Horticulture

What are the axes of this olfactory space? Or, in other words, do odors associated with certain parts of this space have different perceptual or physicochemical properties? Previous studies found axes that correlated with perceptual odor pleasantnes and physicochemical properties such as molecular boiling point and acidity . We checked for associations with all of these properties, and supporting previous findings found that points corresponding to pleasant and unpleasant odors occupied different parts of the space. We note that this analysis of pleasantness rankings was based only on odor components from tomato and strawberry data sets, for which these rankings were available. Thus, the pleasant-unpleasant odor axis can be identified even solely using fruit odor components. The direction most associated with a change in pleasantness value is marked by the red line in Fig. 4 . For odor mixtures produced by individual fruit samples, we use the “overall liking” rating assigned by humans to fruit samples as a measure of pleasantness . To test how well the identified pleasantness axes can predict measured pleasantness rankings for novel samples,procona valencia buckets we regenerated this axis using only strawberry samples and use it to predict pleasantness ranking for tomato samples. The correlation was significant, with correlation coefficient R = 0.34 and P = 0.01 .

The pleasant- ness values could also be assigned to individual odor components based on the correlation between the odor concentration in a mixture and mixture pleasantness computed over samples . This measure of pleasantness produced an even stronger correlation between pleasantness and odor coordinates within the space,R = 0.66 and P = 3×10−7 . We also computed these correlation values using different odor components from those used to generate the pleasant-unpleasant axis for odor components in Fig. 4C. Specifically, we used two-thirds of randomly selected odor components to generate this axis; we computed the correlation value using the remaining components. The pleasantness axis for odor components had a similar orientation to the one for mixtures . In addition to the pleasantness axis, we could also find axes that were strongly associated with two other properties: molecular boiling point—which is probably a reflection of volatility—and acidity, both of which showed significant correlations . We assigned acidity for individual odors as the correlation coefficient be- tween its concentration and fruit sample acidity measurement. Be- cause the space is essentially 2D, the three axes of odor pleasantness, acidity, and molecular boiling point are not independent. In other words, knowing the coordinates along the molecular boiling point and acidity axes, one can predict the position along the pleasantness axis. That is, the identified mapping to a sphere in a hyperbolic space makes it possible to predict, with correlation R = 0.34 for natural mixtures and with R = 0.66 for monomolecular odors, how perceptually pleasant these odors are based on their projections on the acidity and volatility axes.

The observation that the odor mixtures can be mapped onto a continuous metric space is consistent with a previous vector-based model of human olfactory perception . This model posits that the perception of odor mixtures is based on a combination of the mixture components or, in other words, that there is an underlying set of coordinates that can represent olfactory mixtures. Previous analysis of the Dravnieks database containing human perceptual descriptions of > 120 monomolecular odors showed that the perceptual space is likely to be curved. Qualitatively, the points were found to form a “potato-chip” surface . This can be a signature of the hyperbolic space; potato-chip or saddle-like surface have a negative curvature and serve as an everyday example of hyperboloid surfaces. To quantitatively test whether the perceptual space is described by hyperbolic geometry, we applied the Betti curve method to the Dravnieks database. First, we found that Euclidean spaces were not consistent with measured Betti curves . The first Betti curve could not be matched to the data in terms of its area for any dimensionality of the Euclidean space . Second, we found that the full hyperbolic space of varying dimensions could match the area of the first Betti curve. However, only hyperbolic spaces with small number of dimensions could also simultaneously match the area of the second Betti curve . The 3D hyperbolic space produced the best fit, with larger dimensions yielding increasing deviations. Hyperbolic spaces with dimensions nine and above could be excluded with P < 0.034. The third Betti curve was essentially zero and is not shown here.

One may notice that the first and second Betti curves were not as regular as in the case of odorants and contained multiple peaks. It turns out that the biphasic nature of the Betti curve could be explained by the nonuniform distribution of points across the two angles . Unlike in the case of olfactory stimulus spaces that are sampled approximately uniformly, here, the distribution of points obtained using MDS is not uniform and clusters in one-half of the space. Sampling points from this embedding yields biphasic Betti curves that match those derived from perceptual data . Specifically, P values for L1 differences between Betti curves derived from data and MDS fits were P = 0.32 , P = 0.20 , P = 0 , and P = 0.06 . The MDS distances also better correlated with perceptual distances when we carried out MDS in the hyperbolic space compared to Euclidean space . Our results highlight the importance of hyperbolic curved geometry for understanding how natural odors are represented in the nervous system. Overall, we find that both the statistics of natural odor mixtures and human odor perception can be mapped onto hyperbolic spaces. In the natural environment, hierarchical biochemical networks produce odor components. Hierarchical networks can often be approximated by trees and, therefore, by hyperbolic spaces . We find that most natural odor components fall near the boundary of the observed hyperbolic space, corresponding to leaves of the trees . At the perceptual level, we also found hyperbolic organization. However, in this case, the odors selected for the Dravnieks database did not sample the human perceptual space uniformly . Hyperbolic perceptual organization is likely to be general across different sensory modalities. There are two reasons for this. First, neural networks that give rise to perception are hierarchically organized, and as we have seen in Fig. 1, this can lead to hyperbolic geometry. Second, individual neurons have limited response ranges. Because of response saturation, small changes in neural responses near their limit correspond to exponentially large changes in the input values. This compressive mapping is similar to the Poincare disk representation of the hyperbolic space. There is evidence that visual, haptic,procona buckets and auditory perceptual spaces are all hyperbolic . Adding olfactory perception to this list could help explain why humans can map odors to auditory pitch and to colors. Noteworthy is the low dimensionality of both the physical odor space and perceptual odor spaces. In both cases, the curved space contains approximately three dimensionsdespite the fact that the data vary in > 50 dimensions associated with different samples of natural odor mixtures and according > 100 perceptual descriptors. The low dimensionality of the environmental odor space could be a general property of natural odors because it occurred for odors as diverse as fruit and mammalian urine odors. Note that all four natural odor data sets were described by the same 3D hyperbolic space with exactly the same radius . This property could make it easier to represent data from different data sets within the same space. For example, odors from strawberry and tomato could be represented jointly within a single 3D space .

We could not combine data from other data sets because, for example, there were no overlapping components between fruit odors and mouse urine data sets. It is possible that representing all possible natural odors will increase the dimensionality of the overall space. Another possibility is that introducing odors from different sources will “fill in” the inner part of the hyperbolic space. The natural odors considered here mapped onto a surface of a hyperbolic space. Odors produced by biochemical pathways of different complexity are likely to map to surfaces with a different radius, filling in the space. This possibility is especially interesting because it would provide a link to the filled 3D hyperbolic space that we find for perceptual data, which was obtained using diverse classes of odors. At the same time, the perceptual odor mapping reveals that odors tested so far concentrate on one side of the space , whereas natural odor components cover their respective space rather uniformly . These analyses thus suggest perceptual coordinates that are yet to be explored. The match in dimensionality between the environmental and perceptual spaces would not have been expected a priori. The matching dimensionality between the input and perceptual spaces can help avoid nonlinear distortions that would necessarily arise when mapping two nonlinear spaces of different dimensionality. These distortions are known to exist in vision where we perceive distances in a compressed way: The moon appears disproportionately closer to us than would be based on the actual Euclidean distance . We also plot equidistant and parallel lines differently, which is one of the key signatures of the hyperbolic space. Similar distortions arise in the haptic space . The matching geometry between the input and perceptual spaces in olfaction may therefore serve to minimize these distortions in odor perception. Overall, the ability of the perceptual system to resolve points in the low-dimensional odor space would depend on the number and tuning properties of sensory receptors . We followed procedures from to generate Betti curves for samples taken from spaces with different geometries. This renders the algorithm’s results invariant under monotonic transformation of values, for example, due to nonlinearities introduced at the measurement stage. However, this property can also be used to assign a distance between points based on the correlation in the activity of two units in a network or, as in our case, between two odors across different samples. All monotonic functions will yield the same result. We chose Di j = −|Ci j|, where Di j is the assigned distance between odors, and |Ci j| is the absolute value of the correlation coefficient of odor concentrations among a set of points. This definition ensures that stronger correlations corresponded to tighter connections and smaller geometric distances, as in. The first three Betti curves turn out to be quite sensitive measures of the distance matrices and can be used to find underlying geometries consistent with the data. In addition to random spaces, we screened two kinds of geometric structures: Euclidean spaces of different dimensionality and hyperbolic space [we used the hyperbolic ball model with curvature z = 1] with different parameters. In each space, we uniformly sampled points based on the metric of the space. In a d-dimensional Euclidean space, the points were uniformly distributed in a d-cube with Euclidean distance. For a d-dimensional hyperbolic ball model, we used partial space by setting the minimal radius Rmin and maximal radius Rmax for the ball. This choice of the model was motivated by the fact that hyperbolic space approximates hierarchical tree-like networks, with odors reflecting leaves—the neighborhood of the surface. We use the differences between descriptions across odors, because in this case the absolute value of the descriptor matter, unlike in the case of odors where correlations were a more appropriate measure. When fitting the data using geometric models, no noise was added to distances in models. We also tested the sensitivity of the Betti curves to noise in pairwise perceptual distances between odors. This was done by computing perceptual distances based on randomly selected subset of 120 out of the total 146 descriptors. The variability in the resultant distance values was proportional to the mean distance . Importantly, the relative error in the integrated Betti values across these samples was the same as the relative error of the distances themselves . Thus, although the Betti curve construction evaluates data structure globally, it is not driven by variability in larger distances. In the case of the perceptual dataset, we found that the full hyperbolic space better described the data rather than a shell, and therefore the minimal radius was set to zero. We optimized maximal radius of the hyperbolic model, which is a measure of its curvature, to fit the integrated Betti value of the first Betti curve.

Lack of nutrition education for students was the third most commonly cited barrier to successfully providing additional servings of fresh fruit. Although it was intended that the program include a nutrition education component, schools were provided with only 1 cent per meal served for all nonfood expenses related to the program, including nutrition education and promotion. Yet, in spite of the limited funding, over half of the schools did report using some nutrition education or promotional materials.Although slightly more than half of nutrition directors had implemented some form of staff training, 40% reported that staff needed more training in the areas of fruit and vegetable handling and management and nutrition education in order to more effectively market fruits and vegetables to students. Nutrition, Family and Consumer Science advisors are poised to provide this kind of education and training.The 10 cents per meal the program provided was substantially less than the amount required to cover a supplementary serving of fruit. Without considering the labor and other costs associated with serving additional fruit,plastic planters wholesale the cost of the fruit alone was 3 cents higher than the 10 cent reimbursement, 4 cents above the 9 cents designated for food cost.

The cost per serving for apples, oranges and bananas is 12 to 15 cents, whereas the other fruits offered in the program range from 14 to 25 cents per serving . The 1 cent intended for all other costs, including labor, education and administrative expenses, was totally inadequate; labor costs for school food service departments usually account for 30% to 50% of expenses, and costs associated with storage and produce loss from perishability add to the expense of handling fresh produce. If the California Fresh Start Program were to offer a greater variety of fruits and higher-quality fruit, as students would prefer, fruit consumption would likely increase, leading to substantial increases in labor costs and costs of facility modification, as well as produce costs. Most nutrition directors reported that the program reimbursement was inadequate to cover the cost of implementing the program. Those that did report nonfood expenses spent an average of $2,784, primarily on small equipment and educational materials. Nutrition directors identified cost as the main barrier to providing a greater variety of fruits. They tended to serve fruit whole to reduce waste, increase shelf life and reduce labor costs associated with chopping fruit. They served apples, bananas and oranges most frequently because they are the most affordable. Student favorites such as strawberries cost much more . To keep costs down, nutrition directors obtain their fruit whenever possible through the commodity food and Department of Defense programs.

According to the nutrition directors, they could serve a greater variety of produce if these programs offered a more consistent supply and greater variety of fruits and vegetables. Despite the perceived inadequacy of the reimbursement, many nutrition directors thought the program was helpful financially and made it easier for them to provide more fruits because enrollment in the breakfast program increased. Higher participation rates in school breakfast might help to improve the bottom line by bringing in more federal reimbursement dollars. Participation increased slightly during the program, but the increase was too low to be statistically significant; it is not clear what would have happened over a longer period of time. During the program, more California grown fruit was sold and distributed because the quantities of fruit and varieties of fruit purchased by schools increased. Using production records from schools participating in the program evaluation and interviews with selected produce distributors, we were able to estimate the proportion of produce the schools purchased that was California grown .If all schools in California were to increase fresh produce offerings at breakfast, annual school purchases of California-grown fruit would increase by an estimated 26 million servings, valued currently at approximately $4 million per year. These estimates are based on an average increase of at least one-third of a serving of fresh fruit per school breakfast meal served, an assumption that 47% of the fresh fruit served would be California grown, and an average cost per serving of 15 cents. Given this scenario, an additional $8.3 million would be spent on fresh produce, of which approximately $3.9 million would be spent on California grown produce and $4.4 million would be spent on fresh produce from other sources.

If students’ favorite fruits, which are primarily California grown, were served more often, our findings suggest students would take even more fruit at breakfast and therefore the increase in the value of school purchases of California grown produce would be even higher. According to school nutrition directors, produce vendors were a program asset, making it possible to increase fresh fruit servings by providing dependable service, high quality and reasonable prices, and providing access to locally grown produce. While nutrition directors were enthusiastic about using more locally grown produce, some felt that they needed a go-between to procure the produce from local growers. Some reported that there were few, if any, local growers, and even if there were multiple local growers, it would be difficult to work directly with them. Directors expressed concern about supply, distribution, dependability, food safety and cost issues when dealing directly with local growers. Produce vendors were seen as necessary intermediaries between the schools and the growers as some directors felt that food service departments are not equipped to deal with many small growers. They preferred to continue dealing with the major suppliers with whom they have ongoing relationships. Increased purchase of California grown produce can be a win-win for schools, students, distributors and farmers. Food distributors indicate that they prefer to purchase fresh fruit from California farmers, when available, as it is more affordable than fruit imported from out of state. Schools benefit from these savings, and farmers benefit from an increased market demand. The ultimate beneficiary is the student, whose increased consumption of fruit will contribute to long-term health. While the California Fresh Start Program was designed to provide more fruits and vegetables to students, its application to the breakfast program led to an overwhelming emphasis on fruit, and it is therefore impossible to draw conclusions about its potential impact on vegetable consumption from our data. However, other studies have indicated that increasing student vegetable consumption at other times in the school day presents greater challenges than are found with fruit . A systematic review of 27 school-based programs designed to increase fruit and vegetable intake found that although the programs moderately improved fruit intake, they had minimal impact on vegetable consumption. The authors called for additional studies to address barriers to changing dietary behavior, particularly in relation to vegetables . As implemented,plastic plant pot the California program was effective in increasing the amount of fruit, particularly the amount of fresh fruit, offered to and taken by California school children each day. Further, the variety of fruits offered, especially those that were fresh, increased substantially. The success of the program demonstrated that schools can have a positive impact on students’ consumption of fruit, which is particularly important since produce consumption at school is lower than at home. Piloting the program also provided lessons for the future implementation and expansion of such a program. Given the well-documented health risks that poor nutrition poses for California’s school children and, at the same time, the likelihood that a school fruit program may decrease children’s intake of unhealthy snacks at school , it is critical to closely examine those lessons. The significant increase in the number of fruit servings students took at breakfast during the program was observed even in the absence of adequate funding to promote the effort or to upgrade facilities and equipment so that the fruit could be served in a way that would make it more attractive to students. Infusing additional resources for training, technical support and facilities upgrades, including, for example, improvements in storage capacity, adequacy and attractiveness of cafeteria seating, and creative presentation such as the use of salad-type fruit bars and point of-service displays, could lead to even more substantial increases in the servings students take.

The program also was successful at shifting student consumption away from fruit juice toward fresh fruit. When schools serve less juice at breakfast, students take more fresh produce. Fruits are a healthier option than juice because of their higher levels of fiber and associated micronutrients. While the program resulted in a doubling of fruits offered to students at breakfast and a doubling of fresh fruit taken, a limitation of the study is the lack of assessment of the amount of fruit consumed. While not assessed in this study, improved variety and appeal in produce offerings, improved facilities, and more nutrition education could all potentially result in higher total consumption as well. Our evaluation of the program suggested that school food service personnel faced a dilemma: If they took steps to improve the variety, presentation and promotion of fruit, their labor and food costs would increase beyond the 10 cents per meal provided by the program. At the same time, more students would likely take more fruit servings, particularly servings of fresh fruit, thereby further straining food service budgets. Our study suggests that additional financial resources will be required to ensure that most or all students take the recommended two servings of fruits and vegetables at breakfast. If the program reimbursement were increased to 15 cents to better cover estimated actual costs and if all eligible schools participated, the total reimbursement figure would be about $26 million per year. School programs would further benefit from additional funding to make food service facilities adequate to store, prepare and serve fruits and vegetables in a safe and appealing manner. Interestingly, the USDA’s Food and Nutrition Service recently estimated that the final meal pattern ruling mandating increases in whole grain and fruits in the School Breakfast Program would necessarily increase the cost of food and labor by 14 cents, an amount similar to our estimate for fruit . As part of the Healthy, Hunger-Free Kids Act of 2010, an extra 6 cents per meal in reimbursement was provided for school nutrition programs that complied with the mandate to increase the kinds and amounts of fruits and vegetables. As the final provisions of this act are implemented in schools across the nation, it will be important to evaluate school programs. California has recognized the need to reverse the trend toward poor youth diets and has acknowledged the responsibility of schools to promote health. Unfortunately, lack of financial resources led the state to discontinue funding for the California Fresh Start Program after the pilot. However, new programs can benefit from lessons learned from the California program. There are currently changes taking place in school nutrition policy at the federal level. Partnerships among influential organizations and sectors, including growers, schools, public health agencies and others have been suggested as a lynchpin of the National Action Plan of the National Fruit and Vegetable Alliance . The health of future generations depends upon our commitment to ensuring that everything possible is done to help today’s youth adopt healthy food habits.Aging can be modulated by genes and lifestyle. For instance, specific gene variants of insulin-like growth factor-1 receptor and fork head box O3A are associated with longer lifespan in centenarians. In terms of lifestyle, one of the most studied interventions that delay aging is caloric restriction , which can increase lifespan in organisms ranging from yeasts to primates. Diet composition also influences the aging process, with low protein diets and high phytochemical intake being associated with a longer lifespan. Notably, a recent analysis suggests that the heritability of human longevity is below 10% , indicating that lifestyle choices play a major role in influencing aging and longevity. Since interventions such as CR and dieting are difficult to implement and maintain over a long period, interest has focused on identifying molecules that produce effects similar to CR . This endeavor is based on the observation that signaling pathways that are modulated by CR, including 5’ adenosine-monophosphate-activated protein kinase , mammalian target of rapamycin and sirtuin-1, can be targeted by small organic compound. Activation of these pathways induces autophagy, mitochondrial bio-genesis and expression of antioxidant and detoxifying enzymes, which together can improve cellular function. In a manner similar to CR, several organic compounds labeled as CR mimetics promote physiological functions and reduce the development of chronic diseases, thus improving both health and longevity.

Viable and non-viable seeds in yellow starthistle seed heads are easily distinguishable based on color and shape. Because yellow starthistle requires pollination to produce viable seeds , non-viable seeds represent pollen limitation occurring during the 4-hour period that the flowers were exposed to pollinators. All seeds were counted to compare ratios of viable to non-viable seeds. Any seed predation was noted, and when possible, the seed predator was identified.All analyses were done in R 2.15.1 . Because each site had an AM, Mid-Day, and PM observation event, there were a total of 36 observation events, each with unique wind and temperature recordings, and visit observations of the 15 bee morphotypes. From these, we calculated the total number of bee visitors, total number of bee morphotypes, Shannon diversity of morphotypes, and morphotype evenness. Shannon diversity and evenness were calculated using the R package vegan. The spatial autocorrelation of all bee visitor response variables was assessed by Mantel tests in R package ade4,plastic pots large using the average values for each time of day at each site. Spatial autocorrelation was not detected .

To test for the effect of land use type on each of the response variables we used a generalized linear mixed model using the R package lme4. We designated land use type, bloom category of flowering patch, observation time period, wind, and temperature as fixed effects and site as a random effect. Natural land use and AM observation time period were the model baselines for the categorical variables of land use type and observation time. Shannon diversity and evenness were fit with Gaussian distributions while all other variables were fit with Poisson distributions. In comparing the ratios of viable seeds to total seeds vs. the ratio of viable seeds to counted stigmas, we found that there was a strong correlation between these metrics. To look at the effect of land use type on seed-set, we therefore decided to utilize the ratio of viable seeds to total seeds in each seed head that did not experience seed predation, because of error in counting the number of stigmas . We then used a generalized linear mixed model fit with a Binomial distribution, with land use type as a fixed effect and site as a random effect. Finally, we tested for an effect of floral visitor observations on yellow starthistle seed set at each site. We averaged the number of visits from each morphotype across temporal observation events at the same site. Morphotypes that averaged at least one visit per 30 minute observation window were included as fixed effects in alinear mixed model fit with a binomial distribution, with site as a random effect and the ratio of viable to total seeds as the response variable. We also modeled the effects of total bee visitation, morphotype richness, and morphotype diversity on seed set ratios.

Our results show that rates of bee visitation and seed set vary among urban, agricultural, and natural landscapes, demonstrating the importance of land use in the dynamics of plant-pollinator interactions. We suggest that these effects are at least in part explained by floral availability, a vital bee resource, which can be highly variable among different land use types. For example, in August there are few plants in flower besides yellow starthistle in the natural areas of Contra Costa County, California, whereas in urban and agricultural areas there are many exotic plants and supplementary inputs available . From pantrapping of bee specimens in the region , we know that total bee abundance is highest in the spring in natural areas. However, towards the end of the summer when yellow starthistle is in flower, there is little difference in collected bee abundance between human-altered landscapes and natural areas, and human-altered areas may even exhibit overall higher bee abundance. Our results of bee visitation to yellow starthistle support this pattern. Agricultural areas have large populations of managed honey bee colonies, so one would predict visitation to yellow starthistle by honey bees to be positively associated with surrounding agricultural land use. By contrast for native bees , the highest rates of visitation to yellow starthistle were in sites with more surrounding urban land use.

Urban gardens have many exotic plants, often selected for aesthetic purposes, many of which are in flower later in the season than most California native plants. In addition, many of the plants in urban areas both directly and indirectly receive supplementary resources, particularly water, that further extend their flowering time. Even though agricultural areas also have supplementary resources, the main crop in flower in East Contra Costa County later in the season is maize, which is wind pollinated. There may be multiple impacts of exotic plants in urban areas. By filling the phenological flowering gap noted above, they may help attract even larger populations of bees into the urban landscape. In addition, bees in urban sites may be behaviorally more likely to visit non-native plants due to the increased encounters they have with novel plants. In agricultural and natural landscapes, a positive correlation between pollinator visitation and seed set is typical. Surprisingly in our system, in human-altered landscapes, higher total observed bee visitation did not result in higher proportions of seed set, as would be expected. In fact, urban areas, despite receiving the highest rates of native bee visitation, exhibited the lowest rates of seed set. Conversely, natural areas, which received the lowest amount of total bee visitation, had the highest rates of seed set. We suggest 2 possible explanations for this discrepancy between pollinator visitation and rates of seed set: 1) pollinator efficiency; and/or 2) the composition of the local flowering community. Depending on the plant, certain pollinator species are much more effective than other. For example, Osmia, Habropoda, and Apis, have been found to produce varying amounts of seed set as a result of a single visit to blueberry, but these results vary slightly depending on the blueberry variety. In the case of yellow starthistle, it is likely that the most frequent visitors are perhaps not the most efficient. When we directly compared average seed set at each site against visitation rates, we found a significant positive association with the medium hairy leg bees. The medium hairy leg bee morphotype includes those species which fall in both the Tribes Emphorini and Eucerini. Emphorini are known to largely be oligolectic , meaning they specialize on certain plant groups, which theory suggests would make them more efficient pollinators than generalists. The medium hairy leg bee morphotype was not significantly associated with any of the land use typesIt was also the only group that was observed most frequently during morning sampling, perhaps reflecting a difference in when yellow starthistle is most receptive to pollination.

Despite the overwhelming abundance of honey bees in agriculture areas, we did not observe higher seed set in those regions, consistent with the observation that honey bees can be poorer pollinators than other species.It is also important to note that this study used a morphotype classification,black plastic nursery pots and there may be multiple species that fit within the same morphotype that provide varying degrees of pollination service. It is possible there are rare, but highly efficient, pollinators that were rarely observed during the sampling period, or were lumped together with a more frequently observed morphotype. An alternative explanation for the lack of an association between floral visitation and seed set is that higher plant diversity in urban and agricultural areas may decrease pollinator efficiency. Previous research has shown that invasive alien plants can have a negative effect on native plant communities by acting as attractors for pollinators, or decreasing pollinator efficiency by providing a wider range of resources for pollinators to visit, with the consequence that visitors transfer pollen from non con-specifics, potentially clogging stigmas and reducing pollination success. In this case, our target plant, yellow starthistle is indeed considered an invasive alien plant, but the hypothesis of it being in a novel diverse community could lead to a similar effect on the frequency and quality of pollination services that it receives. In sites where there are many other potential plants to visit and accompanying decreased floral fidelity leading to diverse pollen loads, one predicts decreased pollinator efficiency. Abundant sources of exotic plant pollen could occur in areas where there is a greater diversity of nearby plants for pollinators to visit. This explanation might account for the observation that shield-tipped small dark bees were negatively correlated with seed set. We selected yellow starthistle as the target plant for this study because of its ubiquitous distribution, reliance on pollination, and its attraction for a wide set of visitors; it is also a highly invasive and undesirable plant. Previous research on yellow starthistle has found that its invasion can be facilitated other non-native pollinator species such as the honey bee, Apis mellifera, and the starthistle bee, Megachile apicalis, which is included in the medium striped hairy belly bee morphotype. However, the abundance of bees in both of these 2 morphotypes were most closely associated with agricultural areas, which did not have the highest rates of seed set as would be predicted by visitation alone. Our results indicate clearly that bee visitation in human-altered landscapes can be higher than that in comparable natural areas, especially towards the end of the flowering season when there are few resources available in natural landscapes.

Because the response of bee visitors to land use change depends on species specific requirements and these pollinators also have variable effects on plants, understanding the effect of land use change on pollination services requires knowledge not only of which pollinator groups shift to the human-altered landscapes, but also the rate of pollination that those groups have on the plant species in those landscapes. Future research will benefit from looking at a wider range of plants with a different range of target pollinators and that flower earlier in the year to better tease out these hypotheses. If the patterns of bee visitation and seed set that we observed are indeed consistent across other plant species, the novel plant communities created in these human-altered landscapes and the generalist bee species that are favored in such landscapes will lead to a reduction in overall pollination services.Diet and lifestyle patterns play a significant role in the development of Metabolic Syndrome and the diseases it precipitates, namely cardiovascular disease and type 2 diabetes mellitus. In particular, MetS development is associated with the consumption of foods typical of the Western diet, including red meat and fried foods. The Western diet is characterized by excess intake of energy, readily available carbohydrates and fats that result in postprandial hyperglycemia and hyperlipidemia. Three large scale prospective cohort studies support the relationship of postprandial lipemia and risk of cardiovascular disease. Postprandial inflammation and oxidative stress are postulated to be products of hyperglycemia and lipemia and are associated with impaired insulin sensitivity. Impaired insulin responses to Western meals exaggerate postprandial hyperglycemia and lipemia, creating a vicious cycle leading to vascular dysfunction and damage that, over time, likely augments risk for CVD. Reducing transient fluctuations of these postprandial responses is one lifestyle modification for the prevention and management of MetS. The addition of certain foods to the diet may attenuate these postprandial responses. Foods rich in dietary fiber have been shown to modulate postprandial lipemia and aid in glycemic control.Antioxidant-rich foods have been shown to attenuate postprandial oxidative status markers and increase plasma antioxidant capacity. Few studies have investigated the postprandial response to beans and only one has studied black beans. These few studies have demonstrated that different types of beans elicit different biological responses. This may be in part due to dosing differences, polyphenol bio-availability, and endogenous factors. For example, polyphenols modulate digestive enzymes, eliciting beneficial effects on the postprandial responses. Anthocyanidins in particular, which are found in black beans, have been shown to inhibit α–amylase, maltase, and sucrase activity, which lower postprandial blood glucose. Further, metabolic health benefits of beans are likely related to the functional effects of their inherent dietary fiber and polyphenol content, the latter being questioned as an effect of the general antioxidant properties of polyphenols or a specific effect of the polyphenol structure. Nowadays, it is popular to isolate and sell functional components of foods as dietary supplements and many supplements are marketed for their “antioxidant” properties.

At the time of this study, the trees in the control and treatment bioswales were fully established and approaching mature size. Measurements recorded the differences in surface runoff dynamics and pollutant reduction rates, as well as tree and shrub growth. This study provides new information on the long-term effectiveness of engineered bioswales in a region with a Mediterranean climate. The water collection system was installed in 2007 to collect composited samples from natural runoff . In this study, surface runoff samples from the control site were collected at a high frequency using grab samples to better observe pollutant concentration dynamics for each experiment throughout a storm hydrograph. A test run was conducted on 10 October 2013 to determine the optimal runoff sampling time intervals and the number of samples needed to capture the peak and total loadings. For the test run, the water soluble fertilizer was applied to both of the sites at a rate of 2.24 g m−2 . Grab samples were collected at a 10-min frequency over a two hour irrigation period from the control site. Water samples were collected immediately before the runoff was directed to the underground tank.

The water samples were coarse filtered during sample collection with coffee filters to remove large tree leaves,square plant pots grass clippings and large soil particles. Based on the results of the test run, the water sampling frequency for subsequent trials was extended to six hours with a variable sampling interval to better characterize the runoff pollutant pattern. The composite water sample from the treatment site was used for calculating the total loading of the treatment site where little surface runoff occurred in this study. A 5.1 cm diameter and 0.9 m long PVC drainage pipe was vertically installed into the middle of the treatment bioswale to collect a representative water sample for monitoring pollutants concentration dynamics in the bioswale. The treatment site water samples were not affected by successive flow from the control site because the treatment site was located upslope of the control site. The water sample collected from the control site was surface runoff, which was not affected by subsurface flow of the treatment site because of the site’s relatively flat surface. The pH ranged from 7.90 to 8.24 for the control site and 8.07 to 8.20 for the treatment site. The relatively high pH values indicate that alkalinity was not elevated by the ESM used in this bioswale. The high pH and alkalinity results from the relatively poor quality of the irrigation water originating from groundwater. The groundwater sources are from marine sedimentary rocks of the Coastal Range, which have high salt and carbonate levels.

The irrigation water supply had an average pH value of 8.79, and its hardness was as high as 890 ppm. Another factor that contributed to the accumulation of salts in the soil was the 2013–2014 drought, which resulted in very little soil leaching in the year prior to this study . Without leaching from winter precipitation, the salts accumulated in the surface soil layer. The cations were mainly Na+ , Ca2+, and Mg2+, and the anions were mainly Cl−, SO4 2−, and HCO3 −. Concentrations of these major cations and anions did not show significant variation from sample to sample, indicating that their dominant source is the irrigation water. The high TIC in all water samples reflects the high carbonate concentrations of the groundwater used for irrigation. On average, the bioswale with ESM reduced pollutants carried in surface runoff by more than 99.5%. The average pollutant loading reduction rate was 99.6% , 99.5% , and 99.4% for the LPL, MPL, and HPL experiments, respectively . This treatment bioswale had slightly higher pollutant reduction rates as compared to the bioswale with ESM installed adjacent to a parking lot in a previous study. The parking lot bioswale reduced the nutrients by 95.3% and organic carbon by 95.5%. The peak pollutant concentration reduction rates found in this study were a minimum of 53% higher than those reported in the parking lot study. One possible explanation for this difference is that the trees and shrubs in the bioswale were more extensive and older than the tree in the parking lot site. Tree and shrub roots can function as a biofilter, where pollutants are immobilized, transformed, or degraded.

Although data were not available for below ground biomass, a more extensive rooting system and associated microorganisms in this study’s bioswale could be partially responsible for its improved performance. Another possible explanation is the difference in pollutant inflows. The primary pollutant source in the parking lot study was from atmospheric deposition, with lower concentrations when compared to the fertilizer rates applied in this study. Storm water BMPs, such as bioswales, are reported to have higher removal rates when treating storm water with high inflow concentrations. Concentration of Zn, Cd, Ni, Cu, and Pb are key water quality concern parameters. They were excluded from this analysis because their concentrations were below detection levels in the irrigation source waters and these metals are not identified as impairments in the study area.The interpretation of results from this study is subject to some limitations. Pollutants can leave the system via infiltration deeper into the soil and potentially enter the groundwater. Deep leaching can be an important flow path affecting the fate of pollutants, and was not included in the scope of this study. Caution should be taken regarding the potential for groundwater contamination when considering the use of ESM in bioswale projects. In this experiment, the trees were eight years old and their root systems were well established. Trees received excess surface irrigation runoff during the hot/dry summer. Because the ESM in this study was 75% lava rock it may not retain enough moisture for tree roots during long dry periods. Trees in bioswales with ESM may require more irrigation than trees in native soils, especially for establishment. In this study, the pollutants were artificially added to the system by using dissolved fertilizer. Actual storm runoff includes pollutants from atmospheric deposition and has a more complex mixture of pollutants. These factors introduce uncertainty in extrapolating the pollutant reduction efficiency of the bioswale to other sites. It is unclear whether all of the pollutants retained by the bioswale were fully retained by the vegetation and soil, or if a portion of these pollutants were only temporarily immobilized in the system by the soil-tree root system. The bioswale system tested in this study was eight years old, relatively young when compared to its 20 to 30 year life expectancy. Long-term monitoring of system performance is needed to document bioswale performance over longer time periods typical of urban green infrastructure. Additional research is needed that follows the fate and transport of pollutants after infiltration. In particular, chemical analyses of soil and tree samples are needed to understand the fate and transport of the pollutants in the bioswale system. The length of lifespan of a particular normal cell of any organism is predetermined.

Similarly, the length of lifespan of all organisms is pre-determined by their genetic makeup and their external and internal environments and diet-related factors specific to an organism. Therefore, the length of lifespan can be increased or decreased by manipulating the environment, diet and genetic factors only by small extent. The length of lifespan of the organisms can also be impacted by differential rates of senescence of cells and organs that ultimately lead to the death of the organisms. The differential rates of cellular senescence are influenced by several confounding factors,plastic potting pots such as external and internal environments, diet and genetic factors. Because of these confounding factors that can impact rates of progression of degenerative changes in the organs, it is almost impossible to study aging in the absence of organ pathology. Based on numerous studies on aging in vertebrates and invertebrates, a recent informative review has suggested that oxidative stress theory of aging can only be applied to conditions in which age associated pathologies are included. Furthermore, it was suggested that in environment with minimal stress, oxidative damage plays little role in aging. This suggestion can be argued on the fact that little oxidative damage may take longer time to deregulate protective transcription factors, adaptive responses to stressors, and repair mechanisms, and thereby extending the lifespan of the organisms more than that produced by higher oxidative damage which can deregulate above biological functions in shorter time. Using vertebrate and invertebrate models, some major biochemical and genetic factors that are associated with aging processes have been identified. They include increased oxidative stress and chronic inflammation, decreased adaptive response to stressors, post-translational protein modifications, mitochondrial dysfunction, decreased of proteasome and lysosomal-mediated proteolytic activity, shortening of telomeres and transcriptional deregulation. Among these, the theory of oxidative stress is most extensively investigated in various experimental models, using pharmacological agents, antioxidants, anti-inflammatory agents and deletion of one or more antioxidant enzymes as well as of mitochondrial complexes. Depending upon the experimental models, experimental designs, and substrate used to assay oxidative stress and criteria of oxidative stress, the role of oxidative stress in aging has been substantiated or questioned. We hypothesized that increased oxidative stress may be one of the primary early events that causes chronic inflammation, transcriptional deregulation, post-translational protein modifications, mitochondrial dysfunction, decreased of proteasome and lysosomal-mediated proteolytic activity and shortening of telomeres. Invertebrate models, such as Caenorhabditis elegans has been extensively used to evaluate the role of oxidative stress in aging primarily due to shorter lifespan of about 3 days and ease of genetic manipulation. This review analyzes recent published studies on C. elegans on the role of oxidative stress in determining the length of lifespan by generating mutants that show suppression of mitochondrial function or lack of superoxide dismutase .

Caenorhabditis elegans has been extensively used to investigate the role of oxidative stress in aging by measuring the length of lifespan. Mitochondria are considered the major sites for the production Reactive oxygen species , although ROS are also produced outside the mitochondria. In order to demonstrate the impact of oxidative stress, several mutants of C. elegans were generated. They include mutations in four clock genes , mutation in the iron sulfur protein of mitochondrial complex III, mutation in the gene NUO-6 and mutation in the gene daf-2. The effects of mutations on oxidative stress and lifespan are summarized in Table 1. The clk-1 gene encodes an enzyme that is necessary for the biosynthesis of ubiquinone that is required by the mitochondria to generate energy. Mutation in the clk-1 gene increases the life span by slowing down mitochondrial activity due to reduced availability of ubiquinone. This slowing of the electron transport chain would reduce oxidative stress. The role of reduced oxidative stress in extending the lifespan is further supported by the fact that overexpression of clk-1 gene in wild-type C. elegans increased mitochondrial activity and shortened the lifespan. A mutation in the iron sulfur protein of mitochondrial complex III causes low oxygen consumption, reduced oxidative stress and increased lifespan. Mutation in the daf-2 gene which codes for a member of insulin receptor family increased lifespan and enhanced resistance to oxidative stress. In this daf-2 mutant, expression of the SOD-3 gene, which encodes mitochondrial Mn-superoxide dismutase, was much higher than in the wild type. This implies that the increased levels of SOD-3 in the daf-2 mutant reduced oxidative stress and thereby increased lifespan. Mutation in the gene NUO-6 which encodes complex I of mitochondria increases life span of C. elegans by decreasing the mitochondrial function. Mutation in the age-1 increased lifespan by two folds. This mutant worm had increased catalase and Cu/Zn SOD activities which may account for the increased resistance to the paraquat, a superoxide generating chemical. The mutants C. elegans support the view that the levels of oxidative stress is one of the important determinant factors in determining the length of lifespan.Superoxide anions are produced enzymatically outside the mitochondria by different oxidases and nonenzymatically inside the mitochondria. SOD detoxifies superoxide to hydrogen peroxide , which is converted to water and oxygen by catalase. There are five superoxide dismutase isoforms SOD-1, SOD-2, SOD-3, SOD-4 and SOD-5 in C. elegans. However, in most organisms there are only 3 SODs. SOD-1 is present in the cytoplasm and represents the majority of SOD activity, whereas SOD-2 and SOD-3 are present in mitochondria.

As we collect soil metagenome sequence data, we need to improve how we mine such datasets. For example, the way we conduct BLAST searches might overlook valuable information, while the unassembled reads might be too short for annotating genes with confidence. Thus, we might well need to develop new assembly and annotation algorithms. Another challenge is how to integrate different kinds of omics data, including metatranscriptomics and metaproteomics, to better understand functional processes of soil microbial communities. Metagenome sequence data, while informative, provides information about genes with the potential for being expressed, but cannot determine which ones are functional. Also, because we sequence total DNA, it is not possible to distinguish genes from actively growing cells from those in dormant or dead cells. Perhaps some analyses should be reserved for that fraction of DNA from active community members—for example, by extracting DNA that is allowed to incorporate stable isotopes or bromodeoxyuridine during replication. Ultimately,large pots plastic combining these approaches should enable us to gain a better understanding of which microbes are alive and active, and which enzymes and pathways function in soil microbial communities under different conditions.

Then we can begin to truly comprehend soil microbial communities from the microscopic to the global scale.Traumatic brain injury accounts for approximately 90% of brain injuries, and is associated with cognitive dysfunction and long-term disability. As a result of domestic incidents, military combat, traffic accidents and sports, TBI can compromise broad aspects of neuronal function. Patients often experience problems in the domains of learning, memory and affective functions that can profoundly influence quality of life. Existing therapeutic strategies for TBI have not been successful in counteracting the heterogeneous TBI pathology nor improving the quality of life of patients. Hence, identifying interventions with broad applicability seems necessary for effective management of TBI. Dietary polyphenols have significant positive effects on brain health via protecting neurons against injury and enhancing neuronal function. Evidence supports the neuromodulatory effects of flavonoid-rich blueberry, particularly in promotion of brain plasticity, and counteracting behavioural deficits. In the United States, demand for blueberries has increased, with 2017 fresh per capita consumption of 1.79 pounds/person. Several reports indicate that blueberry dietary supplementation improves memory, learning and general cognitive function, and protects against neuronal injury associated with stroke.

Moreover, it has been shown that blueberries possess potent antioxidant capacity through their ability to reduce free radical formation or upregulating endogenous antioxidant defenses. These studies suggest that blueberry supplementation can have the potential to be used to overcome the broad pathology of TBI. Given the lack of information about the effects of blueberry intake immediately after TBI, we have performed studies to assess the effects of blueberry extracts during the acute phase of TBI. Evidence suggests that TBI is characterized by dysfunction in synaptic plasticity, elevated levels of free radicals, plasma membrane dysfunction, which can contribute to the behavioural dysfunction. Oxidative stress is part of the pathology of TBI and compromises neuronal function. In particular, excessive free radical formation leads to accumulation of lipid oxidation by-products such as 4-hydroxynonenal with subsequent impairments in plasma membrane fluidity, receptor signaling across the membrane to deteriorate synaptic plasticity and reduce neuronal excitability. Deficiencies in brain derived neurotrophic factor reduce the brain plasticity necessary to cope with the effects of TBI. BDNF activates cAMP-responsive elementbinding protein , a multifaceted transcriptional regulator involved in synaptic plasticity essential for learning and memory. BDNF is known to bind to TrkB receptors, leading to activation of Ca2+/calmodulin-dependent protein kinase II , required for synaptic processes involved in behaviour. Several observations indicate that the flavonoids exert action through modulation of signaling pathways to promote synaptic and neuronal function.

Accordingly, in the current study, we investigated whether blueberry supplementation would counteract TBI pathology by involving BDNF related pathways involved in synaptic plasticity and oxidative stress to influence cognitive behaviours. Freeze-dried high bush whole blueberry fruit powder . This blend contained bio-active phytocompounds , and other macro- and micronutrients . Diet supplemented with 5% w/w BB was mixed with pulverized standard rodent chow . 1.6% fructose, 1.45% glucose and 0.0009% vitamin C were mixed with the standard rodent chow to match the levels of sugars in the BB supplemented diet and used as the rodent control diet . Sprague–Dawley male rats were purchased from Charles River Laboratories at 10 weeks of age and were acclimatized for vivarium 1 week prior to commencement of experimental procedures. Rats were housed in environmentally controlled conditions with 12-h light/dark cycle in a controlled room with free access to food and water. All procedures were approved by the University of California at Los Angeles Chancellor’s Animal Research Committee and were conducted with adherence to the guidelines set out by the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. After acclimatization, rats underwent fluid percussion injury or sham surgery and were pair housed to a specific diet group with either regular diet or blueberry supplemented diet for 2 weeks immediately. The BB dose was chosen based on previous in vivo studies which demonstrated that administration of blueberry offsets oxidative stress and reverses cognitive impairment. The groups were: Sham-RD as control group, TBI-RD, and TBI-BB. The rats were subjected to fluid percussion injury or sham surgery. Rats were subjected to learning on Barnes maze at post-injury day 14 for 5 days, and after a 2-day interval, memory was assessed at PID 21. Rats were tested for anxiety-like behaviour on elevated plus maze on PID 22 . All behavioural assessments were conducted between 9:00 and 13:00 hours. Rats were provided with diets prepared daily and fed ad libitum in powder form. To determine the voluntary food intake , food was weighed daily to measure consumption in each cage. Since the rats were pair-housed, food intake was divided by two to yield an approximate intake/rat.We employed our standard lateral fluid percussion injury protocol as described earlier. Briefly, 3% isoflurane was provided in a chamber , and then maintained with 2–2.5% isoflurane via nose cone while rats were in a stereotaxic frame. Body temperature was controlled by a heating pad. Under aseptic surgical conditions, a midline skin incision was made to expose the skull. Using a high-speed drill ,square planter pots craniotomy was made 3.0 mm posterior to bregma and 6.0 mm lateral of midline to expose the intact dura. A hollow plastic injury cap was placed over the craniotomy, secured with dental acrylic cement and was later filled with 0.9% saline. When the dental cement hardened, the anesthesia was discontinued and the rat was attached to the FPI device via the head cap. At the first response of hind-limb withdrawal to a paw pinch, rats received a moderate fluid percussion pulse . Upon resumption of spontaneous breathing the head cap was removed and the skin was sutured. Neomycin was applied on the suture and the rats were placed in a heated recovery chamber to be fully ambulatory before being returned to their cages. The sham animals were prepared using the identical surgically procedure but without the fluid pulse. Barnes maze testing was performed 2 weeks after experimental TBI with two trials per day with a 5-min test period . For learning assessment, rats were given two trials per day for five consecutive days at approximately the same time every day. Subsequently, memory retention was assessed at post-injury day 21. Latency to finding the escape hole and search strategies were analyzed for each trial.

Three search strategies were identified using following categorization: spatial, peripheral, and random using data recorded with AnyMaze software. EPM test was performed to assess anxiety-like behavior 2 weeks after experimental TBI with two trials as described in the Supplemental Information. Specifically, rats were individually placed in the closed arm of the EPM apparatus and permitted free exploration for 5 min during which their movements were camera recorded. The behaviors scored were time spent and number of entries into the open arm using automated video tracking system. EPM testing was conducted after Barnes maze memory test. Upon completion of the experiment, hippocampal tissues were harvested frozen in dry ice, and stored at –80 °C until use for immunoblotting. The left side hippocampus were homogenized in a lysis buffer containing 20 mM Tris–HCl , 137 mM NaCl, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonylfluoride , 10 μg/ml aprotinin, 0.1 mM benzethonium chloride, 0.5 mM sodium vanadate. Total protein was then determined using a BCA Protein Assay kit , using bovine serum albumin as standard. Equal amounts of protein were separated by sodiumdocecylsulphate-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes . Membranes were probed with anti-actin or anti-BDNF, anti-pCREB, anti-CREB, anti-CaMKII, anti-4- hydroxynonenal followed by secondary antibody . Immunoreactive proteins were visualized using enhanced chemiluminescence reagents . Band intensities were quantified using Image J32 Software. β-actin was used as an internal control for normalization western blot such that data were standardized according to β-actin values. Blots for each experimental group were normalized to ShamRD values within the same gel. Protein data are expressed as mean ± standard error of the mean . Body weight data expressed as mean ± standard deviation. Statistical analysis was performed by software GraphPad Prism 7.04. A level of 5% probability was considered as statistically significant. The Barnes maze learning data analysis were analyzed by repeated measures analysis of variance . Protein results are expressed as percentage of Sham-RD group.In the present study, we found that BB supplementation can attenuate important aspects of the acute TBI pathology. We report that BB supplementation immediately following TBI mitigates behavioural deficits in spatial learning and memory. BB supplementation counteracted the effects of TBI on proteins associated with the action of BDNF on plasticity and behaviour. In addition, BB supplementation counteracted the increase of the end product of lipid peroxidation, 4-HNE. The results showing that markers of neuronal plasticity and lipid peroxidation change in proportion to memory performance suggest a possible association between these molecular parameters and behaviour. Taken together, the present findings emphasize the beneficial effects of BB supplementation in fostering brain plasticity in the TBI pathology. In agreement with previous reports, we found that TBI impairs spatial learning as evidenced by an increase in latency in the Barnes maze, while BB supplementation decreased latency time to find the escape hole at each training day. We assessed the use of spatial learning strategies in our paradigm to provide a complementary measure of cognitive function less dependent on motor behaviour. Interestingly, we found that BB supplementation appeared to counteract a lost capacity of TBI rats to employ spatial leaning cues. This information together with results of the shorter latencies strongly suggest that BB supplementation protects TBI animals from a loss in spatial learning performance. In this regard, recent functional neuroimaging study in humans has established a connection between BB intake and cognitive function. Further, in the elevated plus maze test, rats exposed to TBI showed a tendency to reduce time spent in the open arms, which encompasses with clinical reports that psychiatric disorders are often observed in TBI patients. TBI-induced behavioural deficits probably stems from the impairments in BDNF-TrkB signaling that has been implicated in various cognitive and affective disorders. We cannot ascertain the cellular identity of the reported protein alterations. Although neuronal cells are the primary locus for learning and memory processing, non-neuronal cell types such as astrocytes and microglia can also contribute to these alterations. Moreover, it known that astrocytes and microglia provide support to synaptic transmission that is fundamental for neuronal function involved in cognitive processing.In the present investigation, we also found that TBI significantly reduced levels of hippocampal BDNF, and that BB dietary supplementation normalized these the levels. Previous report indicated that deficiencies in BDNF signaling is associated with impairments in cognition. Alternatively, cognition is strongly reliant on long-term potentiation and hippocampal BDNF, and the interaction between BDNF and its tyrosine kinase receptor is required for induction of LTP. Previously we have shown the protective effects of BDNF on the TBI pathology. Presently, our findings show that BB supplementation counteracted the BDNF reduction induced by TBI, paralleling improvements in cognitive function. It is well established that BDNF regulates synaptic plasticity and learning through interaction with the transcription factor CREB. Interestingly, our results also showed that BB supplementation normalized levels of CREB in TBI animals, and that these changes were proportional to changes in BDNF levels.

Long-term climatic cycles, such as El Niño Southern Oscillation events, could also play a role, particularly in rainfed systems that we may expect to exhibit stronger variability than their irrigated counterparts. ENSO phases have been shown to induce synchrony in masting systems, but knowledge of ENSO effects on crop plants is largely limited to annual crops. As a primary source of climate variation in Brazil and Iran, ENSO could be a cause of periodic yield in Brazilian tangerine and Iranian apricot . Climate and pest cycles may also interact to produce complex longer-term cycles in crops. If future work confirms a biological basis of long cycles in some crops at a national scale, it would be profitable to understand the extent to which these longer-term crop fluctuations reflect exogenous forcing versus endogenous feed backs . In conclusion, we have found that perennial crops frequently exhibit alternate bearing even at a national scale and that this is especially pronounced in wind-pollinated crops. This pattern is remarkable, given the general assumption that management practices have come to outweigh the ecological drivers that would synchronize country-wide production. Our results suggest that historic yield data present a thus far underused resource for further analyses on the mechanisms of reproductive synchrony across time, space and taxa.

Future work could explore the intraspecific and intraregional differences in synchrony and the degree of overlap and divergence between patterns in natural and managed systems. We encourage strengthened collaborations between theoretical ecologists,drainage gutter applied ecologists and horticulturalists for the mutual benefits of achieving an enhanced understanding of the mechanisms of synchronous interannual variability and promoting stable yields and farmer livelihoods.Bees and other flower-visiting animals provide essential pollination services to many US crops and to wild plant species . Bees contributed an estimated 11% of the nation’s agricultural gross domestic product in 2009 , equal to $14.6 billion per year . Of this, at least 20% is provided by wild pollinators that depend on suitable land for nesting and foraging . As the consumption of specialty fruit and vegetable crops has grown , the demand for pollination services has increased. However, the supply of managed honey bees has not kept pace , due to management challenges and colony losses over the last decade . There is growing evidence that wild, unmanaged bees can provide effective pollination services where sufficient habitat exists to support their populations . They can also contribute to the long-term stability of crop pollination, thereby reducing the risk of pollination deficits from variable supply or activity of honey bees . As a result, wild pollinators should be integrated into crop pollination management plans as a supplement or alternative to managed bees . Despite the agricultural importance of wild bees, there is increasing evidence that multiple species are declining in range or abundance. Some of the most important crop pollinators, such as bumble bees , have declined over past decades in the United States . Among the numerous threats to wild bees, including pesticide use, climate change, and disease , habitat loss seems to contribute to most observed declines .

Indeed, a National Research Council committee on the status of pollinators in North America reported that conserving and improving habitats for wild bees is important for ensuring continued pollination services and food security . Recognizing both the growing need for pollination services and increasing threats to wild bees, a recent presidential memorandum called for a national assessment of the status of wild pollinators and available habitat in the United States . The resulting report sets a goal of 7 million acres of land for pollinators over the next 5 y . However, there has been no assessment at the national level of the current status of native pollinator habitat, where and at what rate this habitat is being degraded, and the impact of these changes on bee populations and the pollination services they provide. A national assessment is challenging because plant–pollinator interactions and dynamics occur at relatively fine spatial scales. Wild bee populations are largely determined by the spatial distribution of habitat resources within their foraging range , and this varies from ∼100–2,000 m . Accordingly, most of our understanding of native bee populations is at the scale of landscapes and local sites. Several field-based assessments of habitat resources for native bee species have been developed at landscape scales . However, the required cost and time to scale this type of field assessment to cover all habitat types and bee species nationwide is logistically challenging and prohibitively expensive. When field observations are lacking, careful use of expert derived data has been shown to provide informative estimates that enable habitat assessments , including studies on pollination . Use of expert opinion may therefore be an efficient path to an initial nationwide assessment of pollinator habitat and abundance in the absence of consistent data across different land categories.

Such an approach must include careful treatment of uncertainty that may arise from differences in expertise among regions, authorities, taxa, and so on . Indeed, a robust analysis of uncertainty, and its implications for assessment findings, is a useful result in itself. It can help orient research toward addressing the most important gaps in our national knowledge of wild bees and their importance for crop pollination. Here, we use a published model of bee abundance and expert knowledge to assess the status, trends, and impact of wild bee abundance and associated uncertainties across the coterminous United States. The spatially explicit model predicts a relative index of wild bee abundance based on local nesting resources and the quality of surrounding forage . We parameterize the model with expert-derived estimates of nesting and forage quality for each of the main land-use types in each of the major ecoregions to construct a probability distribution for each parameter that captures estimates by multiple experts and their uncertainty. We first validate model predictions with bee collections and observations from a variety of landscape settings. We then map bee abundance, its uncertainty, and the agricultural demand for pollination across the United States to address the following questions: What are the current status and trends of wild bee abundance across the coterminous United States? What land use changes have driven these trends over a 5-y period ? Which regions and crops experience relatively low bee abundance compared with crop pollination demands? How does uncertainty in our knowledge affect these predictions? Responses to these questions will inform future research efforts and policy decisions to conserve native bees at the national level and can help guide a coordinated and ongoing nationwide assessment of wild bees.Our model predicts generally high abundances of wild bees in areas rich in resources such as chaparral and desert shrublands,plastic gutter intermediate abundances in temperate forest and grassland/rangelands, and lower abundances in most agricultural areas . Patterns of wild bee abundance and expert uncertainty seem correlated . In fact, whereas most areas with low bee abundance also present low uncertainty, only 5% of areas with high bee abundance have low uncertainty. This suggests that experts are more individually or collectively certain about uniformly poor bee habitats than they are about higher-quality habitats , which can vary in quality over time and space . Between 2008 and 2013, wild bee abundance was consistent in 67% of the US land area . However, our model indicates decreases in 23% of the United States , and these decreases were highly likely in 9% of the United States . Most of the areas of likely decrease occurred in agricultural regions of Midwestern and Great Plains states and in the Mississippi river valley. Eleven states [Minnesota, Texas , Wisconsin , South Dakota , North Dakota , Illinois, Missouri, Nebraska, Oklahoma, Kansas, and Louisiana] collectively accounted for 60% of the areas of predicted decrease in wild bee abundance. Over the 5-y period in these states, corn and grain cropland increased 200% and 100%, respectively, and mostly replaced grasslands and pasture . Bee abundance increased in 10% of the United States and the increase was highly likely in 3% of the country .

Areas of likely increase in bee abundance were found in northern ND, eastern Washington and Pennsylvania , southern Montana, parts of several states in the Great Plains, and in southeastern coastal areas . In these areas, grasslands, pastures, and corn/soy fields were converted to higher-quality habitat, such as shrublands or fallow crop fields .Bee abundance maps can be interpreted as the potential “supply” of pollination services from wild bees. To compare this measure of supply to potential agricultural demand, we calculated the area of pollinator-dependent crops, weighted by each crop’s degree of pollinator dependence, for each US county in 2013 . By comparing the two maps, we identified counties with relatively high supply of wild bees and relatively low demand and, conversely, where high demand occurs in counties with relatively low supply . We identified 139 counties where high demand and low supply coincide and 39 counties where this difference was particularly extreme . All of the 139 counties with a pollinator disparity had relatively low uncertainty for 2013 bee abundance , which indicates that there is high confidence in this mismatch. These counties tend to contain either a significant percentage of area that consists of highly pollinator-dependent crops [e.g., almonds, blueberries, and apples in California , Oregon, and WA, respectively] or large amount of less-dependent crops . To examine changes in the relationship between wild bee supply and pollination demand, we combined the two trend maps . We found that 106 counties have simultaneously experienced increases in demand for pollination services and decreases in wild bee abundance . This represents 54% of the 195 counties that have experienced substantial changes in pollination demand . In 27 of these counties, declines in supply were highly likely , whereas in the remaining 79 counties declines were less certain . In counties of West Coast states and Michigan, increases in demand were mostly driven by increases in specialty crops such as almonds, cherries, blueberries, apples, watermelons, and squash. In contrast, demand increases in the Great Plains and Mississippi Valley were driven by increases in crops, such as sunflower, canola, soybeans, and cotton, with moderate to low pollinator dependency. Trends in our measures of supply and demand vary widely among individual crops . Most crops that require animal pollination have expanded in area between 2008 and 2013, whereas the predicted supply of wild bees in many of these cropped areas has declined. Specialty crops, such as pumpkins, blueberries, peaches, apples, and watermelons, are among the crops that present the strongest mismatch between changes in supply and demand. Others, such as canola, have experienced increases in both supply and demand. Of particular concern for future abilities to meet pollination demands, crops that are most dependent on pollinators tend to have experienced simultaneous declines in supply and increases in demand.Our study is the first to our knowledge to map the status and trends of wild bees and their potential impacts on pollination services across the coterminous United States. By combining a spatial model with expert knowledge, we find highly heterogeneous patterns of both predicted abundance of wild bees and our uncertainty regarding those predictions. We also identify counties and crops of potential concern, where declines in wild bee abundance oppose increased need for crop pollination. These analyses form an important step toward a nationwide understanding of the status of wild pollinators. They can also help focus attention and future research toward regions of high uncertainty and to direct management efforts to areas of major concern. Our mapped index of bee abundance clearly shows that areas of intense agriculture are among the lowest in predicted wild bee abundance. Our predictions are also relatively certain in these areas . This reflects consensus among experts about the low suitability of intensively managed agricultural land for wild bees and is supported by an abundance of previous research on the negative effects of intensive agriculture on bee populations . Areas of bee abundance where declines are most certain tend to have experienced additional conversion of natural land covers to crops, especially corn . These results reinforce recent evidence that increased demand for corn in bio-fuel production has intensified threats to natural habitats in corn-growing regions . For example, a recent land-use simulation found that expansion of annual bio-fuel crops could reduce pollinator abundance and diversity at the state level .

The walnut industry submitted its research results for a heart health claim to the U.S. Food and Drug Administration , and the almond industry submitted its as part of a petition filed by the International Tree Nut Council Nutrition Research and Education Foundation to the FDA for a heart health claim for nuts. The FDA approved a qualified health claim for walnuts, and another for almonds and other selected nuts, on July 15, 2003, which states: “Scientific evidence suggests but does not prove that eating 1.5 ounces per day of as part of a diet low in saturated fat and cholesterol may reduce the risk of heart disease.” Each of these four commodity groups has used news releases and public relations to publicize the nutrition and health benefits of their products. The messages for walnuts and avocados have been communicated almost entirely through third parties such as magazines, newspapers, doctors, nutritionists or other credible sources, rather than paid advertising. The advertising emphasis for walnuts and avocados has been on quality, taste and recipes. The Almond Board of California initially relied on public relations to disseminate its message on the health and nutritional benefits of almonds. Following FDA approval of their qualified health claim, their research results were incorporated into paid advertising and promotion, almost always featuring a health message.

Likewise, in 2003 the California Strawberry Commission introduced a promotion campaign called “Be Well — Get the Red Edge” , which targets health and nutrition professionals,greenhouse ABS snap clamp and consumer and trade media. Commodity groups have found that they can stretch their promotion budgets by partnering with other groups. After FDA approval of the qualified heart health claim for nuts, the Almond Board of California partnered with the American Heart Association and now makes liberal use of the AHA logo in almond advertising. The California Walnut Commission formed a partnership with the Spanish Heart Foundation, and distributed more than 40,000 brochures and samples during “Heart Week” in Spain. The foundation also includes recipes and other information concerning California walnuts on its Web site. The California Avocado Commission is also leveraging its research results by partnering with health organizations, including the American Diabetes Association, UCLA Nutrition Education, American Association of Diabetes Educators, American Dietetic Association and IDEA Health and Fitness Association. Imports can easily create a “free rider” problem for U.S. commodity programs. California avocado producers spent more than $182 million on market development between 1961 and 2003 . They were understandably upset to see producers in other countries taking advantage of state promotional efforts when the national market share of imported avocadoes increased from less than 3% prior to 1990 to about 34% from 1998 through 2002. With the 2002 passage of the Hass Avocado Promotion and Research Order , all Hass avocados sold in the United States, including imports, are assessed 2.5 cents per pound to fund advertising, promotion, research and data dissemination.

Increased avocado demand due to HAPO promotion will offset much of the price impact of increased imports, and importers should enjoy attractive returns from their promotion dollars. Carman estimated that returns for importers’ spending on advertising and promotion ranged from $2.09 to $6.31 per dollar spent, depending on the level of imports and the effectiveness of Hass avocado advertising.An important and often overlooked benefit of mandated marketing programs, in addition to having an organized commodity group, is the value of the information they gather, organize and disseminate. A first-of-its-kind innovation for commodity groups was the establishment, by the Hass Avocado Board , of a Web-based program designed to exchange crop and marketing information among 100 packers and over 20,000 producers from the five HAB members — California, Chile, Dominican Republic, Mexico and New Zealand. The HAB Web site notes: “The program goal is to develop collaborative strategies to achieve an orderly flow of the 750 million pounds of fruit sent annually into the U.S. marketplace.” This Web-based program collects, tracks, analyzes and disseminates information relevant to selling Hass avocados in the U.S. market. It provides all players in the U.S. market with 24-hour access to critical market data that drives decisions about growing, shipping, distribution and marketing. The HAB market information program is an exciting development in produce marketing, made possible by recent worldwide developments in communication networks. It provides a dramatic reversal of trends that have reduced the availability and timeliness of market and price information for produce markets.

The widespread availability of marketing information and data is a theoretical requirement for competitive markets. It will be interesting to see to what degree improvements in marketing efficiency made possible through HAB flow to producers funding the program. If successful, the program template can be extended to other commodities and countries, with benefits flowing to producers and consumers worldwide.Drosophila suzukii , commonly known as the spotted-wing drosophila, is an invasive pest of small fruit and berry production native to Southeast Asia. Starting in 2008, this species underwent a rapid geographic range expansion, spreading across North America, South America, and Europe. Following its arrival to the USA, growers in California, Oregon, and Washington reported yield losses of up to 50% in raspberries and blackberries, 40% in blueberries, and 33% in cherries. Unlike other Drosophila species that typically breed on overripe and rotting fruit, D. suzukii females readily lay eggs into firm, still-ripening fruit using specialized serrated ovipositors. After hatching, larvae continue to feed and develop within the fruit, causing rapid decay and increased susceptibility to subsequent infestation by other drosophilids. Efforts to control D. suzukii and prevent infestation have led to a drastic increase in the use of broad-spectrum insecticide applications in susceptible host crops. Specifically, three main classes of insecticide–organophosphates, pyrethroids, and spinosyns–have shown high efficacy and now form the basis of most conventional D. suzukii management programs. Carbamates and some diamides have also shown moderate efficacy against D. suzukii, but the use of these materials is more limited throughout North America and Europe. The continued reliance on chemical sprays to control this pest, combined with its high fecundity and rapid development time, have elicited concerns that insecticide resistance could quickly develop. For organic growers, the risk of resistance is even greater. Currently, spinosad is the only insecticide approved by the Organic Materials Review Institute with high efficacy against D. suzukii, meaning that growers must rotate spinosad sprays with low-efficacy materials such as pyrethrins and azadirachtin in order to comply with label requirements. Consequently, D. suzukii in organic fields are primarily challenged by only one class of insecticide further increasing the risk of resistance. Indeed, recent work by Gress and Zalom reported low to moderate levels of spinosad resistance in D. suzukii collected from commercial caneberry fields near Watsonville, CA. Specifically, this study utilized dose-response analysis of adult D. suzukii to quantify the degree of resistance and found that Watsonville females exhibited LC50s 5–8 times higher than females from a second, untreated location in California. This finding demonstrates an immediate need to expand our understanding of resistance evolution in D. suzukii and to develop novel tools that aid in the early detection and assessment of resistant populations so that appropriate management actions can be implemented. When decreased susceptibility to an insecticide is first detected in a pest field population,snap clamps ABS pvc pipe clip laboratory selection studies, often referred to as resistance risk assessments, represent a critical first step towards understanding the potential for resistance to develop. These studies are performed by exposing large numbers of individuals from the suspect field population to moderately lethal concentrations of insecticide for multiple generations such that the most susceptible individuals are eliminated and the most tolerant survive. Dose-response analyses are performed both before and after implementing the selection protocol, and the change in LC50 is used to quantify the response to selection and better understand the potential for resistance to develop. This approach, however, is not without its limitations. For example, because laboratory colonies contain limited genetic variation relative to large field populations, these studies can understate the evolutionary potential for resistance to develop in a field setting.

Conversely, selection imposed in laboratory settings may be more intense and consistent than in the field, leading to possible overestimation of resistance development. Nevertheless, an increase in resistance due to laboratory selection provides unambiguous evidence that further loss of susceptibility in the field is possible. Additionally, if laboratory selection is successful and resistant colonies are generated, these strains can assist researchers in the development of novel tools for detecting, monitoring, and managing resistance in the field. For example, resistant colonies can be used to identify the molecular mechanism that produces the resistant phenotype, and these markers can, in some instances, be used to track spatiotemporal patterns of resistance development in the field. Laboratory-generated resistant colonies can also be used to assess whether insecticide resistance in a particular population is associated with fitness costs , and this information can be used to develop programs to manage resistance in the field. The presence of such costs under benign conditions would suggest that efforts to temporarily eliminate the target insecticide from spray programs could successfully maintain susceptibility in the field, at least in the short-term. After decreased spinosad susceptibility in the Watsonville, CA population was identified, Gress and Zalom performed a resistance risk assessment by continuing to expose adults from this population to a discriminating dose of spinosad using a standard glass vial bio-assay protocol developed for D. suzukii . After implementing this technique for 5 generations, Gress and Zalom found that LC50 values increased by 86% for males and 49% for females. This selection protocol, however, is highly labor intensive and costly, making it impractical for long-term selection programs which are needed to generate and maintain resistant lines. Additionally, this method requires that researchers kill off much of their adult population each generation, increasing the risk that selection lines will be lost in the process. Here, we describe a simple larval bio-assay that overcomes these challenges and requires little effort or cost beyond what is already needed for basic laboratory stock maintenance. We then perform a series of larval dose-response bio-assays to calibrate the selection protocol and identify baseline susceptibility for three commonly used insecticides. Finally, we implement the larval bio-assay protocol to perform resistance risk assessments using the same Watsonville, CA field strain previously tested by Gress and Zalom. Changes in the susceptibility at both the larval and adult life stages were quantified, and results from this study are compared to those obtained with the original adult selection method.First, groups of n = 20 mated females were transferred into plastic 6 oz Drosophila stock bottles containing fresh Bloomington standard Drosophila cornmeal diet. Females were allowed to lay eggs for four days before being transferred into new bottles with fresh diet. Once females were removed, 400 μL of insecticide solution was pipetted onto the surface of the diet, and each bottle was shaken sideto-side to distribute the solution. This ensured that all larvae were uniformly exposed to the insecticide, both through external contact and internally while feeding on the treated diet. Control bottles were treated with either ddH2O or acetone, depending on the insecticide treatment , but otherwise were handled using the same protocol as experimental bottles. All bottles were then re-plugged with a cotton stopper and stored in a climate-controlled walk in chamber at 22–23˚C and 14–10 light-dark cycle. Because the first larvae typically crawl out of the diet to pupate after 6 days under these conditions , treatment timing is critical to ensure that all larvae are exposed. Bottles were monitored for up to 18 days following treatment for the emergence of adult D. suzukii, and all newly enclosed adults were transferred to fresh bottles and counted.To quantify baseline susceptibility of D. suzukii larvae, we used the larval bio-assay protocol to perform dose-response analyses for three insecticide AIs, each representing a different insecticide class, commonly used against this pest. This work was conducted using a susceptible laboratory strain of D. suzukii originally collected from USDA Wolfskill Germplasm Repository in Winters, CA in fall of 2017. Wolfskill is an experimental, mixed-fruit orchard, and crops at this location receive no insecticide treatment. This strain of D. suzukii was previously used as a susceptible control by Gress and Zalom when quantifying resistance in the Watsonville population. The three formulated insecticides used in this study were malathion , spinosad , and zeta-cypermethrin .

Four of the orchards contained wild bees such as Bombus vosnesenskii Rad. and Bombus melanopygus Nyl., and the fifth contained the managed native blue orchard bee O. lignaria.In 2009, we categorized 14 almond orchards either as having non-Apis bees or lacking non-Apis bees , based on standard observations of flower visitors . In each orchard, we covered a set of almond branches with mesh bags before flowering to exclude pollinator visits. Once anorchard was in bloom, the bags were removed, and the branches with the previously unvisited flowers were removed from the trees and immediately presented to foraging A. mellifera. After an A. mellifera had visited one of these flowers, the flower was removed from the branch, its petals and anthers were removed, and it was placed in a 1.5 ml microcentrifuge tube containing 0.5 ml of water, such that the stigma did not touch the tube’s surface and the pedicle was in water. These flowers were left at room temperature, away from direct sunlight for 72 h to allow pollen tubes to grow. After 72 h, the pistils were fixed in FAA and stored at 48C until further processing. From each of the 14 orchards, an average of 30 stigmas were processed after a single honey bee visit .

To examine pollen tube growth, pistils were removed from the FAA and the tissue softened by boiling in 5 per cent sodium sulphite for 30 min. They were then soaked in tap water for 20 min,drainage planter pot and incubated for 24 h in a decolourized solution of 0.1 per cent aniline blue dye, dissolved in 0.1 N K3PO4. The softened stained pistils were squashed onto a microscope slide to reveal pollen tubes. The slides were examined using a fluorescent microscope . For each slide, the numbers of pollen tubes initiating growth on the stigma and reaching the base of the style were scored. In 2009, we measured fruit set in each of nine orchards with nonApis bees and nine without non-Apis bees. We marked 1 m lengths of branches on five trees on the outer row of each orchard. To calculate fruit set, the number of flowers on each marked branch section was counted and in July the number of developing fruits on the same section of branch was recorded. In 2008, standardized observations of flower visitors were conducted in the same orchards. In the nine orchards with non-Apis bees , overall 18 different non-Apis bee species/morphospecies were observed visiting almond flowers, with Andrena cerasifolii being the most common non-Apis visitor .In 2011, three large cages were set up in one of the study orchards. Each cage contained two rows of four trees .

One row was the Monterey variety and the other the Carmel variety. The cages were stocked with bees at the initiation of bloom . One cage received a four-frame nucleus A. mellifera colony with two entrances, set up so that one entrance opened into the cage and one entrance to the open orchard. One cage received 32 female and 32 male individuals of O. lignaria. Wooden nesting blocks, water and loose soil were provided for nesting. In the third cage both A. mellifera and O. lignaria were stocked at opposite ends of the cage as described previously. Observational scans were made of the frequency of flower visits in the cages to assess the number of foraging bees within each cage. In each scan, a group of flowers was observed for 20 s, and the number of flowers observed and the frequency of flower visits recorded. Scans were repeated in different sections of each tree on different days . Many of the same methods as detailed above for the open orchards were used in the cages, with the following differences. The movement between adjacent trees was recorded during 1 min observations. Three observations were made down one row, three down the other row and four between the rows. These observations were conducted on 5 days in the A. mellifera cage and 4 days in the mixed A. mellifera/O. lignaria cage . Single-visit pollination effectiveness was measured for A. mellifera in the A. mellifera cage and the mixed cage .

For each visit, the number of pollen grains on the stigma, the number of pollen tubes initiating growth and the number of pollen tubes reaching the base of the style were counted. The fruit set was estimated as above by marking two branches on each of the eight trees per cage. Per visit fruit set was estimated by dividing the fruit set by the average flower visitation rate in the cage.Data from the open orchards on A. mellifera movement , pollen tube growth and fruit set were analysed using generalized linear mixed models . All models were simplified by stepwise deletion. Analysis of variance was used to compare the loss of explanatory power from the removal of an explanatory variable, and if p 0.05 the variable was dropped. A Mann –Whitney U-test was performed on the data from flower visitor observations in the orchards where fruit set was recorded. The visitation rate of A. mellifera at the orchard edge was compared between the orchards with non-Apis bees present and those without. For the cage data, means and standard errors were calculated for informal comparison between the cages, as replications at the cage level were not possible. All analyses were carried out in R v. 2.14.1 .Our findings show that increased pollinator diversity can synergistically increase pollination service through species interactions that alter the behaviour and resulting functional quality of a dominant pollinator species. This highlights a largely unexplored facilitative component of the benefit of biodiversity to ecosystem services. Total bee visitation rates were similar between orchard types, and the visitation rate of A. mellifera was lower in orchards with non-Apis bees. However, A. mellifera pollination effectiveness was greater and fruit set was higher when non-Apis bees were present. Apis mellifera increased their proportion of movement between tree rows when non-Apis bees were present, thereby improving pollination effectiveness. More successful pollen tube growth translated into significantly higher fruit set in the orchards where non-Apis bees were present. The greater proportion of between row movements by A. mellifera individuals most probably resulted in the deposition of more compatible pollen, since pollen from the same variety generally does not set fruit . This synergistic effect of the presence of non-Apis bees suggests that maintaining biodiversity in agricultural ecosystems could provide unrecognized benefits, and it offers exciting opportunities for the integration of more diverse pollination systems to improve the longer-term sustainability of crop production for almond and similar crops. Unlike in sunflower, very few direct interactions were observed between A. mellifera and non-Apis bees. One potential mechanism for the increased proportion of inter-row movement of A. mellifera in the presence of other non-Apis bees is linked to resource depletion. Because some non-Apis bees can fly at lower temperatures than A. mellifera, and therefore earlier in the day, it may be that if A. mellifera begin foraging and find flowers have already been depleted then they increase the distance of their foraging flights. Alternatively, it may be related to the scent marks left by non-Apis bees on the flowers. Apis mellifera have been shown to avoid visiting flowers that have been marked by bumble bees, and it may be that as well as avoiding the flowers they also shift their foraging location. Given that A. mellifera tend to move down the same row, if a response to perceived resource competition is to shift foraging locations, this could be expected to involve movement across rows.

However, at present the mechanism for the increased between-row movements when non-Apis bees are present is unknown. In the controlled cage environment, the steeper increase in the number of pollen tubes initiated with increasing pollen deposition in the mixed cage and the higher fruit set supports the findings from the open orchards that more compatible pollen is being moved when pollinator communities are diverse. Because there was only one cage per treatment,plant pot with drainage the data from the cages are only descriptive, but they do support the findings from the open orchards. Future work should attempt to replicate similar treatments. The greater fruit set in the mixed cage when the visitation rate was lower than in the A. mellifera cage may be due to the slightly higher proportion of between-row movements in the mixed cage. Although the differences in movement were not great, the pollen tube data suggest that more pollen was moving between varieties in the mixed cage. The size of the cages limited the opportunity for between-tree flights. This and the relatively low visitation rate of O. lignaria in the cages compared with the open orchards may explain why the difference in pollen tube formation and fruit set between the cages was less than in the open orchards. The price of California almond in 2011 was approximately $1.79 per pound and production was estimated at 2670 lbs per acre . If a 5 per cent increase in fruit set as calculated from the controlled cage environment translated into an equivalent increase in production, farmers would make $239 more per acre . This estimate from cages may be a lower bound compared with open orchards. Sampling effects and complementarity have been the primary explanations for a positive relationship between biodiversity and ecosystem function. Here, we show a different mechanism, possibly due to interspecific competition, where community composition alters the behaviour of a service-providing organism with a positive knock-on effect for the ecosystem service. Our results show alterations in A. mellifera foraging behaviour when a diverse community of other bees are present, and suggest almond yield can be increased by encouraging wild bees in the orchards. As such, natural habitat near almond orchards should be conserved to protect wild bee communities. The availability of A. mellifera is not predicted to increase at the same rate as demand for their services in agriculture. Thus, increasing the pollination effectiveness of A. mellifera and conserving wild pollinator communities could help increase crop yields.

The synergistic combination of A. mellifera and non-Apis bees represents a sustainable way to improve crop pollination services, but the generality of such effects still need to be tested across multiple crop systems.Mosquitoes rely on the olfactory system to find plants as a source of carbohydrates, hosts for blood meals, and oviposition sites. Because pathogens might be transmitted during a bite by an infected mosquito, there is understandably a great deal of interest in unraveling the olfactory aspects of human-mosquito interactions to explore ways of reducing mosquito bites. However, plant nectar sources are often essential for mosquitoes because they increase mosquito life span and reproductive capacity and long-living mosquitoes are more dangerous . Therefore, understanding how mosquitoes find plants/flowers is also important for reducing the transmission of vector-borne diseases. Previously, we have identified generic and plant kairomone sensitive odorant receptors from the Southern house mosquito, Culex quinquefasciatus . One of these ORs, CquiOR1, belongs to a cluster of 6 ORs from the Southern house mosquito and 2 ORs from the yellow fever mosquito, Aedesaegypti . Specifically, CquiOR2, CquiOR4, AaegOR14, AaegOR15, CquiOR5, CquiOR84, and CquiOR85. Of note, CquiOR2 is not the previously reported oviposition attractant-detecting OR2 , which has been renamed CquiOR121 . We cloned CquiOR2 and the other ORs in this cluster . We then deorphanized these receptors using the Xenopus oocyte recording system and a panel of odorants with physiologically and behaviorally relevant compounds, including oviposition attractants, mosquito repellents, and plant-derived compounds . Here, we report that these receptors, particularly CquiOR4, CquiOR5, and AaegOR15, are very sensitive to plant-derived compounds, including repellents. CquiOR4, for example, which is very specific to female antennae, with high and low transcript levels in non-blood fed and blood-fed mosquitoes, respectively, showed a robust response to the natural repellent 2-phenylethanol. Repellency activity elicited by 2-phenylethanol reduced significantly in CquiOR4-dsRNA-treated mosquitoes, but it was unchanged when these mosquitoes were tested against DEET, which is detected with another receptor . Mosquitoes used in this study were from a laboratory colony of Cx. quinquefasciatus originating from adult mosquitoes collected in Merced, CA in the 1950s and kept at the Kearney Agricultural Research Center, University of California, Parlier, CA. Specifically, we used mosquitoes from the Davis colony, which was initiated about eight years ago with mosquitoes from the Kearney colony. In Davis, mosquitoes were maintained at 27 ± 1 °C, 75 ± 5% relative humidity, and under a photoperiod of 12:12 h.

These tandem duplicates may have evolved new functions , possibly involved in the biosynthesis of novel compounds, and/or were selected to improve the metabolic flux of specific bio-synthetic steps that alter the dosage of certain endpoint metabolites. Future studies are needed to further investigate the possible role of tandem duplications in having modified metabolite levels and composition in wild and cultivated blueberry. Our analyses also revealed that high bush blueberry, a tetraploid, likely arose from the hybridization of two distinct parents, possibly allopolyploidy, based on the sequence divergence, unique transposable element insertions, and sub-genome expression patterns. Our analyses revealed that the sub-genomes in high bush blueberry may be controlling a distinct set of genetic programs . The dominantly expressed sub-genome in most surveyed tissues becomes the lowest expressed during fruit development. This observation is similar to findings in allopolyploid wheat where developmental and adaptive traits were shown to be controlled by different sub-genomes.

For example,10 liter drainage pot cell type- and stage dependent sub-genome expression dominance was observed in the developing wheat grain. We argue that both high bush blueberry and hexaploid wheat, each now with high-quality reference genomes, make excellent systems to further investigate these underlying mechanisms of sub-genomes dominance. sub-genomes dominance has far-reaching implications to numerous research areas including breeding efforts. For example, marker-assisted breeding needs to target the correct set of dominant homoeologs given the trait in polyploids that exhibit sub-genomes dominance. Thus, we anticipate that this genome, combined with improved insights into sub-genomes dominance, will greatly accelerate molecular breeding efforts in the cultivated high bush blueberry.Cardiovascular disease remains the leading cause of mortality in the United States, and after decades of decline, is rising coincident with the increase in obesity, insulin resistance, and diabetes that characterize cardiometabolic risk. Notwithstanding hereditary predisposition, reduction in identified, modifiable lifestyle risk factors can reverse CMR and CVD. It is estimated that 45.4% of all cardiometabolic deaths in the United States due to heart disease, stroke, and diabetes are associated with sub-optimal intakes of 10 dietary factors. Fewer than 1% of American children and adolescents meet full recommended metrics of heart healthy nutrition, falling especially short of recommended intake in the categories of fruits, vegetables, fiber and essential fatty acids.

Intensive pediatric lifestyle interventions for obesity are effective in achieving significant reduction in body mass index but do not elicit stable changes in nutrition habits in children and adolescents. These studies suggest a critical need for developing innovative tools to improve diet quality in youth. We have previously shown that twice daily consumption for two weeks of a whole food based nutrient bar composed of a blueberry, dark chocolate, red grape, and walnut matrix, soluble and insoluble fiber, with supplemental vitamins, minerals and essential long chain fatty acids, significantly increased high density lipoprotein cholesterol , due primarily to a 28% increase in large HDL particles, in generally healthy and insulin sensitive lean and overweight adults. In a subsequent 2 mo study of the effects of the nutrient bar on CMR markers in individuals across a range of BMIs, only those with low inflammation at baseline as assessed by high sensitivity C-reactive protein < 14.3 nmol/L responded comparably to those in the earlier trial, with not only increased HDL cholesterol and large HDL particles but also a trend toward increased high molecular weight adiponectin and a decrease in other CMR factors at 2 weeks, sustained at 2 months . In particular, a shift in low density lipoprotein particle subfractions toward a less atherogenic profile was evident in the non-inflamed group . Although the participants with overweight or obesity and CRP > 14.3 nmol/L did not show this response, they did experience an upward trend in adiponectin by 2 months. These results suggest that there may be a continuum of metabolic responsiveness to this nutrient supplement that is slowed in the face of the chronic low-level inflammation commonly observed with obesity and insulin resistance. A 6 month study of this nutrient bar in obese adolescents with non-eosinophilic asthma showed improved lung function at 2 months, but favorable movement in cardiometabolic biomarkers only began to emerge at 6 months.

It is not known whether more sensitive biomarkers of early metabolic change may be capable of detecting short-term effects of the nutrient bar supplementation in persons at CMR. Past metabolomics studies in obese adolescents and adults have identified strong positive associations between baseline levels of branched chain, aromatic, sulfur, and gluconeogenic amino acid metabolites and parameters of inflammation and insulin resistance. Similarly, elevated levels of specific ceramide species have been shown to associate with inflammation, dyslipidemia and insulin resistance in both adult and adolescent obesity. The relative sensitivity of these biomarkers to reflect moderate changes in dietary intake of polyphenols, essential lipids, fiber and vitamin/minerals, remains incompletely understood. In the present randomized, controlled, non-blinded trial, a two month intervention with exercise and nutrition counseling alone or with nutrient bar supplementation was performed in a high CMR cohort of adolescent /parent adult caretaker family units to determine 1) cross-sectional relationships in both adolescents and adults between traditional CMR biomarkers and amino acid and ceramide metabolites and 2) longitudinal changes within groups in the same CMR biomarkers following the lifestyle +/- nutrient bar intervention.This study was approved by the University of California Benioff Children’s Hospital Oakland Human Subjects Review Board, under approval number 2011022 entitled: The Impact of a Nutritional Supplement on Weight and Metabolic Health in a Parent-Child Intervention . All participating adolescents signed an assent document and all parent /adult legal guardian participants signed a written consent for their own participation and a separate consent for their participating adolescent. The study cohort was recruited from the UC San Francisco Benioff Children’s Hospital Oakland weight management program .

Teen participants were eligible for inclusion if they had a BMI greater than the 95th percentile, were between 14 and 18 years of age, fluent in English , and willing to eat the nutrient bar twice daily, after having tasted a sample, with one or both PACs also willing to participate. PAC inclusion criteria were the same except that there was no adult weight threshold. Exclusion criteria for both Teens and PACs included the diagnosis of diabetes mellitus or hypertension , and use of glucocorticoid, weight loss, insulin-sensitizing, lipid lowering, or anti-hypertensive medication. With Institutional Review Board approval, PAC consent for self and adolescent, and adolescent assent, 17 dyads and 1 triad were enrolled in May and June, 2011. One INT PAC dropped out before study measures were initiated but the rest of the family unit continued participation. All family units were seen within two weeks of the baseline group counseling session and within 2 weeks of the final session on the Clinical and Translational Research unit for pre-post blood testing. Participants were randomized at the baseline visit 1.25:1 to INT:CONT groups that met on separate days in the late afternoon after school and work for eight identical lifestyle counseling sessions in July and August 2011; 30 minutes of weekly group nutrition counseling followed by 30 minutes of supervised group exercise. In addition,25 liter pot the INT group was given a one week supply of nutrient bars at each visit through the seventh week and advised to return wrappers at the following session. Bar composition has been previously described , and was designed to supplement prevalent nutrient deficiencies in the American diet up to daily recommended intake , including 515 mg polyphenols, 4 gm whey protein, 9 gm of total fiber and 200 mg of long chain omega 3 fatty acid docosahexaenoic acid . Most vitamins and minerals added to the whole food matrix of the bar are in amounts representing 10% to 50% of their corresponding recommended daily intake, with the exceptions of vitamin C and vitamin D both added above RDI. Additional vitamin C serves as an antioxidant preservative for other bar ingredients, notably the omega 3 fatty acid DHA. Additional vitamin D addresses the prevalent deficiency in persons at high CMR, and the relatively low blood levels in the study cohort . Consumption of 2 bars each day was advised, with the first to be eaten before noon and the second in either the afternoon or evening, together with a minimum of 8 ounces of water with each bar due to the high fiber content. Compliance with eating the supplement and home lifestyle adherence were evaluated with phone call or text communication to each participating family every other day. Pre-post change in 25 hydroxy vitamin D levels served as an objective measure of compliance with the nutrient bars.Assessment of physical , behavioral and activity, and metabolic status was conducted at baseline and study completion on all Teen and PAC participants.Anthropometric and clinical evaluation: All anthropometric measures were performed in duplicate in the clinical research center and if not within 10% agreement, were repeated a third time. The reported measure is an average of the two closest numbers. Height was measured with a stationary stadiometer.

Weight was measured using a digital electronic scale and the BMI and waist to height ratio were calculated. Waist circumference was measured at end expiration to the nearest mm with a Gulick II Plus tape midway between the lowest border of the rib cage and the upper border of the iliac crest.Blood pressure and Resting Heart Rate: Each was measured in triplicate after 5 minutes sitting quietly with readings taken at least one minute apart. An automatic digital blood pressure monitor was used with cuff size adjusted for arm size. Traditional CMR biomarkers: Fasting blood samples were drawn and processed in the UCSF Benioff Children’s Hospital Oakland Clinical Research Center and sent to ARUP Diagnostic Laboratories for: standard lipid profile [total triglyceride, total cholesterol, and cholesterol within HDL and LDL , glucose, insulin, 25 hydroxy Vitamin D level and CRP. TG to HDL ratio and non-HDL were calculated. Fasting insulin and glucose were used to calculate the Homeostasis Model Assessment of Insulin Resistance Index according to the formula: fasting insulin x fasting glucose /22.5. Lipoprotein particle subclasses were analyzed by an ion mobility procedure that sensitively and directly measures concentrations of lipoprotein particle subfractions. High molecular weight adiponectin was measured by solid-phase sandwich ELISA . Metabolomic analyses: 1) Targeted analyses of 42 amine-containing metabolites consisting of 20 major amino acids, and secondary metabolites of arginine and cysteine whose levels are sensitive to inflammation and oxidative stress were performed on stored samples preserved at -70o Fahrenheit. Briefly, plasma was acidified with 5% perchloric acid containing 8 stable isotope internal standards. Acid-soluble supernatant was used for strong-cation exchange solidphase extraction to capture cationic amine- containing metabolites. Extracted metabolites were further derivatized with isopropylchloroformate. Derivatives of metabolites were resolved using Agilent 1260 ultra-high pressure liquid chromatography and eluted with a gradient of water and isopropanol . An Agilent 6490 triple quadrupole mass spectrometer was used to detect resolved analytes and quantify them using authenticated external and internal standards. 2) Sphingolipidomics by electrospray tandem mass-spectrometry by validated techniques was used to identify sphingolipid metabolites, including ceramides.De-identified baseline and study completion data points, paired by participant study ID, were entered into SPSS . Descriptive analyses of the study cohort were summarized and results for Teens and PACs, as well as for combined CONT and INT groups, were compared by unpaired Student t-test. Continuous physical and metabolic variables were tested for normality by examining the skewness, kurtosis and the Shapiro Wilk tests and transformed as necessary before analysis. Most of the variables in our data were normally distributed. Log transformations were conducted for the continuous physical and metabolic variables that were skewed to make them as normal as possible. The Shapiro-Wilk tests show that all of the transformed variables except two are approximately normal. The measures of metabolite concentrations used for principal component analysis were Z-transformed to render them normally distributed on the same scale with mean of zero and standard deviation of one. Pre-post change in absolute metabolite levels were compared by repeated samples paired t-test. Baseline z-scores of metabolites were subjected to principal component analysis without rotation. PCA is an unsupervised analysis that aims to decrease the complexity of data by reducing variables to a smaller number of principal components . A direct oblimin rotation was used and 6 factors before the bend in the scree plot , and eigenvalues >1 were retained. Component scores for each participant were calculated with a standardized scoring coefficient.

Recent outbreaks of X. fastidiosa diseases in Europe and Asia and also in new plant hosts such as olive , blueberry , and pear suggest the great adaptation potential of this pathogen. In a number of plant species, X. fastidiosa is believed to live as a harmless endophyte without inducing disease symptoms . Coexistence in the same xylem system of different strains for a long time without killing the host represents a fertile environment for exchange of DNA material. Several MLST-based studies detected inter subspecific recombination among strains of X. fastidiosa and proposed recombination as the mechanism of new allele acquisition, leading to plant host shift and disease emergence. Intersubspecific recombination was described to generate strains that infect citrus and coffee , mulberry , and blueberry and blackberry . A recent study also showed intersubspecific recombination between coffee-infecting strains in plants intercepted in France . Natural competence could be an explanation for the frequent recombination events detected in X. fastidiosa. Natural competence in X. fastidiosa was recently described in vitro , the rate of homologous recombination was shown to be higher when the cells were growing exponentially in solid agar plates than in batch culture tubes, and minimal medium was more conducive than rich medium .

With a plasmid as a donor DNA, 96 bp of flanking homology was sufficient to initiate recombination . Moreover,vertical farm tower some competence-related and type IV pili genes were shown to be involved in the process . Although some of those studies were performed using plasmids as donor DNA, two strains were also shown to recombine in coculture conditions , although the capacity of these strains to act either as a donor or a recipient for DNA exchange was not determined in those studies. The objective of this study was to test the hypothesis that natural competence in X. fastidiosa occurs under flow conditions . Associated with this objective was the aim of elucidating whether previous observations of high frequencies of X. fastidiosa natural competence in vitrowere dependent on batch culture conditions , which allow cell-tocell contact for longer times without replenishing of nutrients or removal of secreted molecules. Although natural competence and recombination are assumed to occur in natural habitats based on field surveys and DNA sequence data, experimental indications of its occurrence in the plant or insect host are not yet available for X. fastidiosa. Therefore, to circumvent the limitation of X. fastidiosa recombination tests in the natural hosts that are affected by uneven bacterial distribution and low populations , we performed recombination experiments in a microfluidic chamber system that mimics the natural environment of xylem vessels and insect foreguts.

The MC system allows continuous media flow conditions and formation of biofilms and has been previously used to study the behavior of X. fastidiosa . The biofilm fraction of the MC and the planktonic and detached cell fraction can be collected separately, and the behavior of cells in the two fractions can be determined. Two strains used in all of the previous publications on this topic were used in this current study to facilitate comparison with the literature and to further our understanding of natural competence in X. fastidiosa. The results presented here show that growth under flow conditions supports natural competence in X. fastidiosa, with recombination frequencies equivalent to that on solid media, previously described to be the most conducive environment for natural competence in vitro . These findings support the hypothesis that recombination occurs at high rates under flow conditions, representing the natural habitats of X. fastidiosa.Xylella fastidiosa subspecies fastidiosa mutants NS1-CmR and pglA-KmR were used in this study. The mutants were cultured in periwinkle wilt agar medium , modified by omitting phenol red and adding 1.8 g liter 1 bovine serum albumin and supplemented with the respective antibiotics. PD3 medium and modified X. fastidiosa medium were used when stated. Pectin was added to a final concentration of 0.01% as previously described . Kanamycin was used at 30 g m1 and chloramphenicol at 10 g ml. Inocula were prepared by streaking cultures from the 80°C freezer stocks on PW agar plates and incubating the plates for 5 to 7 days at 28°C. Cultures were then rest reaked onto new plates and incubated for another 5 to 7 days before use.To select a medium to test the occurrence of natural competence in MCs, three media were first tested in solid agar plates.

XFM and PW, used in previous studies , were selected as positive- and negative-control media, respectively, for recombination. Natural competence experiments were performed according to the method of Kung et al. with some modifications. Briefly, cells of the NS1-CmR and pglAKmR mutants were prepared in liquid media by scraping the cultures from PW-antibiotic plates. Ten microliters of each strain was spotted on top of each other on the agar plates of PW, XFM, and PD3 without antibiotics, and the spots were allowed to dry for 1 h. The plates were then incubated at 28°C for 3 days. Next, two spots from the same plate were scraped off and suspended in 1 ml of PD3 to make one replication, and 3 to 4 replicates were included for each media type per experiment. The experiments were repeated independently twice for XFM and at least three times for PD3 and PW. Single mutant strains were included as controls. The suspensions were then serially diluted, and100 l aliquots of appropriate dilutions were plated on PW agar plates in triplicate supplemented with both antibiotics to recover recombinants at the antibiotic-resistant site and with a single antibiotic to check for the growth of both parents in the mixture. Appropriate dilutions also were plated onto PW plates without antibiotics for enumeration of total viable cells. Plates were incubated at 28°C for at least 14 days before CFU were enumerated. The recombination frequency at the antibiotic-resistant site was calculated as the ratio of recombinant CFU to total CFU in equal volumes of suspension. After selection of the media that supported recombination in the agar plates, the media were tested in the MCs for cell attachment and biofilm formation.To test for the specific components that may influence natural competence, an initial screen was performed by removing or adding components to PW and PD3 in solid agar plates as described above. The components tested were sodium citrate dehydrate, succinic acid, and starch ; BSA and L-glutamine ; and pectin.

The effect of BSA was further tested by supplementing PD3 and PD3 plus L-glutamine with BSA and removing BSA from PW and XFM. Experiments were repeated three times independently with three replicates each time, except for PD3 plus L-glutamine treatment that was performed once with three replicates. The twitching motilities of both mutants were determined in media with and without BSA, according to previous studies with few modifications. Briefly, for PD3 and PW with and without BSA, media plates solidified with agar or Gelrite were divided into two halves, 10 to 12 spots of each mutant strain were made using a sterile toothpick, and plates were incubated at 28°C for 4 to 5 days. For XFM with and without BSA, plates solidified with agar were used and incubated for 10 to 12 days before measurements were recorded. Colony peripheral fringes were observed under 10 magnification using a Nikon Eclipse Ti inverted microscope ,vertical planter tower and fringe widths were measured for six colonies per plate per strain, with at least seven measurements per colony using a Nikon DS-Q1 digital camera connected to a Nikon Eclipse Ti inverted microscope and controlled by NIS-Elements imaging software version 3.0. Twitching experiments were performed at least three times independently for PD3 and PW with and without BSA and once for XFM with and without BSA.Three growth conditions were used: solid agar plates , liquid culture tubes , and continuous liquid flow . PD3 without antibiotics was the medium used, and the initial inocula of the NS1-CmR and pglA-KmR mutants were prepared as described above. Competence in tubes. Twenty-five-milliliter glass test tubes containing 3 ml of PD3 were inoculated with 100 l of each of the OD adjusted strain suspensions as donor and recipient cells . Tubes containing single strain inoculations were included as control treatments. Tubes were then incubated with shaking . After 3 days, the tubes were vortexed well to mix the biofilm formed on the air-liquid interface with the rest of the suspension and serially diluted and plated as described above. Three independent experiments were performed, and three replications were included in each experiment .MCs were prepared as previously described . Briefly, two parallel channels with separate inlets for bacterial cells and growing media were etched on a silicon wafer. The channels were modeled into polydimethylsiloxane and sandwiched between the PDMS layer and a glass cover slide. The inlets and outlets were then connected to tubings that were connected to syringes . The syringes were connected to pumps which control the media flow rate in the MC. The MC was mounted onto a Nikon Eclipse Ti inverted microscope to observe cell attachment and biofilm formation using phase-contrast and Nomarski differential interference contrast optics. Time-lapse video was taken using a Nikon DS-Q1 digital camera connected to the microscope and controlled by NIS-Elements imaging software version 3.0. For preparing the inocula for MCs, equal volumes of the strain pairs were mixed and inoculated into the cell inlet syringes, and growing medium was injected in the media syringes. MCs were run for 5 to 7 days with a media flow rate of 0.25 l min1 until abundant growth of biofilm was observed.

At the end of the experiment, the fraction of cells collected in the outlet syringe was harvested, and the fraction formed inside the channels was detached and pushed to the outlet collection syringe by increasing the flow rate to 30 to 40 l min 1 . Serial dilution, plating, CFU counts, and the frequency of recombination calculations were done as described above. Four independent experiments were performed with seven replicates in total .Confirmation of homologous recombination occurring via natural competence was performed by using heat killed donor cells in the solid agar plates. Suspensions of the donor cells were incubated at 90°C for 15 min for heat killing. The heat-killed donor and live recipients were then spotted on PD3 plates as described above. For confirmation of homologous recombination at the desired genome region, randomly selected recombinant CFU were rest reaked onto new double-antibiotic PW plates, and colony PCR was performed using the primers targeting the flanking region of the construct used to generate the mutants according to Kung et al. . Sequences of the flanking regions of antibiotic cassette insertion sites between the parent strains were compared using the muscle pairwise alignment algorithm within the Geneious 9.0.3 platform .Several hypotheses have been proposed to explain the existence of natural competence in bacteria. One explanation is that starvation signals induce competence, and the incoming DNA serves as a nutrient source under poor nutrient conditions as demonstrated in H. influenzae , Pseudomonas stutzeri , and R. solanacearum . Based on the results with a minimal medium and a rich undefined medium , a previous study speculated that growth in a low-nutrient medium favors natural competence inX. fastidiosa. However, the results of this study with these two media and PD3, another undefined rich medium, demonstrated that growth in PD3 significantly increases the recombination frequency. This suggests that starvation is not necessary to induce competence in X. fastidiosa. Further investigations of the differences between PD3 and PW were performed by either removing or adding these components to/from one another. Initial screening with the components showed a pronounced effect of BSA on the number of recombinants recovered. Additional experiments confirmed that BSA significantly reduces the recombination frequency when present in PD3, PW, and XFM. Since both XFM and PW contain BSA, this may explain the lower recombination frequencies in these media. In a previous study, BSA had been found to reduce the surface attachment and twitching motility of X. fastidiosa . In fact, natural competence and twitching motility are dependent on the activity of type IV pili in X. fastidiosa . Therefore, in this study the correlation between twitching movement and natural competence in different media was investigated. Interestingly, PD3 allowed the highest fringe width, and the presence of BSA significantly reduced twitching motility in all three media.