Month: May 2024

The total tonnage of fertilizer applied to corn increases by 484.45 tons, an 26.6 percent increase, statistically significant at the 1 percent level. The impact congregational mergers have on corn fertilizer use is not only statistically significant, but also represents an economically meaningful change. In sum, the information in Table 2.3, Table 2.4, and Table 2.5, demonstrates that congregational mergers increase fertilizer use, in keeping with Proposition 2. Next, we test Proposition 3: that congregational mergers will not affect technology adoption when all potential adopters are fully informed. Strip cropping, irrigation, and orchards are technologies for which we expect no effect. Table 2.6 demonstrates the impact congregational mergers have on the number of farms using strip cropping and the acres under strip cropping; the number of farms reporting irrigation use and the total number of irrigated acres; and the total number of acres in fruit orchards, groves, vineyards, and nut trees. Table 2.6 confirms our hypothesis: we see no statistically significant impacts of congrega- tional mergers on strip cropping, irrigation, or orchard acreage. Furthermore, the magnitudes of the coefficients we do see are small, and in the case of irrigation, have negative signs. The absence of results in this table further supports the fact that congregational mergers are driving the changes in input uses observed above, because mergers are affecting farmers on the dimensions we would expect, but we are not seeing impacts on agriculture that should not be affected by mergers.With any regression analysis,blueberries in containers growing it is important to ensure that the results are robust. We use three pieces of evidence to demonstrate that the results we present in this paper hold up to robustness checks. First, we saw impacts of congregational mergers where we expected them: on fertilizer and lime use and on corn. We did not see impacts where we expected them not to be: on strip cropping, irrigation, and orchards, as presented above.

In addition, our results are robust to a variety of different land use variables provided in the census of agriculture data: the outcomes measured in number of farms do not change significantly if instead of total farms, we use commercial farms or cash-grain farms; the outcomes measured in acreage and tonnage do not change if we use acres in the county or acres harvested rather than acres in farms. In this section of the paper, we perform a placebo test. We also investigate channels other than information through which congregational mergers might be driving fertilizer adoption, and provide evidence against these other possible explanations.Table 2.7 has the placebo analysis. We run this check in order to see whether mergers that occurred from 1964 to 1967 impact our outcomes in 1964, before these mergers actually occurred. We use the time period 1964 to 1967 because it includes the same number of years as our actual treatment period. Column looks at farms using fertilizer; at acres fertilized; at tons of fertilizer used; at corn acres fertilized; at farms using lime; and at acres limed. We expect to see no statistically significant impacts of “future” congregational mergers on these outcomes. Indeed, Table 2.7 shows that, with the exception of the number of farms using lime, there is no statistically significant effect of future congregational mergers on 1964 input outcomes. In addition, comparing these effects to those in Tables 2.3, 2.4, and 2.5, the magnitudes of the coefficients are quite small. This helps confirm that the effects we are observing above are real and driven by the congregational mergers we observe, rather than by something unobserved. To assuage concerns about small-sample inference, we also run a permutation test, in which we randomly assign 39 counties to treatment 10,000 times. Using each of these treatment assignment vectors as a placebo treatment, we find our actual effect on farms using fertilizer is larger than all but 4 percent of the randomly drawn treatment vectors. Figure 2.7 displays the results of this procedure. It is still possible that our results are being driven by something other than a congregational merger driven information effect. Here, we explore two other possible explanations for our results. The first is the presence of agricultural extension.

Agricultural extension, formally introduced in the United States by the Smith-Lever Act of 1914, plays a major role in information dissemination in agriculture. There is a large literature on the effect of agricultural extension, both in the United States and elsewhere, on agricultural productivity and technology adoption ; Huffman ; Birkhaeuser et al. ; Dercon et al.. Despite the importance of extension, we argue that it is in fact congregational mergers and not extension services that generate the results we find in this paper: because of the fixed effects strategy, in order for agricultural extension to be driving these results, we would need to see agricultural extension services changing differently over time in treatment counties than in control counties, having removed the state time trend, only over the 1959 to 1964 time period. This is potentially plausible, but seems unlikely, especially because extension funding and the number of extension agents allowed is governed by state laws, which do not change often. For example, the Minnesota statutes outlining extension were first passed in 1923, updated in 1953, and were not revised again until 1969. The law allows for “the formation of one county corporation in each county in [Minnesota]” to act as an extension agency, with in most cases one extension agent and a specified budget, based on the number of townships in the county. While county extension offices documented their activities for mandatory state reports, these reports were inconsistent across different counties and years. Also, many of the variables measured were endogenous, such as the number of phone calls received or the number of attendees at extension events. As a result, it is impossible to credibly measure the intensity and efficacy of extension efforts over our sample period.Another plausible explanation would be that the mergers also facilitated increased access to capital. In order to provide evidence against this possibility, we estimate Equation again, this time with the number of farms with each of a variety of capital-intensive technologies as outcome variables. Table 2.8 shows the impact congregational mergers have on the number of farms with cars, trucks, tractors, bailers, and freezers.

As expected, we find no statistically or economically significant effect of congregational mergers on capital-intensive inputs: the standard errors are quite wide, and the effect sizes small: the coefficient on cars, for example, is only a 0.01 percent increase relative to the control group mean, and the standard error is almost one hundred times the size of the coefficient. This suggests that congregational mergers did not substantially increase access to capital, and provides additional evidence that information is the main channel through which congregational mergers impacted technology adoption. Finally, one might worry that by only using TALC congregational mergers in our analysis, we are understating the true treatment effect. We argue above that the TALC mergers are exogenous, and, due to the heavily Lutheran populations in these regions, the mergers where we would expect to see an effect. Indeed, the congregations that are merging in these data have, on average, 492 baptized members, so seeing an additional 35 farms begin to use fertilizer is an entirely reasonable effect size. There is another major Lutheran church branch, the Lutheran Church – Missouri Synod ,planting blueberries in containers that was not directly involved in the TALC merger, but whose mergers could be attributed to increased discussion about merger surrounding TALC. We collected data from Concordia Historical Institute, the LCMS seminary, on congregational mergers between LCMS churches during the sample period. There is only one merger that occurs in a non-metropolitan county during this time period, and the inclusion of said merger does not produce a statistically distinguishable result from using only the TALC mergers. Ultimately, given the range of tests that we perform, we have confidence that our results are robust and that we are correctly attributing them to the information effect of congregational mergers. Since the early 2000s, US ethanol production has exploded in response to federal policies incentivizing the production of renewable fuels. In 2005, Congress passed the Energy Policy Act introducing a Renewable Fuel Standard mandating that 2.78% of gasoline sold in the US be from renewable sources. In 2007, Congress passed the Energy Independence and Security Act setting annual renewable fuel mandates for US production with an ultimate goal of 36 billion gallons by 2022. Of these 36 billion gallons, 15 billion are to be conventional bio-fuels – corn-based ethanol in particular. The US ethanol industry has clearly responded to the Renewable Fuel Standards established in the EPAct and EISA. Between 2002 and 2014, US ethanol production has increased from just over 2 billion gallons per year to over 14 billion gallons per year . In order to produce such quantities of ethanol, the number of corn ethanol refineries in the US has increased from 62 in 2002 to 204 in 2014 . The striking increase in US corn ethanol production has raised several important questions about its unintended consequences. One strand of research has explored how increased demand for ethanol has affected land use in the US corn belt as aggregate demand for corn increases . Another strand of research has been more concerned about the environmental externalities of changing agricultural patterns, particularly focused on nitrate runoff and water pollution . In this chapter, I explore both the land use change effects and environmental effects of expanding ethanol production. In particular, I study the geospatial effect of ethanol re- fineries’ placement on nearby land use change and use my results to estimate environmental consequences. I am specifically interested in how the location of ethanol refineries spatially affects agricultural land, and I do not attempt to identify the full general equilibrium effect of the 14 billion gallon US corn ethanol industry. Put another way, I study how the distribution of ethanol refineries differentially affects different agricultural areas net of the ethanol industry’s aggregate effect on corn prices. I find that within a population of almost 114 million acres of agricultural land in Illinois, Indiana, Iowa, and Nebraska, nearly 300,000 more acres of corn were grown in 2014 than in 2002 due merely to ethanol refinery location effects.

This represents approximately 21,000 tons of nitrogen applied as fertilizer. Almost all the 300,000 acres of increased corn acreage exist within 30 miles of an ethanol refinery, suggesting that these refineries have strong local effects on land use change and nitrogen use. There is clear economic intuition for why ethanol refineries would differentially affect nearby and faraway agricultural land. When a corn-fed ethanol refinery is built, it represents a new terminal market for corn. Since refineries operate continuously, they have an inelastic demand for this input. And since transportation costs are significant for grains, one would expect an ethanol refinery to source its corn from the nearest producers. Thus, by reducing transportation costs for nearby producers , ethanol refineries essentially subsidize corn production for nearby farmers. On the margin, this subsidy incentivizes farmers to grow more corn – or grow corn more often – than they otherwise would. As corn production increases, so will nitrogen fertilizer use. Corn requires higher levels of nitrogen fertilizer than other Corn Belt crops, and particularly high levels of fertilizer when grown successively corn-after-corn. Thus, economic intuition suggests ethanol refineries would have a localized effect increasing corn production and nitrogen fertilizer use. Consequently, these refineries would also have an effect on localized nitrate runoff due to the increased nitrogen fertilizer use. Researchers have previously addressed different components of the ethanol industry’s effects on land use change and nitrate runoff. One line of research has explored whether the hypothesized local corn subsidy provided by nearby ethanol refineries actually exists. In a frequently cited paper, McNew and Griffith find that corn prices at an ethanol refinery are 12.5¢ higher than average, that the effect is slightly stronger for “upstream” refineries than for “downstream” refineries, and that price effects can be detected up to 68 miles from a refinery. However, Katchova and O’Brien both fail to find such a subsidy. Gallagher et al. highlight that locally-owned and non-locally-owned refineries have different effects on corn prices: the authors find that corn prices are increased by proximity to a non-locally-owned refinery, but not by proximity to a locally-owned refinery. Finally, Lewis finds different results in different states: ethanol refineries in Michigan and Kansas affect local corn prices, but refineries in Iowa and Indiana do not.

A study conducted on diabetic Zucker rats supplemented with an anthocyanin-rich extract from purple potato and bilberry, showed a reduction in dysbiosis of colonic microbiota and anthocyanins gut microbiota-derived metabolites, increased cecal sugar levels together with the increase in the abundance of Peptostreptococcaceae sp. and Parabacteroides spp. in colon. These results demonstrate that anthocyanins by modulating the gut microbiota can affect gut function and consequently prevent T2DM . Our RNAseq analysis showed for the first time that an anthocyanin-rich BB extract can affect the expression of large number of genes in adipose tissue, not only protein coding but also non-coding genes like miRNAs or lncRNAs. According to our knowledge, very few studies have reported the multigenomic effect of BB anthocyanins, as well as isolated anthocyanins, on adipose tissue. It has been observed that cyanidin 3-glucoside, anthocyanin from BB, can up-regulate the expression of GLUT4 gene in vitro in 3T3-L1 cells . The BB extract rich in flavonoids and saponins was also shown to exert the capacity to modulate the expression of key genes involved in lipogenesis, such as SREBP1c, ABCG5, CPT1, SREBP2, FAS, HMGCR, INSIG1 and INSIG2 . Supplementation of mice diet with BB has been reported to modulate the expression of the SCFAG-protein coupled receptors in colonic tissue as well as the expression of genes regulated the integrity of the epithelial barrier: occludin, JAM-A, E-cadherin and the junction/cytoskeletal connector ZO-1 . Recently it has been observed that black bean concentrate improves hepatic steatosis by reducing lipogenesis and increasing fatty acid oxidation in rats fed a high fat-sucrose diet . However, these studies present an important drawback,large plastic pots for plants which is use of targeted approach to assess the expression of few specific genes.

Rare recent studies have shown that anthocyanins can affect large number of genes presenting multi-modal action. For example, anthocyanins from blackberries affected the expression of over 600 genes, including miRNAs, in circulating immune cells in human volunteers . Another study has demonstrated the capacity of anthocyanin-rich bilberry extract to modulate the expression of over 1,600 genes in hippocampus of ApoE-/- mice . Enrichment pathway analysis in adipose tissue were performed using 406 differentially expressed genes following BB extract intake. This allowed us to show that the black bean extract can affect genes regulating cell signaling pathways in adipose tissue from diabetic rats, pathways related with phosphatase activity, phospholipid metabolism, phosphatidyl inositol 3-kinase binding, phosphatidylserine binding, and ER organization. On the other hand, we also found down-regulated pathways that participate in the modulation of NIK/NF-κB signaling, regulation of response to extracellular stimulus, positive regulation of cell junction assembly, negative regulation of cell population proliferation and negative regulation of cytokine-mediated signaling pathway. Remarkably, while regulation of NIK/NF-κB signaling is up-regulated in the DB group, we observed that the treatment with BB extract down-regulated this pathway in the BB group, suggesting the capacity of the extract to counteract the effect of T2D. The biological relevance of this pathway on adipose tissue is the critical function in reprogramming the fat cell transcriptome to inflammation in response to over nutrition and metabolic stress. The inhibition of NF-κB signaling has metabolic advantages for adipose tissue inflammation caused by obesity . On the other hand, NF-κB inducing kinase is a key controller of immunity and inflammation at local and systemic levels on metabolic processes.

In adipose tissue, NIK promotes adipogenesis by activating non-canonical NF-κB pathway, demonstrated by Pflug et al., when NIK deficient mice fed with a high fat diet showed decreased overall fat mass, increased insulin sensitivity, and energy expenditure . Moreover, anti-inflammatory effects have also been attributed to anthocyanins from Hibiscus syriacus L. by the capacity to inhibit TLR4 in LPS-induced cells, followed by the decrease of the phosphorylation of MyD88 and IRAK4, which resulted in NF-κB inactivation . Black soybeans also demonstrated to inhibit IκB phosphorylation that impede NF-κB translocation resulting in the inhibition of iNOS transcription and iNOS and COX-2 translation . Taken together, these results suggest that extract from BB can exert health properties by countering the genomic modifications induced by T2DM. Elevated levels of FFAs, inflammation, excess of nutrients, inadequately folded proteins, and local hypoxia, are characteristics of obesity and can lead to ER stress. This results in increased oxidative stress in adipose tissue of obese animals . ER plays an important role in adipose tissue inflammation due to their participation in the activation of NF-κB signaling by the phosphorylation of JNK, IKK, and JNK-mediated phosphorylation of IRS1/2 triggering the unfolded protein reaction and involving pathways like PERK , IRE1 , and ATF6 . Exposure of J774A.1 macrophages to a cyanidin-3-O-galactoside-rich aqueous extract of Sambucus ebulus L. resulted in suppression of LPS-induced transcription of pro-inflammatory genes like IL-1β, IL-6, TNF-α, Ccl2, Icam-1, Fabp4, COX2, iNOS, Noxo1, IL-1ra and Sirt1. SE also showed to produce the decrease in protein levels of iNOS, peIFα, ATFα and C/EBP Homologous Protein . Similarly, Punica granatum L. a high-content polyphenols Chinese plant, was administrated to diabetic rats for 4 weeks. It was observed that doses between 50 and 100 mg/kg can improve ER stress signals including IRE1, activation of XBP-1, as well as lower levels of IREα, XBPs, and CHOP . Therefore, anthocyanins present in our extract also have the capacity to modulate the expression of genes regulating ER stress, presenting another important molecular target underlying the anti-diabetic effects.

Our bio-informatic analysis also revealed that phosphatidylinositol 3-kinase binding signaling pathway was up-regulated in BB group. The importance of this pathway in adipose tissue is due to its role in adipose hypertrophy which rises tissue immune cell infiltration, fibrosis, and lipolysis. Decreases IRS-1 activation and AKT-induced glucose uptake, as well as exacerbates systemic insulin resistance and the development of T2DM. Different factors like adipocytokines and adipose hypertrophy produce insulin resistance by obstructing PI3K/AKT-mediated inhibition of lipolysis reducing the capacity of glucose utilization and diminishing the capacity of SREBP to stimulate lipid synthesis . Recent research concluded that sweet potato leaf polyphenols might up-regulate the important mediators of the insulin-mediated PI3K/AKT/GSK-3β signaling pathway in a dose-dependent way in diabetic mice by regulating the mRNA expression of genes IR, IRS-1, PI3K, AKT and GLUT-4 . Bioinformatic analysis of differentially expressed genes permitted to identify potential transcription factors whose activity can be affected by BB extract and result in the detected nutrigenomic modifications. Among these transcription factors are GATA2, POU2AF1, IRF3, GATA1, NR2F2 or PPARA. Interestingly some of these transcription factors have been identified as playing a role in adipose tissue development and/or diabetes. For example, it has been shown that GATA2 plays an important role in diabetes development and associated diseases . It has been described that hyperinsulinemia, observed during Type 2 diabetes, can activate NR2F2 which can induce development of different diseases . PPARs have central role in lipid and glucose homeostasis, pathogenesis of insulin resistance and metabolic syndromes and are active in adipose tissue. It has been shown that binding phenolic and other molecules to PPARs result in significant changes in their activities and present important mode of treatment type 2 diabetes mellitus. . Similarly, IRF3 has been described as a main transcriptional regulator of adipose tissue inflammation and is involved in preserving systemic glucose and energy homeostasis . One study also observed that IRF3 in adipose tissue promotes adipose inflammation and insulin resistance . These observations from our bioinformatic analysis reveal significant regulators of BB extract underlying their observed health properties. Moreover, our genomic analysis revealed that black bean extract rich in anthocyanins can also modulate the expression of microRNAs. MicroRNAs are short, non-coding RNAs which can bind to mRNAs,plant pots with drainage resulting in modification in translational repression. It is estimated that there are approximately 2200 miRNA genes in the human genome that can regulate the transcriptional levels of over 60% of genes. Consequently, they can regulate many major cellular functions such as development, differentiation, growth, or metabolism . Moreover, it has been shown that modifications in their expression produce an important role in progress of diseases. Among these, it has been demonstrated that miRNA might have an important role in development of diabetes and metabolic disorders . On the other hand, several previous studies have shown that anthocyanins can modulate the expression of these non-coding RNAs. For example, it was shown that a mixture of anthocyanins as well as their gut microbiome derived metabolites at physiologically relevant concentrations can affect the expression of miRNAs in isolated human primary endothelial cells . Also, a study showed that supplementation of mice diets with different polyphenols at nutritionally relevant doses can significantly affect global miRNA expression profile in liver . Among the miRNAs identified as differentially expressed by black bean extract are miR-152, miR-219a1, miR-384 or miR-615.

It has been observed that miR-152 is expressed in adipocytes and can stimulate lipid accumulation in preadipocytes accompanied by higher expression of some pro-adipogenic genes, adipogenesis and intramuscular fat formation . Its expression is also altered in patients with T2D with or without medication . miR-219a1 has been suggested to show a role in fat development. Similarly, it has been demonstrated that miR-615 is expressed in adipose tissue in humans and that its expression changes with the obesity . Moreover, the expression of this miRNA has also been identified in patients with diabetes . Together with miRNAs, our analysis also revealed changes in the expression of long non-coding RNAs. Biogenesis of lncRNAs is different than mRNAs and can interact with DNA, RNA, and proteins, modulating chromatin function, changing the stability and translation of cytoplasmic mRNAs and interfering with signaling pathways . Because of their large mode of action, they affect numerous cellular and consequently physio-pathological processes. It has been shown that they regulate adipogenesis and adipose tissue function and diabetes . Only few studies have suggested that polyphenols are potent modulators of the expression of noncoding RNAs. For example, epicatechin metabolites can regulate the expression of these miRNAs in human brain endothelial cells in physiologically relevant conditions . Therefore, our study provides novel and original data about the capacity of legumes and their bio-actives to affect the expression of this class of RNAs. However due to the lack of known biological functions of these lncRNAs as well as their targets, regulated cellular and molecular processes remain to be identified. Taken together, our results suggest that black bean extract rich in anthocyanins exert protective properties by modulating the expression of miRNAs and lncRNAs in adipose tissue in diabetic conditions.From a microbiological perspective, soil is largely unexplored even though we know it has a rich diversity of microbial life. Depending on its physical and chemical properties, soil can contain 109-1010 microbial cells per gram, including tens of thousands of different bacterial, archaeal, and fungal species, plus viruses and protists. Soil microbes carry out life-sustaining functions for our planet, including cycling of nutrients and promoting plant growth. Respiring soil microorganisms, producing metabolic byproducts such as carbon dioxide and methane, cycle enormous volumes of carbon-containing gases from the terrestrial ecosystem into the atmosphere. Perturbing this ecosystem—for example, when global warming raises temperatures—potentially alters the flux of these gases. Despite the immensity of the carbon reservoir in soil , its fate in the face of climate change is not known. The high diversity of soil microbial communities makes them difficult to study. Exacerbating this difficulty, few soil microorganisms are amenable to isolation and cultivation, steps that in conventional terms are crucial for elucidating microbial physiology and biochemistry. Although most isolated soil bacteria are “weeds” such as pseudomonads and actinobacteria, 16S rRNA gene surveys reveal that few species from the great bulk of soil bacteria have cultured representatives. For example, members of the Acidobacteria phylum are widespread in soil but are notoriously difficult to cultivate. To address these issues, microbial ecologists are now using a metagenomics approach, sequencing soil DNA to develop a better understanding of the microbial identities and their potential functions in soils. Initially, investigators who used metagenomics to study soil microbial communities cloned DNA extracted from soil into bacterial artificial chromosome vectors or fosmid vectors. One advantage of this cloning-based approach is that it permits investigators to screen for expression of particular phenotypes, including production of antibiotics and enzymes.

A national-scale analysis also allows for broad comparison with masting species where such synchrony has been observed. In this study, we characterized alternate bearing at national scales using three complementary metrics: lag-1 auto correlation , coefficient of variation in seed production and bimodality of seed production . Classic alternate bearing would have all three metrics at a population level; in our study, we test whether they are detectable at a national scale. Throughout, when we discuss alternate bearing at a national scale, we refer to any or all of these variables. We had strong a priori expectations that wind-pollinated species would show stronger alternate bearing at national scales, i.e. they would have stronger negative lag-1 autocorrelation, higher CV and possibly stronger bimodality than insect-pollinated species. After assessing these patterns at national scales, we discuss the socio-ecological implications of our findings and explore future research directions.We downloaded 58 years of data collected by the FAO on yield and production for 27 perennial crop taxa that have been reported in the horticultural literature as having a tendency toward alternate bearing at the individual level. For each crop, we selected up to 10 countries worldwide, each with at least 20 years of data, for inclusion in the analysis by their highest total production. The included nations were filtered for historical consistency and the crop names were modified for interpretability . The resulting dataset was comprised of 236 crop–country combinations . We performed the country selection and all subsequent analyses twice, once including only the past 25 years and once including all data on record . Patterns did not differ between the two time series; the results and discussion below describe the ‘long’ time series .

To confirm that the FAO dataset reflected historical reproductive output of agricultural perennials,drainage for plants in pots we scanned horticultural and economic online publications on focal crops that mentioned environmental shocks that occurred in exceptionally low yielding years at national scales and looked for signals of these in the detrended FAO time series . This validation exercise was done as a qualitative check of how well these aggregate time series represent on-the-ground experiences of growers; therefore, we scanned the available online information, rather than attempting a systematic review of trade publications.All statistical analyses were performed in R Studio Software 3.5.0. To focus on interannual variation in yield, rather than broader patterns of increasing or decreasing yield, we detrended all time series using generalized additive models fit to time series of yield through time. These were calculated with defaults from the mcgv package and appeared to provide reasonable fits to the data based on visual inspection . To confirm that the results were not dependent on the detrending method, we repeated the analysis using locally estimated scatterplot smoothing and differencing as alternative detrending methods. The observed patterns persisted; we report the GAM-detrended results below . While national production was used for selecting the top-producing countries included in the analysis, yield was used for time series analysis because annual yield estimates should be less sensitive than production to additional variability from factors like farm expansion or contraction that are irrelevant to the present study. We characterized each time series with three common mast seeding metrics that provide complementary information about alternate bearing. The lag-1 autocorrelation is a measure of alternate bearing that captures the tendency of high years to follow low ones.We also inspected the autocorrelation function at lags up to 17 years as a descriptive tool to understand patterns of yield that differ from alternate bearing.

Our second metric, the CV, is typically the standard deviation divided by the mean. However, because the mean of a detrended time series is 0, we used the standard deviation of the detrended time series divided by the mean of the raw time series, a technique commonly applied in climate modelling. Third, we used Hartigans’ dip statistic to test for bimodality. D is a measure of deviations from a unimodal distribution of values; significant differences would indicate that yield is multimodal . D was rarely significant , so we did not perform further tests to distinguish bimodality from other multimodal distributions. We analysed whether each masting metric differed as a function of pollination syndrome . We treat pollination syndrome as a binary variable as in previous masting studies, though relative dependence on insect pollinators may vary in more nuanced ways . Of the 27 crops, 6 were wind-pollinated and 21 were insect-pollinated . Because alternate bearing could be driven in part by phylogenetic relatedness, we conducted these tests using phylogenetically corrected linear mixed models with the trait as the predictor, the masting metric as the response, and random effects of plant phylogeny and country using the lme4 package . The plant phylogeny was constructed using the function ‘phylo.maker’ in the R package V.PhyloMaker using the GBOTB.extended mega-phylogeny as backbone and the default option ‘scenario 3’, in which the tip for a new genus is bound to the 1/2 point between the family root node and basal node. The resulting phylogeny had 35 tips because six of the crop categories contained multiple species. The phylogenetically corrected LMMs were run using the ‘phylo_lmm’ function on all possible combinations of FAO crop species of these six categories . Because the results were consistent across all iterations and varied minimally , the mean Chi-square and p-values are reported below . The only observed differences across iterations were driven by species in the ‘mango, mangosteen and guava’ category, which are grouped by the FAO despite distant phylogenetic relatedness.

We also performed a phylogenetically corrected LMM to compare the relationship between AC-1 and CV.A negative lag-1 autocorrelation is significantly more common in wind- than insect-pollinated plants. Yield variability is also significantly greater in wind than insect-pollinated crops. These results are in line with previous findings in natural plant systems that masting is more common and more pronounced among wind-pollinated taxa. Bimodality was rare in these time series and did not differ between pollination syndromes, which is also consistent with natural systems where strong bimodality is uncommon and ‘partial masting’ prevails. Given that we observed a strong signal of alternate bearing despite limitations of the FAO dataset , our findings offer encouraging support that enhanced integration of the alternate-bearing and masting literature could offer mutual insight. Unsurprisingly, the CV of alternate-bearing crops reported here at national scales is smaller than has been observed for both masting and alternate-bearing taxa at population scales. In general, alternate bearing leads to lower CVs than mast-seeding at longer intervals . More importantly, we expect that in crop plants, as in wild plants, synchrony should decay with distance, and, at the present time, the scale of synchrony in crop plants is largely unknown. To explore this spatial scale somewhat quantitatively, we compared our results to data presented by Noble et al., one of the few published studies of yield variability at multiple scales in a crop plant. Noble et al. provide data on pistachio yield in four Californian counties. In their data, farm-level yield was more variable than county-level yield . These are all higher than estimates of pistachio CV at a national scale from the FAO data presented here . This very limited exploration suggests that synchrony across crop yields decays with distance, and that further exploration of farm- to regional-scale data for crop plants could be valuable for understanding spatial synchrony, especially in insect-pollinated species. Yield stability is frequently the goal of farmers and horticultural researchers, yet our results suggest that alternate bearing persists even at a national level. At the present time,growing raspberries in pots studies on drivers of synchrony in agricultural settings are scarce and would benefit from integration with ecological theories about the causes of mast-seeding. For example, numerous studies of crop pollination are based on the premise of enhancing yield in a single year. However, if the alternate bearing is due to resource depletion after high-seed years, then maximizing yield in 1 year could lead to greater variability in yield, an undesirable outcome. The long-term impact of increased pollination will be affected by whether or not the crop tends to bear alternately at the floral initiation stage or in the flower-to-fruit conversion stage and by the relative cost of seeds to flowers. These aspects of plant development are rarely integrated into models of crop yield but would be straightforward to measure and implement to better align insect conservation and farmer priorities. We observed signals of numerous environmental shocks in crops at a national scale, but their role as a driver of synchrony in alternate bearing is largely unknown and presents another opportunity for masting theory to inform horticultural understanding. Environmental vetoes—external conditions that prevent seed set—have been well-supported as a driver of synchrony in masting systems. As a recent example, Schermer et al. studied frost-induced fruit losses in relationship to flower phenology in oaks.

They concluded that a delay in flowering would lead to a more deterministic pattern of seed production characterized by a lower CV at the population level and a more pronounced lag-1 autocorrelation, which strongly resembles an alternate-bearing pattern . By contrast, an advancing flower phenology was predicted to increase the stochastic component of interannual variation characteristic of masting. Agricultural studies have offered stronger support for the latter scenario, i.e. advances in flower phenology with climate change, but have not explored the implications of advancing phenology on yield patterns. Greater stochasticity in natural systems can be advantageous as a pest control agent ; however, farmers are unlikely to experience a net benefit from increased variability given their reliance on a steady income. Our understanding of mast-seeding would similarly benefit from enhanced integration with agricultural crop data. First, given their direct socio-economic implications, long-term yield datasets are widely available for crop plants and can scale from individual plants to farms to national scales. Our results suggest these data present an underused resource for understanding perennial variability and synchrony. While Gleiser et al.’s recent analysis of yield variability across all crops in the FAO data found that increasing pollinator dependence was positively associated with yield variability, their inclusion of annual plants prevents meaningful inference for perennials. However, they observed greater interannual variability in woody than herbaceous plants, which is consistent with alternate bearing in long-lived perennials. Another difference is that we restricted our analysis to plant species known to be alternate bearing at the individual level. To the best of our knowledge, ours is the first study to use FAO data to assess patterns of yield variability specifically in alternate-bearing plants. Second, the horticultural literature has focused extensively on the genetic and hormonal bases of alternate bearing in an effort to achieve more stable yields. By contrast, genetic studies in wild-masting trees are scarce but could be valuable for understanding drivers of masting in the wild . Finally, the existing variability in farm practices and the potential to manipulate them through space and time offer novel opportunities to explore the roles of resource availability, habitat structure, phenology, pollination dynamics and genetics in synchronous perennial plant reproduction. We observed a national signal of alternate bearing in some insect-pollinated crops , despite a stronger tendency toward alternate bearing in wind-pollinated crops. Further work could explore whether crop alternate bearing emerged from a similar pathway across pollination syndromes or as a convergent trait. In wind-pollinated crops, breeding and cultivation may have served to shift the reproductive pattern from stochastic masting toward a relatively more deterministic biennial pattern of reproduction. For insect-pollinated crops, the alternate bearing could be a symptom of agricultural intensification if plants in their native habitat rarely ‘over invested’ resources in reproduction and instead produced a steady, low number of seeds. Monocultures and high-density planting, for example, can make crops more susceptible to pest outbreaks and more exposed to environmental disasters, which would induce synchrony if there were an endogenous resource-driven mechanism involved in alternate bearing. Coffee illustrates this well; the plant is native to the shaded understory of Ethiopian rainforest but is now often cultivated in sun monocultures that have been shown to exaggerate alternate bearing and resource tradeoffs . Data on the reproductive patterns of wild/ancestral lineages of contemporary perennial crops are scarce but would shed valuable insight on the basis of contemporary alternate bearing. Unexpectedly, some crop series appeared to display longer-term cycles . Such a pattern could arise at farm scales as a result of pruning and subsequent recovery, but we would not anticipate these farmer practices to be synchronized at a national scale.

A walnut protein hydrolysate administered to rats exposed to ultraviolet radiation significantly reduced skin photoaging and enhanced skin elasticity. Supplementation with ellagic acid, a compound found in many berries, prevented ultraviolet B -related inflammation and collagen degradation related to skin wrinkling and aging in a murine model. More human studies, using objective measures of skin wrinkles, skin elasticity and response to low-dose UVB radiation exposure are warranted. Monitoring skin responses to a UVB radiation challenge has been used as a marker of whole-body antioxidant status in response to almond consumption. The response to a UVB challenge has also been used to monitor oxidant defenses and changes in skin microbiome following the intake of pomegranate juice .Age-related macular degeneration is the third leading cause of vision loss worldwide. Anthocyanins, carotenoids, flavonoids, and vitamins C and E, found in many berries, have been shown to reduce risk of eye-related diseases. Goji berries, containing the highest amount of zeaxanthin of any known food, hold particular promise since this compound binds to receptors in the macula to offer protection from blue and ultraviolet light. Regular supplementation with 28 g/d of goji berries for 3 mo increased macular pigment optical density, a biomarker for AMD, as well as the skin carotenoid index. Nuts may also be protective against AMD since they are a rich source of vitamin E and essential fatty acids. Regular intake of nuts has been associated with a reduced risk and slower progression of AMD in 2 epidemiological studies, thought to be due to the beneficial role of polyunsaturated fatty acids .Identification of new cultivars with traits desirable for growers, processors,square plastic plant pot and consumers is a continuous effort. As researchers continue to produce new varieties by both conventional and molecular-driven approaches, assessing these varieties for nutritional value is a challenge.

A combination of broad targeted and untargeted metabolomic approaches, along with defined functional phenotyping could be used for rapid screening and defining of mechanistic pathways associated with health. However, consumer preferences for new cultivars are often driven by size and appearance of the berry or nut and flavor, rather than its nutritional value. This would further confirm the need to balance improvements to nutritional profiles with enhancement of consumer-driven traits, maintaining the marketable nature of the berries and nuts.Although the USDA competitive grants program offers funding for outstanding research projects, budget limitations favor animal or in vitro study proposals. Compelling pilot data is needed to be competitive for clinical studies funded by the USDA or NIH, so many researchers submit their initial ideas to commodity groups representing specific nuts or berries. Commodity groups represent farmers, processors, and distributors and have been instrumental in supporting fundamental and applied research focused on their specific berry or nut. The perception that studies funded by nut and berry commodity groups are inherently biased in favor of the test food is an issue sometimes raised by critics, journalists, and the general public. As in all nutrition research, ethical considerations regarding the structure of research questions, hypotheses, study design, outcome measures, interpretation of data, and conclusions must be rigorously considered. The food and beverage industries have played a key role in providing funds and supporting nutrition research on individual foods and beverages, including berries and nuts. Although this draws scrutiny regarding scientific integrity and data reporting, collaboration between academia and industry compared to exclusive corporate funding may help offset some of these concerns. For example, in multiple reported studies, matching funds were also provided by non-industry sources, including institutional and federal agencies.

In other cases, while the food industry provided the test agents, key research personnel and staff were not supported by the same funding source. The academia-industry collaboration has also led to the formation of scientific advisory committees that evaluate and recommend proposals for funding, a peer review process that helps ensure rigorous study designs, data reporting, and dissemination of results. Human studies of sufficient statistical power are expensive, labor-intensive efforts requiring sophisticated and costly laboratory equipment and supplies. In order for research proposals to be competitive for funding from the USDA or NIH, pilot data is required, and for nuts and berries, the only realistic source of funding for these exploratory trials is from industry sources. Critics of industry support for nutrition research have yet to propose realistic alternatives for funding needed to generate initial data. Further, ongoing industry funding of nuts and berries research has yielded important insights into the molecular and physiological understanding of mechanisms of action. Without industry support, provided in an ethical and transparent manner, advances in our understanding of the role of nuts and berries in a healthy dietary pattern would be limited. A risk-of-bias study of 5675 journal articles used in systematic reviews published between 1930 and 2015, representing a wide variety of nutrition topics, concluded that ROB domains started to significantly decrease after 1990, and particularly after 2000. Another study examined the incidence of favorable outcomes reported in studies funded by the food industry in the 10 most-cited nutrition and dietetics journals in 2018 . Of the 1461 articles included in the analysis, 196 reported industry support, with processed food and dietary supplement manufacturers supporting 68% of the studies included. Studies supported by any nut or berry commodity group were not considered due to an incidence lower than 3% of qualifying articles. Studies with food industry support reported favorable results in 56% of their articles, compared to 10% of articles with no industry involvement. The authors offer a number of suggestions to help minimize real or perceived bias, calling on research institutions to enforce strict, regularly updated, and transparent oversight of all research projects involving industry. Suggestions in support of research transparency and integrity have also been advanced from guidelines adapted from the International Life Sciences Institute North America. This served as the basis for the development of consensus guiding principles for public-private partnerships developed by a group of representatives from academia, scientific societies and organizations, industry scientists, and the USDA, NIH, US Centers for Disease Control, and the US Food and Drug Administration.

These provisions include full disclosure of funding and confirmation of no direct industry involvement in the study design, data and statistical analyses, and interpretation of the results and only minimal, if any, involvement of industry coauthor, often given as a courtesy to acknowledge funding and logistical support by the investigators with no intellectual involvement by the study sponsor. This is in contrast to industry-initiated research, where the industry office or commodity group sets predetermined research objectives, provides intellectual collaboration, and often has input on the study design, interpretation of results, and decisions regarding publication. Although some critics may argue that repeated industry funding in support of research groups that report favorable results on a particular nut or berry shows a bias toward positive outcomes, other interpretations are also possible. First, few labs have the infrastructure, detailed methodology and analytical equipment, and trained personnel to conduct clinical studies in an efficient and timely manner. Industry funded studies conducted at major universities have layers of review and accountability within their organizations to guard against malfeasance, and while these layers may not focus directly on precise elements of research design and interpretation of results, faculty members at such institutions generally have a level of integrity and accountability, knowing that administrative review exists. Calls for industry-funded research are often broad in scope, which allows researchers to generate proposals, research questions, and hypotheses that do not have preconceived outcomes. A third consideration is that the nuts or berries under study may simply have sufficient bio-activity to produce favorable outcomes, independent of potential researcher bias.California’s government-mandated marketing programs covered commodities accounting for over $21.18 billion of California crop and livestock production in 2004. While the framework for these marketing programs is set by legislation,25 liter square pot the specific provisions are proposed by producers, approved by the secretary of agriculture or the legislature and governor , and enacted by a super majority vote of producers covered by the program’s provisions. Once enacted, all producers are subject to program provisions and all must pay assessments to cover program costs, with enforcement based on the police and taxing powers of government. This paper reviews the nature, importance, extent and changing use of common program provisions for mandated marketing programs utilized by California producers, and the development of new research, promotion and information initiatives. Not included in the data tables are California producers’ participation in federal promotion programs, commonly referred to as national check-off programs, nor activities of the California Department of Food and Agriculture’s Dairy Branch in administering and enforcing provisions of the California Milk Marketing Order. In 2004, mandated marketing programs covered California commodities ranging from 96.6% of the value of fruit and nut production to 8.3% of nursery and floral production .

As of June 2006, California’s 63 active marketing programs included 11 federal marketing orders, 29 state marketing orders and agreements, 20 commissions, and 3 councils . Since then the Pistachio Commission has been terminated by an industry vote, and a California Leafy Green Products Handler Marketing Agreement has been approved to certify the safe handling, shipment and sale of leafy green products to consumers . Individual commodity marketing programs have been terminated, consolidated and initiated in response to changing marketing issues, with the total number increasing over time. Notable trends have been a decrease in the number of federal marketing orders applicable to California crops and an increase in the number of commodity commissions. More than half of these programs have been established since 1980. In addition, 12 commodity commissions have been established since 1990, with several replacing marketing orders. While all mandated marketing programs are subject to government approval and oversight, commissions and councils tend to enjoy the most autonomy. Mandated marketing programs can include one or more provisions for research, minimum quality standards, regulation of packaging and containers, quantity controls, and/or generic advertising and promotion. Generic, as contrasted with brand advertising and promotion, speaks to general commodity characteristics rather than referring to a specific producer, brand name or processor. The purpose of generic programs is to increase the total demand for a commodity , while brand programs seek to increase market share . Federal and state marketing orders established during the 1930s and 1940s emphasized the use of supply controls to improve prices. Now the provisions that are most often used by federal programs are minimum grades and sizes, pack and container regulations, research and generic promotion. Likewise, the emphasis of state marketing orders has shifted, with more than three-quarters of California programs including provisions for generic promotion and research by 1960. In addition, California commodity commissions emphasizing promotion and research have taken the place of many state marketing orders.While producer support for promotion programs is strong, it is not unanimous, and litigation over mandatory assessments for advertising and promotion has been essentially continuous since the 1980s. The majority of lawsuits have been filed by large growers for various reasons, including philosophical opposition to government interference in marketing their products, a belief that they could obtain a better return promoting their own brand, and basic disagreements with the promotion message or operation of the program. Three cases concerning the constitutionality of generic promotion programs have been heard by the U.S. Supreme Court . In the 1997 case of Glickman v. Wileman Bros. & Elliott, Inc., et al., the Supreme Court ruled that federally mandated generic advertising for California peaches, plums and nectarines did not violate the First Amendment of the U.S. Constitution. In the five-to-four ruling, the Court noted that the business entities that are compelled to fund generic advertising do so as part of a broader collective enterprise in which the freedom to act independently is already constrained by the regulatory scheme. This ruling seemed to take much of the legal pressure off generic promotion programs, until a contrary decision was issued in 2001. In U.S. v. United Foods, the Supreme Court ruled that the national Mushroom Promotion Act of 1990 violated the First Amendment. This ruling set off a flood of litigation against other promotion programs, with lower courts striking down a number of them. Then, in 2005 the Supreme Court agreed to hear a third promotion program case on an Eighth Circuit Court ruling that the national beef checkoff program was unconstitutional. In Livestock Marketing Association v. USDA, the Supreme Court ruled that the national beef check-off program is constitutional.

These results suggest that blueberry components may support angiogenesis under normal physiological conditions, but attenuate abnormal angiogenesis induced by overactive growth factors. Blueberries exert protective effects on endothelial models by reducing oxidative stress and monocyte adhesion to the endothelium and modulating angiogenesis. The direct impact of blueberry compounds on endothelial dysfunctionin vivo remains to be elucidated, with few studies focusing on the molecular pathways involved in atherosclerosis.Inflammation in the brain can be generated by the dysregulation of inflammatory pathways, molecular signals released by injured neurons, or the accumulation of protein aggregates . Brain inflammation has been associated with an impairment of neuron regeneration and an increase in the incidence of neurodegenerative diseases such as multiple sclerosis , and Alzheimer’s and Parkinson’s diseases . Mechanisms involved in neurodegeneration have been reviewed in detail by Jellinger . The sensitivity of brain muscarinic receptors to oxidative stress increases with aging , and abnormal signaling is implicated in neurodegenerative diseases. Blueberry extracts alleviate oxidative stress in neuronal models through a variety of mechanisms , including scavenging of ROS and downregulation of iNOS gene expression . In COS-7 cells transfected with muscarinic receptors, treatment with a whole blueberry extract reduced activation of the cAMP response element-binding pathway, which can be activated by protein aggregates and is involved in oxidative stress in neurons . Several blueberry fractions exerted antioxidant effects,black plastic plant pots although via different mechanisms: a blueberry extract polar fraction and a nonpolar fraction both reduced oxidative stress in TNF-α– and PMA-induced human neuroblastoma . The polyphenolic-rich fraction exerted scavenging activity of ROS, whereas the nonpolar fraction disrupted NOX assembly, a mechanism that generates the production of ROS.

Blueberry treatment also reduced proinflammatory cytokines in LPS-induced BV-2 cells, through modulation of COX-2 gene expression and the inhibition of NF-κB translocation into the nucleus . The phenolic extracts increased concentrations of Arg-1 , a marker of the M2 phenotype of macrophages, which promotes a return to homeostasis necessary to avoid chronic inflammation. In a model of microglial cells subjected to inflammation in different glycemic conditions, a blueberry extract was effective at inhibiting inflammatory markers, with a comparable effect to the insulin treatment used in the model . Protein aggregations in the brain result from the abnormal deposition of misfolded proteins, leading to the accumulation of fibrils, including α-synuclein or amyloid-βinto plaques . Accumulation of Aβ aggregates is associated with impairment of normal physiological activity of the neurons, increased production of ROS , and activation of microglia and astrocytes . Immune cells also generate Aβ , therefore upregulating the inflammatory cascade. A blueberry extract and an anthocyanin fraction conferred protection against protein aggregate-induced cytotoxicity in mouse neuroblastoma and rat hippocampal neurons challenged with Aβ species , most likely through the inhibition of the CREB pathway but with no effect on ERK activation . αSyn-challenged mouse microglia were also protected by polyphenol extracts, but not the cells challenged with glutamate, which is sometimes found in excess in pathological conditions . Based on results in cell models, it is possible that the active compounds of blueberries could dampen the development of neurodegenerative diseases by reducing oxidative stress and inflammation, which limits the formation of protein aggregates and associated cell damage.

It is important to remember that although cell culture studies of neurons and glial cells provide useful information to understand inflammation mechanisms in the brain, these models may be oversimplified due to the lack of a blood-brain barrier, which is critical in maintaining homeostasis .The skin is subjected to constant challenge, including pollution, physical damage , and light irradiation, that play a role in the initiation of inflammation . Cell models include keratinocytes, found in the epidermis , and fibroblasts that synthesize the extracellular matrix, used alone or in coculture . Recently, 3D skin models and a human skin equivalent have been developed to provide more complex models for testing toxicity, absorption, and metabolism with the presence of several epidermal layers and stratum corneum . Blueberry extracts were used to treat human keratinocytes and a human skin model challenged with O3 exposure, a toxic pollutant that can alter the redox status of the skin and induce inflammation . O3 impaired wound healing of the cells, but function was recovered with a blueberry polyphenol treatment by activating cellular antioxidant defense systems, reducing H2O2 production . A blueberry polyphenol extract was compared with 2 anthocyanin and proanthocyanidin-rich extracts to investigate wound healing in human fibroblasts , and anti-inflammatory and antioxidant effects on murine macrophages. The fractions were both effective at reducing ROS production and alleviated inflammation by reducing the expression of COX-2 and iNOS in the macrophages, but the protective effects of proanthocyanidins were more pronounced than for the other blueberry polyphenols . Blueberry treatments, obtained through several extraction methods, were also effective at reducing NO production of human foreskin fibroblast induced by IL-2β . The major risk factor for developing skin cancer is sun exposure. DNA absorbs UV-B, which can induce the formation of dimeric pyrimidine bases . These mutations are usually repaired by nucleotide excision, although certain mutations, notably in the p53 gene, result in the loss of this repair function , possibly leading to the development of squamous or basal cell carcinoma .

In cells exposed to UV-B, responsible for DNA damage and initiation of proinflammatory pathways, a blueberry extract protected DNA against strand break formation . Blueberry extracts also demonstrated anti-inflammatory effects in UVB-challenged cells and reduced cytokine gene expression through inhibition of NF-κB and MAPK activations . UV-A, in contrast, is less absorbed by DNA, but can interact with exogenous chromophores and induce lipid peroxidation, which generates ROS . These reactive species can lead to the oxidation of nucleic acid, DNA strand breaks , and increased MMP-1 activity , potentially increasing the risk of aggressive cancer. The increase of oxidative stress by UV-A on keratinocytes was partially reduced by a blueberry phenolic extract through decreased ROS production and lipid peroxidation . The third type of sunlight irradiation, UV-C, has the weakest penetration rate in the atmosphere, but has very high energy, possibly contributing to oxidative stress, inflammation, and DNA damage in the skin cells. A blueberry extract applied to HaCaT keratinocytes and HFF fibroblasts irradiated with UV-C showed protective effects against oxidative stress and DNA damage. The anti-inflammatory effects of the treatment were, however, only characterized in the keratinocyte and fibroblast coculture and the activation of MMP-1 expression by fibroblasts after irradiation was only observed in the presence of keratinocytes, suggesting interactions between the 2 types of cells . These observations support the need to use more complex models to investigate mechanisms mediated by the interaction between cell types in the skin. Limiting the studies to one type of cell may leave out crucial information regarding the physiological effects of the treatments on the skin. The results to date in cell models suggest that blueberry derivatives could have a protective effect on skin damage in response to sun and other stressors; however, the extent to which these molecules are stored and present in skin cells or associated tissue layers in vivo is not clear.Artificial lights, environment, diet deficiencies, pathologies, and aging can impact oxidative stress and inflammation in the eyes, and may contribute to diabetic retinopathy or age-related macular degeneration . The eye tissues and fluids contain little extracellular SOD, hence they have less protection against superoxide radical damage . Despite having higher SOD activity, the retina is at more risk of oxidative stress due to its high content of unsaturated fatty acids and high exposure to light leading to lipid peroxidation, which can, in turn, react with DNA, proteins, and lipids, inducing cell damage . Studies on blueberry compounds applied to cell-based models relevant to eye function have typically used light irradiation , a chemical challenge ,black plastic planting pots or a combination of both to induce inflammation . The protective effect of blueberry compounds on oxidative stress induced by light in ocular models has been tentatively explained through a decrease in ROS production and lipid peroxidation. In murine photoreceptors induced with UV and blue light, blueberry extracts exerted protective effects through regulation of ROS production, limiting cell death induced by the light through inhibition of p38-MAPK, JNK, and NF-κB pathways . The effects of blueberry extracts on alternatively challenged cells were also reported , but more research is needed to confirm the mechanisms behind their antioxidant and anti-inflammatory effects. Oxidative stress was alleviated by blueberry compounds in chemically challenged retinal pigment epithelial cells through an increased production of antioxidant enzymes . Blueberry anthocyanins modulated oxidative stress through a decrease in ROS production and increase in glutathione peroxidase activity in ARPE-19 cells induced with H2O2 . In 661 W cells challenged with high glucose, anthocyanins reduced ROS secretion and NOX protein expression, but limited anti-inflammatory effects were reported with no clear modulation of NF-κB .

Blueberry anthocyanins tested on light-induced ARPE-19 cells helped mitigate lipid peroxidation , whereas malvidin 3-glucoside, purified from a blueberry extract, decreased ROS and VEGF production in irradiated cells . However, a blueberry flavonoid fraction and whole polyphenol mixture were more effective than the anthocyanin and phenolic acid fractions at reducing lipid peroxidation . These observations support the evidence for a general effect to reduce cell stress markers in ocular cell models when using multiple blueberry fractions and phytochemicals, with a potentially stronger activity of the total polyphenol extracts as opposed to individual compounds. Thus, testing the effects of dietary blueberries or their derivatives on eye function is an interesting avenue for future research.Inflammatory bowel diseases encompass chronic inflammation-related disorders in the gastrointestinal tract. Chronic inflammation, associated with oxidative stress, increases the risk of developing colorectal cancers . Even though genetic predispositions have been documented , environmental factors such as the gut microbiota composition, influenced by lifestyle and diet, and its interaction with the intestinal mucosa, are key factors in increasing the prevalence of IBD . The effects of blueberry compounds on intestinal models are reported in Table 7, along with experimental conditions. IBDs are characterized by a loss of intestinal barrier integrity through the alteration of tight junction proteins . Although it is not clear if this loss of permeability is a cause or consequence of IBD, it promotes the chance of pathogens to enter the mucosa, triggering inflammation . Blueberry extracts were tested on Caco-2 cell permeability using a transepithelial electrical resistance measurement. The anthocyanin and total polyphenolic fractions decreased permeability of the cell monolayer induced by Escherichia coli challenge, but the proanthocyanidin fraction did not restore TEER values. The authors suggested an interaction between proanthocyanidins, cell surface proteins, and the ability of E. coli to adhere to the cells . Regarding oxidative and inflammatory markers, a number of cytokine gene expressions have been found to increase in colonic mucosa of ulcerative colitis patients due to excessive immune response and immune cell reactivity . In T84 cells, the increase in TNF-α and IP-10 gene expressions by a cytokine cocktail were counteracted by a blueberry extract and the production of IL-8 protein was slightly, but not significantly inhibited . ROS production in a similar cell model was also decreased by blueberry compounds . However, the effects of blueberry compounds on the NF-κB pathway remain unclear, with contradictory effects reported in intestinal models of inflammation. The response induced by cytokines was dose-dependently alleviated by a blueberry anthocyanin fraction in Caco-2 cells through the inactivation of NF-κB, but a water extract from the same fruits did not modulate the inflammatory response . An anthocyanin extract used on T84 human colon cells decreased the activation of the STAT pathway, upregulated during IBD, but also upregulated the NF-κB pathway . A strong advantage of using an intestinal model is the possibility to study both the effect of polyphenol compounds and their colonic metabolites in a more physiologically relevant situation than for other systems in the body, as both parent molecules and metabolites are present in the intestinal epithelial cell environment despite the low bio-availability of polyphenolic compounds. The limited number of studies on blueberry phytochemicals and cell culture models of intestinal inflammation, the diversity of cell lines used, and parameters measured speak to the need for more studies to determine how blueberries modulate gut function and health.Because both inflammation and oxidative stress are frequently associated with the development of chronic diseases , it is important to understand how dietary factors impact these outcomes. Here, we have reviewed studies reporting the effects of blueberry phytochemicals on cell culture models of inflammation and/or oxidative stress .

The cuticular and epidermal layers contain nearly all of the phytonutrients in the fruit such as anthocyanins, proanthocyanidins, and flavonols. Previous studies on blueberry have reported that these groups of compounds may have diverse health-promoting properties, including controlling diabetes, improving cognitive function, and inhibiting tumor growth. With the growing awareness of the potential health benefits of blueberry and increasing consumer demand, a primary goal of the blueberry research community is to develop cultivars with improved antioxidant levels along with other important fruit quality traits. However, despite its economic importance and health benefit potential, breeding efforts to improve fruit quality traits in blueberry have been slow due, in large part, to the lack of genomic resources. A draft genome for a wild diploid species of blueberry was previously assembled. However, that draft genome consists of a large number of scaffolds , high percentage of gaps in a ∼393.16 Mb assembly, and, most importantly, does not reflect the genome complexity of the economically important and cultivated tetraploid high bush blueberry. Here, we present the first chromosome-scale genome assembly of tetraploid high bush blueberry. The haplotype-phased assembly consists of 48 pseudomolecules with ∼1.68 Gb of assembled sequence, ∼1.29% gaps, and an average of 32,140 protein coding genes per haplotype . A haplotype is the complete set of DNA within the nucleus of an individual that was inherited from one parent. We leveraged this genome to examine the origin of the polyploid event, gain insights into the underlying genetics of fruit development,square pot and identify candidate genes involved in the biosynthesis of metabolites contributing to superior fruit quality. Furthermore, we examined gene expression patterns among the four haplotypes in high bush blueberry.

This analysis uncovered the presence of spatial-temporal specific dominantly expressed sub-genomes. These findings and the reference genome will serve as a powerful platform to further investigate ”sub-genomes dominance” , facilitate the discovery and analysis of genes encoding economically important traits, and ultimately enable molecular breeding efforts in blueberry.Our goal was to obtain a high-quality reference genome for the high bush blueberry cultivar ”Draper,” which is widely grown around the world due to its excellent fruit quality. We sequenced the genome using a combination of both 10× Genomics and Illumina , totaling 324X coverage of the genome . These data were assembled and scaffolded using the software package DenovoMAGIC3 . The genome was further scaffolded to chromosome-scale using Hi-C data with the HiRise pipeline . The total length of the final assembly is 1,679,081,592 bases distributed across 48 chromosome-level pseudomolecules . The final assembly size falls within the estimated genome size of ”Draper” based on flow cytometry . The genome was annotated using a combination of evidence based and ab initio gene prediction using the MAKER-P pipeline. RNA sequencing data from 13 different gene expression libraries, representing unique organs, developmental stages, and treatments , and publicly available transcriptome and expressed sequence tags data of V. corymbosum in theNational Center for Biotechnology Information were used as transcript evidence. Protein sequences from Arabidopsis thaliana , Actinidia chinensis, and UniprotKB plant database were also used as evidence for genome annotation. We predicted a total of 128,559 protein-coding genes. Benchmarking Universal SingleCopy Orthologs analysis v.3 was performed to assess the completeness of the assembly and qual-ity of the genome annotation. The annotated gene set contains 1,394 out of 1,440 BUSCO genes .

Functional annotation was assigned using Basic Local Alignment Search Tool 2GO to reference pathways in the Kyoto Encyclopedia of Genes and Genomes database. Comparative genomic analyses assigned genes to 16,909 orthogroups shared by six phylogenetically diverse plant species including five eudicots , each with distinct fruit types, and Zea mays as the outgroup. Transposable elements , both Class I and II, were identified and classified in the genome using the protocol described by Campbell et al.. Overall, 44.3% of the blueberry genome is composed of TEs . Consistent with previous reports, the most abundant Class I TEs were long terminal repeat retrotransposons , specifically the super family LTR/Gypsy followed by LTR/Copia, while for Class II transposons, the miniature inverted repeat super family hAT was the most abundant. The quality of the genome was further assessed by examining the assembly continuity of repeat space using the LTR Assembly Index deployed in the LTR retriever package. The adjusted LAI score of this blueberry genome is 14, and based on the LAI classification, this score is within the range of ”reference” quality . Estimation of the regional LAI in 3 Mb sliding windows also showed that assembly continuity is uniform and of high quality across the entire genome.The origin of high bush blueberry from either a single or multiple diploid progenitor species is a long-standing question. Previous reports have suggested that high bush blueberry may be an autotetraploid based on the segregation ratios of certain traits. However, an analysis of chromosome pairing among different cultivars revealed largely bivalent pairing during metaphase I, similar to patterns observed in known allopolyploids. To gain further insights into the polyploid history of high bush blueberry, we calculated sequence similarity and synonymous substitution rates between genes in homoeologous regions across the genome. The average sequence similarity is ∼96.3% among syntenic homoeologous genes. The average Ks divergence between syntenic homoeologous genes is ∼0.036 per synonymous site. The average Ks divergence between homoeologous genes can be used to not only identify polyploid events but also to estimate the divergence of the diploid progenitors from their most recent common ancestor .

The Ks divergence between homoeologs in high bush blueberry is six times higher than that between orthologs of two A. thaliana lines that diverged roughly 200,000 years ago. Based on the relatively high Ks rate between homoeologous regions across the genome, this suggests that tetraploid blueberry is unlikely an autopolyploid that was formed from somatic doubling or failure during meiosis involving a single individual . Furthermore, comparative genomics revealed that homoeologous regions are highly collinear, except a few notable chromosome-level translocations . These translocations were manually inspected and verified with both the raw sequence and Hi-C data. Rapid changes among homoeologous chromosomes is known to occur in newly formed allopolyploids. We also assessed the level of similarity and content of LTR transposable elements among the four haplotypes. As the most prevalent transposable elements in plants, LTR-RTs undergo continual ”bloat and purge” cycles within most plant genomes, resulting in a unique signature that may distinguish sub-genomes in an allopolyploid. To examine the evolutionary history of LTR-RTs in the high bush blueberry genome,drainage collection pot we calculated the mean sequence identity of LTR sequences among each of the four haplotypes . This analysis revealed that the majority of more recent LTRs are sub-genomes specific in high bush blueberry. In other words, the data suggest that LTRs proliferated independently in the genomes of each diploid progenitor , following the divergence from their MRCA, but prior to polyploidy. The pair-wise LTR difference of the two ancestors is 2.4%–2.6%. With Jukes-Cantor correction and synonymous substitution rate of , the estimated time of divergence for the diploid progenitors from their MRCA is between 0.94 to 1.02 million years ago. These date estimates and the average speciation rate for temperate angiosperms suggests that high bush blueberry is either an allopolyploid derived from two closely related species or an autopolyploid derived from the hybridization of two highly divergent populations of a single species. To date the most recent polyploid event in high bush blueberry, we analyzed the unique LTR insertions present in each haplotype. Based on the pair-wise LTR difference between the four haplotypes, which is of 0.81%–0.89%, the polyploid event occurred approximately 313 to 344 thousand years ago. The substitution rate of LTR sequences is likely different from that of protein coding genes. Thus, more accurate date estimates will be possible once the LTR substition rate in high bush blueberry becomes available from future studies. After allopolyploidization, one of the parental genomes often emerges with significantly greater gene content and a greater number of more highly expressed genes. The emergence of a dominant sub-genomes in an allopolyploid is hypothesized to resolve genetic and epigenetic conflicts that may arise from the merger of highly divergent sub-genomes into a single nucleus. However, classic autopolyploids, formed by somatic doubling, are not expected to face these challenges or exhibit sub-genome dominance since all genomic copies were contributed by a single parent. This was recently supported by genome-wide analyses of a putative ancient autopolyploid. It’s important to note that sub-genome expression dominance could still be observed in intraspecific hybrids and autopolyploids formed by parents with highly differentiated genomes. To explore this in high bush blueberry, we compared gene content and expression-level patterns between homoeologous chromosomes .

While gene content levels were largely similar among homoeologous chromosomes, with a few notable exceptions , gene expression levels were highest for one of the four chromosome copies in the majority of gene expression libraries . Noteworthy, in the three fruit libraries, the most dominantly expressed often became the least expressed among the four homoeologous chromosomes or among the two lowest expressed copies . The most dominantly expressed in other tissues remained so in developing fruit for only two of the chromosomes . These homoeologous chromosome sets have undergone the most structural variation, which may have modified gene expression patterns . These analyses are based on a single biological replicate from a plant grown in a growth chamber. Thus, the findings reported here should be considered as preliminary. Future studies should further explore sub-genome expression dominance in high bush blueberry, including at the individual homoeolog level, with additional biological replicates and across multiple environments.The progression of fruit development in blueberry is marked with visible external and internal morphological changes including in size and color . We profiled gene expression in fruit across seven developmental stages from the earliest stage through the final stage to identify genes differentially expressed during fruit development. Distinctive transitions in gene expression were observed between early fruit growth to start of color development and complete color change to ripened fruit. We found that the majority of genes upregulated during early fruit development were involved in phenylpropanoid biosynthesis, nitrogen metabolism, as well as cutin, suberin, and wax biosynthesis . In contrast, genes involved in starch and sugar metabolism were highly expressed at the onset of and during fruit ripening . Moreover, principal component analysis showed the first two components accounted for 84% of the variation and separated the developmental stages into three groups: early developmental stages, petal fall and small green fruit; middle developmental stages, expanding green and pink fruit; and ,late developmental stages, complete fruit color change, unripe and ripe fruit . Genes associated with cell division, cell wall synthesis, and transport were found to be expressed the highest during the earliest developmental stages , which is consistent with previous work on other fruit species. In addition to genes regulating cell proliferation, defense response-related genes were also highly upregulated during the earliest developmental stages. During the middle developmental stages, genes regulating cell expansion, seed development, and secondary metabolite biosynthesis were highly expressed. During late developmental stages and as the berry transitions to ripening, late embryogenesis, transmembrane transport, defense, secondary metabolite biosynthesis, and abscisic acid related genes were highly over represented. Blueberry is considered a climacteric fruit; however,unlike the ethylene-driven fruit ripening in other climacteric species, abscisic acid has been demonstrated to regulate fruit ripening in blueberry. In summary, global gene expression patterns mirror the morphological and physiological changes observed during blueberry development .We assessed the total antioxidant capacity in mature fruit across a blueberry diversity panel and the abundance of secondary metabolites responsible for its antioxidant activity in developing fruit. A diversity panel, composed of 71 high bush blueberry cultivars and 13 wild Vaccinium species, was evaluated for total antioxidant capacity in mature fruit using the oxygen radical absorbance capacity assay. Similar to previous reports, we observed a wide range in antioxidant capacity across cultivars, with ”Draper” having the highest levels of antioxidants . The observed variation in antioxidants among high bush blueberry, consistent with our results, were previously shown not to correlate with fruit weight or size. However, in another study, a correlation between fruit size and total anthocyanin levels was identified within a few select high bush blueberry cultivars but not across other Vaccinium species or blackberry. This inconsistency is likely due to sample size differences between studies.

Colibactin has been shown to crosslink with DNA, producing double-stranded breaks. Furthermore, pks+ E. coli strains have been shown to be prevalent in CRC patients. In one study, nearly two-thirds of CRC patients had pks+ E. coli strains in their intestinal bacteria. In the same study, pks+ E. coli also existed in about 20 percent of healthy individuals. Colibactin, however, is a reactive and short-lived protein, requiring close contact with epithelial cells to cause DNA damage. A healthy mucosal barrier keeps colibactin at a distance and reduces the chance of affecting the intestinal epithelium. Evidence for the pathogenic relationship between diets, Fusobacterium nucleatum, and CRC has been emerging. The F. nucleatum levels have been shown to be higher in CRC than in adjacent normal mucosa. Utilizing the molecular pathological epidemiology paradigm and methods, a recent study has shown the association of fiber-rich diets with decreased risk of F. nucleatum-detectable CRC, but not that of F. nucleatum-undetectable CRC. Experimental evidence supports a carcinogenic role of F. nucleatum, as well as its role in modifying therapeutic outcomes. The amount of F. nucleatum in CRC tissue has been associated with proximal tumor location, CpG island methylator phenotype , microsatellite instability, low-level CD3+ T cell infiltrate, high-level macrophage infiltration, and unfavorable patient survival. The amount of F. nucleatum in average increased in CRC from rectum to cecum, supporting the colorectal continuum model. Future studies should examine the role of diets, microbiota, and CRC in detailed tumor locations. Dietary prevention of CRC, then, has two intertwined aims: to reduce inflammation and to promote a healthy intestinal microbiota. As already discussed, preclinical evidence implies that dietary bio-active compounds,vertical tower for strawberries particularly anthocyanins, can reduce symptoms of low grade chronic inflammation as well as oxidative stress. It can also aid in balancing the intestinal microbiota by promoting the growth of beneficial bacteria and by reducing the populations of pro-inflammatory bacteria.

Clinical trials have had mixed results, but anthocyanins and some polyphenols have shown to counteract against CRC actively. More research, however, is necessary for conclusive results. How, then, are individuals to consume enough bio-active compounds to have an effect on health? Some answers may be found in the food consumption practices of cultures with historically low CRC incidence. Parts of India, for example, have had some of the lowest CRC incidence rates in the world; however, this status has been changing. In recent decades, increasing urbanization and similar factors have led to progressively Westernized diet patterns and lifestyle. CRC incidence rates are similarly rising, lending weight to the hypothesis that the traditional Indian diet may help prevent CRC. Furthermore, Indian immigrants to Western countries have a much higher incidence of CRC compared to Indians in India. Typical components of traditional Indian meals include a broad variety of flavors, as promoted in Ayurvedic medicine, and a variety of other foods. Both are facilitated by using a thali platter to serve the meal. The traditional American main meal includes an entree , one or more carbohydrates , and one or more vegetables. This basic structure can potentially be adapted with inspiration from thali meals by reducing the size of the main dish and serving more vegetables, legumes, pulses, herbs, and spices to accompany it. A unique component to thali is the combination of many tastes and colors. The inclusion of multiple colors in a meal is desirable, because certain bio-active compounds, particularly anthocyanins are also pigments. Blue, purple, and red-purple colors in plant foods indicate high anthocyanin content. Purple-pigmented potatoes can be prepared in the same way as traditional white potatoes, but the anthocyanin content is significantly higher in the pigmented varieties. Purple sweet potatoes also contain more anthocyanins than the more common orange varieties and can be easily substituted for them. Other vegetables with red or purple cultivars include carrots, cauliflower, and cabbage. Different colors can indicate the presence of other bio-active compounds, such as orange , yellow , and red/pink . Thus, healthy bio-active compound consumption may be increased by selecting colorful vegetables.

Another way to increase consumption of bio-active compounds is to increase their presence in available foods. The agricultural industry could greatly impact health by adopting food plant cultivars that produce bio-active compounds in larger amounts than is currently common. New cultivars may need to be developed that retain desirable characteristics such as large size, pest resistance, reduced spoilage, etc., but also have high bio-active content at the time of consumption. bio-active compounds, with some exceptions, tend to deteriorate during storage. Even when compounds have not deteriorated, storage may reduce the anti-inflammatory/antioxidant activity of bio-active compounds to affect health. A second systemic change that would promote increased bio-active compound consumption involves reworking how fruits and vegetables are currently stored and processed, as well as reducing the average storage time and adapting processing to optimize the amount of bio-active compounds. Presently, “nutritional adequacy” does not consider many of the bio-active compounds discussed in this paper. Further clinical studies are needed to support and elucidate the role of bio-active compounds in the prevention and treatment of disease.The vast nutritional benefits of a diet containing a wide variety of plants have long been known. However, benefits distinct from simple nutrition, such as phytochemicals have recently become clear. Diets rich in a plethora of phytochemicals can promote a healthy and diverse gut microbiota, reduce intestinal and systemic inflammation, and decrease the risk of colorectal cancer and type 2 diabetes mellitus. Some of these benefits can be observed around the world. Many parts of India have historically low colon cancer incidence rates. The Indian subcontinent has been continuously settled for millennia. Ancient cities inthe Indus valley have been dated to the third and fourth millennia BC and some sites are even older. Archaeological evidence of grain cultivation, including several varieties of barley and wheat, has been found in excavations dated to the sixth millennium BC. Wheat is still a staple crop in northern India, and many other grains, including barley, were commonly cultivated and eaten until the 1950s, when wheat and white rice became dominant. Although the country has many diverse cultures, some customs remain common and conventional throughout the nation. One such tradition is the form of main meals where a large round platter, the thali, holds rice or bread and several smaller bowls, or katori, which hold a separate condiment or curry to be eaten with the rice or bread at the diner’s preference.

Typical dishes include, but are not limited to, dal , yogurt , and assorted spices and vegetables. The development of agriculture early in its history has allowed India to develop rich traditions around food. These traditions have been deeply influenced by Ayurveda, the ancient Indian system of medicine. In Ayurvedic practice, food is a source of nourishment and medicine, used to both prevent and treat illness. Maintaining a proper balance of Ayurvedic elements through diet is considered an effective way to live a healthy lifestyle. According to the Ayurvedic principles,container vertical farming each meal should contain a balance of the six major flavors. This calls for the many small portions of a thali meal which also easily incorporate variety. A variety of flavors in a meal often indicates the presence of many classes of bio-active compounds . Although these substances may not be macro-nutrients, vitamins, or minerals, they still impact human health. Polyphenols are perhaps the largest class of bio-active compounds, containing sub-classes such as flavonoids, isoflavones, stilbenes, lignans, and tannins. As a flavonoid subgroup, anthocyanins are included in this class. Anthocyanins are of interest in the food industry as nontoxic and water-soluble pigments, as most are colored red, purple, or blue, and many display antioxidant and anti-inflammatory activity. A class of phytochemicals called polyphenols is also found in virtually all plant foods, though their quantity may be reduced by preparation methods. Rich sources of anthocyanins include deeply colored fruits and vegetables, such as blueberries, eggplants, and certain carrot and potato cultivars. Given that many phytochemicals exert anti-inflammatory activity via promoting gut bacterial diversity, there is a growing interest in a food-based approach to countering the growing epidemic of inflammation-promoted chronic diseases such as colon cancer.We have learned that no discussion of diet is complete without consideration of the intestinal microbiota. Trillions of bacteria, distributed throughout the gastrointestinal tract from mouth to anus, facilitate digestion and intestinal homeostasis. Structural factors greatly impact the overall makeup of each community. For example, low pH prohibits many pathogenic bacteria from colonizing the stomach and the upper small intestine. The depths of the large intestine, on the other hand, is an ideal habitat for many anaerobes. The gut microbiota is a dynamic community, composed of living organisms that can alter in response to diet, disease, and other environmental pressures. Changes in the intestinal microbiota were first correlated with illness in 1681 when Anton van Leeuwenhoek recorded that the microbial composition of his diarrhea differed from normal fecal samples. Since then, the intestinal microbiome has been closely studied to show how it can be implicated in a variety of conditions ranging from obesity to colon cancer. A great deal of investigation into microbiota has been accomplished in the last decade. Many of these observed changes result in an overall loss of bacterial diversity in the microbiota, indicating that species diversity is associated with health. However, the opposite may be true for cause-consequence relations, but not enough research has been brought to light. High-throughput technologies have driven advances in identifying the trillions of microbes and the metabolic functions that live in the colon. This led to a critical insight that gut plays as dynamic of a role in metabolism as the liver. The proximity of these microbes to the intestinal mucosa and gut lymphoid tissue explains the critical role they play in health and disease. Indeed, dysbiosis plays a significant role in the development of inflammatory bowel disease, obesity, and colon cancer.

Emerging evidence suggests that diet can directly influence the content and composition of gut microbiota. Thus, understanding the complex interactions between diet, gut microbiota, and the host are crucial in prevention and treatment of chronic diseases that plague our society. Studies in murine models have shown rapid changes in the gut bacteria of mice being switched quickly from a standard diet to a high-calorie diet back to a standard diet. In humans, surveys show that diets high in fiber correlate with higher microbial diversity and reduced populations of Enterobacteriaceae, including Escherichia and Shigella species. Marked differences are also seen during consumption of animal- vs. plant-based diets. While nutrients in the diet will affect intestinal microbes, other substances present in food may also have an effect. For example, most anthocyanins are not absorbed into the bloodstream in the small intestine, and so they stay in the gastrointestinal tract until they reach the colon. There, they can affect the colonic microbiota in multiple ways. Firstly, anthocyanins have antioxidant activity that can reduce inflammation-induced oxidative stress on the gut bacteria. Secondly, anthocyanins are a potential carbon source, which bacteria can metabolize, resulting in increased growth of certain microbes. Lastly, bacterial metabolism of anthocyanins produces a variety of metabolite byproducts, some of which have antimicrobial effects on enteric pathogen species including Escherichia coli.Chronic intestinal inflammation is a hallmark of certain bowel disorders, such as ulcerative colitis and Crohn’s disease, which are two major forms of inflammatory bowel diseases , and IBD is also considered a risk factor for colorectal cancer. In the latter, inflammation is generally low-grade but persists over a long period of time. Diet composition can promote or suppress chronic inflammation. Low-fiber high-calorie diets, which are typical in Western countries, may directly promote inflammation, or as already discussed, indirectly promote this through dysbiosis. Indeed, some dietary patterns associated with chronic inflammation are also linked to the reduction of total microbial diversity and imbalances in intestinal microbial groups. Furthermore, some bacteria, including E. coli, can flourish during low-grade inflammation, where thinning of the intestinal mucus layer occurs and allows for more direct interaction between the host’s cells and the intestinal bacteria. This condition can cause a feedback loop in which contact between bacteria and epithelial cells leads to dysregulation of mucosal immune response. This contact can lead to a bacterial biofilm, formed when bacteria attach themselves to the surfaces of the aqueous environment in the gut and begin to secrete substances that allow them to affix onto the epithelium.

Climate change has brought warmer temperatures, shifts in precipitation patterns, sea level rise, and an increase in extreme weather events to agricultural regions globally . Coastal regions are particularly at risk, since they often feature micro-climates conducive to the development of high-value crops that are difficult to grow in other locations. Rising temperatures and increased variability in precipitation can reduce the agricultural productivity of these delicate products, especially if the security of water resources is unknown. Water supply issues are magnified in coastal locations, as groundwater resources are subject to seawater intrusion, exacerbated by the over pumping of water . If a region is reliant on groundwater that suffers from seawater intrusion, there are a limited number of strategies available to improve salinity conditions. Options hinge on actively reducing the amount of groundwater pumping or increasing the recharge of higher quality water into the groundwater basin. This may involve pricing groundwater or setting limits on extraction, building infrastructure that improves recharge, or finding additional sources of irrigation water. One emerging tool is to use treated municipal wastewater, or recycled water, to reduce the reliance on groundwater pumping and increase recharge to the underlying aquifer. As of yet, this is not a well-studied option, because there are limited micro-level water-use data available to credibly estimate individual grower impacts. In addition, recycled water itself is not typically of the highest quality, and is an expensive, “last-resort” solution that has not been implemented in many locations. However,hydroponic vertical farm in the uniquely profitable climates of coastal agricultural regions, recycled water has started to emerge as a potentially economically feasible adaptation strategy. Moreover, the possible benefits are expected to increase under climate change.

This paper rigorously investigates the viability of recycled water in a high-value coastal agricultural region as a mitigation strategy for drought and over-pumping of groundwater. I use predicted crop choices to estimate welfare changes due to recycled water access, using a panel mixed logit choice model. The crop choice model is also used to estimate the damages associated with high salinity conditions. I then examine the impacts that recycled water has on improving the underlying water quality of the aquifer, using staggered difference-in differences and event studies. I evaluate the effects for growers that receive recycled water, as well as those who do not have access to recycled water, but farm in the same region. I then discuss conditions under which recycled water may be economically viable. To my knowledge, this is the first economic study of a real-world implementation of recycled water in agriculture. The Pajaro Valley, located on California’s central coast, offers this critical opportunity to estimate the effectiveness of recycled water. Best known for its berries and vegetables, this region has documented seawater intrusion issues since the 1950s, due to its dependence on groundwater for irrigation and its proximity to the coast. With its foggy, temperate climate, growers in the highly productive valley are motivated to find solutions that allow them to continue growing high-value, salt-sensitive produce. The local water management agency developed a groundwater pricing scheme to fund a recycled water program, delivering municipal treated wastewater from the nearby town to growers along the coast experiencing high salinity. As part of their duties, the agency has been extensively monitoring groundwater quality, pumping, land use, and delivered water. This includes a rich network of monitoring wells, which enables the observation and interpolation of water quality across space and time. While their production of especially valuable crops means that Pajaro Valley is an early adopter in using recycled water, this analysis provides a useful template for other coastal agricultural regions likely to suffer from seawater intrusion in the coming decades. I find that small quantities of recycled water provide substantial benefits to the Pajaro Valley.

Growers who receive recycled water deliveries are able to grow higher-value, salt sensitive crops at increased yields. Their direct benefits, at $16 million annually, are higher than the management agency’s annual program costs. In addition, the groundwater quality beneath parcels that receive recycled water deliveries substantially improves, primarily in years where groundwater salinity is otherwise much higher than average. Neighboring parcels that do not directly receive recycled water deliveries also see their groundwater quality improve in years of high basin-wide salinity, although the effects attenuate quickly. Conservatively, these water quality benefits add up to an additional $10.8 million in high salinity years. While all growers benefit from the recycled water program’s prevention of future seawater intrusion, the current beneficiaries of the recycled water program are growers located nearest to the coast. Overall, this paper has two major contributions: the first quasi-experimental, empirical assessment of the welfare effects from the implementation of a recycled water program, and the first study to propose and analyze recycled water as a mitigation strategy for salinity or groundwater overdraft. While there is no current economic literature on the implementation of a recycled water program, Ziolkowska and Reyes discusses socioeconomic factors that influence desalinization plant development. There are also several studies using survey methods to elicit a willingness to pay for recycled water or for products grown with recycled water. A few studies explore consumer concerns about the use of treated wastewater in agricultural production . Menegaki, Hanley, and Tsagarakis surveys agricultural producers on their willingness to pay for recycled water of various quality in Greece, when faced with no restrictions in freshwater supplies. More closely linked to our work, Iftekhar, Blackmore, and Fogarty use contingent valuation and contingent behavior methods to elicit willingness-to-pay estimates for recycled water in water-constrained Perth, Australia, finding that agricultural users and horticulturalists have the highest valuation at $91 AUD/acre-ft. Seawater intrusion is a growing problem for coastal agriculture, affecting many regions globally . There is a small but growing literature on the economic damages from saline irrigation water. Mukherjee and Schwabe conduct a hedonic analysis of farmland sales in California’s Central Valley to estimate the marginal value of changes in groundwater salinity to irrigated agriculture. Rabbani, Rahman, and Mainuddin use survey methods in Bangladesh, examining severe damage to rice production due to a cyclone-induced seawater intrusion event. They find that average households lost 43-45% of their annual income, and salt-tolerant crops were not able to overcome the acute damage. Other research estimates salinity damages using structural approaches , since high-quality seawater intrusion data is limited.

Currently, little research has been done to study ways to mitigate damages from salinity. There has been some work done to estimate the optimal groundwater extraction under seawater intrusion , as well as under saline soil conditions . Direct damages from groundwater overdraft can be tricky to measure, since depletion of an aquifer typically occurs over a long time horizon. There is excellent work on the externalities associated with extraction and water supply reduction . Other research is focused on the economic damages from land subsidence, where the land surface sinks due to reduced groundwater tables. Wade et al. studies land subsidence in Virginia, finding that coastal pumping invokes the greatest externality, but inland rural communities experience the highest damages. Several policies have been proposed to overcome this market failure,nft vertical farming including water prices and markets or restrictions on groundwater pumping . While often effective mechanisms, water prices and restrictions are politically unpopular. This work proposes a new policy mechanism to reduce groundwater overdraft: recycled water as an alternative water supply. Results provide valuable insights for coastal regions experiencing seawater intrusion, but also for other locations affected by water quality or supply constraints. With sufficient treatment, recycled water programs can provide an additional clean source of water to also combat soil salinity, or other types of groundwater contamination. In fact, 20% to 50% of irrigated agriculture worldwide is already negatively impacted by salinity . Currently, there are recycled water facilities operating in California, Arizona, Texas, Florida, and Australia, and programs are being considered in water-stressed regions globally. More broadly, this analysis has important policy implications for groundwater regulation. Many water basins around the world have already been stressed by persistent over-pumping of groundwater . In California, groundwater issues are at the forefront of water policy debates, where on average groundwater accounts for 40% of the state’s agricultural water supply. California’s Sustainable Groundwater Management Act of 2014 requires over drafted basins throughout California to reach and maintain long-term stable groundwater levels and correct undesirable outcomes associated with pumping over the next 20 years. The legislation includes specific mandates to local groundwater agencies to address seawater intrusion. Evaluating the possible benefits of an alternative water supply is critical to informing optimal groundwater regulation. The paper proceeds as follows. Section 2 describes the background and policy context, while Section 3 describes the data and descriptive statistics. Section 4 outlines the crop choice model and results for estimating the direct benefits of recycled water. Section 5 presents the specifications and results for the indirect benefits of recycled water. Section 6 evaluates Pajaro Valley’s program and discusses the feasibility of recycled water in other contexts. Section 7 concludes. In coastal regions, underground freshwater aquifers and seawater are not typically separated by an impermeable boundary. Instead, they coexist, with the seawater underlying the freshwater, since the salts in seawater give it a higher density. The seawater “toe” describes how far inland the saltwater layer extends below the freshwater aquifer.This frequently occurs when irrigation tube wells are drilled and users pump groundwater at a rate faster than the rate of recharge; i.e. more water leaves the aquifer than enters from rainfall or agricultural runoff.

When groundwater is over pumped, the pressure causes cones of depression to form, and seawater starts to enter the freshwater zones. This issue is exacerbated with sea-level rise, because the increased ocean pressure extends the seawater toe further inland, putting more of the aquifer at risk of seawater intrusion. In the Pajaro Valley, seawater intrusion has been documented since 1951, shortly after irrigation tube wells were introduced in the region. With little rainfall during the primary growing season, and surface water making up 1.6% of irrigation water sources, almost all irrigation water is from groundwater pumping. On average, 55,000 acre-ft of water is pumped annually. This is nearly twice the sustainable yield of the basin, meaning that only half of the extracted groundwater is replenished through rainfall or from irrigation runoff. These groundwater withdrawals, combined with the proximity to the coast, have resulted in severe seawater intrusion. The extent of the intruded region has increased seven-fold since it was first documented. Seawater intrusion in the Pajaro Valley, on average, moves inland approximately 200 ft/year, and renders 11,000 acre-feet of water unusable annually . The over pumping of groundwater and resulting seawater intrusion has led to salinity issues that currently impact crop production and threaten the stability of the basin’s future water supplies. In 1980, the California Department of Water Resources listed Pajaro Valley as one of 11 water basins threatened by severe overdraft, out of 447 total basins . The severity of the overdraft led to the development of the Pajaro Valley Water Management Agency in 1984, to develop conservation programs and manage water resources. Under the Sustainable Groundwater Management Act, PVWMA has been tasked with bringing the groundwater basin into “balance” by 2040, such that groundwater extraction does not exceed water recharge into the aquifer. While the management agency is encouraging water conservation in the form of improved irrigation efficiency, their main projects are in the development of alternative sources of water and promoting its recharge into the basin. The primary source of alternative water supplies to combat seawater intrusion comes from a treated municipal wastewater facility built in the town of Watsonville, along with limited runoff from nearby wetlands. Both of these projects are limited in scale, and are described in more detail below. In total, they have the capacity to provide approximately 7500 acre-feet of water annually , which is equivalent to 13% of the annual groundwater pumping in the region, although annual deliveries have not yet exceeded 5500 acre-ft. The total annual quantity of recycled water delivered can be found in Figure 3.1. Since the recycled water program can only provide a limited amount of the irrigation water requirements of the Pajaro Valley, as a means to allocate the limited recycled water supplies, the agency created a “Delivered Water Zone” .

Five adaption configurations using Mini-ImageNet, three PV settings, and AFD are proposed. As shown in Figure 4, S1 uses a general dataset in base-training and meta-learning, then uses target dataset in test, which is the adaptation from one domain to another, denoted in Formula 3. S2 uses a general dataset in base-training, target dataset in meta-learning and test, which is denoted in Formula 4. S3 uses target dataset in three stages, which is denoted in Formula 5. S4 uses general dataset in base-training, similar-target dataset in meta learning, and target dataset in test, which is denoted in Formula 6. When AFD is used in test, PV is considered as a similar domain as the target domain, because they are both associated with leaf diseases of the plants. S5 uses the similar target dataset in base-training and meta-learning, and target domain dataset in test, which is denoted in Formula 7. S1, S4, S5 are cross-domain, and S2, S3 are intra-domain. According to the definitions of SD and TD, e2, e3, e5, e6, e8, e9 are intra-domain experiments, because the data used in meta-learning and test is from the same dataset. The results are shown in Table 4 and Figure 5A. In PV-Split-2, the accuracy of e5 is better than e4 and e6. In PV-Split-3, the accuracy of e8 is better than e7 and e9. What the two settings have in common is that the disease classes belong to different plants. To the diverse species cases, S2 is better than S1 and S3. Especially when the number of species is bigger, the superiority of S2 is more obvious. As listed, e6 gets close to e5, but e8 is much better than e9, which means that the general dataset is better supported when the testing data is more diverse. A broad prior knowledge is very useful for adapting to diverse target. However, in PV-Split-1, e3 is the best one by using S3 because the testing data belongs to the same plant. So, the features of testing data are intensive and the general date in base-training is not helpful.

Oppositely, the data belonging to the same dataset is easier for adaption. In short, to the intra domain cases,hydroponic nft gully if the testing classes are of super-classes, S2 is the best strategy. If the testing classes are sub-classes, S3 is the best strategy. Experiments e1, e4, e7, e10, e11, e12 are cross-domain cases. e1, e4, e7, e10 are the experiments with the worst results in their respective data settings by using S1, due to the big gap between the general domain and target domain. Comparing e10, e11 and e12, e11 has the highest accuracy by using S4, which are shown in Table 4 and Figure 5B. e12 is not as good as e11 because too intensive features extracted from monotonous samples leads to weaker adaptation. S4 is the best training strategy for cross-domain cases, which uses general dataset in base-training to learn the prior knowledge in a wide range, and uses similar-target dataset in meta-learning for adapting to new domain smoothly.Ablation experiments e13–e22 are conducted to show the positive effects of CMSFF module and CA module, respectively. The results are listed in Table 5. Under four data configurations: PV-Setting-1, PV-Setting-2, PV-Setting-3, and AFD, we execute 8 experiments. The training settings are listed: Mini-ImageNet is used in base-training; backbone network is Resnet12; distance metric is cosine similarity; training strategy is S2 and S4. Taking e2, e5, e8, e11 as the baseline, the CMSFF module is added and the results of e13, e15, e19, e21 show the improvement of CMSFF. e14, e18, e20, and e22 indicate that CA has further improved the performances on the basis of CMSFF. e15 and e17 are used to compare the PMSFF module with the CMSFF module, and the results show that CMSFF outperforms PMSFF.Sub-class is defined as the classes belong to the same entry class. The PV-Setting-1 and AFD are sub-class classification examples. Sub-class classification is also named as fine-grained vision categorization which aims to distinguish subordinate categories within entry level categories. Because the samples belonging to the same super-class are similar with each other, sub-class classification is a challenging problem. In Table 4, the PV-setting-1 is the lowest accuracy group among the three PV-settings, as the samples all belong to tomato and are indistinguishable.

The results of AFD group are worse than PV-Setting-1, which is not only because of Subclass reason, also due to cross-domain and in-wild setting of images. Even if the images of AFD are already pre-processed, the backgrounds of images are still different from PV. Also, the illumination condition, resolution, photography devices are all different. Intuitively, the gap of features from SD to TD causes the accuracy declining.N-way and K-shot are the configurations of the task that indicate the difficulty of the task. Given a fixed K, the accuracy decreases as N increases. The result of PV-split-1 with N-way, 10-shot is shown in Figure 5C. The accuracy drops down from 85.39% to 64.35% as N-way increases from 3 to 10. All experimental results listed in Table 4 are executed with fixed 5-way, which indicates that regardless of the data configurations, all experiments follow the common trend: accuracy increases with the number of shots. The accuracy sharply increases as the Shot increases from 1-shot to 5-shot, and tends to be stable when the Shot is larger than 10. After the shot is larger than 20, the growth is not significant. From 1-shot to 50-shot, the increase of accuracy ranges from at least 10% to a maximum of 32%. The results show that the accuracy increases with the number of shot and decreases with the number of way. More ways means higher complexity, and more shots means more supporting information. In existing researches, the N−way is set to 5 generally. In application scenarios, the N is determined by the number of target categories and should not be limited to 5. For example, a plant may have more than five diseases, then the ways should the same as the number of diseases that may occur in the specific scenario. N-way and K-shot are a pair with trade off relationship. When expanding novel classes, we can increase the number of shots as compensation to maintain accuracy. For a new class to be identified, it is acceptable to collect 10 to 50 samples as its support set. However, the positive relationship of shots and accuracy is not linear. The increase of accuracy as Kshot has ceiling. When the K is larger than 30, the accuracy is still growing but very slowly. In this work, we compared three distance metrics: dot product, cosine similarity, and Euclidean distance.This is because even if there is no parameter to be trained in this module, the losses calculated from the distance measurement still affect the parameter updates in the iterations.

An appropriate distance metric significantly helps in improving the performance of classification, clustering process etc. Cosine similarity hits the best performance, as shown in Table 6 and in Figure 5D. The reason is that the vectors obtained from encoder are high dimensional vectors. The cosine similarity has often been used to counteract the problem of Euclidean distance in high dimensional space. The normalization in cosine similarity also has positive effect. In this work, we compared different backbone networks: Convnet4 , AlexNet , Resnet12, Resnet18, Resnet50, Resnet101 , DenseNet , MobileNet-V2 . The Convnet4 is the classical architecture used in FSL which stacks four blocks of convolutional calculation. Different networks include different sizes of trainable parameters. The trainable parameters are more in base-training than in meta learning because the base-training classifier is removed in meta-learning. The size of trainable parameters, learning rate , training time, and epochs in the two training stages are listed in Table 7. e25–e31 are conducted with the configuration: Mini-ImageNet is used in base-training and PV-2- 22 is used in meta-learning. The different number of iterations is due to the different convergence speed in meta-learning. The performances of the backbone networks are listed in Table 8. Resnet12 and Resnet50 outperform the other networks,aeroponic tower garden system with Resnet12 being more efficient. In base-training and meta-learning, we use the validation data to test the accuracy of 5-way, 1-shot tasks which is shown in Figure 6. The black numbers on the black lines are the best accuracy in base-training, and the black numbers on the red lines are the best accuracy in meta-learning. The lifting ranges of accuracy in meta-learning are marked in red numbers. It is shown that the model trained in base-training stage already has the identification ability with few shots to some extent, even without training with tasks in meta-learning. However, in base training, the model is already convergent by training with image wise data, and the accuracy of task testing no longer increases. In fact, the model still has space to improve. Based on this, in meta learning, by using task-wise data, the accuracy has been further promoted around 20% to 30%. In recent years, the architectures of networks go deeper and deeper. Some researchers proposed a question that do we really need so deep networks? Our results show that a medium sized network outperforms other networks in this task. We summarized two reasons: In CNNs, the simpler and more basic features are learnt in shallower layers, the more abstract and complex features are learnt from deeper layers. From shallower layers to deeper layers, the features transition from edges, lines, and colors, to textures and patterns, to complex graphics, even to specific objects. For our specific task, even humans rely more on color, shape, and texture for disease identification. Hence, the too deep networks may be not critical meaningful. 

FSL is the kind of learning task with limited data-scale. For a deeper network, it always has large number of parameters needed to be updated. In the data limitation condition, too deep network could meet insufficient updating of parameters in back propagation due to the too long back propagation path. In parameter updating, shallower networks are more flexible, while the deeper networks look bulky. In short, it does not mean that deeper networks always outperform shallower networks. The size of network should match the specific task and data resources.In order to show the superiority of our method, we conducted several experiments to compare with some recent related researches. Argüeso et al. used Siamese Network, Triplet Network, and PV as their experimental material. They set a different data splitting: 32 classes are used for training and the rest six classes for testing. They listed results of three methods: transfer learning, Siamese Network, and Triplet Network. Their backbone network is Inception-V3. In order to be comparable, we executed the experiments with the same data setting as their work. Mini-ImageNet is used in base-training, 32 classes of PV are used in meta-learning, and the rest 6 classes are used in test. The results of e32–e34 are shown in Table 9. We also compared with Li and Chao . They proposed a Semi-supervised FSL approach. The baseline is a typical fine-tuning model. The Single SS adds Semi-supervised step on the top of baseline. The Iterative SS adds one more Semisupervised step on the top of Single SS. PV was also used as their experimental material and set to three splits. Each split has 28 classes for training and the rest 10 classes for testing. They compared with Argüeso et al. too. We also conducted experiments by our methods with the same data settings as Li and Chao . The results of e35–e43 are shown in Table 9. All the comparison results are shown in Figure 7. The data settings of the two references are different from our data settings. The results indicate that our method outperforms the existing works with all data settings, which means that our method is superior and robust.The method learning from few samples is very promising in plant disease recognition, which has wide range of potential application scenarios for its saving of cost on data. When expanding the range of application, a well established model of FSL can easily generalize to novel species or diseases without retraining and providing large scale training data. However, some existing limitations of the FSL itself and the specific applied areas are needed to be considered.

To our knowledge, however, gibbons have never been tested with these quantities. Nevertheless, in our scenario gibbons did not necessarily need to discern between these two quantities because the two amounts were never presented at the same time. Thus, although it is possible that this difference could have played a role in their performance, it is more parsimonious to think that their motivation to pull in direct food test trials was due to the high probability to eat the extra reward while pulling the handle. Future studies may use different reward constellations varying in quantity and/or quality to continue shedding light on gibbon socio-cognitive performance. Finally, given the quasi-experimental nature of our task, we did not always capture the social dilemma scenario we envisioned. Future tasks should implement designs in which cooperative acts are clearly costly for those individuals willing to volunteer. In addition, given our restricted sample size we could not test species differences or the presence of individual biases . Te present study advances our understanding of how tolerance may allow primates to solve potential conflict over food rewards. In our study gibbons exhibit high degrees of social tolerance . Passive partners tolerate that actors obtain higher benefits in a majority of trials while actors often actively forego opportunities to maximize rewards . Relatedly, gibbons engaged in cofeeding events relatively often. One possibility is that such a high degree of social tolerance towards conspecifics results from gibbons’ unique pairliving social system compared to other great apes,hydroponic dutch buckets although future studies should inspect this relationship in more detail. Overall, the inclusion of gibbons in studies exploring the nature of primate socio-cognitive abilities is critical.

It will help to elucidate the nature of our prosocial motivations and their relationship to specific socio-ecological pressures and ultimately to understand how they have evolved since the last common ancestor with all living apes.One experimenter interacted with the apes during a test session while a second experimenter recorded the session and scored the subjects behavior . Each experimenter tested half of the dyads. We used high quality rewards that would be easily visible to the subjects. Blueberries were not part of their daily diet but were sometimes presented as enrichment in puzzle feeders and were highly desirable for all gibbons housed at the GCC. Te apparatus was composed of a plastic folding table with a square wooden plank clamped to the top. At one end of the plank a transparent plastic bin was taped so that it could be lifed up or hang down. Te bin, at rest, would hang down and remain unmoved on the top of a wooden ramp. A hole big enough to ft blueberries was drilled on the back side of the bin so that when at rest on the ramp, the experimenter could place five blueberries into the bin. A thin purple rope was tied to the far end of the plastic bin and was routed back to the opposite end of the wooden plank. This was set up so that pulling on the purple rope would reliably life the plastic bin, so blueberries could fall down the wooden ramp and be easily accessible for subjects to obtain. Te extreme end of the rope was attached to the mesh of the enclosure. To allow reaching and pulling the rope, we attached a small, handheld, opaque white handle . At the right tension, pulling on the handle would reliably life the plastic bin. Te handle could contain a single blueberry inside depending on the condition presented. We used two handles of the same dimensions and appearance to avoid contamination of blueberry lefovers after the trial. Te table with wooden plank would be set up at a distance so that it could not be grabbed by subjects and the ramp was placed underneath so that blueberries would roll down and land in front of the enclosure gate.

E2 would then distract the two subjects to an opposite or adjacent side of the subjects’ enclosure with a handful of cereal pieces while E1 tied the end of the purple rope with the handle onto the mesh gate of the enclosure, roughly at the experimenter height, approximately 2 m to the right or left. Te distance and location of the rope was kept constant for all trials of each dyad; however, because the enclosures differed in layout, the rope would go to the most convenient side. This way, we ensured that the rope had proper tension to be pulled by gibbons and life the plastic bin as well as be distant enough from the ramp so that a subject could not easily pull on the rope and obtain food from the ramp at the same time.Individual solo pre-testing of the mechanism of the apparatus was not possible because the separation of the dyads was prohibited. However, gibbons had had experience with ropes before as part of their enrichment and several individuals had participated in pilot sessions where they had to pull from different ropes and handles. Tree conditions were tested: direct food test condition, indirect food test condition and no food control condition. In the direct food test condition, the following procedure was performed. E1 would place five blueberries in the plastic bin on the apparatus. To gain the attention of the subjects, E1 would call the subjects names and show the food, if they were not already focused on the food/experimenter. Once both subjects had observed the five blueberries placed in the plastic bin, E1 would squeeze a single blueberry on top of the handle, so that the blueberry would be clearly visible. Te rope and handle would be set up so that the handle was just far enough from the enclosure in order for subjects to need to pull on the rope to obtain access to the handle and blueberry. Consequently, pulling the rope would also life the plastic bin and drop five blueberries down the ramp, accessible to subjects. Te experimenter would also call the names of the subjects when placing the single blueberry in the handle. A choice was recorded when one of the subjects pulled the rope. If no subject pulled the rope within 90 s, the trial ended and was recorded as no pull. If an experimenter error was made , up to 3 repetitions of the trial would be completed.

Environmental conditions such as rain would also end test sessions to be continued the next day. In the indirect food test condition, there was no single blueberry placed in the handle. To compare conditions, we followed the same procedure as in the direct food test condition. Instead of inserting a blueberry inside the handle, we approached it with the first close and then we touched it with the fingers. In the no food control condition, no blueberries were used in the trial. In order to control for time and actions, we used the same procedure of calling the subjects and touching both the box and the handle.Two cameras on tripods recorded footage concurrently. One was placed to the side of the experimenter in order to capture a wide view of the trials, specifically to show the positions of the subjects, their choices and if they obtained blueberries. Te other was placed close to the ramp to accurately count the quantity of blueberries obtained by each subject. For all trials we coded the act of pulling or not pulling and the ID of the puller and non-puller . We also coded the number of blueberries each subject ate and whether the actor subject ate the blueberry from the handle. Next,bato bucket we coded whether a passive subject was present in front of the ramp or within one meter from it at the moment the plastic bin was lifed and at the moment the actor arrived at the release location. Additionally, we coded instances of cofeeding and displacements. Cofeeding was coded when individuals feed within a distance of 1 m of one another. Displacements occurred when an individual left her spot due to the partners’ arrival. Additionally, we calculated the latency to pull from the start of the trial until the individual releases . All analyses were conducted with R statistics . We used Generalized Linear Mixed Models to investigate gibbons’ choices . Covariates were z-transformed. Every full model was compared to a null model excluding the test variables. We controlled for session and trial number in all our models. We controlled for the length of the dyad in models 1 to 3 given the larger dataset compared to models 4 to 6. In addition, in model 3 we included individuals’ age and sex as control predictors. When the comparison between the full and the null model was significant, we further investigated the significance of the test variables and/or their interactions. We used the “drop1” function of the lme4 package68 to test each variable significance including interactions between test predictors. A likelihood ratio test with significance set at p<0.05 was used to compare models and to test the significance of the individual fixed effects. We ruled out collinearity by checking Variance Infation Factors . All VIF values were close to 1 except for age and length of dyad in model 3. Te two variables were slightly collinear . For every model we assessed its stability by comparing the estimates derived by a model based on all data with those obtained from models with the levels of the random effects excluded one at a time. All models were stable. We also fitted a mixed-effects Cox proportional hazards model to analyze gibbons’ latencies to act. For this purpose, we used the “coxme” function from the coxme package. Te results of Model 2 are reported as hazard ratios .

An HR greater than one indicates an increased likelihood of acting and an HR smaller than 1 indicated a decreased hazard of acting. In addition, to obtain the p-values for the individual fixed effects we conducted likelihood-ratio tests.The human brain requires a constant movement of blood through a network of cerebral arteries and veins to deliver oxygen, glucose, and other essential nutrients, but also to remove carbon dioxide, lactic acid, and other metabolic products. CBF in adults represents approximately 15% of the total cardiac output, while the brain accounts for only 2% of total body weight. Regional blood flow, which is tightly regulated to meet the metabolic demands of the brain, varies significantly between gray and white matter, and among different gray matter regions. After adolescence, cerebral blood flow stays relatively stable for a long period, after which it steadily declines. In fact, in middle-aged and elderly adults, aging accounts for a decrease of approximately 0.45% to 0.50% in global CBF per year. It has been shown that, likewise, perfusion through both cortical regions of the cerebral cortex decreases with age, especially in the frontal, temporal, and parietal lobes, and subcortical regions. Aging is also a main risk factor for cognitive impairment and dementia. In elderly subjects, regional CBF in the superior temporal gyrus was positively associated with global cognitive performance. Furthermore, lifestyle factors increased global and regional CBF, and these lifestyle-induced changes in cerebral perfusion may improve cognitive functioning. These relationships are schematically depicted in Figure 1. Differences in CBF between elderly subjects are related to vascular risk factors as well as risk factors for dementia. Bangen and colleagues have observed that the presence of multiple vascular risk factors may add to the already diminished cerebral perfusion that results from aging. It has also been shown that mean gray matter CBF was 15% lower in late middle-aged subjects suffering from metabolic syndrome than age-matched healthy subjects, and was associated with lower cognitive function. Moreover, in the elderly, reduced cerebral perfusion correlated with the volume of white matter hyper intensities and cortical microbleeds, which are established risk factors for dementia. Neurovascular coupling is another critical component that affects CBF and consequently cognitive function. This phenomenon refers to the close temporal and regional relationship between neural activity elicited, for example, by a cognitive task and subsequent changes detected in cerebral perfusion. In particular, aging impairs the mechanisms that match oxygen and nutrient delivery with the increased metabolic demands in active brain region.