietnam is formed as Eq :LnCatcThe North and the South of Vietnam were united in 1975. Therefore, the time-series data can only be collected uniformly from 1976 until recently, i.e., 2018. Annual data is employed due to the seasonal characteristics of capture production. Data are mined from their most reliable sources: data on catch yield, capacity, and labors are collected from the Vietnam General Statistical Office; climate data such as sea surface temperature, rainfall, and Southern Oscillation index are exploited from the National Oceanic and Atmospheric Administration (USA) database and the World Bank climate knowledge portal; data on typhoons and their maximum wind speeds are from the Joint Typhoon Warning Center (USA) database. Variable descriptions and sources of data are described in The Autoregressive-distributed lag (ARDL) model is applied to capture the lag impacts of the previous year(s) on total catches. ARDL is appropriate for regression with few observations . The lonst century, i.e., 2018-2035, 2046-2065, and 2080-2099, based on the RCPs 4.5 and 8.5 for 32 coastal provinces of Vietnam.Vietnam’s climate change scenarios developed by the Ministry of Natural Resources and Environment in 2016  and updaThere are two major types of supply-demand analysis: general equilibrium and partial equilibrium. In the general equilibrium analysis, the market system includes many separate but connected markets, of which the fishery market is one component. In the market system, all goods and factors of production are interrelated so that prices in all markets are set at the same time. Partial equilibrium analysis is limited to the study of supply-demand balance in individual markets . The primary feature of partial equilibrium analysis is the use of supply and demand curves of a particular commodity to determine its prices and quantities.P is fish price; QD and QS are demand and supply quantities, respectively; AD and AS are constants; and εD and εS are price elasticities of demand and supply, respectively. Since the demand curve slopes downward and the supply curve slopes upward, we have εD < 0 and εS > 0.This study focuses on the welfare of fishery consumers and producers. Hence, we have chosen the partial equilibrium analysis  to evaluAt the initial state, the price equilibrium is determined ata and b, respectively , the new supply and demand functions are as follows:When the relative changes in demand and supply are The new equilibrium price is identified atSolve the system of Eqs from  to 7); ; 7); we Change in consumer surplus:Change in producer surplus:Change in social surplus:About 85% of Vietnam’s total catches are for domestic use, and the rest, 15%, is for export . It is aWe recall the demand function from Eq :lnQD=lIn microeconomics, the consumer’s Marshallian demand depends on its price, consumer income, and prices of substitutes. Household demand also varies on household size, number of children, and other factors such as region, urban/rural location, occupation, employment, education, age, race, religion, crop, and availability of fishery shops .Data from Vietnam Household Living Standards Survey (VHLSS) in 2018  are explThe dependent variable is fishery consumption quantity in 12 months in logarithm form, irrespective of capture or culture. In Vietnam, substitutes for fishery products are often pork and chicken. Therefore, the fishery demand function has the following form:Qfish is household fish intake in 12 months (kg);Pfish is fish price paid by household (thousand dongs per kg);Y is the household income per capita (thousand dongs);Ppork and Pchicken are prices of substitutes, which are pork and chicken (thousand dongs per kg);Xi is a set of other independent variables. Following Cheng and Capps [Xi include dummies for five geographical regions: Red River Delta, North Central and Coastal Central, Central Highland, Southeast, and Mekong Delta (Northwest region is the base category); coastal provinces, number of household members, and characteristics of household head such as gender, age, education, marital status, and occupation ;nd Capps , Xi inclβk are coefficients, in which β1 is the own-price elasticity, and εt is the error term.The long-run form of the ARDL model indicates the long-run impact of climate change on catch fisheries. The results are presented in SOI is positive, indicating that catch yield decreases when El Niño occurs and increases in the case of La Niña. However, the long-term impact of SOI is neglectable, as 1 point decrease in SOI leads to a merely 0.06% decrease in capture yield.The regression results  show thast century under the RCP8.5. Under the RCP4.5, the typhoon frequency is not likely to change. Therefore, we will not consider the typhoon frequency and Southern Oscillation to pay attention to assess the impact of change in temperature, rainfall and maximum wind speed of typhoons on capture fisheries in the long run.IPCC  reports st century, i.e., 2018-2035, 2046-2065, and 2080-2099, are estimated based on the RCPs 4.5 and 8.5 for coastal provinces of Vietnam [ Vietnam , 77.As shown in The regression results  indicateɛD = -0.16, i.e., when the price goes up by 1%, the demand drops by 0.16%. Similar elasticities are obtained when data from VHLSS in 2012 and 2014 are used [The own-price elasticity of fishery demand is are used , indicatare used  or the Uare used . Howeverare used , where pɛS = 0.2, as capture in Vietnam is over the maximum sustainable yield [The International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT) of the IFPRI  uses a sle yield ; consequQD = QS0 = 3.2 million tons, the average fish price P0 = 24.5 million dongs per ton in 2018 [εD = -0.16 and εS = 0.20 into the demand and supply functions (2) and (3), we obtain AD = 6.0 and AS = 1.4.Replacing the total catches b < 1. The value of b is estimated as followings:Based on the Eqs –9, the cb∈ for RCP4.5; b∈ for RCP8.5;By 2035, b∈ for RCP4.5; b∈ for RCP8.5.By 2065, a in Eq 11) show Since the consumer loss is higher than producer gain, the net social loss occurs, from 3.3 to 4.3 billion USD in 2035 and 4.5 to 8.5 billion USD in 2065. All the currency values are fixed in 2018, in which 1 USD = 7465 VND in PPP (World Bank databank).Aquaculture production is proliferating in Vietnam and worldwide . In bothFrom Eq :P1P0=(From both Eqs  and 15)15):CS1Eq  indicateFrom Eq :PS1PS0a indicates that if the demand for wild fish decreases by 70%–80% of the supply reduction, producers start to suffer from loss of profit.Eq  indicatea = b, then, from Eq  by 2035 and 2065, equal to 9%–11% of total capture value in 2018.Eqs  and 19)19) depicFrom Eq :P1P0=(From Eq :CS1CS0From Eq :PS1PS0Eqs  and 21)21) show From Eq :P1P0= catches in Northern Australia. Alnafissa, Kotb [The negative impacts of sea surface temperature, rainfall, typhoons, and wind speed on catch yields are also found in various literature in Vietnam  and othesa, Kotb  realize The welfare impacts of climate change on producers and consumers depend significantly on the change in wild fish demand.If the demand curve remains unchanged, then the drop in supply induces fishery price increase, creating tremendous loss to consumers, from 7 to 9 billion USD by 2035 and from 10 to 18 billion USD in 2065 . If the demand decreases, mainly due to the substitution effect of the aquaculture expansion, consumers also suffer from loss. If the demand increases, primarily owing to the growing population and improving income, consumers may benefit if the demand surpasses a certain level. However, the latter case is not likely to happen since aquaculture is escalating rapidly, and consumption is more likely to shift from wild to farmed fish.In contradiction to many concerns that climate change will affect producers adversely as it reduces capture revenues , 36, 85,These findings suggest that although climate change hinders fishing production, fisher’s welfare is more likely to increase, and consumer welfare is more likely to decrease.In Vietnam, the capture fishery sector is a pro-poor economic sector. The fishery policies usually aim to maximize social welfare, helping people escape poverty. Therefore, wild fish is open access, leading to the spread of small-scale fishers along the seaside, escalating stress on nearshore fish stock. No specific actions limiting small-scale capture have been implemented in the Vietnam fishery sector. The application of quotas for inshore and offshore fishing vessels, newly introduced since 2019, is controversial and faces many obstacles, especially from fishers. The research findings imply that limiting capture effort do not harm fishers’ welfare in the long run.The Vietnam Fisheries Development Strategy for 2021-2030  sets itsS1 Data1(RAR)Click here for additional data file.


Aspergillus westerdijkiae, the producer of ochratoxin A (OTA), which is of worldwide concern, is an import fungal species in agriculture, food, and industry. Here, we got the uridine auxotrophic mutant of A. westerdijkiae by deleting AwpyrG. The ΔAwpyrG could be used for bio-transformation with exogenous AfpyrG expression cassette as a selection marker. In order to enhance the efficiency of gene targeting, Awku70 and Awlig4 were homologously deleted from ΔAwpyrG. The efficiencies of homologous replacement for ΔAwku70 and ΔAwlig4 were 95.7 and 87.0% in the deletion of AwAreA, respectively, demonstrating a drastic increase from 4.3% of the wild type (WT) strain. Furthermore, the function of AwAreA was identified with AwAreA deletion mutant and the control strain ΔAwku70. AwAreA regulated the growth and conidiation of A. westerdijkiae in response to nitrogen sources. The concentration of OTA for ΔAwku70 was in the range of 19.4 to 186.9 ng/cm2 on all kinds of nitrogen sources. The OTA production influenced by the deletion of AwAreA was different based on nitrogen sources. Pathogenicity assays on pears, grapes, salted meat, and cheese showed that AwAreA acted as a negative regulator in the infection of food substrates. Therefore, the genetic methods and engineered strains enable us to substantially expand the use of A. westerdijkiae, one of more than twenty OTA-producing fungi, in the study of mycotoxin biosynthesis and regulation, and consequently to aim at providing new ways for controlling this pathogen. In 2005chraceus . Since tchraceus , 2019. Uchraceus . Homologduce OTA . They coSaccharomyces cerevisiae shows a high homologous integration rate for the domination of the HR system for DNA repair. In contrast, some organisms like filamentous fungi seem to prefer to repair DNA by NHEJ, resulting in the random integration of exogenous DNA into chromosomes. Therefore, inhibiting or eliminating the NHEJ pathway is a strategy to increase gene targeting frequency. The process of the NHEJ pathway is mediated by the Ku70-Ku80 heterodimer, the DNA ligase IV (Lig4)–Xrcc4 complex, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs)  and non-homologous end-joining (NHEJ) are important pathways to repair DNA double-strand breaks . HR is aNA-PKcs) . The conossoides .A. ochraceus (Aspergillus niger (Aspergillus carbonarius (Penicillium verrucosum (pyrG/URA3 orthologs are genes that encode orotidine 5′-monophosphate decarboxylases (OMPdecase) or orotidylate decarboxylase that catalyzes the decarboxylation of orotidine 5′-monophosphate to form uridine monophosphate. That is the key step for the biosynthesis of uridine triphosphate pyrimidine nucleotides. pyrG/URA3 can also catalyze 5′-fluoroorotic acid (5-FOA) to the toxic 5′-fluoro-UMP, which inhibits fungal growth. Therefore, a pyrG/URA3 defective strain has often been isolated by positive selection via resistance to 5-FOA in the presence of uridine.The usability of the selection marker is another important factor for genetic transformation. Until now, the genetic manipulations of all the OTA-producing fungi, such as chraceus , Aspergius niger , Aspergibonarius , and Penrrucosum , have utrrucosum . pyrG/URAspergillus flavus when grown in Czapek Dox medium  and morphological identification. As shown in A. westerdijkiae Fc-1 was cultured at 33°C, which was not observed for strain A. ochraceus ITEM 7043. This was consistent with the description for the morphological differences in Aspergillus section Circumdati  supported with 15 kinds of nitrogen sources at the concentration of 35 mM were used to study the phenotype of A. westerdijkiae and mutants. Nitrogen sources for testing included (NH4)2HPO3, (NH4)2SO4, ammonium citrate, glutamine, sodium glutamate, NaNO3, arginine, aspartic acid, cysteine, histidine, methionine, phenylalanine, threonine, tryptophan, and urea. Each experiment was performed three times as biological replicates.Potato Dextrose Agar  was routinely used to culture A. westerdijkiae mutants was performed using protoplast genetic transformation as we described previously (ef1a (elongation factor 1) was used as the single-copy reference gene. Primers used in this study are listed in The generation of eviously . Positiveviously . The reaeviously . BrieflyAwpyrG was similar to the process of fungal transformation. Actually, we identified the appropriate concentration of 1.6 mg/ml for 5-FOA  in advance. The two DNA fragments, upstream and downstream of AwpyrG, were fused as the deletion cassette. 5-FOA (1.6 mg/ml), uridine , and uracil  were added to the selection media. The surviving colonies were selected and examined by diagnosed PCR.The deletion of A. westerdijkiae, 1 μl of conidia suspension (106 conidia/ml) was inoculated into the plate and incubated at 28°C under dark conditions. The growth rate was evaluated by checking the diameter of each colony. For the conidiation study, each plate was washed with 0.1% Tween-80 solution, and the number of conidia was calculated under a microscope with a hemocytometer.To assess the growth of To investigate the OTA production, six agar plugs (1-cm diameter) from equivalent zones of the fungal surface of the plate were collected and extracted with 2 ml of methanol. After ultrasonic vibrating (30 min) and vortexing (3 min), the supernatant solution was filtered through a 0.22-μm filter into a vial. An Agilent high-performance liquid chromatography (HPLC) system  was used to analyze OTA concentration, with a mobile phase of acetonitrile/water/acetic acid .Pyrus bretschneideri Rehd), grapes (Vitis vinifera L.), cheese, and salted meat were selected as food substrates to test the pathogenicity of wild type (WT) and ΔAwAreA. The surface of food substrates was disinfected three times with 0.1% NaClO for 10 s and rinsed with sterilized water for 30 s. For pears and grapes, the surface was punctured by a sterilized needle to make a wound with a diameter of 2 mm for inoculation, and 2 μl of conidia suspension (106 conidia/ml) was injected into the wound. For cheese and salted meat, 2 μl of conidia suspension (106 conidia/ml) was inoculated on the surface directly. Inoculation of sterilized water served as control. Each treatment was incubated at 28°C under dark conditions.Fresh pears  and Duncan’s test. It was considered statistically significant when A. westerdijkiae and other ochratoxigenic fungi, BlastP searches were performed using pyrG, ku70, and lig4 from A. nidulans as probes, toward several fungal genomes  are present in  genomes . As showand lig4  was in ationship . The homAwpyrG deletion cassette (combining upstream and downstream of AwpyrG) was constructed by the fusion PCR method, followed by transforming to the protoplast of A. westerdijkiae  was inserted in the deletion cassettes as a selection marker. A total of 23 transformants that exhibited resistance to hygromycin B were randomly selected for PCR identification. Finally, only one transformant was verified to be positive, for which the amplification of gene AwAreA (KO) was absent, and the amplification of UP and DOWN fragments were both positive, indicating the hygR cassette integrated at the target loci  had one copy of reference gene ef1a. The WT strain possessed one copy of AwAreA, while the three mutants had lost the gene. The hygR cassette could not be detected in the WT strain. Two mutants possessed one copy of hygR, while one of the mutants had two copies of hygR, which indicated a hygR cassette happened during ectopic insertion in the genome. For ΔAwku70 and ΔAwlig4, all of the generated ΔAwAreA strains had one copy of hygR 2SO4, and phenylalanine was obviously slower than that of others, and the colony edge showed an irregular shape. We could also observe the color alterations and different patterns of haloes in colonies when ΔAwku70 was cultivated with different nitrogen sources. For example, the colony was yellow on glutamine medium while white on tryptophan medium, and a white halo was produced around the colony on arginine medium. These morphological characteristics seemed to be correlated with the production of conidiospore, pigments, and other secondary metabolites 2SO4 and methionine media but decreased on sodium glutamate medium 2SO4 and methionine, the concentration of OTA dramatically increased after AwAreA deletion. The production of OTA significantly decreased in the case of glutamine and aspartic acid as nitrogen sources. Especially, the biosynthesis of OTA was completely suppressed in ΔAwAreA-ku70 for glutamine. These results indicated that the biosynthesis of OTA was affected by nitrogen sources and modulated by gene AwAreA.OTA is the most important and toxic secondary metabolite produced by AwAreA deletion on the pathogenicity of A. westerdijkiae toward pear, grape, salted meat, and cheese. The assay revealed that the loss of AwAreA had varying degrees of effect on fungal infection. As shown in A. westerdijkiae had infected and formed a colony on grapes at 12 and 16 days after inoculation. There was no significant difference between ΔAwku70 and ΔAwAreA-ku70. On pear infection, the lesion of ΔAwAreA-ku70 demonstrated an increase compared with ΔAwku70, indicating that AwAreA is a negative regulator in A. westerdijkiae for pear infection  fusion based on high efficiency of gene target have been reported in nidulans . Here, wus fungi . Until nng fungi , we specAwpyrG, ΔAwku70, and ΔAwlig4 were evaluated. These data showed the strains could be used to study the regulatory function of genes toward the development of A. westerdijkiae. The virulence ΔAwku70 and ΔAwlig4 in pears enabled them to be used in exploring the interaction between plants and fungi.Furthermore, the growth rate and conidia number of ΔAwAreA, the major regulator of nitrogen utilization, was characterized in this study. Similar phenotypes were demonstrated among the ΔAwAreA mutants generated from WT, ΔAwku70, and ΔAwlig4. This demonstrated that ΔAwku70 and ΔAwlig4 could be used to study gene function in the future. Therefore, the subsequence studies of AwAreA were performed in a ku70 deletion background strain. A. westerdijkiae showed different abilities in the utilization of different nitrogen for growth, conidiation, and OTA biosynthesis. AwAreA deletion led to better utilization of ammonium citrate, glutamine, arginine, etc., for growth but worse utilization of threonine by examination of the colony diameter. The ability of A. westerdijkiae to utilize nitrogen for conidiation was also affected by AwAreA deletion. A recent study reported that the number of conidia significantly decreased by disruption AreA in A. flavus when cultivated in a nutritious yeast extract sucrose medium 2SO4 and methionine media. AwAreA may employ a complicated mechanism in the regulation of OTA biosynthesis accompanied by the change of nitrogen sources.The biosynthesis of mycotoxins is influenced by nitrogen availability and nitrogen regulators. umonisin . The GATin vitro, it is reasonable that AwAreA plays various roles in the infection of food substrates containing different nitrogen sources. In A. flavus, ΔAreA was significantly impaired in conidiation compared to the WT  can be found below: GW and FX designed the experiment. GW, YL, BY, WW, and PS performed the experiments. EL and GW analyzed the data. GW wrote the manuscript. FX revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Solanum melongena, its wild relative Solanum insanum, and their interspecific hybrid, grown in the presence of 200 and 400 mM of NaCl, and in adult plants in the long-term presence of 80 mM of NaCl, in order to assess their responses to salt stress. Cultivated eggplant showed a relatively high salt tolerance, compared to most common crops, primarily based on the control of ion transport and osmolyte biosynthesis. S. insanum exhibited some specific responses, such as the salt-induced increase in leaf K+ contents (653.8 μmol g−1 dry weight) compared to S. melongena (403 μmol g−1 dry weight) at 400 mM of NaCl. Although there were no substantial differences in growth in the presence of salt, biochemical evidence of a better response to salt stress of the wild relative was detected, such as a higher proline content. The hybrid showed higher tolerance than either of the parents with better growth parameters, such as plant height increment (7.3 cm) and fresh weight (240.4% root fresh weight and 113.3% shoot fresh weight) at intermediate levels of salt stress. For most biochemical variables, the hybrid showed an intermediate behaviour between the two parent species, but for proline it was closer to S. insanum (ca. 2200 μmol g−1 dry weight at 200 mM NaCl). These results show the possibility of developing new salt tolerance varieties in eggplant by introducing genes from S. insanum.Soil salinity is becoming one of the most critical problems for agriculture in the current climate change scenario. Growth parameters, such as plant height, root length and fresh weight, and several biochemical stress markers , have been determined in young plants of  TThe effects of soil salinity on plants vary widely depending on multiple factors, but salt tolerance is mainly controlled by genotype ,8. An op+ deficiency [Salinity induces detrimental effects in glycophytes, such as (i) reduced water availability, (ii) ion toxicity, (iii) oxidative stress and (iv) Kficiency ,14, whicficiency ,16.Salt stress alters the normal metabolic processes of the plant: photosynthesis and energy production, lipid metabolism, nutrient acquisition, the integrity of cellular membranes and the activity of enzymes . Plant g+ and Cl− in the vacuoles [+ is generally accompanied by K+ deficiency by competition between the two cations because of their similar physicochemical properties. Thus, maintaining Na+/K+ homeostasis is crucial to develop normal metabolic processes in the cytoplasm, such as enzymatic reactions and protein synthesis [To maintain intracellular osmotic balance, some plants accumulate toxic ions such as Navacuoles ,19. An eynthesis . In addiynthesis ,20,21,22Toxic ions absorbed by roots move into photosynthetic organs, causing harmful nutritional imbalances and the generation of oxidative stress by an increase in the production of ROS. Through the oxidation of fatty acids, amino acid residues in proteins and the DNA bases, ROS accumulation leads to membranes degradation, protein inactivation and DNA mutations, causing cellular damage, and eventually, cell death ,24,25. TSolanum melongena L.) is one of the most economically important crops worldwide, reaching 1.86 × 106 cultivated hectares and an annual production of over 54 × 106 tonnes [Solanum melongena can be crossed with many wild relatives from the primary, secondary and tertiary gene pools, adapted to a wide range of environments [Solanum insanum L. to salt stress have recently been studied, and the wild genotype seems more tolerant than the cultivated eggplant, as it can stabilise its growth and photosynthetic rate under saline conditions [S. insanum, toxic Na+ and Cl− ions are transported to the leaves at high external salinity, where they most likely accumulate in vacuoles, according to the ‘ion compartmentalisation hypothesis’ [+ concentrations are maintained. In the presence of high salt concentrations, compared with cultivated eggplant, the wild species also shows a higher accumulation of proline, an excellent marker of a stress tolerance phenotype [Eggplant , a wild relative (S. insanum L.) and their interspecific hybrid, under salt stress conditions in short- and long-term treatments by determining growth parameters and the levels of several biochemical stress markers—such as ions, osmolytes or antioxidant compounds—and the activity of antioxidant enzymes. This work represents the first step in assessing the possibilities of success of the introgression of salt tolerance traits from the wild relative into the cultivated species.This study compares the response of cultivated eggplant (1:5) of the substrate in the pots was measured at the end of the short-term salt treatments of young plants. The obtained values were in concordance with the treatments applied: 4.9 dS m−1 on average for the control, 23.5 dS m−1 on average for irrigation with 200 mM of NaCl and 31.5 dS m−1 for irrigation with 400 mM of NaCl. No significant differences were found in these levels among the three genotypes within each treatment  plants apparently grew faster than the S. melongena (MEL) counterparts, as indicated by a larger increase in LN  was homogeneous in all plants and treatments, around 80%, indicating that the salt stress did not cause dehydration in the treated plants, except for slight water stem loss for INS . In facta (ChlA) showed significantly higher values in the hybrid than in the parents in the presence of 400 mM of NaCl. The carotenoid (Caro) contents did not vary between the three genotypes, although the values calculated for INS plants were significantly lower at 400 mM of NaCl than in the control or at 200 mM of NaCl.In general, no significant differences were found between treatments or genotypes in the major pigments under most experimental conditions tested . For pho+ contents significantly increased with increasing external salinity in a concentration-dependent manner; the patterns of Na+ accumulation were roughly the same in roots and leaves and for the three genotypes  had no effect. In the hybrid, leaf K+ concentrations neither decreased nor increased in response to the salt treatments and were maintained around 500 μmol g−1 DW in the control and the stressed plants  were statistically significant  is a reliable stress marker in most species of the Solanaceae family. Leaf Pro contents increased in response to the salt treatments in the three genotypes but reached absolute values about 25-fold higher in INS and the hybrid than in MEL . In the −1 DW in all cases (2O2) and total phenolic compounds , in general, did not significantly increase in response to the salt treatments, except for H2O2 in INS plants watered with 400 mM of NaCl  contents did not vary significantly between treatments or genotypes, with values around 200 mg equiv. glu gll cases . Similar of NaCl . Total f of NaCl .The salt treatments did not increase the specific activity of relevant antioxidant enzymes, such as superoxide dismutase (SOD), aspartate peroxidase (APx) or glutathione reductase (GR) in any of the studied genotypes , as shouTo complement the previous data, adult plants at the pre-flowering stage were subjected to longer (eight weeks) treatments with lower (80 mM of NaCl) salt concentrations. After the treatment, growth parameters, root and leaf ion contents, and leaf Pro levels were determined in the plants of MEL, INS and their hybrid.Regarding root length (RL), the salt treatment caused a small but significant increase in MEL plants, whereas it did not affect INS; on the other hand, RL was significantly reduced in HYB plants in the presence of 80 mM of NaCl a. The meNo effect of the salt treatment on biomass accumulation was observed at the root level a. Howeve+ concentrations were much higher in roots than in leaves; K+ and Ca2+, on the contrary, accumulated to significantly higher levels in leaves than in roots, although the differences between organs were relatively small compared to those observed for Na+  and their hybrid (HYB), with the aim of establishing possible differences in salt tolerance between the tested genotypes and the inheritance of those tolerance mechanisms. Our general assumption was that both parents, closely related genetically, would not differ in the type of responses to salt stress but could differ in the magnitude or efficiency of those responses.Recent studies have revealed that, even though genotype ,36,37,38genotype , althouggenotype ,39. The S. melongena cultivar and S. insanum. However, in a previous study, another S. melongena cultivar was shown to be less salt-tolerant than the wild relative at high salinities [High salt concentrations cause growth inhibition in plants ,36,40, wlinities , confirmlinities ,42,43,44linities ,46. Hetelinities ,48,49, alinities . This hya and b (ChlA and ChlB), and carotenoid (Caro) contents, might be used as stress biomarkers [Salinity is generally associated with the degradation of photosynthetic pigments, which is more pronounced in less tolerant species, reducing photosynthesis activity in the plant. Consequently, chlorophylls omarkers ,52. In t+ and Cl− contents accumulated in leaves and roots when salinity increased, with similar patterns for S. melongena, S. insanum and the hybrid, as previously reported in different eggplant cultivars [S. insanum, with a more significant Na+ accumulation in leaves. This result supports the idea that in S. insanum, Na+ is transported from roots to leaves and is sequestered in leaf vacuoles with a somewhat higher efficiency than in the cultivated eggplant.Naultivars ,54. Howe+ contents in response to increasing salinity would generally promote a decrease in K+ concentration, as both cations compete for the same transport proteins of the membrane [+/K+ ratio has been reported as a possible indicator of salt tolerance in plants [+ accumulation in eggplant leaves to maintain lower Na+/K+ ratios, reducing Na+ uptake or compartmentalising it into root vacuoles [+ levels under salt stress, which likely contributes to salt tolerance in eggplant, its wild relative and their hybrid. Changes in leaf K+ concentrations were also not detected in salt-treated HYB plants, which in this respect showed an intermediate behaviour between its parent species. The Na+ contents measurements indicated that ion transport in response to salt differs in both parental species. In INS, Na+ levels were higher in leaves, whereas Na+ predominantly accumulated in roots in MEL and the HYB, which in this respect was more similar to the cultivated eggplant than the wild relative.An increase in Namembrane ,53,54. Fn plants ,55. Somevacuoles ,57. In t2+ contents increased in the presence of salt, with higher accumulation in roots than in leaves, supporting the notion that Ca2+ is beneficial for maintaining Na+ and K+ homeostasis, at least partly via the SOS pathway [2+ in the leaves of salt-stressed plants is important to maintain essential metabolic and cellular processes.The Ca pathway . Moreove pathway , increasS. melongena or S. insanum, suggesting a constitutive mechanism of stress response. The salt treatments led to significant, concentration-dependent increases in Pro accumulation in all three genotypes; however, the absolute concentrations reached were much higher, around 25-fold, in INS and HYB than in MEL. Therefore, regarding Pro biosynthesis and accumulation in response to salt stress, the hybrid was much closer to the wild relative than the cultivated eggplant.Proline (Pro) is a reliable marker of abiotic stress in many plant species, increasing its concentration in stressed plants in relation to its background levels in non-stressed controls ,60. In aSoluble sugars (TSS) play a role in osmoregulation under stress environments in some plant species, amongst other multiple functions unrelated to specific responses to stress ,66. This2O2 contents was detected, in agreement with the maintenance of low ROS levels, but for a slight increase in INS at 400 mM of NaCl. These data suggest that salt stress responses based on the control of ion transport and Pro accumulation are efficient enough to avoid the generation of oxidative stress in salt-treated MEL, INS or HYB plants under our experimental conditions. In the absence of oxidative stress, the activation of antioxidant enzymes or the synthesis of antioxidant metabolites, such as phenolic compounds, was not detected, as should be expected.Malondialdehyde (MDA), a product of lipid peroxidation, is an excellent marker of oxidative stress generated as a secondary effect of high salinity and other abiotic stresses due to the increase in cellular ROS levels . TherefoThe responses of adult plants to lower salt concentrations during longer treatment were qualitatively similar, in most cases, to those described above for younger plants, albeit generally weaker. This behaviour could be explained by the increase in stress tolerance with plant age, which has been observed in many plant species ,72,73. T+ contents significantly increased in salt-treated plants with respect to the non-treated controls, in roots and leaves. However, contrary to what was observed in younger plants, Na+ concentrations were much lower in leaves than in roots. It seems that adult plants can efficiently block the transport of the toxic ion to the aerial part of the plants. On the other hand, K+ and Ca2+ concentrations were slightly (but significantly) higher in leaves than in roots and did not vary in response to the salt treatment or between genotypes. Compared to young plants, where Ca2+ is accumulated primarily in roots, Ca2+ concentrations were slightly but significantly higher in leaves in adult plants. Moreover, Na+/K+ ratios were significantly lower in adult hybrid roots compared to young hybrid roots. The relatively low accumulation in the leaves of Na+ ions, and the higher contents of K+ and Ca2+, partly counteracting the deleterious effects of salt stress, most likely contribute to the higher tolerance of adult plants.The Na+ accumulated in leaves at much lower levels than in young plants, it seems logical to assume that lower Pro concentrations are also required for osmotic adjustment.Finally, Pro contents increased in the plants treated with 80 mM of NaCl, although the relative increase over control levels and the maximum values reached were lower than in young plants. In this case, however, the lowest Pro concentrations were measured in INS plants and the highest in MEL, with the hybrid showing intermediate values. Since toxic NaSolanum melongena (MEL), Solanum insanum (INS) and their hybrid S. insanum × S. melongena (HYB), originally obtained from Sri Lanka and maintained in the COMAV eggplant collection  [3). The seeds were incubated at 4 °C covered with aluminium foil for seven days, followed by 24 h at 37 °C, before placing them in the germination chamber .Seeds of , Spain) , were so, Spain) . The seePlants with four leaves were transplanted in the greenhouse into pots of 14.5 cm in diameter, 12.5 cm in height (1.3 L Series CD Alto Thermoformed Pots) with commercial soil (Neuhaus Huminsubstrat S), and a 1 cm layer of vermiculite; the pots were placed in plastic trays distributed in groups of ten. One week later, treatments were started.2), and the electrical conductivity of the substrate (EC) (dS m−1). Once these parameters were determined, the plants were taken into the laboratory to measure the leaf fresh weight (g) (LFW), shoot fresh weight (g) (SFW) and root fresh weight (g) (RFW). Part of the fresh leaf material was deep-frozen in liquid N2 to perform some of the biochemical assays. Part of the fresh material was weighed and dried for three days at 65 °C and then weighed again. The water content (WC) percentage was calculated using Equation (1) [Five plants of each genotype were watered with solutions containing either 200 or 400 mM of NaCl, or with water for the controls, every three days for four weeks (n = 5). At the beginning of the treatments, the number of leaves (NL) and plant height (PH) (cm) were measured in all plants. At the end of the experiment, the same parameters were measured, plus the following: stem diameter (SD) (mm), length of the primary root (LR) (cm), leaf surface (LS) (cmtion (1) .WC (%) 1:5 (dS m−1) was measured with a Crison Conductivity-meter 522 .The EC of the substrate was measured at the end of the treatments using a 1:5 soil:water suspension prepared in deionised water and stirred for two h at 600 rpm and 4 °C. ECa (ChlA) and chlorophyll b (ChlB) were measured following the procedure of Lichtenthaler and Wellburn [g, the supernatant was collected, and its absorbance was measured at 663, 646 and 470 nm. Pigment concentrations were calculated following the equations given by Lihtenthaler and Wellburn [−1 DW.Total carotenoids (Caro), chlorophyll Wellburn , as modiWellburn . PigmentWellburn , and pig2O, according to the original protocol of Weimberg [g. Cations  were quantified with a PFP7 flame photometer , and Cl- was measured using a chloride analyser Corning 926 .The ion contents were determined in samples of 0.05 g of dry leaf, stem and root material after extraction in 15 mL of HWeimberg , with miWeimberg . The samw/v) sulphosalicylic acid and quantified according to the ninhydrin method [−1 DW.Proline (Pro) was extracted from 0.05 g of fresh leaf material with two mL of 3%  methanol and mixed overnight. The extracts were centrifuged for 10 min at 13,300× g, concentrated sulphuric acid and 5% phenol were added to the supernatant, and the absorbance was measured at 490 nm. The TSS contents were expressed as equivalents of glucose, used as the standard (mg eq. gluc g−1 DW) [The total soluble sugars (TSS) were quantified using 0.05 g of fresh leaf material. The samples were extracted with 3 mL of 80% ( g−1 DW) .v/v) were supplemented with 0.5% thiobarbituric acid (TBA) in 20% trichloroacetic acid (TCA), or with 20% TCA without TBA for the controls, and incubated for 15 min at 95 °C in a water bath. The reaction was stopped on ice, and the absorbance was measured at 440, 600 and 532 nm. The MDA concentrations were calculated using the equations described in [The malondialdehyde (MDA) contents were determined following the method described by Hodges et al. , with soribed in .2O2) contents were quantified using the method described by Loreto and Velikova [w/v) TCA solution and centrifuged for 10 min at 13,300× g. The supernatant was mixed with one volume of potassium phosphate buffer  and two volumes of 1 M potassium iodide. The absorbance was measured at 390 nm, and H2O2 concentrations were calculated against a standard calibration curve and expressed as μmol g−1 DW.The hydrogen peroxide (HVelikova . An amou−1 DW). TF were measured following the method described by Zhishen et al. [3 at basic pH. The absorbance was measured at 510 nm, and TF contents were expressed in equivalents of the standard catechin (mg equiv. cathechin g−1 DW).Total phenolic compounds (TPC) and total flavonoids (TF) were determined in the same methanol extracts used for the TSS measurement. TPC were quantified by reaction with Folin–Ciocalteu reagent . The absn et al. , based oCrude protein extracts were prepared from leaf material, as previously described , and stoSuperoxide dismutase (SOD) activity was determined by monitoring, at 560 nm, the inhibition of nitroblue tetrazolium (NBT) photoreduction in reaction mixtures containing riboflavin as the source of superoxide radicals ,90. One 2O2 added to the extracts by the decrease in absorbance at 240 nm [2O2 per min at room temperature.Catalase (CAT) activity was measured following the consumption of Ht 240 nm ,91. One Ascorbate peroxidase (APx) activity was determined following the decrease in absorbance at 290 nm, caused by oxidation of the ascorbate present in the plant extract ,93. One Glutathione reductase (GR) activity was quantified by the decrease in absorbance at 340 nm, the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH), and the cofactor in the GR-catalysed reduction in oxidised glutathione (GSSG) ,95. One Before the salt treatments, ten plants per genotype were transplanted into pots of 33 cm diameter and 28.5 cm height with coconut fibre. A controlled-release fertiliser, CoteN Mix, with NPK = 20−5−10, was added to each pot. Fertilisation was repeated after four weeks. Five plants were maintained until the pre-flowering stage when a long-term salt treatment started, by watering with a solution containing 80 mM of NaCl (n = 5). Automatised irrigation was set to water each plant with 1.65 litres daily. Once a week, the plants were irrigated with a control solution without salt to avoid salt accumulation in the coconut fibre and the pot.After eight weeks, growth parameters  were determined, as well as the DW and WC of leaves, stem and roots, as described above for young plants.+, K+ and Ca2+ were measured using 2 g of dry leaf or root material in an Agilent ICP-EOS 710 photometer . Pro contents were determined following the protocol described in 4.7.Considering the results obtained for young plants, only ions and Pro were quantified in adult plants. NaData were analysed using the software Statgraphics Centurion v.XVII . A factorial analysis of variance was performed for all parameters analysed in the plants, considering two factors of variability—treatments and genotypes—and their interaction. An analysis of variance was carried out separately for each species. Differences between the treatments were evaluated by the Student–Newman–Keuls test.Our results confirm the relatively high tolerance of eggplant to moderate salinity compared to most common crops, primarily based on the control of ion transport and Pro accumulation. These mechanisms seem efficient enough to avoid the generation of oxidative stress and, therefore, the activation of antioxidant systems as an additional defence mechanism.S. melongena x S. insanum showed a heterotic effect on plant growth, being more salt-tolerant than either parent. It appeared closer to the wild species in some crucial responses, such as the NaCl-dependent Pro accumulation in young plants. Wild relatives have been exploited to introgress novel genes that increase the fitness of the cultivated species. The results presented here suggest that S. insanum and the hybrid can be used as new sources of variation for breeding programmes, in order to obtain eggplant varieties that are better adapted to salt stress and, consequently, more sustainable.The hybrid


Most women diagnosed with VAIN3  had a previous history of hysterectomy for CIN2+. At multivariate analysis, colposcopic grade G2 , papillary lesion  and vascularity  were significantly associated with a greater risk of VAIN3. The risk of high-grade VAIN should not be underestimated in women with a history of smoking and previous hysterectomy for CIN2+, especially when colposcopic findings reveal vaginal lesions characterized by grade 2, papillary and vascular patterns. Accurate diagnosis is crucial for an optimal personalized management, based on risk factors, colposcopic patterns and histologic grade of VAIN.Colposcopic patterns of Vaginal Intraepithelial Neoplasia (VAIN) are not definitively related to histological grade. The aim of the present study was to investigate any correlation between clinical and colposcopic features and the development of high-grade VAIN. Two hundred and fifty-five women diagnosed with VAIN  at the European Institute of Oncology, Milan, Italy, from January 2000 to June 2022, were selected for a retrospective analysis. Multivariate logistic regression was performed to estimate the association of risk factors and colposcopic patterns with VAIN grade. Smoking was associated with the development of VAIN (34.1%,  Vaginal Intraepithelial Neoplasia (VAIN) is a rare premalignant lesion of the female lower genital tract, approximately 100-fold less common than cervical squamous intraepithelial lesions ,3, with HPV infection is the fundamental etiological factor for the development of VAIN. However, other risk factors have been identified and investigated over time, including young age at first intercourse, a large number of sexual partners, cigarette smoking, immunosuppression, past or concurrent diagnosis of cervical or vulvar preinvasive or invasive lesions, previous hysterectomy for cervical intraepithelial neoplasia (CIN) or cervical cancer, prior radiotherapy, and a history of in utero exposure to diethylstilbestrol ,7,8. RecAccording to the depth of vaginal epithelium involved by dysplasia, VAIN is usually classified into grades 1, 2 or 3. The 2014 WHO classification of VAIN replaced the previous three-tiered system and recognizes only two categories: low-grade VAIN  and high-grade VAIN  ,11. VAINVAIN mostly occurs in women over 60 years of age, who are commonly asymptomatic but sometimes report vaginal discharge or bleeding . FurtherThe diagnosis is usually made by colposcopic-guided biopsy of suspicious vaginal lesions. After an abnormal cervical screening test with no lesion identified on the cervix, great attention should be paid to the complete evaluation of the vagina. Vaginal colposcopy is quite challenging, often due to vaginal dystrophy in post-menopausal women. In addition, colposcopic patterns of VAIN are highly heterogeneous and not very specific, thus resulting in a lack of correlation between colposcopy and histology, unlike CIN ,18. NeveThe aim of the present study was to identify the potential risk factors for the development of VAIN to evaluate the diagnostic accuracy of colposcopy in relation to the histological grade of VAIN and to investigate any correlation between clinical and colposcopic features and high-grade VAIN.All women affected by VAIN and who were attending the Preventive Gynecologic Unit of the European Institute of Oncology, Milan, Italy, from January 2000 to June 2022, were retrieved from hospital file archives and selected for a retrospective analysis.The local Institutional Review Board approved the study protocol  and written formal consent for the use of data for scientific purposes was signed by each subject.Patients were included if the following criteria were met: (a) age at diagnosis of 25 years or older; (b) colposcopic-guided vaginal biopsies because of an abnormal pap smear or a previous history of any HPV-related lower genital tract diseases; (c) histologic confirmation of any grade of VAIN, including VAIN3 with stromal microinvasion; (c) available data about colposcopic findings. Patients were excluded in the case of (a) denied informed consent; (b) negative histology; or (c) diagnosis of invasive vaginal carcinoma.Data regarding sociodemographic, clinical, laboratory and pathological characteristics of patients were recorded in a dedicated database.Colposcopies were performed by staining with a 5% acetic solution and a 3% Lugol’s solution (Schiller test), by expert colposcopists working at the Preventive Gynecologic Unit of the European Institute of Oncology. Abnormal colposcopic findings were described as grade 1 if minor  or grade 2 if major , according to the 2011 Colposcopic Terminology of the International Federation for Cervical Pathology and Colposcopy (IFCPC) ,22. All Single or multiple colposcopic-guided biopsies were taken from suspicious vaginal lesions with the worst colposcopic characteristics. Dedicated gynecological pathologists working at the Pathology Division of our Institute performed all pathologic diagnoses. In the case of multifocal lesions and different grades of VAIN, the worst pathologic diagnosis and the related colposcopic pattern was considered for our analysis.When possible, the Cobas 4800 HPV test , an HR-HPV DNA assay with concurrent partial genotyping, was performed on liquid-based cervical (LBC) specimens at the time of colposcopy. The Cobas test is a Real-Time PCR-based assay able to detect HR-HPV genotypes 16 and 18 in separate channels, as well as a group of 12 other HR-HPV types  in another channel. It is a fully automated test and includes an internal control (B-globin) as a marker of sample adequacy.Categorical patients’ characteristics at diagnosis were summarized by counts and percent, age by mean and standard deviation and cross-tabulated by VAIN grade. Between VAIN grade groups, comparisons were done by using Fisher’s exact test for categorical variables and the F-test for age . Lesion type and vascularity were significantly associated with colposcopic grade and then entered two separated multivariate logistic regression analyses in order to estimate their association with VAIN grade as risk factors. Results are presented as Odds Ratios (OR) with 95% Confidence Intervals (CI). All tests were two-tailed and considered significant at the 5% level. All analyses were done using SAS 9.4 .After applying inclusion and exclusion criteria, 255 women affected by VAIN and attending the Preventive Gynecologic Unit of the European Institute of Oncology, Milan, Italy, from January 2000 to June 2022, were selected for our retrospective analysis.VAIN 1, 2 and 3 were diagnosed in 52, 55 and 148 women, respectively.The main clinical characteristics of patients are summarized by VAIN grade at diagnosis in p = 0.01) and more than half of patients reported previous pregnancies . Both variables were significantly associated with the diagnosis of VAIN, even according to histological grade.The mean age of women at first diagnosis was 52.4 ± 12.8 years, with no significant difference among patients diagnosed with different histological grade of VAIN. About a third of cases was a current or former smoker . Prior cervical cancer occurred in 49 patients undergoing hysterectomy , diagnosed as FIGO stage IA, IB and IIA-B in 29.4, 52.9 and 17.7% of cases (p = 0.17), respectively.Previous hysterectomy for CIN2+ was reported by 38.0% of women affected by VAIN, especially VAIN3  between cytology and histological grade of VAIN: 59.1% of VAIN1 were preceded by ASCUS-LSIL, whereas 73.1% of VAIN3 by ASCH-HSIL.There was a significant correlation  or AIN , were significantly associated with VAIN and histological grade.p = 0.05), probably due to the large number of missing data regarding the Cobas result in our population (n = 95).Interestingly, the HPV test was positive for HR-HPV with 16 and/or 18 and other HR-HPV not 16–18 in 44.4% and 39.4% of patients, respectively. Most women affected by VAIN3 were positive for HR-HPV with 16 and/or 18 (53.5%), however borderline significant  (p < 0.001) (p < 0.001). Indeed, about one-third of VAIN3 (31.4%) showed a vascular pattern (Most VAIN1 (88.2%) were characterized by colposcopic grade G1, and most VAIN3 (66.9%) were characterized by colposcopic grade G2 (< 0.001) . A flat < 0.001) . A vascu pattern .p = 0.36) and vaginal localization (p = 0.09) by VAIN grade at diagnosis.No significant association was found for multifocal lesions , papillary lesions (90.0%) and vascular pattern (44.4%) with a However, a sensitivity analysis showed that these colposcopic variables were still statistically significantly correlated with VAIN grade even after excluding all cases with microinvasive VAIN3 .n = 22, p = 0.03) and vascular pattern  were significant. In particular, all missing data regarding vascular pattern were in VAIN3 category. However, the arbitrary imputation of the missing data to the presence of the vascular pattern did not change the significance of the association with the histological grade of VAIN (data not shown).Old medical reports of women firstly diagnosed with VAIN farther away from current times had some missing clinical and colposcopic data. The distribution of missing data by VAIN grade at diagnosis was evaluated as a possible selection bias for different variables . Only thSince lesion type and vascularity were significantly associated with colposcopic grade , two sepp = 0.01) and VAIN3 .As shown in p = 0.03), whereas a previous hysterectomy for CIN2+ , papillary lesion  and vascular pattern  significantly led to a higher risk of VAIN3.When excluding the colposcopic grade from the multivariate logistic regression , only paOur findings confirmed that smoking, parity, previous hysterectomy for CIN2+ and abnormal cytology should be considered as potential risk factors for VAIN, and a significant association is maintained by histologic grade. In addition, abnormal colposcopic findings, including grade G2, papillary and vascular patterns, are predictive of the development of high-grade VAIN, even at multivariate analysis.According to our results, current or former smoking was significantly associated with the risk of VAIN, as already well-known in previous literature ,24,25. SIn our analysis, parity was related with a significantly increased risk of developing VAIN and, in particular, high-grade VAIN, as opposed to previous findings . HoweverIt is well-established that women with a previous history of CIN or cervical cancer, who underwent hysterectomy, remain at a higher life-time risk of VAIN and should be carefully screened for HPV-related vaginal and vulvar disease throughout their lives ,27. Our Unlike other authors , we did Even immunosuppression was not associated with the development of VAIN in our cohort, as opposed to previous studies , probablMost diagnoses of VAIN were preceded by an abnormal pap smear result, thus supporting the assumption that cytology, in combination with a HR-HPV test, is an effective tool for early diagnosis of VAIN, even after hysterectomy, since its sensitivity is not inferior to that for CIN2+ detection . We did Regarding the diagnostic accuracy of colposcopy in relation to the histological grade of VAIN, our study confirmed that colposcopic grade G2 and vascularity were significantly associated with VAIN3, including VAIN3 with stromal microinvasion. These associations have been widely demonstrated by other authors ,19,20, tThe correlation between vascularity and high-grade VAIN has been already explained by Boonlikit et al., as a consequence of the lack of vascular structure in very mature squamous vaginal epithelium. Thus, vascular patterns appear later, as distinct from the cervical dysplastic process, in which vascular punctuation appears early due to the immature squamous metaplasia of the transformation zone .Conversely, our results showed a significant association between papillary lesions and VAIN3, and in particular, microinvasive VAIN3. This is totally different from the evidence of other authors who detected micropapillary patterns more frequently in women affected by low-grade VAIN ,20,22. TNotably, we did not find any significant difference among VAIN grade groups regarding lesions number, as against that sustained by Zhou et al. . Even vaTo the best of our knowledge, this is the first study to investigate and portray colposcopic characteristics of not only low- and high-grade VAIN, but also VAIN3 with stromal microinvasion, that should always be correctly identified before choosing a therapeutic approach.The main strengths of the study are related to the higher proportion of high-grade VAIN in our population and the data homogeneity, because all colposcopies were performed at a referral oncologic center, only by trained colposcopists, with particular expertise in the diagnosis and treatment of vaginal lesions. On the contrary, limits of the study include selection bias related to the single center retrospective design of the study and the amount of missing data in old medical reports.A better defined and standardized application of the 2011 ICFPC colposcopic terminology for vaginal lesions could be useful for correct diagnosis and management of VAIN. Indeed, identifying risk factors and colposcopic patterns predictive for high-grade VAIN would help the colposcopist to sample the area most likely to contain VAIN3 or stromal invasion, especially in large and multifocal lesions, which could simultaneously hold different grades of VAIN.Appropriate diagnosis of VAIN3, with or without stromal microinvasion, is mandatory to choose the optimal management, which still remains challenging and controversial for high-grade VAIN. Several therapeutic regimens, including conservative surveillance, ablative procedures and surgical excisions, have been proposed over time, due to a high recurrence rate of VAIN2-3 despite the type of treatment ,36,37,38The risk of high-grade VAIN should not be underestimated in women with a history of current or former smoking and previous hysterectomy for CIN2+, which also represents a risk factor for recurrence and progression to vaginal cancer. Colposcopic findings, including grade 2, papillary and vascular patterns, are predictive factors for VAIN3 with or without stromal microinvasion. Accurate colposcopic and histologic diagnosis is crucial for the optimal management of vaginal pre-cancers and cancers. In addition, HPV genotyping could be a helpful tool for risk stratification and prompt identification of women with VAIN3 at higher risk of persistence, progression and recurrence.