Vitis davidii Foex) are an important wild grape species. Here, the quality characteristics of one white and three red spine grape clones were evaluated via targeted metabolomic and transcription level analysis. Xiangzhenzhu (XZZ) had the highest soluble sugar and organic acid content. Malvidin-3-acetyl-glucoside and cyanidin-3-glucoside were the characteristic anthocyanins in spine grapes, and significant differences in anthocyanin composition between different clones were detected. Anthocyanins were not detected in Baiyu (BY) grapes. The transcript levels of VdGST, VdF3′H, VdOMT, VdLDOX, and VdUFGT were significantly related to the anthocyanin biosynthesis and proportions. A total of 27 kinds of glycosidically bound volatiles  were identified in spine grapes, with Gaoshan #4 (G4) and BY grapes having the highest concentrations. The VdGT expression levels were closely related to glycosidically bound volatile concentrations. These results increase our understanding of the quality of wild spine grapes and further promote the development and use of wild grape resources.Native to China, spine grapes ( Vitis davidii Foex) are native to China and belong to the East Asian Vitis spp. They are so named because the canes are densely covered with 1–2 mm thorns [V. vinifera grapes, spine grapes are mostly consumed as fresh fruits, so there are good commercial prospects for the development and use of spine grapes. More study on the characteristics of spine grapes is needed to provide a theoretical basis for their commercial use.Spine grapes  anthocyanin profiles and the expressions of key genes, such as VvPAL, VvC4H, VvCHS, VvF3′H, VvF3′5′H etc., involved in anthocyanin biosynthesis; (3) the composition and content of GBVs and the expression levels of VdGT5, VdGT6, VdGT7, VdGT9 and VdPNGer, which are associated with glycosidically bound volatiles biosynthesis; (4) the correlation among metabolites and gene expression levels.Four representative spine grapes clones in South China were therefore identified in the present study for analysis of their (1) soluble sugar content, organic acid content, and the expression levels of sugar unloading-associated genes, such as Vitis davidii Foex, including three red clones—Gaoshan #2 (G2), Gaoshan #4 (G4), and Xiangzhenzhu (XZZ)—and one white clone, Baiyu (BY), were collected at commercial maturity from a commercial vineyard . For each sample, collection was carried out by picking small groups of 4–6 grape berries from different parts of each cluster, for a total mass of about 1000 berries. All samples were frozen in liquid nitrogen immediately and stored at −80 °C before analysis.Four samples of All of the standards for soluble sugars, organic acids, anthocyanins, and GBVs were purchased from Sigma-Aldrich . The purity of these standards was ≥97%. Formic acid and acetonitrile (HPLC grade) were purchased from Fisher Co. . All other chemicals used were analytical grade or above and were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. .A digital Atago PAL-1 m  was used to analyze the total soluble sugar (°Brix) content of the samples. Titratable acid was determined according to the methods of Wang et al. . The pH 18 column  were used to determine soluble sugars. Injection volume was 10 μL. The column temperature was 40 °C. The phase was acetonitrile/water (v/v 80/20), the flow rate was 1.0 mL/min, and it was analyzed for 20 min. To determine organic acid content, a diode array detector (DAD) and Thermo Hypersil COLD aQ column  were used. Injection volume was 20 μL. The column temperature was 25 °C. The phase was 0.01 mol/L KH2PO4 (pH:2.55)/methanol (v/v 97/3), the flow rate was 0.5 mL/min, and it was analyzed for 20 min. Identification and quantification of soluble sugars and organic acids were according to the calibration curve of their standards. The concentrations of the stock solutions of fructose  and glucose  were 100 g/L, and the concentrations of the working solutions of fructose and glucose were 1 g/L, 2.5 g/L, 5 g/L, 10 g/L, 25 g/L, 50 g/L and 100 g/L, respectively. The linear ranges of fructose and glucose were from 2.5 g/L to 100 g/L. The concentrations of the stock solutions of tartaric acid  and malic acid  were 100 g/L, and the concentrations of the working solutions of tartaric acid and malic acid were 0.5 g/L, 1 g/L, 2.5 g/L, 5 g/L, 10 g/L and 20 g/L, respectively. The linear ranges of tartaric acid and malic acid were from 1 g/L to 10 g/L. The concentration of the stock solution of citric acid  was 1 g/L, and the concentrations of the working solutions were 0.03 g/L, 0.06 g/L, 0.12 g/L, 0.24 g/L, 0.48 g/L and 0.98 g/L, respectively. The linear range was from 0.03 g/L to 0.48 g/L. Three technical replicates of each sample were analyzed.About 100 berries from each sample were deseeded, and the juice was squeezed out of them; 10 mL of juice was then centrifuged at 8000 rpm, 4 °C for 10 min. After centrifugation, 5 mL supernatant was filtered through a 0.45 μm inorganic filter and directly subjected to high-performance liquid chromatography  for further analysis. A differential refraction detector (RID) and ZORBAXSD-C18 column  was used to analyze the anthocyanin profiles. The conditions were as follows: mobile phase A: formic acid: acetonitrile: water (v/v/v) = 7:10:83; mobile phase B: formic acid: acetonitrile: water (v/v/v) = 2:54:44. The elution procedure was as follows: 0–15 min, 0–30% B; 15–25 min, 30–50% B; 25–35 min, 50% B. The flow rate of the mobile phase was 1.0 mL/min; the column temperature was 30 °C; the detection wavelength was 525 nm; the wavelength was scanned at 200–600 nm; the injection volume was 20 μL. Malvidin-3-glucoside  was used as the internal standard to quantify the anthocyanins. The concentration of the stock solutions of standard was 400 mg/L, and the concentrations of the working solution were 0.054 mg/L, 0.13 mg/L, 0.27 mg/L, 0.54 mg/L, 0.9 mg/L, 1.5 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 400 mg/L, respectively. The linear range was from 0.27 mg/L to 400 mg/L.The extraction of anthocyanins from spine grapes was completed according to our previous methods , the berGBVs were extracted according to the methods of Wen et al. . BrieflyA Thermo-Finnigan TRACE1310-ISQLT GC–MS instrument  was employed to analyze the GBVs. The TraceGOLD TG-5MS column  was used. The GBVs were extracted using headspace solid-phase micro-extraction fiber  in the headspace of the vials at 40 °C for 30 min. The fiber was desorbed for 4 min at 250 °C. The oven temperature was 50 °C (for 1 min), then increased to 130 °C at 3 °C/min and held there for 5 min, and then increased to 220 °C at 3 °C/min and held for 5 min. The transfer line temperature was 250 °C, and the ion source temperature was 280 °C. The mass spectrometer was scanned from 20 m/z to 450 m/z with an electron impact (EI) mode at 70 eV.GBV identification was according to the NIST02 library and retention indices of the authentic standards. The internal standard was 2-octanol. The concentration of the stock solution of 2-octanol was 1 g/L, and the concentration of the working solution was 4 μg/L. The GBV calibration curves were built using the area ratio of target compounds to the internal standard against the concentration ratio. GBV quantification was carried out using these calibration curves.TM5 Connect Real-Time System . The specific primers used in this study are shown in VdGAPDH was used as the internal reference gene to analyze the gene expression levels. The 2−ΔΔCT method was used to analyze the qRT-PCR data. All samples were analyzed in triplicate.The relative expressions of the genes involved in the pathways for sugar unloading, anthocyanin biosynthesis, and GBV biosynthesis were determined by qRT-PCR. Total spine grape RNA was extracted using the extraction kit  . First-sp < 0.05. Data were analyzed using Excel and SPSS 21.0 software . Multivariate statistical analysis was performed by MetaboAnalyst 3.0  [All data are shown as mean ± SD. One-way ANOVA was performed to assess differences among samples using Duncan’s multiple range test with a significance level of yst.ca/) .Vitis vinifera cultivars such as Cabernet Sauvignon (about 21 g/L–27 g/L in China), Merlot (about 21 g/L–25 g/L in China), and Chardonnay (about 20 g/L–25 g/L in China). This was consistent with the report by Meng et al. [Vitis vinifera cultivars such as Cabernet Sauvignon. The pH value of grape must does not change significantly during the fermentation process, so the pH of the grape berries determines the pH of the wine. In general, the pH value of V. vinifera wine ranges from 2.8 to 3.8, and appropriate pH value is important for the stability of the color of the wine [V. vinifera wines [To examine the differences between spine grape clones, physicochemical parameters—including soluble solids content (°Brix), pH, titratable acid, and total anthocyanin content—were analyzed. As g et al.  also fouthe wine . Anthocythe wine . In the the wine ,25. XZZ ra wines .Vitis vinifera cultivars.The soluble sugar and organic acid profiles of spine grapes are shown in VdHT1, VdHT3, VdHT4, VdcwINV, VdGIN1, and VdGIN2) involved in sugar unloading pathway were measured. As shown in VdHT1, VdHT3, and VdGIN2 in XZZ grapes were significantly higher than those in other varieties, while the expression levels of VdHT4 and VdcwINV in XZZ grapes were significantly lower than those in other varieties. VdHT genes are monosaccharide transporter proteins, and VdcwINV, VdGIN1, and VdGIN2 are invertase genes [VdHT1 and VdHT3 might increase the glucose content in grapes [VdHT3 and VdGIN1, with the lowest levels of VdHT1 and VdGIN2. The expression level of VdcwIVN was higher in BY grapes than in other varieties. Previous studies revealed that VvSK1, a sugar inducible protein kinase, may regulate the expressions of VvHTs and thus the accumulation of sugar in grape cells [VdcwINV and VdGINs might regulate the accumulation of sucrose in spine grapes, so sucrose was not detected. Similar results were also reported for Cabernet Sauvignon grapes [The sugar in grape berries is transported in the form of sucrose from the source organs (leaves) through the phloem to the sieve element/companion cell (SE/CC) complex in the phloem of the fruit vascular bundle, and then unloaded from the sieve–companion cell complex and enters the sink cells . The expse genes . The hign grapes . G2 grappe cells . These rpe cells ,35. The n grapes .Vitis vinifera grapes [Vitis vinifera and Vitis quinquangularis cultivars is high, while the Mv derivative content is very low [Vitis davidii cultivars might have different anthocyanin component characteristics than Vitis vinifera and Vitis quinquangularis varieties. More research is needed to reveal the anthocyanin properties of Vitis davidii cultivars. A recent study reported that some grape species native to China, including V. davidii and V. amurensis, had higher total anthocyanin contents (TAC) than V. vinifera grapes [V. vinifera grape wines. In the present study, we also found that the anthocyanins contents in spine grapes were significantly higher than V. vinifera grapes, this might indicate that spine wines had richer anthocyanins and a more attractive color for consumers than V. vinifera wines [The anthocyanin profiles of grapes may be affected by many factors, such as variety, management, temperature, and light . In the a grapes . The Mv very low ,25. Thesa grapes . These ira wines .VdPAL, VdC4H, VdCHS, VdF3′H, VdF3′5′H, VdDFR, VdLDOX, VdUFGT, VdOMT, and VdGST) involved in the anthocyanin biosynthesis pathway were analyzed. As VdPAL, VdC4H, VdCHS, VdF3′H, VdDFR, VdLDOX, VdUFGT, VdOMT, and VdGST genes were highest in XZZ grapes, followed by G4 grapes. The transcript level of the VdF3′5′H gene was the lowest among the 10 genes in all spine grape varieties. Although anthocyanin biosynthesis genes were also expressed in white varieties (BY), these levels were very low. The product of PAL, C4H, and CHS acts upstream of the phenylalanine metabolic pathway, and its expressions were significantly different among grape varieties [VdGST and VdOMT were the highest among the 10 genes in all spine grapes, especially in XZZ grapes. These two genes play an important role in the biosynthesis of anthocyanins, so the high expression levels of VdGST and VdOMT may be the main reason for the high anthocyanin content of these grapes [To better understand the mechanism of the varietal differences in anthocyanin profiles, the transcript levels of 10 key genes  were determined. As VdGT5, VdGT9, and VdPNGer were higher in G4 grapes than in the other varieties. BY had the highest transcript level of VdGT6 but lower expressions of VdGT6, VdGT9, and VdPNGer. XZZ had the highest expression level of VdGT6 but the lowest expression levels of VdGT5 and VdGT9. The expression levels of VdGT9 and VdGT7 were the highest among the five genes in all spine grapes, especially in G4 and G2 grapes, respectively. The GTs (monoterpenol β-d glucosyltransferases) proteins could catalyze the GBV biosynthesis in grapes [GTs genes and the concentrations of GBVs [VdGTs gene expressions between varieties might be the main reason for variation in GBVs between varieties [To analyze the expression patterns of key genes involved in the GBV biosynthesis pathway in spine grapes, the transcript levels of five genes (n grapes . Many st of GBVs ,44. The arieties ,45.To better understand the relationship between metabolites and gene expressions determined in the present study, multivariate statistical analysis was performed. As Vitis vinifera grapes [To better analyze the differences in metabolites and gene expressions among the four varieties, 15 metabolites or genes with significant differences among the varieties were screened. Interestingly, the anthocyanin content and related gene expression levels were the most significant among the four varieties, especially malvidin-3-acetyl-glucoside and cyanidin-3-glucoside. These results further indicate that the characteristic anthocyanins in spine grapes were malvidin-3-acetyl-glucoside and cyanidin-3-glucoside, which is different from a grapes ,17. The VdGT9 and bound volatiles contents, such as eugenol, 1-heptanol, 3-methyl-3-buten-1-ol, and 3-methyl-1-butanol, were detected. The expressions of VdGT5 and VdGT6 were significantly related to the concentrations of mequinol, benzyl alcohol, 1-hexanol, isogeraniol, citronellol, and linalool. Our results were consistent with those of Yue et al. [GTs genes were significantly related to the concentrations of GBVs. In addition, the concentrations of malvidin-3-acetyl-glucoside and cyanidin-3-glucoside were significantly related to the expressions of anthocyanin biosynthesis genes, especially VdGST, VdF3′H, VdOMT, VdLDOX, and VdUFGT. These genes play important roles in the biosynthesis and proportion of individual anthocyanins [Correlation analysis revealed the relationship between metabolites and related gene expressions. The positive correlations between the expression level of e et al. , which rocyanins . The difocyanins .V. vinifera wines. Spine grapes are rich in GBVs, which give wines more aroma type, such as floral, fruity, etc. G4 and BY grapes had the highest GBV concentrations. Multivariate statistical analysis revealed a close relationship between metabolites and gene expression in spine grapes. The results of this study help remedy the lack of knowledge about wild spine grape resources. Similar studies will ultimately benefit the promotion of spine grapes, and our data might be useful for the development and use of wild grape resources. In addition, considering rich anthocyanin and GBV contents, spine grapes might have potential for winemaking.In conclusion, targeted metabolomics and transcription level analysis revealed that there were significant differences in the soluble sugars, organic acids, anthocyanins, GBVs, and gene expressions among the four spine grape clones. The XZZ grape had the highest soluble sugar and organic acid content. The characteristic anthocyanins in spine grapes were malvidin-3-acetyl-glucoside and cyanidin-3-glucoside, and the XZZ variety had the highest individual anthocyanin content. The rich anthocyanins might give spine wines richer anthocyanins and a more attractive color for consumers than


Nesidiocoris tenuis. Here, we determine whether there is variation in these two traits and the proportion of that variation that has a genetic basis. Our results revealed the presence of genetic variation in both traits for this population. Genetic variation observed in zoophagy was larger than that found in phytophagy. These findings are relevant to the field of genetic improvement of biocontrol agents, since it reports quantitative values necessary for potential breeding programs. Moreover, our results suggest that if such variation is maintained in larger populations, it might be possible that selection for more zoophagous individuals could entail a reduction of the damages derived from its phytophagy. Furthermore, this study sheds light on genetic tools that could be applied to improve the biocontrol practice and, consequently, contribute to a more sustainable agriculture.Breeding of species for the benefit of humans is a well-known practice in plants and some vertebrates. However, its application to invertebrates, specifically biocontrol agents, is rather new and there is still a lack of information on breeding parameters of key traits. In this study, we address two important traits for biocontrol, i.e., the phytophagy and zoophagy of a population of the zoophytophagous predator Nesidiocoristenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator widely used in tomato crops to suppress key pests. We compared nine isofemale lines on their capacity to produce necrotic rings and wilting on tomato plants as a proxy for phytophagy, as well as their efficacy to prey on Ephestia kuehniella Zeller  eggs, as a proxy for zoophagy. Differences between isofemale lines in phytophagy and zoophagy indicated a genetic basis. Variation found in the zoophagy levels was larger than that in phytophagy levels. Our results showed that there is a genetic basis for the variation observed in the feeding behavior of isofemale lines of N.tenuis, highlighting the potential importance of selective breeding for such traits of biocontrol interest.Zoophytophagous predators provide biocontrol services in various major crops of modern horticulture due to the combination of its predatory capacity and the induction of plant defenses derived from its phytophagy. However, under certain conditions of prey scarcity, these natural enemies can inflict plant damage. Exploitation of genetic variation and subsequent selective breeding on foraging traits is a potential alternative to overcome this inconvenience. In this study, we quantified the genetic variation of phytophagy and zoophagy of  Campylomma verbasci (Meyer) (Hemiptera: Miridae), specifically in the zoophagy of the two different prey species this zoophytophagous feeds upon in apple orchards. These results shed light on the possibility to explore and exploit intraspecific genetic variation of interesting biocontrol traits in commercially available zoophytophagous species.The importance of zoophytophagous species to suppress pests in agroecosystems has increased over the last decades ,5,6,7,8.Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is a cosmopolitan zoophytophagous predator that is extensively used in tomato crops in the Mediterranean basin to control different key pests [Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and the invasive South American pinworm Tuta absoluta (Meyrick) (Lepidoptera: Gelichiidae) [N. tenuis was suggested as a means to improve its predatory efficiency, while mitigating the detrimental effects derived from its phytophagy [ey pests . Its majchiidae) ,13,14,15chiidae) ,18,19,20ytophagy .Most natural enemies currently used in augmentative biological control programs are selected based on differences between species (interspecific variation) to effectively control pests. Nevertheless, there is a growing interest in the exploitation of genetic differences in the traits of interest within species (intraspecific variation) of natural enemies ,23,24,25N. tenuis [A first step for selection to be feasible is the presence of variability in the target trait, but more relevant is the existence of a genetic basis for at least part of the variation observed in that trait ,30. Once. tenuis  is one oN. tenuis, i.e., phytophagy and zoophagy, by using an isofemale line approach. Necrotic rings and wilting inflicted in plant tissue, as well as consumption of E. kuehniella eggs, were used as a proxy for phytophagy and zoophagy, respectively. We expected to find variation among isofemale lines for phytophagy and zoophagy, indicative of genetic variation for these traits. Our study was the first to investigate the genetic basis of the phytophagy and zoophagy of N. tenuis, and the results shed light on the potential to use the genetic variation in these two traits to enhance the biocontrol services of this zoophytophagous predator.In this study, we aimed to investigate whether there is genetic variation in two important traits of the feeding behavior of a wild population of N. tenuis were collected with an aspirator from two outdoor tomato farms located in Peñíscola, Castellón (Spain)  (ca. 2 km distance between fields) in September of 2016. The tomato plants in these farms were grown organic and no previous releases of N. tenuis were done before the time of collecting, but wild N. tenuis was reported to appear in these crops every season. The province of Castellón is considered a transition zone between the two regions where mirid predators are used in tomato farms—the warmer Southern Spanish territories, where mostly N. tenuis is released in tomato crops for pest control, and the temperate Eastern Spanish territories, where Macrolophus pygmaeus (Rambur) (Hemiptera: Miridae) is the predominant species used in tomato crops for pest suppression [N. tenuis were scarce during the season of 2016 and only a few insects (adults and nymphs) were captured , hence, it was necessary to mix them to secure the establishment of the laboratory colony with the maximum diversity available at the moment of collection. Individuals collected in both farms were brought to the laboratory, mixed, and placed in a plastic cage (30 × 30 × 30 cm) with green bean pods (Phaseolus vulgaris L.) and eggs of Ephestia kuehniella Zeller  provided twice a week as oviposition substrate and food source, respectively. This colony was kept in the laboratory during five generations at 25 ± 2 °C, 50% ± 10% RH, and 14L:10D h photoperiod . Individuals from the colony were allowed to randomly mate and increase the population numbers necessary to found the isofemale lines. After five generations in the colony, the isofemale lines were initiated as follows—one male and one virgin female, both less than three-days-old, were allowed to mate in a Petri dish with mesh on the lid for ventilation (90 mm on diameter), with an entire green bean pod (ca. 15 cm long) as oviposition substrate and E. kuehniella eggs ad libitum supplied twice a week until the female died. Fifteen isofemale lines were founded but only nine of them survived . Each bean pod bearing eggs laid by the founder females was then placed in an individual muslin cage (25 × 25 × 25 cm), with new bean pods and E. kuehniella eggs. Insects of the same isofemale line were allowed to mate randomly but the parents and progeny were kept separated at all times by removing the bean pods bearing the laid eggs twice a week and placing them in a new cage. E. kuehniella eggs were supplied twice a week as food source. The isofemale lines were kept at 25 ± 2 °C, 50% ± 10% RH, and 14L:10D h photoperiod. Frozen E. kuehniella eggs for all rearings and experiments were provided by Koppert Biological Systems .Wild adults and nymphs of pression . Wild poN. tenuis, hence, it was selected for this study to allow for the visualization and measurement of the phytophagy parameters explained below.Tomato plants cv Raf Supermarmande  used in this experiment were in vegetative stages V5 to V7 (ca. 30–40 cm height). Plants were grown in pots (8 × 8 × 8 cm) filled with soil and vermiculite (3:1) and watered every two days. Plants were kept in pest-free climatic chambers until the start of the experiment, at 25 ± 2 °C, 50% ± 10% RH, and 14L:10D h photoperiod. This cultivar was known to be susceptible to the feeding damage of The isofemale line approach is convenient to study the genetic variation in traits of interest. It consists of the establishment of several strains or lines, each from a single mated female, and the quantification of the trait in several offspring produced by each female ,34. ConsN. tenuis [young leaf was the closest to the apical bud and the old leaf was the second-to-last from the apical bud. The apical bud was not used to test for phytophagy because of (1) the heterogeneity in their size and structure and (2) limitations on the maximum number of individuals available in each isofemale line, at a given generation. Each leaf was enclosed in a muslin bag (15 × 21 cm) and ten adults of the corresponding isofemale line were placed inside the muslin bag without a supplementary food source or prey. The insects were allowed to feed for five days. The dead individuals were removed daily from the muslin bags to prevent necrophagy during this experiment, and mortality per isofemale line was recorded. Dead individuals were not replaced with new individuals. After the feeding period, the remaining insects were removed, and the level of phytophagy was evaluated. Necrotic rings visible in the petiole and rachis were recorded. For wilting evaluation, each leaflet  was assigned the percentage of wilting in a 20% of the total leaf surface.As a proxy for phytophagy, we quantified the number of necrotic rings and wilting percentage produced by ten adults of each isofemale line per replicate, on two types of leaves of a tomato plant. Adult insects used in this experiment were three-five days-old. The age of the experimental individuals was chosen to allow sufficient time for the females to mature, mate, and oviposit, in order to secure enough progeny and survival of the isofemale lines for the following zoophagy experiment (see below). The phytophagy parameters were evaluated in leaves attached to entire plants. During the experiment, the plants were placed in trays inside a pest-free climatic chamber and watered every two days. The space between plants in the trays was enough to avoid differences in development or leaf quality due to competition for light, as well as to reduce the possibility of plant injuries during manipulation of the plants and trays. Provided that the apical part of the tomato plant is preferred by . tenuis ,37, the young leaf and one on the old leaf, and replicates on the same plant never belonged to the same isofemale line to exclude any possible plant effect. Each block consisted of similar-aged adults (three-five days old) and plants of similar developmental stage, as described above. Similar-aged adults were obtained from the bean pods, which were replaced in the cages twice a week, as previously described. This experiment was carried out in a pest-free climatic chamber at 25 ± 2 °C, 50% ± 10% RH, and 14L:10D h photoperiod.This experiment was carried out in ten consecutive blocks . In each block, all isofemale lines were included with a minimum of one replicate per isofemale line  . A repliE. kuehniella eggs preyed upon by the adults of each isofemale line for 24 h, as a proxy for zoophagy. This experiment was carried out in generation 15 because of the decline in the number of individuals in all isofemale lines after the phytophagy experiment, in which most adults of each isofemale line were used to achieve enough replicates. Isofemale line 5 was excluded from the zoophagy experiment because the number of individuals were not enough for the minimum of replicates needed for this experiment. A total of 193 individuals were used in this experiment, with 22–26 individuals tested per isofemale line. For this, females and males of N. tenuis less than five-days-old were starved for 24 h, with a moist cotton plug for water supply. After the starvation period, the insects were placed individually in a Petri dish (55 mm on diameter), and E. kuehniella eggs (n = 120) were offered to each individual in a piece of sticky cardboard. Insects were allowed to feed for 24 h. After the feeding period, the N. tenuis adults were removed and the number of preyed eggs was counted with the help of a dissecting microscope. Experimental conditions were 25 ± 2 °C, 50% ± 10% RH, and 14L:10D h photoperiod.In a separate experiment, we quantified the number of H2) in isofemale lines [The variation for necrotic rings, wilting, and zoophagy was analyzed in two steps. For necrotic rings, we first fitted a linear mixed-effect model (LMM) on the square-root-transformed data to estimate the genetic variation between isofemale lines. In this model, ‘leaf type’ (young or old) and ‘mortality’ were entered as fixed effects, and ‘block’ and ‘line’  were entered as random effects. This model estimated the variance components by REML, which allowed for the estimation of the broad-sense heritability ; models with the lowest AIC were selected as the best-fitted models  Table 1Table 1. H2) was calculated, as the ratio of the total genetic variance (VG)  to the total phenotypic variance (VP) [G/VP expresses the extent to which the phenotype of an individual is determined by its genotype, hence, it is also known as the degree of genetic determination [G was represented by between-line variance and VP was represented by the sum of between-line variance and environmental variance . For zoophagy, VG was represented by between-line variance and VP was represented by the sum of between-line variance and within-line variance. Additionally, we calculated the coefficient of genetic variation (VG) for the phytophagy and zoophagy traits from the estimated genetic components, with the formula:G is the genetic variance and G) is another parameter used as an indication of the ability of a population to respond to natural or artificial selection, i.e., the evolvability of a trait [As an estimate of the genetic variance, the broad-sense heritability (nce (VP)  Table 2H2 was camination . For nec a trait .1221 = 17.86, p < 0.0001), with ‘young’ leaves showing more necrotic rings (2.1 ± 0.2) than ‘old’ leaves (1.4 ± 0.2). The number of necrotic rings inflicted on the tomato plants also differed across the isofemale lines . Isofemale line 14 inflicted the highest number of necrotic rings for both types of leaves, with an average of 2.9 ± 0.5 for ‘young’ leaves and 2.1 ± 0.4 for ‘old’ leaves. The isofemale line producing the least necrotic rings on both types of leaves was isofemale line 10, with an average of 1.5 ± 0.3 for ‘young’ leaves and 1.1 ± 0.2 for ‘old’ leaves (H2 = 0.16 (likelihood-ratio test: p = 0.005) (The number of necrotic rings was significantly different between the ‘old’ leaves and the ‘young’ leaves (F’ leaves . The bro= 0.005) .1220 = 27.78, p < 0.0001). In the ‘young’ leaves, the percentage of wilting reached an average of 42.3 ± 5.9%, whereas in the ‘old’ leaves, the wilting average was 25.5 ± 5.5%. Differences were also observed between the isofemale lines . Isofemale line 13 produced the highest wilting proportion on both ‘young’ and ‘old’ leaves , whereas the lowest proportion of wilted leaves was produced by isofemale line 15, with 28.6 ± 12.2 for ‘young’ leaves and 14.4 ± 7.7 for ‘old’ leaves , with the highest observed mortality in isofemale line 8 (5.79 ± 0.35) and the lowest in isofemale line 15 (2.85 ± 0.36). The estimated broad-sense heritability for wilting was H2 = 0.18 (likelihood-ratio test: p = 0.006)  .E. kuehniella eggs in 24 h significantly differed between sexes . For females, isofemale line 15 showed the overall highest predation rate with 99.2 ± 5.0 eggs preyed in 24 h, whereas isofemale line 13 preyed the lowest rate with 44.3 ± 5.2 eggs  (H2 = 0.37 (likelihood-ratio test: p < 0.0001) (The consumption of 5.2 eggs A. In the5.2 eggs B. The am 0.0001) A,B. Broa 0.0001) .p < 0.0001) (p = 0.0041) and there was no significant correlation between the necrotic rings and zoophagy .The number of necrotic rings was positively correlated with the wilting percentage  . WiltingN. tenuis. Our study adds to the growing body of literature reporting quantitative values of genetic variation, needed to enable successful genetic improvement of the biological control agents [N. tenuis to produce necrotic rings and wilting on tomato plants differed across the isofemale lines and the leaf types. Similarly, differences were also observed for zoophagy across isofemale lines in their consumption of E. kuehniella eggs.The presence of genetic variation in a trait is fundamental for selection processes to take place. In biocontrol agents, the existence of natural genetic variation in traits of interest, provides the opportunity to select for lower or higher values in those traits that could be useful in the biocontrol practice ,25,28,29N. tenuis [Necrotic rings in the stems and petioles of tomato plants are the most visible injuries produced by . tenuis . Our res. tenuis ,42. ThusH2 values. Besides the drawbacks commonly experienced by isofemale lines maintained for a number of generations in the laboratory, such as genetic drift, [Our results showed variability in the levels of necrotic rings and wilting percentage inflicted across the isofemale lines. However, the genetic component in both of these phytophagy parameters is rather limited, as suggested by the low c drift, , other fc drift,  pointed N. tenuis is a zoophytophagous insect that needs plant tissue for both feeding and oviposition [N. tenuis. Thus, it is possible that the low genetic variation observed in the phytophagy-related traits is a trade-off resulting from selection acting on other related traits [H2 values observed for phytophagy might be an indication of strong selection controlling this specific feeding strategy. Furthermore, genetic variation of species associated with cropping systems tends to be limited as a consequence of a homogeneous habitat  that favors certain genotypes of the populations [G) for the phytophagy traits reveal that in the case of necrotic rings, the low H2 value could likely be the consequence of a larger environmental variance and not necessarily due to low genetic variance. Thus, some degree of response to selection might still be expected for the necrotic rings.For instance, as in many hemipterans, position . Phytophposition , and secposition , especiad traits , such asd traits . It is sd traits , whose hulations . The indN. tenuis is essential for its development [N. tenuis isofemale lines. Larger differences between isofemale lines for this trait and the higher H2 values, suggest that a greater proportion of variation is due to variation in genotypes for zoophagy than in the phytophagy traits. This larger genetic variation is consistent with the conditions of constant fluctuations in prey densities and prey species faced by N. tenuis in cultivated systems. Other studies also argued the spatial and temporal changes in prey levels in the agricultural systems as an important cause for the genetic variation in other predatory invertebrates [H2 values observed in this trait suggests that zoophagy is probably under less strong selection pressure [G) for zoophagy is low, relative to a moderate value of H2. This is likely explained by a lower environmental variance relative to the genetic variance observed for this trait.Zoophagy in elopment ,50. In ttebrates ,51. In apressure , hence, N. tenuis showed that plant damage decreases when prey availability increases in tomato plants [N. tenuis could indeed entail a reduction in plant damage. However, in this study, N. tenuis did not have access to plant and prey simultaneously. A choice experiment would be necessary to confirm whether this increased zoophagy is indeed the result of diet specialization in certain isofemale lines , or if it is due to individuals with increased voracity that can also perform well in absence/scarcity of prey. In the event that the increased zoophagy was due to diet specialization, breeding programs on these individuals could bring important benefits in terms of plant damage for the biocontrol practice. Nevertheless, such breeding attempts should also consider the potential drawback that zoophagy-specialized individuals might not survive periods of prey scarcity, which could mean an increase in the number of releases/introductions of this biocontrol agent in the crops.Previous studies with natural populations of o plants , and greo plants ,42. In tN. tenuis, and of the isofemale lines approach, as a tool to be exploited in genetic improvement programs of biological control agents. Although the broad-sense heritability (H2) calculated on isofemale lines can sometimes lead to overestimations of the actual heritability in natural populations [H2 and the smaller differences between isofemale lines observed in phytophagy, in addition to the diverse nature of the factors influencing its variation, suggest that this trait could be more challenging to target inbreeding programs. Although the evolvability (CVG) estimate observed for necrotic rings is still a good sign for potential selection against this trait and should be further investigated. Conversely, the larger variation observed in zoophagy represents a positive output for biocontrol improvement. The existence of isofemale lines showing higher predation rates could favor a decrease in the predator—prey ratio currently used in crops [The results of our study suggest an interesting potential of the genetic variation in the feeding behavior of ulations , the exiin crops . Hence, N. tenuis. Phytophagy-related traits might be more challenging to select against, due to the interaction of several factors, whereas the variation found for zoophagy-related traits is promising for the biocontrol practice. As such, our study adds to the growing body of literature reporting quantitative values of genetic variation, needed to enable the successful genetic improvement of biological control agents.The presence of genetic variation in traits of biocontrol interest is fundamental for selection of improved biocontrol agents. Our study was the first to quantify the genetic variation of the feeding behavior of


L. uvarum LUHS245, L. casei LUHS210, L. curvatus LUHS51, L. plantarum LUHS135, P. acidilactici LUHS29, L. plantarum LUHS122, L. coryniformins LUHS71, L. paracasei LUHS244, P. pentosaceus LUHS183, L. faraginis LUHS206) and MP with (AppMP) or without (MP) the addition of 8% (w/w) apple by-products (App). Two groups of prototypes of beverages were prepared: fermented MP and fermented MP with App (AppMP). Acidity parameters, LAB viable counts, lactose and galactooligosaccharides (GOSs) content, antimicrobial properties against 15 pathogenic and opportunistic bacterial strains, overall acceptability and emotions induced of the final fermented beverages for consumers were evaluated. Results showed that all LAB grew well in MP and LAB strain exhibited a significant (p ≤ 0.05) influence on galactobiose and galactotriose synthesis in the fermentable MP substrate. The highest total content of GOS (26.80 mg/100 mL) was found in MPLUHS29 fermented beverage. In addition, MPLUHS245, MPLUHS210 and AppMPLUHS71 fermented beverages showed high antimicrobial activity, inhibiting 13 out of 15 tested microbial pathogens. The overall acceptability of AppMP fermented beverages was 26.8% higher when compared with fermented beverages without App (MP), and the most intensive “happy” emotion was induced by MPLUHS71, MPLUHS24, MPLUHS183 and MPLUHS206 samples. Finally, very promising results were also attained by the bioconversion of MP with selected LAB and App addition into the prototypes of antimicrobial beverages enriched with GOS.The present research study aims to prepare prototypes of beverages from milk permeate (MP) using fermentation with 10 different strains of lactic acid bacteria (LAB) showing antimicrobial properties ( World’s industry is moving towards “zero waste economy”, which promotes the use of by-products as raw materials for the creation of new functional products with high added value. The objective of this industrial switch, based on the circular economy approach, is to reduce losses of valuable functional and bioactive materials and, simultaneously, to overcome management, economic and environmental handicaps during technological implementation through the utilization and valorization of agro-food industry by-products . Most ofOne of the challenges of dairy industry is to find an answer to the large quantities of dairy by-products, e.g., the milk permeates received considerable attention as suitable carbon sources, since they are substrates which do not require extensive purification, and may even be the most appropriate option for the development of novel products with added value. Many of cheese whey and second cheese whey products  have been extensively studied and reviewed over time as possible solution for cheese whey valorization because of their high industrial interest . HoweverLAB strains are used as microbial starter cultures for food fermentation because they display important beneficial effects with high interest in food industry for several reasons. Primarily, LAB can enhance flavor, texture and nutritional value of food products ,6. In adOne of the main functions of LAB in industrial dairy fermentation is lactose utilization ,19. BesiThe nutritional value of fermented milk permeates can be further improved by adding natural functional additives. An example of functional additives are the preparations of apple juice by-products. Our previous studies revealed that apple juice by-product preparations are suitable ingredients to design antimicrobial food and feed products with a wide spectrum of inhibition capacity against pathogenic and opportunistic bacterial strains ,11,24. FLactobacillus uvarum LUHS245, L. Lactobacillus casei LUHS210, Lactobacillus curvatus LUHS51, Lactobacillus plantarum LUHS135, Pediococcus acidilactici LUHS29, Lactobacillus plantarum LUHS122, Lactobacillus coryniformins LUHS71, Lactobacillus paracasei LUHS244, Pediococcus pentosaceus LUHS183 and Lactobacillus faraginis LUHS206) and with (AppMP) or without (App) apple juice by-product preparations. During the research experiments, acidity parameters, LAB viable counts, lactose concentration, content galactooligosaccharide with different degrees of polymerization (2 to 4), antimicrobial properties, and overall acceptability and emotions induced for consumers by testing the novel developed beverages were evaluated.The aim of the current study was to prepare prototypes of milk permeate-based (MP) functional beverages and resorting to LAB strains with antimicrobial properties  and grown in the Man, Rogosa and Sharpe (MRS) broth  under anaerobic conditions, at 30 °C for 48 h prior to use. Anaerobic conditions were attained by incubation the Petri dishes in anaerobic jars , with GasPak Plus™ .The eristics . Pure LA−3 mPa. Dried samples were ground in an ultra-centrifugal mill ZM 200  mounted with a stainless steel sieve of 0.75 mm-Φ, and stored at room temperature (22 ± 2 °C) in light- and air-protective bags.Milk permeate was obtained from agricultural cooperative “Pienas LT” , and stored at −18 °C before use. Furthermore, apple (variety ‘Auksis’) by-products were received from the Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry  in 2019. The whole press cake of apple by-products from juice production were composed seeds, peel and lignocellulose components. After reception in the lab, the apple by-products, obtained after juice preparation, were immediately vacuum dried in a vacuum dryer XF020 . Vacuum drying was performed at 45.0 ± 2.0 °C and under a pressure of 6.0 × 10L. uvarum LUHS245, L. casei LUHS210, L. curvatus LUHS51, L. plantarum LUHS135, P. acidilactici LUHS29, L. plantarum LUHS122, L. coryniformins LUHS71, L. paracasei LUHS244, P. pentosaceus LUHS183 and L. faraginis LUHS206 were incubated and multiplied in MRS broth culture medium  at 30 °C under anaerobic conditions. A total of 3% (vinnoculum/vmilk permeate) of LAB with a cell concentration of 9.2 log10 CFU/mL (LAB count determined by method described in The wdried apple by-products/wfermented beverages) of apple by-products. Evaluation of the overall acceptability is described in After fermentation, different amounts of dried apple by-products in fermented milk permeate-based beverage prototypes were tested and the most favorable formulation found  was selected, chiefly 8% (w/w) apple by-products [App—with 8% of apple by-products] giving rise to a total of 20 (10 + 10) samples, viz.: LUHS245—fermented with LUHS245 (L. uvarum); LUHS210—fermented with LUHS210 (L. casei); LUHS51—fermented with LUHS51 (L. curvatus); LUHS135—fermented with LUHS135 (L. plantarum); LUHS29—fermented with LUHS29 (P. acidilactici); LUHS122—fermented with LUHS122 (L. plantarum); LUHS71—fermented with LUHS71 (L. coryniformins); LUHS244—fermented with LUHS244 (L. paracasei); LUHS183—fermented with LUHS183 (P. pentosaceus); and LUHS206—fermented with LUHS206 (L. faraginis). Non-fermented MP samples were tested as control (NF—non-fermented).Finally, 10 samples of MP fermented with each of the previous 10 selected LAB strains were prepared without (MP) and with (AppMP) the addition of 8%  of the beverages were determined throughout fermentation time (0–48 h), where 0 h corresponds to the non-fermented MP and 48 h to the final fermented beverage. The pH values of the beverages were measured and recorded using a pH electrode . Total titratable acidity (TTA) was determined in a 10 mL of sample homogenized with 90 mL of distilled water, titrated with a volume (mL) of 0.1 mol/L NaOH to obtain a final pH value of 8.2, and expressed as Neiman degrees (°N). The measurements were undertaken in triplicate and the average values and standard deviations (SDTV’s) calculated.−4 to 10−8 with the same saline solution were prepared for inoculation. Sterile MRS agar  of 5 mm thickness was used for bacterial inoculation and growth onto Petri dishes. The Petri dishes were separately inoculated with the sample suspension using the spread plate technique and were incubated under anaerobic conditions at 30 °C for 72 h. All results were expressed in log10 CFU/mL, taking into account the dilution factor and the amount of sample, and the average values and STDV’s of three analytical determinations were calculated. In microbial viable counts, the logarithmic transformation is required for stabilization of variance and normalization of residuals.For the evaluation of LAB viable counts throughout fermentation time (0–48 h), where 0 h corresponds to the non-fermented MP and 48 h to the final fermented beverage, 10 mL of the beverages were homogenized with 90 mL of aqueous saline (9 g/L) solution. Serial decimal dilutions of 104[Fe(CN)6]∙3H2O} and 2.5 mL Carrez II (250 mM ZnSO4∙7H2O) solutions, and made up to 100 mL with distilled/deionized water. After 15 min, the samples were filtered through a 90 mm-Φ Whatman No. 2 filter paper of 8 µm-pore size  and were further filtered through a 25 mm-Φ disposable syringe filters of 0.22 µm-pore size  before analysis. A sugar standard mixture was prepared by dissolving 0.2 g each of fructose , glucose , sucrose  and lactose  in 100 mL of deionized water. A 2 mg/mL standard sugar mixture and several decimal dilutions were prepared with deionized water, so the chromatographic quantification could be performed after separation and identification of the peaks. Test samples were diluted to fit the linear zone of the calibration curve.Lactose was determined in the initial non-fermented MP (0 h) and final fermented beverages (48 h) by normal-phase (NP) high-performance liquid chromatography (HPLC) with evaporative light-scattering detection (ELSD) (NP-HPLC-ELSD). To that purpose, 0.5–1.0 g of the final beverages were diluted with approximately 70 mL of deionized water, heated to 60 °C in a water-bath for 15 min, clarified with 2.5 mL Carrez I {85 mM K2O) , a flow-rate of 1.2 mL/min, and an injection volume of 20 μL. The chromatographic separation was undertaken in an YMC-Pack Polyamine II 250 length × 4.6 mm I.D. , 5 μm-particle size  column mounted in series with a guard holder . The column temperature was set at 28.0 °C for the chromatographic separation. The detection was performed using an Evaporative Light-Scattering Detector (ELSD) LTII . The ELSD temperature was kept at 55 °C and 600 V. The nebulizer gas (purity of 99.9%) was air compressed at 1 bar. The photomultiplier sensitivity was adjusted to a gain of 6.An LC 1090 Hewlett Packard HPLC system  was employed for separation was employed. The HPLC conditions were as follows: the mobile elution system consisted in acetonitrile:deionized water  was determined in the final fermented beverages (48 h) and performed by normal-phase High-Performance Anion-Exchange Chromatography (NP-HPAEC) with Pulsed Amperometric Detection (PAD) (NP-HPAEC-PAD) method as described by Courtin et al.  with somd-Galactobiose , 3a, 4b-Galactotriose  and 3α, 4β, 3α-Galactotetraose (Santa Cruz Biotechnology Inc) were used [As standard references, 1.4-β-ere used . Furtherere used .1 = +0.05 V, t1 = 400 ms; E2 = +0.75 V, t2 = 200 ms; and E3 = −0.40 V, t3 = 400 ms. All flow-lines of HPLC system and the injection cell (Flexcell) were passivated before connecting the column by flushing at 1.0 mL/min with the following sequence: ethanol (10 min), deionized water (5 min), nitric acid 1N (HNO3) (10 min), deionized water (5 min), 0.1% ethylenediamine tetraacetic acid disodium salt (EDTA-2Na) solution (30 min) and, finally, deionized water (30 min).The following measurement potentials (E) and time periods (t) were selected: Elactose/100gsample and mgGOS/100mLsample.Each sample was analyzed by HPLC and HPAEC in triplicate and the average values and standard deviations (SDTV’s) determined. Results were expressed gKlebsiella pneumoniae, Salmonella enterica 24 SPn06, Pseudomonas aeruginosa 17–331, Acinetobacter baumanni 17–380, Proteus mirabilis, methicillin-resistant Staphylococcus aureus (MRSA) M87fox, Enterococcus faecalis 86, Enterococcus faecium 103, Bacillus cereus 18 01, Streptococcus mutans, Enterobacter cloacae, Citrobacter freundii, Staphylococcus epidermis, Staphylococcus haemolyticus and Pasteurella multocida) by the agar well diffusion method and in liquid medium.All the initial non-fermented MP (0 h) and final fermented beverages (48 h) were assessed for their antimicrobial activities against a variety of 15 pathogenic and opportunistic bacterial strains  agar  l., 2012  and 2020l., 2012 . Wells ov/v) ratio with physiological (sodium chloride) solution. Then, to the 0.5 mL of the diluted beverages, 0.1 mL of the pathogenic and opportunistic bacterial strains, grown in a selective medium, was added and incubated at 35 °C for 24 h. After incubation, the viable pathogenic and opportunistic bacterial strains in beverage prototypes were assessed by plating them on selective culture medium. The results were interpreted as negative or positive if the pathogens did not grow or grow on the selective culture medium, respectively. Experiments were performed in triplicate.To evaluate the antimicrobial activity of beverages in liquid culture medium, the final prototypes were diluted in a 1:3  and fermented beverages (48 h) was determined by 50 judges, according to the International Standards Organization method 8586-1 , using aThe beverage prototypes were also tested by applying FaceReader 6.0 software  , with a The valence was calculated as the intensity of ‘Happy’ minus the intensity of the negative emotion with the highest intensity. Valence scores ranged from −1 and 1. For each sample, the section of intentional facial expression  was extracted and used for statistical analysis. The FaceReader contained an image quality bar , which gn = 3) and were expressed as the average ± standard deviation. Results were analyzed via one-way analysis of variance (1-way ANOVA) using statistical package SPSS for Windows XP version 15.0 . The factors considered independently were the fermentation time  and type of formulations (n = 21) in the final fermented beverages and the dependent variables are summarized in α-value of 0.05 was used thus whenever p-values were equal or below to 0.05 (p < 0.05), the means were considered statistically different.All analysis was performed at least in triplicate (p ≤ 0.05) on average by 25.2 and 32.6% in 24 and 48 h fermented beverages, respectively (except in 24 h fermented MPLUHS210), compared to the control . The lowest pH value of MP (on average by 35.4%) was determined in 48 h fermented MPLUHS183, compared to MPNF. However, significant differences between pH values of all fermented samples with the same fermentation time were not found.Data presented in p ≤ 0.05) higher TTA (on average by 1.2-fold) after 24 and 48 h of fermentation, compared to MPNF. The highest TTA (11.0°N) was found for the sample MPLUHS183 after 48 h of fermentation. As expected, there was a strong negative correlation in the tested samples between pH and TTA (r = −0.85).Fermented MP samples showed a significant  to 7.4 (spring water). The presence of organic acids contributes substantially to the flavor profile, giving to the beverage a distinctive taste and providing a tartness and tangy taste that helps to balance the sweetness of sugar present in the beverages. Indeed, they are key-factors towards the taste and aroma of beverages ,29. Our LAB viable counts in final fermented functional beverages after 48 h of fermentation are tabulated in 10(CFU/mL) in fermented MP. The highest LAB viable cells count was found in MPLUHS210 after 48 h of fermentation. However, every LAB strain had demonstrated any significant influence on LAB growth in fermented samples with the same fermentation time. As predicted, a very strong negative correlation between the samples pH and LAB viable counts was found (r = −0.90), as well as a very strong positive correlation between the samples.The LAB viable counts increased throughout fermentation time, and ranging from 6.34 to 8.82 logr = 0.95), meaning that LAB had a major role on the production of metabolites (mainly organic acids) with high impact on the acidification of the beverages . Milk permeate, as a microbial growth medium, can be used by a wide range of bacteria to yield processed permeate products [Correlation between TTA and LAB viable count was verified  (or simply oligosaccharides) with different number of β-d-galactose monomers [viz. galactobiose , galactotriose  and galactotetraose   . GOS protrimer, G and galap ≤ 0.05) on average by 37.7% after 48 h of fermentation, except for MPLUHS183, showing the ability of these LAB strains to metabolize lactose. In comparison with the MPNF, the highest decrease of lactose content (by 58.1%) was found in the fermented MPLUHS71.The lactose and GOS concentrations in the initial milk permeate (0 h) (MP) and its final fermented beverages (48 h)  on GOS synthesis in fermented MP—thus highlighting the importance of LAB fermentation of milk permeate-based by-products, due to the ability to metabolize lactose and the concomitant production of metabolites with antimicrobial and antioxidant properties, as well as to the fact of being beneficial microorganisms and their metabolites are classified as Generally Regarded as Safe (GRAS) and Qualified Presumption of Safety (QPS) [GOS/100 mLsample) was obtained in fermented MPLUHS29, while the lowest concentration (8.7 mgGOS/100 mLsample) was noticed in fermented MPLUHS135. Galactobiose (G2) in combination with galactotriose (G3) were formed in MPLUHS245, MPLUHS210, MPLUHS29, MPLUHS71 and MPLUHS244. In the remaining samples only G2 was synthesized. Galactotetraose (G4) in fermented MP samples were not detected at all.LAB strains had a significant influence (ty (QPS) . The higr = 0.22, r = −0.15, r = −0.09, r = −0.04, respectively). GOS are produced through transgalactosylation reaction and are characterized in terms of their prebiotic, immunomodulation and functional properties in foods [Lactobacillus spp., P. pentosaceus and P. acidilactici to synthesized GOS in fermented dairy media was reported by other authors [In this study, only weak or very weak correlations between GOS content and pH, TTA, LAB viable counts or initial lactose content were obtained , Pasteurella multocida (DIZ ranged from 14.3 ± 0.3 to 23.3 ± 0.3 mm) and Streptococcus epidermis (except MPLUHS135 and MPLUHS29). Additionally, in MP the antimicrobial activity of MPLUHS210 against Salmonella enterica, Pseudomonas aeruginosa 17–331, Bacillus cereus, Enterobacter cloacae and Staphylococcus haemolyticus were observed. The latter bacteria strain was also inhibited by MPLUHS135 and MPLUHS51.The antimicrobial activities of the final fermented beverage prototypes with apple by-products (AppMP) and without (MP) against a variety of pathogenic and opportunistic bacterial strains are given in Pasteurella multocida. Comparing AppMP beverages, Pseudomonas aeruginosa 17–331 was inhibited by 4 samples of a total of 11 analyzed: AppMPLUHS122 (DIZ 12.0 ± 0.4 mm), AppMPLUHS244 (DIZ 12.3 ± 0.5 mm), AppMPLUHS183 (DIZ 13.4 ± 0.6 mm) and AppMPLUHS206 (DIZ 11.6 ± 0.2 mm). The methicillin-resistant Staphylococcus aureus MRSA was inhibited by AppMPLUHS245 (DIZ 11.0 ± 0.7 mm), AppMPLUHS210 (DIZ 12.06 ± 0.3 mm), AppMPLUHS122 (DIZ 11.03 ± 0.2 mm), and AppMPLUHS71 (DIZ 12.4 ± 0.5 mm). Bacillus cereus 18 01 was inhibited by AppMPLUHS210 AppMPLUHS122, AppMPLUHS71, AppMPLUHS244, AppMPLUHS183 and AppMPLUHS206 (average DIZ of 11.6 ± 0.29 mm). Streptococcus epidermis was inhibited by AppMPLUHS245, AppMPLUHS210, AppMPLUHS51, AppMPLUHS135 and AppMPLUHS29 (DIZ ranged from 11.2 ± 0.1 to 18.9 ± 0.6 mm). It should also be mentioned that Streptococcus mutans was inhibited by AppMPLUHS210, AppMPLUHS29 and AppMPNF .It must be emphasized that a broader inhibition spectrum of the samples prepared with fermented MP with apple by-products (AppMP) was unfolded. All of AppMP beverages inhibited The antimicrobial activities of the tested beverage prototypes against pathogenic and opportunistic bacteria in liquid medium  revealedLactobacillus is mainly related with production of organics acids, including lactic and acetic acids [LAB have been used in food processing because of their capability to increase the healthiness and sensory properties of food . Howeveric acids . These aic acids ,15,16,17ic acids .Bacillus cereus, while the organic peel demonstrated antimicrobial activity against Escherichia coli 0157:07 [Streptococcus epidermis and Streptococcus mutans found din our research data. However, Liya and Siddique (2018) [The increasing number of research studies have found that apples can possess some antimicrobial activity. Conventionally produced apples inhibited  0157:07 . It was  0157:07 . In the  0157:07  wild appe (2018)  reportedp < 0.05) among most of the fermented MP beverages  were found when compared to the control MPNF. Likewise, in comparison with AppMPNF almost all AppMP beverages (except MPLUHS135 and AppMPLUHS245) were significantly different in terms of OA.The results of overall acceptability (OA) and emotions induced by the final fermented beverage prototypes are given in r = −0.53) and a strong positive (r = 0.72 and r = 0.73) correlations between AppMP acceptability and pH, TTA or lactose content, respectively, were obtained in this study—which shows a trend for acceptance of higher contents of acidity and lactose. Similar trends were reported by Pereira and Rodrigues (2018) [As could be perceived from our results, the fermentation with different LAB and addition of apple by-products improved the sensory perception of the final fermented beverages and increased the acceptability. The positive changes in OA could be explained on the basis of the slight variation in the composition of the fermented products. Moreover, a higher level of the flavor compounds from apple by-products provided a more expressive taste of fermented drinks that the sensory analysis panel enjoyed better. A negative moderate (s (2018) , Janiasks (2018) , and Balp ≤ 0.05) in “happy” emotion among the tested beverages were observed. MPLUHS71, MPLUHS24, MPLUHS183, MPLUHS206 and MPNF intensity scores of “happy” were significantly higher when compared to the other samples. However, it was noticed that other emotions showed ambiguous results, because not all of them were statistically reliable. It is important to mention that a negative moderate (r = −0.54) and positive weak (r = 0.3) correlations between beverages acceptability and emotions “happy” or “angry” were respectively encountered. Examination of emotional response to food products could provide additional information about consumers’ perception of food and acceptability of new products [The analysis of facial expressions with FaceReader software showed a diverse distribution of emotions between each tested beverage. Besides “neutral”, which is usually expressed as the most intensive facial expression, “happy”, “sad” and “angry” were elicited more intensely than the rest during this research. Significant differences (e r = −0. and posie r = −0. and posiproducts  and Dannproducts .GOS/100 mLsample) and possessed important antimicrobial activity against most of the tested bacterial pathogens. Fermented MP showed a significantly (p ≤ 0.05) lower pH and higher TTA compared to non-fermented MP. The type of LAB strains had a significant (p ≤ 0.05) influence on GOS synthesis in fermented MP. The highest total content of GOS  was found in fermented MPLUHS29. The antimicrobial activity of all MP beverages against Streptococcus mutans and Pasteurella multocida was observed, and the latter one was also inhibited by all AppMP beverages. MPLUHS245, MPLUHS210 and AppMPLUHS71 beverages inhibited the highest number of tested pathogens, chiefly 13 of the 15. The addition of apple by-products in fermented MP increased the overall acceptability of the final fermented beverages (by 26.8% in comparison with beverages without App). AppMPLUHS183 was the most acceptable beverage. The diverse distribution of emotions between each tested beverage was noticed. MPLUHS71, MPLUHS24, MPLUHS183 and MPLUHS206 intensity scores of “happy” were significantly higher compared to other samples. This research study highlights the real potential of the bioactive prototypes of MP-based fermented beverages (with and without the addition of apple juice by-products), in the commercial point of view, as well as in the promotion of healthy eating habits and a circular economy.The functional beverages are a healthy alternative in human nutrition, whereas their production can simultaneously lead to the sustainable valorization of dairy industry by-products. The results of this study showed that fermented MP beverage prototypes with selected LAB strains contained GOS (from 8.7 to 26.8 mg


The present study aimed to gain insight into the genetic origin of the Shina population from Gilgit-Baltistan, Pakistan. We partially performed the mitochondrial DNA (mtDNA) control region of healthy unrelated individuals of Shina tribe residing in the remote area of Gilgit-Baltistan to investigate their maternal lineages. The present study is the first report about Shina’s genetic structure, origin, and relationship with the surrounding north-western Pakistani population. The mtDNA sequences of the Shina samples were compared with the revised Cambridge Reference Sequence (rCRS) and the HVR-1 D-loop region was covered. The comparison with rCRS identified overall 38 haplotypes and 08 haplogroups for Shina samples. Among these haplotypes, 18 were shared by more than one individual of the Shina tribe. The obtained mtDNA sequences of Shina were compared with surrounding north-western Pakistani population groups, i.e. Kho, Kashmiri, and Pathan. The genetic diversity (i.e. 1.0424) and power of discrimination (i.e. 0.9266) of the Shina was found equivalent to surrounding north-western groups. The haplogroups frequencies, phylogenetic tree and network analysis identified the west Eurasian ancestral origin of Shina group with nearby maternal ancestral relationships with the Kashmiri population. However, no close genetic relationship of Shina was depicted with nearby residing Kho population group. Pakistan is located at the crossroads of Asia on the intersection of the Middle East, Central Asia, and Southeast Asia. The early human migration from Africa to South Asia and Australia occurred across the Pakistani corridor around 50,000–70,000 years ago  from the east and north-east and India-administered Kashmir to the southeast. The linguistic groups of Gilgit-Baltistan are Shina, Balti, Wakhi, Domaki, Khowar, Burushaski and Gojri . Informed consent was obtained from all the study participants. The collected blood samples were preserved in EDTA vacutainers. DNA was extracted from collected blood samples according to the published phenol/chloroform method .About 3 ml blood samples were collected from healthy unrelated 07 male and 02 female individuals of Shina tribe belonging from Gilgit-Baltistan  . These sequences were trimmed according to Shina high-quality mtDNA sequences to perform comparative population genetic analyses.The DNA sequence reads trace quality check was performed using Staden package  was constructed using MEGA7.0.9 software. The neighbour-Joining (NJ) and maximum-likelihood (ML) methods were followed with 10,000 bootstrap replicates during this analysis  to confirm the sequence variants. The high-quality nucleotide draft sequences of 504 bp were generated covering the 16,066–16,569 positions of the rCRS mtDNA D-loop region (HVR-1). The mtDNA sequences were downstream analyzed for possible mtDNA haplotypes and haplogroup composition. A total of 38 mtDNA haplotypes were identified for Shina’s mtDNA sequences among which 18 were shared by more than one individual. The haplotypes based on C16073A (18.42%) and T16519C (13.15%) SNPs were found frequently. Besides, 08 different haplogroups were observed for Shina’s mtDNA sequences. A haplogroup H2a was found shared in Shina samples . In compWe performed an additional comparative analysis of the Shina mtDNA sequences to know their maternal lineage relationship with closely residing north-western Pakistani populations . The mtDNA HVR sequences of these population were acquired from GenBank, NCBI database. The Shina samples were found to have few shared haplogroups and haplotypes with surrounding northwestern Pakistani populations . The basThe haplogroup ‘H’ was found predominant in Shina samples with a total frequency of 44.4%. This haplogroup ‘H’ is the most common European haplogroup that originated in Southwest Asia approximately 20,000–25,000 YBP. The haplogroup HV is the ancestral haplogroup of ‘H’. The HV and R0a are the descendant of haplogroup ‘R’ . The overall low confidence clustering pattern in these analyses mainly arose due to the small sample sizes of the population groups.During the neighbour-joining (NJ) and maximum-likelihood (ML)-based phylogenetic trees reconstruction the Shina and the rest of three surrounding north-western Pakistani populations’ samples formed separate major tree clades indicating their distinct genetic structure. The Shina samples were somehow found to share the phylogenetic tree clades with the Kashmiri in both MP and ML trees . AdditioWe performed haplotypes networking analysis of Shina and the rest of available northern population groups’ samples. During this analysis, Shina samples were found to share two nodes with Kashmiri and single node with Kho population. The Kho and Kashmiri samples were also found to share few haplotype nodes. The close mtDNA based genetic relationship between these populations groups have previously been reported  residing in the Gilgit-Baltistan and close regions somehow shared their maternal lineages. However, the current study based on low sample size and the findings need to be validated with a larger sample size from Shina tribe. It would be noteworthy to plan genome-wide markers based population genetic investigation of Shina to better infer their genetic origin, demography and micro-evolutionary pattern shaped against the environmental burden.Click here for additional data file.


Puma concolor) populations in California are genetically structured and exhibit extreme variation in population genetic diversity. Although human development may have fragmented gene flow, we hypothesized the quantity and quality of remaining habitat available would affect the genetic viability of each population. Our results indicate that area of suitable habitat, determined via a resource selection function derived using 843,500 location fixes from 263 radio‐collared mountain lions, is strongly and positively associated with population genetic diversity and viability metrics, particularly with effective population size. Our results suggested that contiguous habitat of ≥10,000 km2 may be sufficient to alleviate the negative effects of genetic drift and inbreeding, allowing mountain lion populations to maintain suitable effective population sizes. Areas occupied by five of the nine geographic–genetic mountain lion populations in California fell below this habitat threshold, and two (Santa Monica Area and Santa Ana) of those five populations lack connectivity to nearby populations. Enhancing ecological conditions by protection of greater areas of suitable habitat and facilitating positive evolutionary processes by increasing connectivity  might promote persistence of small or isolated populations. The conservation status of suitable habitat also appeared to influence genetic diversity of populations. Thus, our results demonstrate that both the area and status  of suitable habitat influence the genetic viability of mountain lion populations.Jointly considering the ecology  and genetics  of a species can increase understanding of current conservation status and inform future management practices. Previous analyses indicate that mountain lion ( We report a strong relationship between amount of habitat and genetic diversity in a charismatic umbrella species. We also demonstrate habitat thresholds needed to maintain genetic integrity of the species. Our work defines current conservation needs and adds to general ecological theory concerning genetic diversity and amount of available habitat. We hypothesized that increasing areas of suitable mountain lion habitat would be correlated with higher levels of genetic diversity  in California , and used linear regression to evaluate relationships between habitat areas and Ne Table . We usedp ≤ .05 to be statistically significant.We used Program R version 3.4.3  and areas of overall and protected habitat (Table r2 = 0.60) and allelic richness (r2 = 0.87), and in both cases explained more variation that did the area of overall suitable habitat (Table eN (r2 = 0.88), the area of overall suitable habitat had even more predictive power (r2 = 0.97). Likelihood‐ratio tests demonstrated that the logarithmic regression model using area of protected habitat had a significantly better fit  for the given data than the same model using area of overall habitat for allelic richness . Likelihood‐ratio tests demonstrated there was no significant difference  between the two logarithmic regression models for expected heterozygosity, although the model using area of protected habitat had higher likelihood (17.1) than the model using area of overall habitat (15.7). Further, a likelihood‐ratio test demonstrated that the linear regression model using area of overall habitat had significantly better fit for the given data than the same model using area of protected habitat for eN . Lastly, the minimum area of suitable habitat estimated to maintain eN ≥ 50 was 14,591 km2 for overall suitable habitat and 7,923 km2 for protected suitable habitat.We observed statistically significant relationships between all of the population genetic metrics , steelhead salmon (Oncorhynchus mykiss), and California condors  may fall under the umbrella and benefit from conservation of mountain lion habitat  as much is required to maintain a mountain lion population with allelic richness ≥ 3.00 than if suitable, mixed‐status  habitat is maintained. High allelic richness in protected habitat compared to unprotected/mixed‐status habitat is likely a result of less habitat fragmentation and higher landscape permeability, which can increase facilitate gene flow  and genetically diverse ; Data curation ; Formal analysis ; Funding acquisition (lead); Investigation (lead); Methodology ; Project administration (lead); Resources (lead); Software ; Supervision (lead); Validation ; Visualization (lead); Writing‐original draft (lead); Writing‐review & editing . Kyle Gustafson: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Resources ; Software ; Validation ; Writing‐review & editing . Daniel Gammons: Conceptualization (supporting); Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Resources ; Supervision ; Validation ; Writing‐review & editing (lead). Holly Ernest: Data curation ; Formal analysis ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Writing‐review & editing . Steven Torres: Conceptualization ; Formal analysis ; Funding acquisition (lead); Investigation ; Project administration ; Resources ; Supervision (lead); Writing‐review & editing (lead).This study was overseen by the California Department of Fish and Wildlife (CDFW) which is the state agency in California with wildlife trustee authority. Captures led by CDFW personnel were conducted with the approval of a CDFW wildlife veterinarian and under the scope of CDFW’s animal care and use policy . Captures led by non‐CDFW personnel  were conducted under the approval of CDFW  or an affiliated animal care and use committee .


Agave plantations for pulque production have disappeared or been abandoned in recent decades. To create strategies for the conservation and production of pulque agaves, it is necessary to first determine their taxonomic identities and to better understand their genetic and morphological diversity. Despite the historical importance of pulque in Mexico, little attention has been placed on the study of Agave plants used for its production. Therefore, we analyzed the morphological diversity of vegetative characters of nine landraces of two Agave species  which are widely cultivated for pulque production in Tlaxcala, Mexico. The analysis of morphological characters showed that the landraces largely clustered based on classic taxonomic relationships. One cluster of landraces associated with Agave mapisaga var. mapisaga and another with A. salmiana subsp. salmiana, but with the exception of A. salmiana subsp. salmiana “Ayoteco”, which is more closely related with A. mapisaga var. mapisaga. Additionally, we analyzed the genetic relationships between 14 landraces and wild individuals using molecular markers (trnL and ITS). The identified genetic variants or haplotypes and genetic pools mainly corresponded with the species. In the case of “Ayoteco”, incongruence between markers was observed. Low selection intensity, genetic flow events, and the plasticity of morphological traits may explain the high number of landraces without clear differences in their morphological diversity (vegetative characters) or genetic pools. The use of reproductive traits and massive sequencing might be useful for identifying possible morphological and genetic changes in the Agave landraces used for pulque production.Pulque is one of the oldest fermented beverages, with its origins dating back to pre-Hispanic Mexico. Recently, public consumption has increased. However, the majority of  Agave L. genus has great economic, cultural, and ecological importance in Mexico and beyond. For over 10,000 years, agaves have provided many products, such as food, fiber, construction materials, and beverages  are enormous plants that can measure more than 5 m in diameter and 10 m in height, including their inflorescence. These plants are mainly adapted to the elevation and climatic conditions of central Mexico. The main species utilized in pulque production are Agaveamericana L., Agave inaequidens Koch, Agave mapisaga Trel., Agave salmiana Otto ex Salm-Dyck subsp. salmiana, and Agave salmiana Otto ex Salm-Dyck subsp. tehuacanensis  García-Mend. , and an intense smear campaign  and red worm (Hipota agavis Blásquez); and maguey mushrooms , beans (Phaseolus vulgaris L.), barley (Hordeum vulgare L.), and potato (Solanum tuberosum L.). Nearly 41% of the land area is comprised of grasslands, and another 9% of forests, wherein the main arboreal species are Pinus teocote Schiede ex Schltdl., Juniperus depeana Steud., Abies religiosa (Kunth) Schltdl & Cham., and Quercus laurina Humb & Bonpl  and has a population of 1,272,847. Its climate can be characterized as semi-arid and cool temperate, with an annual precipitation of 720 mm . The mai & Bonpl .Agave landraces used for pulque production in Tlaxcala and three wild individuals (plants from wild populations that are not used for pulque production) were analyzed . The number of landraces and analyzed individuals depended on the availability of the number of mature Agave plants close to initiating inflorescence emergence.Nine analyzed Figure 2Twenty morphological variables were measured Figure 3R = 0.11 appear collapsed in the same group, all of them providing redundant information. Based on correlations and a PCA , while the y-axis is the value of the specific finite Fourier series (defined in each situation) when evaluated over the interval from –π to π  intron (c–d) of cpDNA (ITS) 1–4 (trnL(UAA) primer was amplified by polymerase chain reaction (PCR) under the following protocol: 95°C/7 min and 35 cycles of 95°C/1 min, 52.4°C/60 s, and 72°C/90 s followed by one cycle of 72°C/7 min and one cycle of 4°C/10 min. The nuclear ribosomal DNA ITS 1–4 were sequenced under the following protocol: 94°C/2 min followed by 30 cycles of 94°C/1 min, 57°C/90 s, 72°C/2 min; an elongation period of 72°C/10 min; and one cycle of 4°C/10 min. The PCR was performed for both pairs of primers, each reaction had a final volume of 25 µL and contained: 2 µL of DNA (10–100 ng), 1 µL of 10 µM for each pair of primer, and 21 µL (1X) of GoTaq Hot Start Colorless Master Mix . The PCR products were run in an electrophoresis chamber in agarose gel and purified with QIAquick PCR purification kits (Qiagen). The sequences were obtained in the Biodiversity and Health Genomic Sequencing Laboratory of the Institute of Biology, Universidad Nacional Autónoma de México (UNAM). To prepare the samples, we used 0.4 µL of big dye terminator v. 3.1, 2 µL of 5x buffer, 4 µL of water, 1 µL of primer (10 µM), and 3 µL of the purified amplificate. The samples were placed in the PCR, and the following program was used: 30 cycles at 96°C/10 s, 50°C/5 s, and 60°C/4 min. Once the cycle series ended, the samples were purified with Centri-Sep (Thermo Fisher Scientific) following the manufacturer’s specifications. To the purified samples, 25 µL of EDTA (0.5 mM) were added, and the samples on the plate were run in an Applied Biosystems 3730xL sequencer (ThermoFisher Scientific). The generated sequences were uploaded to GenBank (see Ten primers previously used to screen for interspecific variation in e tested Table S2of cpDNA  and the ITS) 1–4 . The trnSequences were cleaned and aligned by eye with the support of BioEdit 7.2.6.1. Haplotype networks per marker and per combined matrix were obtained, and a parsimony analysis was performed using the TCS 1.21 program . The gapFor each marker and combined matrix, a Bayesian clustering method was applied with the support of the STRUCTURE v. 2.3.4 program. For these analyses, an independent allelic frequencies linkage model was used . A totalA. salmiana subsp. salmiana, with the exception of “Ayoteco”, were mainly observed in one of the clusters, and the other cluster was mainly composed of A. mapisaga var. mapisaga and A. salmiana subsp. salmiana “Ayoteco”. This confirms that the analysis of only the vegetative characters can distinguish the Agave species but not landraces of commonly used for pulque production. However, the landrace “Ayoteco” recognized in classical taxonomy as A. salmiana subsp. salmiana is closer to A. mapisaga var. mapisaga based on morphometric evidence, as provided below.In the different analyses carried out under the unsupervised learning method, we mainly observed two clusters related with each species. All of the landraces of A. salmiana subsp. salmiana, with the exception of “Ayoteco”, and cluster 1 contains mainly A. mapisaga var. mapisaga and A. salmiana subsp. salmiana “Ayoteco”. The dendrogram Figure 5A. mapisaga var. mapisaga and A. salmiana subsp. salmiana “Ayoteco”) had, on average, shorter average teeth length, shorter spine length, and thinner cuticles; they also have a longer distance to the first tooth, greater leaf thickness at the base, and longer leaves. On the other hand, the means of the individuals in cluster 2 and 3  showed some differences yet tended to be more similar.A. mapisaga var. mapisaga and A. salmiana subsp. salmiana “Ayoteco”) is dominated by individuals with curved spines and straight teeth. Cluster 2  is basically formed by individuals with straight spines and straight teeth. Meanwhile, in cluster 3 , all individuals have straight spines and curved teeth. Morphological differences between clusters are also evident in the percentage of rigid leaves. Ninety-five percent of individuals in cluster 3 had at least 90% rigid leaves, but only 66% of individuals in cluster 1 had rigid leaves. Meanwhile, cluster 2 is between clusters 1 and 3 in terms of percentage of rigid leaves  could be expressing these characters of interest. Notably, we also observed that the subspecies salmiana, in particular the “Manso” landrace, is still cultivated in a very traditional manner in Tlaxcala. On the other hand, A. mapisaga and “Ayoteco” could be the landraces most modified by growers. However, these differences could also be due to pre-existing differences between species resulting from their evolutionary histories. The shape of the spine and teeth could be explained by the latter, as these do not appear to be characters of interest but rather distinctive characters of the landraces  intron of cpDNA at the 1,209 position in the total matrix for A. mapisaga and A. salmiana subsp. salmiana “Ayoteco”. Therefore, in building our trnL haplotype network  at position 602 in a 635-bp-long matrix, and polymorphisms were observed in A. mapisaga. Thus, the ITS haplotype network  separated by only one mutation from “Ayoteco” (H2) and the remaining A. salmiana taxa (H3). This latter haplotype is separated from the other two by various steps.A 13 base-pair (bp) inversion was found in the trnL intron and combined matrices . In the case of the ITS marker, only one cluster was identified with a K = 1  tends to be different because of its origin, evolutionary processes, and manner of inheritance. Chloroplasts are haploid with maternal inheritance, almost nonexistent recombination, and a lower mutation rate compared to the nuclear genome, but higher than nuclear ribosomal DNA . Many pltrnL intron could be more plausible than the ITS marker. Ultimately, these approaches represent hypotheses that can be re-addressed in future studies to search for further phylogenetic resolution for the taxa in question, as the genetic data presented herein are not conclusive. It is necessary to explore a larger number of Agave species , A. inaequides  shows high genetic diversity and low structuring and that genetic diversity was mainly associated with the environment or the samples’ region of origin. This could imply that there is variability among populations of landraces or throughout their geographic distribution, which highlights the need to carry out further studies on their morphological and genetic diversity throughout their geographic distribution. Later, once again using microsatellite molecular markers (SSR), Agave inaequides, Agave hookeri, and Agave cupreata) tended to form genetic pools corresponding with the species level, even though A. hookeri is mixed with cultivated A. inaequides plants. In the present study, A. salmiana and A. mapisaga could similarly represent separate genetic pools, with the exception of “Ayoteco”  are useful for testing hypothesis on the hybridization between species or populations . It is also recommendable to perform population analyses of Agave landraces throughout their geographic distribution and compare these to their wild relatives.The identity of Agave landraces with a local distribution can be strengthened through their taxonomic identification and continued local use. However, the conservation of their vast richness also depends on avoiding their replacement with other landraces that could lead to their extinction, such as in the Yucatan Peninsula, where local landraces (henequen) have been displaced, or in Jalisco, where traditional varieties once used for tequila production have been substituted by the blue agave.In Tlaxcala, the conservation of The datasets generated for this study can be found in the GenBank see Table S2LT participated in collecting, taking morphological characters measurements, obtaining sequences, analyzing kin relationships, writing and editing the article. MR and DC-T did the statistical analysis of morphological characters, and participated in writing and editing the article. ER-G took part in collecting, taking morphological characters measurements and making some of the figures. LM-C also took part in collecting and measuring morphological characters.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.


Petunia species to ask what fate the interspecific hybridpopulation has met over time. We analyzed the genetic diversity in twogenerations from two contact sites and evaluated the effect of introgression. Todo this, we collected all adult plants from the contact zones, includingcanonicals and intermediary colored individuals, and compared them with purebredrepresentatives of both species based on seven highly informative microsatelliteloci. We compared the genetic diversity observed in the contact zones with whatis seen in isolated populations of each species, considering two generations ofthese annual species. Our results have confirmed the genetic differentiationbetween the species and the hybrid origin of the majority of the intermediarycolored individuals. We also observed a differentiation related to geneticvariability and inbreeding levels among the populations. Over time, there wereno significant differences per site related to genetic diversity or phenotypecomposition. We found two stable populations kept by high inbreeding andbackcross rates that influence the genetic diversity of their parental speciesthrough introgression.Interspecific hybridization has been fundamental in plant evolution.Nevertheless, the fate of hybrid zones throughout the generations remains poorlyaddressed. We analyzed a pair of recently diverged, interfertile, and sympatric The dispersal of organisms and their gametes leads to the spread of genetic variants.The exchange of genetic variants between diverged lineages is referred to ashybridization, and the geographic areas where hybridization is localized are hybridzones .Interspecific hybridization has played an essential role in plant evolution, and geneexchange between divergent species can result in new phenotypic or geneticdiversity, adaptive variation, and, in some cases, contribute to speciation  due to tPetunia to ask what fate the interspecific hybrid populationmeets over time.Nevertheless, regardless of the importance of interspecific hybridization and thefact that it is frequently studied, the fate of hybrid zones across generationsremains poorly addressed. Some controlled experiments have revealed that hybrids canevolve adaptive traits faster than non hybrid under the same conditions, despite thelower initial fines of hybrids . Aiming Petunia contains 14 species that are narrowly distributedin southern South America , where this last species is endemic. Sandstone towers with 300–500 m inelevation interspacing mosaics of open fields in middle of the Pampas biomecharacterize the Serra do Sudeste region , individuals with the typical morphology ofP. axillaris have been found outside shelters, whereas plantswith typical P. exserta morphology are found inside shelterstogether with a variable number of individuals with intermediary corolla color, hereafter referred to as isolated sitesPaIS1 to PaIS3 and PeIS1 to PeIS5, respectively. We considered these to beisolated sites because they are localities where only one species can be found,and all individuals display all morphological traits that characterize eachspecies based on the description by We collected young leaves of all adult individuals in two flowering seasons (2011and 2015) from the two known contact zones between TableS2): inside cavities, P.exserta - red-colored individuals or hybrid classes A to E -intermediary colored flowers; outside cavities, P. axillaris -white-flowered plants  and their flower color asvisually inspected against a Red-Green-Blue chart. Microsatellite loci were amplified following theprotocol of DNA extraction followed a CTAB-based method (omosomes . Characthttp://www.microchecker.hull.ac.uk/) to estimate genotypingerrors due to stutter bands, allele dropout, and null alleles. Basic frequencystatistics [number of alleles, allele richness, number of private alleles, andNei's unbiased gene diversity , P. exserta , and hybrids . As we did not observe the Wahlund effect, we run all analysesconsidering collection sites as populations . We estimated the observed and expected heterozygosity underHardy-Weinberg equilibrium after Bonferroni correction and analysis of molecularvariance  and a Kruskal-Wallis test tocompare the diversity indices among populations using the IBM SPSS Statistics24.0 package .We used Micro-Checker software for each contact zone in each generation through ta = (1 -FIS)/(1 + FIS), assuming that predominantly inbreeding populations have ta ≤0.2, populations with mixed mating systems show 0.2 <ta ≤ 0.8, andpredominantly outcrossing populations have ta >0.8 . We ran two independent analyses usingJeffrey's priors with uniform priors that included 100,000 steps as burn-infollowed by 1,000,000 MCMC interactions to assure the convergence of chains andhomogeneity across runs. Analyses were performed without previous information onpopulation or taxonomic identity . As expected, from all isolated sites ofP. axillaris, the individuals were found outside shelterand displayed pure white corollas. Similarly, from isolated sites of P.exserta, individuals were uniformly red-colored, and all of themwere found inside shelter. Pairwise fluctuations in the number of individualsper site and color class were compared through Student's t-test(TableS4), and the number of P.axillaris individuals was different between the contact zones andover time, whereas the P. exserta number was constant betweengenerations in CO1. Among intermediary colored individuals, only those in classC were constant in number over time and across collection sites. Considering allintermediary classes (A to E) as one unit, we observed significant fluctuationin numbers of individuals in all comparisons.All collected adult individuals were classified in the field based on the corollacolor and continuously numbered (Table S5). The frequency of null alleleswas low (< 0.5%) across all loci and we did not found genotyping errors.Considering the total diversity per collection site per year . Allele richnessand genetic diversity did not differ between sites between years, whereasobserved heterozygosity and inbreeding coefficient varied between sites andyears. The highest FIS was observed in PeIS, whereas this value significantly decreasedin CO1 over the years with the opposite situation in CO2 , and all loci and collection siteswere polymorphic , whereas when the backcrosses wereconsidered, we observed purebred individuals of each species and hybrids.Individuals from contact zones across generations displayed a varied pattern ofthresholds, including values from those of each species to those as expectedfrom F1, F2, and backcrosses and the observed thresholdwas not always concordant with the visual classification of corolla color.Considering both contact zones and the two generations, ca. 86% ofwhite-flowered individuals had the P. axillaris geneticcomponent, whereas ca. 83% of red-colored flowers were assigned to theP. exserta genetic group. Intermediary colored individualswere preferentially (71%) assigned as P. exserta(TableS2).The simulated genotypes with Hybridlab, independent of whether it was from theF2 hybrids,independent of collection site and reproductive season, and considered PP ≥ 0.50to assign individuals to each genotypic class. All individuals except onecollected in isolated sites of P. axillaris were classified aspurebred P. axillaris individuals. That F2individual displayed a white corolla color, was found outside shelter, andshowed a threshold q ≤ 0.20 in Structure analysis. Allindividuals from PeIS were classified as purebred P. exsertaindividuals. The composition of the contact zones was different with purebredP. axillaris individuals found more frequently in CO1 inboth generations, whereas in CO2, we observed a higher number of purebredP. exserta individuals. Only one individual remainedunclassified based on PP, and it showed a hybrid threshold in Structure andcorolla color class A . Concordance among phenotypes(corolla color), Structure, and NewHybrids classifications was observed morefrequently  than discrepancies. Corolla color and Structureclassifications were concordant for 70% of individuals, whereas color class andNewHybrids agreed in 65% of cases. Structure and NewHybrids classifications wereconcordant 83% of the time (Table S2).Based on the NewHybrids analysis ,we onlyP. axillaris and P. exserta occur,considering two generations of crosses that resulted in the production ofindividuals with atypical morphology and intermediary corolla color between theparental species. We based the analysis on highly variable and informativemicrosatellite loci , at least forP. axillaris . Mixedmating systems and high levels of inbreeding have also explained, at least in part,the genetic diversity ,indicating long-term hybridization, back-crosses, and historic introgression betweenr parent .P. axillaris andP. exserta has been observed through plastid haplotype sharingamong individuals of both species collected in the same towers, despite finding themin different patches inside or outside shelters (P. axillaris,pollen flow is restricted to short distances (P. axillaris and P. exserta (Some individuals from allopatric populations presented signals of hybridization,which can be interpreted as a consequence of historical gene exchange, retention ofancestral polymorphisms or even a small degree of long-distance gene flow throughthe pollen. Ancestral polymorphism sharing between P. axillaris and P. exserta (P.axillaris and P. exserta could reflect theirpreferences for landscape and habitat characteristics, likely as a consequence ofdistinct evolutionary histories rather than simple ancestral polymorphism sharing aspreviously thought based on plastid markers (Interspecific hybridization in zones of secondary contact after allopatric divergenceis frequently observed between closely related species with overlappingdistributions , suchasPetunia species in an area of more than 3,000km2 and dozens of populations of each species occupying towers (Furthermore, the existence of only two contact zones with a low frequency of hybridsbetween two P. axillaris and P. exserta by secondaryor non-specific pollinators.In sum, we are facing two stable hybrid populations that influence the geneticdiversity of their parental species. These sites are mainly maintained by highinbreeding and backcross rates and could initially be formed through pollen exchangebetween


Salmo trutta caspius population is an important species for aquaculture and livestock industry. Moreover, these species are used for studies on molecular markers. Mitochondrial genomic is also beneficial for phylogenetic studies in salmonid species. They are applied for maternal traits whereas paternal traits are related to nuclear genomics. Mtgenomics will be genetically highly divergent indicating that they may represent distinct and potentially locally adapted gene pools. Evolutionary history of the salmo taxa such as brown trout, salmo salar, and s. trutta populations has been studied. In the present study, 50 samples of the Iranian S.t. caspius population were collected from three regions . The PCR product was carried out for machinery sequencing. Mtgenomic in S.t. caspius was deposited in GenBank under accession no. LC011387.1. Evolutionary analyses were conducted using MEGA7 software. The analysis involved 50 nucleotide sequences. A close relationship was observed between the samples. Salmo trutta caspius population is an important species for the aquaculture and livestock industry. Moreover, these species are used for studies on molecular markers. Mitochondrial genomic is also beneficial for phylogenetic studies on salmonid species. They are applied for maternal traits whereas paternal traits are related to nuclear genomics. Mitochondrial genomics are genetically highly divergent indicating that they may represent distinct and potentially locally adapted gene pools , Ramsar (Safa Roud) in Mazandaran province and Talesh (Nav Roud) in Guillan province]. The distance between Cheshmekileh Roud and Safa Roud to NavRoud is approximately 249 km.In the present study, 50 samples of s.t. fario was deposited in GenBank accession no. LC011387.1. Evolutionary analyses were conducted using MEGA Software Version 7 (Tamura and Nei s.t. caspius marked by black underline and other salmonids like Hucho taimen, s.t. fario, etc. A close relationship was observed between the samples (The sample salmonids caught in fall 2015 were three years old. For the next stage, total genomic DNA was isolated from blood taken from fresh specimens stored in −20 °C in the refrigerator. DNA isolation was carried out using the phenol–chloroform protocol (Sambrook and Russel  samples .