If, in contrast, gibbons would react competitively as other great ape species did in previous social dilemmas, we would expect them to try to maximize their food rewards by hesitating to act and by taking advantage of their passive role—placing themselves in front of the release mechanism. In that sense, we would expect passive partners to benefit from their position and obtain more rewards than the actors. Furthermore, we would also expect gibbons to be more likely to act in direct food test trials given that they could easily obtain at least one direct reward from their actions.Therefore, actors were still able to obtain the majority of the five released rewards in both conditions. Furthermore, actors obtained the extra rewards in 87% of the direct food test trials in which they acted. In line with this finding, Fig. 2 shows the percentage of trials in which individuals release food against the number of rewards that each individual obtained in indirect and direct food test trials. Interestingly, in only one dyad the more active individual obtained less benefits than the passive partner in both conditions. We also found significant effects of our control variables age and length of dyad suggesting that younger individuals obtain more food and that dyads who have been longer together distributed the five rewards more equally. Next, we evaluated whether passive gibbons acted strategically by placing themselves in front of the ramp at the moment the actor released the five food rewards and whether they would take advantage of their position in direct food test trials compared to indirect food test trials given that the actor gibbon would spend some time retrieving the food baited inside the handle.We also explored whether gibbons would take an advantageous position after the food had been released.

For that we measured whether the passive individual was in front of the ramp by the time the actor arrived at the location where the food had been released. After releasing the food,hydroponic nft the actor moved to the food location in 237 of those 287 trials . Passive individuals were in front of the ramp by the time the actor arrived in 76 occasions . We found no differences between conditions . We found that actors did not approach the released food in 50 trials in which they released it. A majority of those trials were direct food test trials . This makes sense since actors spent some time obtaining the food reward placed inside the handle. Nevertheless, in a great majority of these trials the actors ended up obtaining the food reward from the handle before the passive individual finished her last reward. Interestingly though, this behavior predominantly occurred in three of the six dyads. Furthermore, we only found one case in which the actor showed an intention to approach the partner by getting closer to him.Te results of the study suggest that dyads of gibbons managed to solve a competitive food task where one dyad member had the opportunity to pull and activate a release mechanism containing five potential food rewards for both partners. When gibbons had the opportunity to obtain an extra reward from their actions , they almost always acted, avoiding mutual defection. In contrast, in an indirect food test condition where gibbons could not obtain the extra rewards from their pulling efforts, their likelihood to pull and release potential food rewards dropped significantly in comparison to the direct food test condition. In our opinion, two primary reasons could explain this pattern of results. First, it is possible that gibbons were more motivated to pull when they could directly benefit from the extra reward inside the handle. Similarly, gibbons would have significantly pulled more often in the indirect food test condition compared to the no food control because they could still obtain some rewards. Clearly, gibbons showed that they did not just act for the sake of pulling the rope. Most likely, their actions were motivated by the prospect of obtaining rewards, especially when those were easier to access. While this reason is very plausible, it does not necessarily account for why gibbons did not pull for 90 s in almost 40% of the indirect food test trials.

This is especially surprising if we consider that actors benefitted more than passive partners in this condition. In other words, gibbons did not seem to interpret the situation as beneficial for actors. If that were the case, we would have expected gibbons to pull more often in indirect food test trials. Thus, the second possible explanation for our results is that gibbons interpreted the situation as a conflict of interest and hesitated to pull to avoid losing rewards in favor of the passive partner. This interpretation would be in line with previous findings in chimpanzees, bonobos and common marmosets. Thus, given the two non-mutually exclusive explanations, it remains unclear whether gibbons defected in indirect food test trials due to a reduced motivation to act because they could not access the extra reward attached to the handle or because they wanted to avoid the possibility of losing rewards to their partner. So far, we have mainly discussed gibbons’ decisions whether to pull or not. The next question concerns whether gibbons strategized when they took a passive role. In other words, did they try to maximize their own rewards when their partners pulled? The main source of potential conflict between participants lied on the possibility that passive individuals could position themselves in front of the release mechanism during direct and indirect food test trials. In direct food test trials, this could be particularly beneficial for passive partners given that actors could lose some valuable seconds trying to obtain the extra food reward attached to the handle. However, we found that actors actually obtained most of the rewards in both conditions, with a special advantage over passive partners during indirect food test trials. In fact, in only one dyad of gibbons the passive individual obtained more rewards than the actor in both test conditions. Furthermore, passive individuals rarely took advantage of the situation and they did not distinguish between conditions. That is, during direct food test trials passive partners did not benefit from the time that the actors spent trying to obtain the reward located inside the handle. In that sense, gibbons did not interpret the situation as a social dilemma in which they could benefit more than their partner.

Yet, gibbons also did not solve this task cooperatively. For that to be the case, dyad members would have needed to benefit more or less equally on both conditions and they would have not hesitated to manipulate the handle in indirect food trials. It is also very unlikely that gibbons’ decisions were driven by proactive prosocial motivations such as releasing food rewards to favor their dyad members given that actors benefited the most from their own actions. One possible explanation to understand why actors obtained more rewards than passive partners is that both individuals approached the handle during direct food test trials to obtain the reward, although only one individual pulled the handle. This occurred in 27.6% of the direct food test trials in which an individual pulled the handle. This possibility could partially explain why passive gibbons rarely position themselves in front of the release mechanism during direct food test trials, but it cannot explain why in indirect food test trials they did not take advantage of their passive role. In fact, in indirect food test trials passive partners approached the handle in 10% of trials. However, there are numerous reasons to suggest that a dominance component cannot fully explain our pattern of results. First,hydroponic channel only three of 12 individuals released rewards in less than 20% of the direct and indirect food test trials. Te result suggests that although they did not solve the task cooperatively, in half of the dyads both individuals exchanged roles relatively often. Furthermore, our results are also in line with literature suggesting that there is no clear dominance of one sex over the other and that gibbons engage in food sharing in both captive and natural settings. Importantly, conflict avoidance does not seem to be relevant here either. Cofeeding events occurred three times as often than displacements events, suggesting that individuals were usually tolerant with each other, as it has been observed in other populations. Relatedly, gibbons never hoarded the blueberries. They ate them as soon as they got them. Furthermore, these tolerance may be mediated by the length of time that dyads had spent together. In our study, the length of dyad predicted how equally gibbons divided the five food rewards. A third alternative explanation is that once a gibbon decided to manipulate the handle, the other one totally disengaged from the task, as if acknowledging a sort of property claim. This could explain why passive partners did not take an advantageous position in front of the release mechanism and why, as a consequence, actors did not face a social dilemma in many trials. After all, actors benefit more than passive partners despite the costs to pull the rope, understood as the possibility to lose rewards due to the distance they had to cover and the time lost to return to the location where the food was released. Nevertheless, this is unlikely because passive individuals still obtained a significant fraction of the food rewards .

All these previous arguments cannot fully explain why passive partners rarely took advantage of their position, especially given how successful this strategy was: passive partners obtained almost 75% of rewards when they positioned themselves in front of the release mechanism by the time the actor manipulated the handle. We thus propose that passive gibbons did not always take advantage of their partner actions because of the combined processes of motivation from the side of the actor and a general high level of social tolerance towards inequities. Tat is, individuals that were more motivated to obtain food and more attentive were also more likely to take the actors’ role in our task. Trough participation, they could become more aware of the situation as a whole and react faster to obtain the rewards despite the potential costs of pulling the rope. This gave them an advantage over their passive partners, whom at the same time tolerated actors obtaining the majority of rewards . Importantly, tolerance towards reward inequities also came from the perspective of the actors. Indeed we found that in fly trials individuals from five of the six dyads seemed to adopt a prosocial attitude towards their passive partners by letting them access all the released rewards. Future studies should improve different aspects of our setup to continue exploring gibbons’ decision-making strategies when individuals’ interests collide. Te main weakness of our design is that we were not able to separate dyads of gibbons before the experimental sessions. In that sense we could not train them with the different task contingencies as it is usually the case in this type of settings but see. It is thus possible that some individuals were more skillful than others, and that might have affected our results. Nevertheless, all individuals approached the apparatus and obtained rewards and only one never pulled from the handle during the course of the study. Moreover, despite the fact that gibbons distinguished between conditions with food and the no food control, their latencies to act did not differ between the indirect food test trials and the no food control trials. This could be interpreted as strategic behavior in the indirect food test trials if the gibbons were waiting for their partners to act. It is possible that with longer trials we would have found a significant difference between gibbons’ latencies in indirect food test trials and no food control trials. In that sense, future changes in the trial time or the food rewards can better assess whether gibbons strategize to solve conflicts of interest. Relatedly, it is possible that gibbons were more motivated to act during direct food test trials compared to indirect food test trials because there were more rewards in play . It has been found that at least great apes are able to distinguish between these two amounts when they are presented at the same time.

Using standardized observations of floral visitation and seed set measurements of yellow starthistle, we test the hypotheses that increasing urbanization decreases 1) rates of bee visitation, 2) viable seed set, and 3) the efficiency of pollination . In addition to contributing to a better understanding of how change in landscape use, particularly urbanization, affects pollination-plant interactions, the study illustrates the importance of use of neighboring lands for pollination services.Our study system was located around Brentwood, in east Contra Costa County, California, where natural, agricultural, and urban areas intersect with each other within a 20620 km region . A county water district , regional park district , and California state park all fall within the region, leaving large areas of land protected from development. This protected land consists mainly of grasslands and oak woodlands, some portions of which are managed for grazing. East Contra Costa County has had a farming community presence since the late 19th century. The agricultural areas of Brentwood, Knightsen, and Byron mostly consist of orchards , corn, alfalfa, and tomatoes. A housing boom in the 1990s led to massive residential growth in the area. The city of Brentwood has grown from less than 2500 people in the 1970s to over 50,000 today , and nearby Antioch has now over 100,000 residents .We selected 12 sites dominated by yellow star thistle in a stratified design to span the different land use types . Yellow starthistle is a common weedy plant that forms homogenous flowering patches in grassy areas throughout this region. Many different bee taxa in a range of functional groups and size classes have been observed to visit yellow starthistle, in part because it flowers late in the season relative to other floral resources. Despite being considered a serious introduced weed, yellow starthistle is unusual as an invasive species in that it depends on animal pollinator visits in order to set seed. Within each site we selected a 50 m650 m plot such that each plot was at least 2 km away from all others,hydroponic gutter a distance larger than the maximum assumed typical bee foraging ranges.

Although certain bee species have been recorded foraging as far as 1400 m, most bees in this type of habitat have nesting and foraging habitat within a few hundred meters of each other. Within each plot we estimated number of flowering yellow starthistle blooms by randomly placing 10, 1 m61 m quadrats and counting the number of flowering blooms in each. We also measured the spatial area of yellow starthistle patches within each 50 m650 m plot to obtain an estimate of total flowering blooms within each plot. We categorized total blooms/plot on a log scale: ,103 , 103 –104 , and .104 . Using NOAA’s 2006 Pacific Coast Land Cover dataset , a 500 m buffer was created around each plot, and the number of pixels classified as agricultural, urban, natural, water, or bare land was extracted. These categories were obtained by lumping finer categories in NOAA’s classification scheme using the following definitions: Urban–‘‘High Intensity Developed’’, ‘‘Medium Intensity Developed’’, ‘‘Low Intensity Developed’’, and ‘‘Developed Open Space’’; Agricultural–‘‘Cultivated’’, ‘‘Pasture/ Hay’’; Natural– ‘‘Grassland’’, Deciduous Forest’’, ‘‘Mixed Forest’’, ‘‘Scrub/Shrub’’. Each plot was classified as a proportion of each of the 3 different land use categories, as well as for the category that was dominant. By this latter measure, of our 12 sites, 4 of each were classified as ‘‘urban’’, ‘‘agricultural’’, and ‘‘natural’’.We observed visits by all bee species to yellow starthistle at all sites 3 times for a 30 min period for a total of 90 min of total observation time per site within the same 2 wk period in August 2011. AM was defined as being between the hours of 9:30–11:30, Mid-Day as between 11:30–13:30, and PM as between 13:30–15:30. Also recorded at each observation period were approximate number of blooms, and wind and temperature simultaneously . Bees were not netted for later identification as we did not want to interfere with visitation to starthistle during this study. Instead, we used a modified protocol of citizen scientist observation surveys with 15 expected bee morphotypes that correspond to 30 possible genera known to occur in the region .

The observer slowly walked through the yellow starthistle patch, and upon reaching patch edge, returned on a path at least 3 m away from the previous, and recorded the morphotype classification of all bee visitors within 1.5 m on either side of the transect.Yellow starthistle has composite flowers, which are aggregations of anywhere from 20–80 florets. At each site, 12 yellow star thistle buds were randomly selected from different plants and covered with a mesh bag. Yellow starthistle blooming cycles have been described in detail in other publications. We selected buds at stage BU-4, when buds had no yellow petals exposed, but had well-developed straw-colored spines. When in full flowering, 10 bags were opened for a 4 hour period from 10 am to 2 pm, while 2 were kept closed as controls to verify that self-pollination was not occurring. At the opening and re-closing of the bags, the number of florets that had their stigmas extended were counted. Later, when flowers were fully mature , seed heads were collected, and later dissected in the lab. 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 vega. The spatial auto-correlation of all bee visitor response variables was assessed by Mantel tests in R package ade4, using the average values for each time of day at each site. Spatial auto-correlation 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,u planting gutter 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 others. 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, and there may be multiple species that fit within the same morphotype that provide varying degrees of pollination services.

Going forward, as researchers across various scientific domains and sectors come closer to a unified definition of resilience and perhaps agree to the use of a standard checklist for designing and reporting on resilience studies, there is greater opportunity to harmonize the science and develop more empirical evidence of resilience outcomes.Optimizing performance also includes building resilience in order to enhance the ability to perform tasks and ensuring resilience in order to prevent illness, injury, and disease. Within the US Department of Defense, researchers are able to study different models of physical and psychological stress and the application of different nutritional interventions with Service Members throughout their careers. Various models of stress are introduced, including initial military training , advanced military training courses , service academies , and extreme environments , along with examples of various interventions and outcome measures collected to date. The importance of nutrition on readiness and resilience was identified in military populations more than a decade ago and continues to be of interest. Two specific examples are provided to further explore nutrition interventions aimed at optimizing performance in the Department of Defense. The first, a completed double-blind, randomized, placebo-controlled trial, used a calcium and vitamin D fortified food product to optimize bone health during initial military training of Marine Corps recruits. Using a supplement or food intervention for calcium and vitamin D, participants received 2000-mg calcium and 1000-IU vitamin D per day. The primary outcomes of the study showed that bone markers and vitamin D status improve, but the supplementation did not affect skeletal parameters. A second,bato bucket forth coming study aims to evaluate the effectiveness of adding spices and herbs to increase vegetable intake among junior-enlisted Service Members.

Their current research is focused on the stressors and challenges to enhance resilience. Using a cycle of basic science/discovery that advances to clinical trials with various review steps helps move the field of nutrition science forward in a “total force fitness” approach. Total force fitness was introduced as a framework to help Service Members, their families, and military units reach and sustain optimal, holistic health, and performance in a way that aligns with their mission, culture, and identity. Other examples of frameworks focused on a holistic approach to research include Whole Person Health proposed by the National Center for Complementary and Integrative Health, Whole Health developed by the Department of Veterans Affairs, and a recent consensus study report by the National Academies entitled Achieving Whole Health. A focus on improving resilience as a model outcome highlights the opportunities and complexities of conducting optimal health and nutrition research in this space.As the number and proportion of older adults in the population increase, the prevalence of age-related deficits in mobility and cognition also increases. Such deficits may be because of normal aging or to pathologic processes. For instance, cognitive impairments like declines in memory and speed of processing may result from normal brain aging or neurodegenerative diseases like dementia. When considering hallmarks of optimal nutrition and health, improving resilience from cognitive decline has strong promise and impact. Although the etiology of age-related mobility and cognitive changes is multifactorial, it is well established that vulnerability to oxidative stress and inflammation increases as we age. Strategies that target oxidative stress and inflammation may improve resilience to processes that lead to cognitive decline. For example, a healthy diet may help combat both oxidative stress and inflammation in the body, but a diet rich in bio-active polyphenolics from fruit, vegetables, walnuts, and coffee may be especially important in improving resilience and health outcomes. Polyphenols have antioxidant and anti-inflammatory activities, so consuming them could slow or prevent age-related changes.

As previously shown, foods high in polyphenols, e.g., dark-colored berry fruits, prevent age-related neuronal and behavioral deficits in animal models of aging. In particular, studies from animal models of aging have found that polyphenolic compounds from walnuts and berries hold promise in slowing—and perhaps even reversing—age-related motor and cognitive declines. These polyphenolics possess antioxidant and anti-inflammatory properties and may also influence the brain directly through various mechanisms, including altered cell signaling and increased neurogenesis, arborization of dendrites, and autophagy in the brain. In recent randomized, double-blind, placebo-controlled pilot studies in healthy older adults , blueberry or strawberry supplementation was able to improve some aspects of cognitive performance, but not gait or balance. In a randomized, double-blind, placebo-controlled trial in 44 healthy older adults , supplementation with freeze-dried blueberry powder for 3 months improved 1 measure of executive function and 1 measure of learning and memory. In a similarly designed trial, supplementation of freeze-dried strawberry powder in 39 healthy older adults for 3 months improved 2 measures of learning and memory compared with placebo but had no effect on executive function. Both trials found that berry powder supplementation did not affect mobility, including measures of balance and gait, likely because the study subjects had no mobility detriments at baseline. Berry supplementation did not decrease serum levels of inflammatory biomarkers compared with placebo, but when serum from berry-supplemented subjects was applied directly to cultured microglia cells, there was a reduction in LPS-induced inflammatory markers relative to placebo-treated subjects. Interestingly, the serum was protective when taken during fasting as well as post prandially. Although these studies are preliminary, they add to the evidence that berry supplementation may help protect against age-related cognitive declines. In addition to single nutrients, healthy dietary patterns have been shown to slow the rate of cognitive decline. In particular, the Mediterranean-DASH diet intervention for neurodegenerative delay diet, which highlights increased intake of plant-based foods, such as berries and green leafy vegetables, is associated with lower risk of cognitive impairment in older adults.

Further investigations examined mechanisms and other factors involved in the beneficial effects of berry fruits. For example, changes in circulating levels of specific phenolic compounds were correlated with changes in cognition. Furthermore, cognitive performance and inflammation were related, as serum collected from berry-supplemented animals reduced LPS-induced inflammatory-stress-mediated signals in stressed highly aggressively proliferating immortalized microglia in vitro relative to serum from placebo-fed controls, and nitrite levels following supplementation were positively correlated with cognitive performance. Therefore, the inclusion of additional servings of polyphenolic-rich foods, such as nuts and berries, in the diet may be one strategy to forestall age-related neuronal deficits, perhaps via decreases in inflammation and suppression of microglial activation, to help increase cognitive resilience and preserve cognitive function. Other non-nutritive natural compounds derived from plants should also be considered as bio-active compounds that contribute to optimal health and improving resilience. The BENFRA Botanical Dietary Supplements Research Center at Oregon Health & Science University studies Botanicals Enhancing Neurological and Functional Resilience in Aging. Two botanicals of interest are Centella asiatica and Withania somnifera . The Center has considerable experience with Centella asiatica, which is used in Ayurvedic medicine to improve memory. It is a popular dietary supplement for “brain health” and shows potential to be developed as a FDA approved “botanical drug” for the treatment of Alzheimer’s disease. The rational use of botanicals, whether as dietary supplements or botanical drugs, requires their evaluation through optimized clinical trials. These trials must be based on sound preclinical studies providing evidence for functional effects, mechanisms of action, and active compounds. The use of preclinical models is critical to inform the optimal design and implementation of future nutrient or botanical clinical intervention trials in healthy older adults and in patients with neurologic diseases, such as Alzheimer’s disease. However, due to the limitations of preclinical models in representing human health, disease, and responses, evaluation of the efficacy of an intervention through clinical trials in humans is essential. Research needs to focus on product authentication, identification of the biologically active compounds and their mechanisms of action, and detection of relevant biomarkers that translate to humans. Preclinical studies also need to address efficacy and safety of the botanical to advance translational research in cognitive resilience. Preclinical studies at Oregon Health & Science University have confirmed the cognitive effects of Centella asiatica in aged mice and that the antioxidant response gene Nrf2 is a molecular target of this herb. Triterpenes and caffeoylquinic acids have been identified as active compounds in C. asiatica and may account for its neuroprotection.

A phase I clinical trial examining the pharmacokinetics of Centella asiatica compounds in older adults with mild cognitive impairment was recently published. A recently initiated clinical trial will examine safety of C. asiatica and also characterize the biologic signatures of its cognitive effects in a population of cognitively impaired older adults. In the case of Ashwagandha, work at the BENFRA Center has focused on water and hydroethanolic extracts of the root,dutch bucket hydroponic as these preparations are commonly used in dietary supplements and in previously reported scientific studies. In one study, the effects of aqueous and hydroethanolic extracts of Ashwagandha root were compared in Drosophila melanogaster models of sleep, cognition, locomotion , and stress-induced depression. Treatment with the hydroethanolic extract improved age-related sleep fragmentation in male flies. Surprisingly, Ashwagandha root aqueous extract showed stronger effects than the hydroethanolic extract in Drosophila melanogaster models of cognition and locomotion and a model of stress-induced depression. Treatment with the aqueous extract of W. somnifera improved age-related locomotor declines in females at lower doses than the hydroethanolic extract. The aqueous extract also provided some resilience against stress-induced depression both when given prophylactically and continuously in a Drosophila model of depression. By contrast, the hydroethanolic extract was only effective when given continuously. The withanolides, commonly regarded as Ashwagandha’s active compounds, are present in greater amounts in the hydroethanolic than aqueous extracts. Together, this suggests that different Ashwangandha compounds may modulate the botanical’s effects on cognition, mood, and sleep and that compounds other than the well-known with anolides may be involved in some of its biologic effects. Studies are underway to explore these unknown active compounds in resilience to age-related cognitive decline and stress. Knowledge of the bioactive compounds associated with each potential clinical use of Ashwangandha will be important in optimizing products for clinical trials of Ashwangandha for those conditions. In summary, as we pivot to emphasize the promotion of optimal health, we need alternative indices of health besides disease outcomes. An individual’s ability to be resilient, including the ability to respond to stressors and to thrive and retain functionality while maintaining a high quality of life, should be considered. Moreover, both essential nutrients as well as other nonessential bioactive compounds should be considered as key factors that promote optimal health.As we define optimal health, it is clear that the potential solutions will vary depending on many individual and environmental factors. Nutritional interventions in healthy adults are known to produce a variety of responses, and work is underway to identify and characterize the different phenotypes that result in unique metabolic needs, with the goal to design personalized dietary approaches to maximize individual health. Although reasonable skepticism regarding the consumer readiness for precision and personalized nutrition exists, efforts to better illuminate the goals and challenges to this emerging technology are being openly discussed in the research community. For instance, in August of 2021, the National Academy of Science, Engineering, and Medicine held a public workshop titled “Challenges and Opportunities for Precision and Personalized Nutrition”. At this workshop, participants raised important perspectives on current opportunities and information gaps in our understanding and approaches to variability in nutritional responses, the shift in the personalized nutrition industry, and numerous studies demonstrating the potential utility for research in this area to aid in our understanding of variable nutritional responses as well as how in certain circumstances they may be beneficial in tooling both dietary guidance for glucose control and therapeutic interventions for weight loss. Addressing these knowledge gaps and refining our expectations and applications for individualized nutrition will be critical to realizing the full potential of this knowledge as another tool in our arsenal to improve human health. In 2020, the NIH also launched a new strategic plan for nutrition that emphasizes Precision Nutrition. In 2022, the NIH invested $170M in a new Precision Health program that leverages the All of Us research program and will allow unprecedented opportunities to combine metabolism, microbiome, diet assessment methods, and data sciences together to provide new insights in precision nutrition. Instead of a one-size-fits-all, we can envision a time where a person’s unique characteristics will be effectively used in a proactive approach to health promotion and disease prevention, and importantly, allowing personalized strategies based on these characteristics.

The family Betaflexiviridae comprises twelve different genera , including Vitivirus, Trichovirus and Foveavirus. After in-silico and in-vitro analyses of trichoviruses and foveaviruses, we did not find evidence for cross-reaction by the universal assay. A single test for all known grapevine vitiviruses can be a useful tool for improving efficiency and reducing costs of large-scale surveys. Potentially, this generic assay may detect novel Vitivirus species in grapevine and other hosts given its unbiased nature. Similar assays have been developed for carlaviruses, nepoviruses and different members of the family Betaflexiviridae. Grapevine is clonally propagated, consequently, to prevent the spread of vitiviruses, it is critical to use virus tested material. The assay developed here will be made available to diagnostic labs and will facilitate the production of certified virus-tested propagation material and the effective control of vitiviruses.As people age, their risk for cognitive decline increases. An estimated 40% of people 65 years and older have age-associated memory impairment characterized by self perception of memory loss and a standardized memory test score demonstrating lower objective memory performance compared with young adults. While genetic factors play an important role in age-related memory decline, such nongenetic lifestyle factors as diet and exercise contribute as well. In particular, a growing body of evidence indicates that accumulation of oxidative damage to macromolecules increases progressively during the aging process and contributes to neurodegeneration in Alzheimer’s disease. Epidemiological studies have suggested a link between antioxidant consumption and cognitive protection,blueberry packing boxes and studies using a rodent model of Alzheimer’s disease suggest that tau phosphorylation occurs as a compensatory response to oxidative stress. Several clinical trials of antioxidant use in subjects with normal aging or Alzheimer’s disease suggest memory benefits, while others yielded negative results.

While many fruits are rich in various antioxidants, including ascorbic acid, carotenoids, and phenolics, commonly consumed fruits show large differences in antioxidant capacity, as determined by the ferric reducing/antioxidant power assay. In particular, there is special interest in the pomegranate polyphenols, which have been studied extensively in animal models of Alzheimer’s disease. The emerging data on pomegranate fruits and their inherent polyphenols suggest positive benefits ranging from neuroprotective effects to staving off effects of senescent neurodegeneration in animal models of Alzheimer’s disease. Previously, we found that pomegranate juice has the highest antioxidant capacity among fruit juices using five different in vitro antioxidant assays. In comparison, apple juice, which is low in antioxidant capacity, was less effective than pomegranate juice in affecting antioxidant capacity based on FRAP assays in 26 elderly subjects consuming pomegranate juice or apple juice over a 4-week period. Over expression of antioxidant enzymes or supplementation of some antioxidants appears effective in extending the life span in several animal models, and a few studies have specifically found memory-enhancing effects of polyphenols in mice . However, evidence for memory enhancement from polyphenols in human-controlled trials is limited. One preliminary study found beneficial effects of grape juice on memory in five older adults with ageassociated memory impairment compared to seven placebo controls; to our knowledge this small study is the only placebo-controlled human trial of polyphenols that assessed memory change. Therefore, more well-controlled studies are needed to determine potential beneficial effects of antioxidants that occur naturally in various food sources and to elucidate the possible underlying mechanisms. Moreover, studies that examine possible brain mechanisms of polyphenol treatment in humans are limited. One potential method for identifying mechanisms of recovery in drug trials is functional magnetic resonance imaging .

While structural MRI studies measure long-term changes in metrics, such as cortical thickness and volume , fMRI measures blood flow changes in response to memory challenge, which has proven to be effective in determining brain differences among subjects with Alzheimer’s disease, mild cognitive impairment, or non-demented subjects at risk for Alzheimer’s disease. Further, fMRI has proven to be sensitive in measuring changes in neural activation patterns among older subjects in response to memory-enhancing drugs, such as donepezil and memantine, with results showing increased activation or restoration of resting state brain networks following treatment. One investigation used fMRI to examine the effects of antioxidants in young healthy controls and demonstrated increased fMRI activity to cognitive challenge with flavonoid administration; however, no studies to date have examined fMRI effects of antioxidant therapy in older adults with or without memory complaints. To address this knowledge gap, we explored potential neuroprotective effects derived from polyphenols in pomegranate juice in older volunteers with mild memory complaints in a placebo-controlled, randomized, and double blind trial and measured three aspects: metabolites of pomegranate juice using blood biomarkers; effects of pomegranate juice on memory performance; and evidence of functional MRI changes during memory activation. We hypothesized that pomegranate juice compared to placebo would increase metabolites, improve memory, and increase task-related brain activation in the left and right hemispheres for verbal and nonverbal memory tasks, respectively. Together these metrics provide preliminary evidence of bio-availability, clinical efficacy, and brain mechanisms of pomegranate juice in older adults with memory complaints.Thirty-two volunteers were initially enrolled into the study out of 39 who were screened; of these, 28 completed the clinical trial . Subjects were recruited via advertisements through local newspapers, flyers, posters, lectures, and word-of-mouth. We specifically recruited nondemented, older right-handed subjects with self-reported age-related memory complaints. The subjects did not carry a diagnosis of mild cognitive impairment and were screened using the mini-mental state examination . Subjects randomized to placebo versus control groups did not differ in mean age, percentage of women, mean MMSE scores, or baseline memory performance .

Potential subjects were excluded who had documented neurological, psychiatric, or major medical conditions, including heart disease, diabetes, and gastrointestinal disease, that might affect cognitive function or interfere with study procedures. We also excluded subjects who routinely ate more than three servings per day of fruits and vegetables or were taking vitamin supplements, any medication, or dietary supplement that interferes with the absorption of polyphenols. Potential subjects participating in regular vigorous exercises other than ordinary daily walks or who were unwilling to maintain a sedentary lifestyle during the 28-day study were excluded, as were those for whom MRI was contraindicated . Seven potential subjects were screened out based on one or more of these exclusion criteria. Subjects were instructed that they would be removed from the study if they failed to follow the prescribed low polyphenol diet during the one-week run-in period based on self-report in-person interview with a study dietitian. The study was approved by the UCLA Institutional Review Board; all subjects gave informed consent to participate in all procedures and were reconsented for followup evaluations. Funding for this study was provided by the UCLA Center for Human Nutrition from a “various donors” account. The investigators were blind to the contributors to this fund, and there was no communication or input from donors or commercial entities at any stage of the study.Following phone screening, volunteers participated in an initial clinical evaluation,package of blueberries which included an explanation of the study procedures, consent, medical screening, MMSE, and routine CBC and chemistry panel. Prior to the intervention phase, each subject met with a registered dietitian and was instructed on a low polyphenol diet, which required that they restrict their intake of several fruits and vegetables, onions, tea, chocolate, and dried beans, for one week prior to the baseline visit and for the duration for the study. At baseline, subjects underwent neuroimaging and neurocognitive testing . We also acquired blood samples to perform the antioxidant assay and to verify compliance with the protocol. Subjects were then randomized to either the pomegranate or placebo groups; investigators and subjects were blind to group membership. After these procedures, subjects were given a one-week supply of 8-ounce containers of pomegranate or placebo juice with instructions to consume 8 ounces of juice daily. The pomegranate drink was the commercial Pom Wonderful product. The flavor-matched drink contained sugar, citric acid, and food color to match the calorie, taste, and color of the juice. Each week, subjects returned their empty juice containers to ensure compliance, picked up their juice supply for the following week, and met briefly with the dietitian to ensure compliance with the low polyphenol diet. At the end of the study , subjects again underwent neuroimaging, cognitive assessment, and blood draws. A placebo drink using sugars and natural colorings and flavoring to imitate the taste and appearance of pomegranate juice was used. The research personnel were blinded as to the assignment of subjects to placebo or pomegranate juice.Each subject underwent fMRI and brief memory testing immediately prior to beginning the trial and following 28 days of pomegranate juice or placebo. Subjects received a memory testing battery and underwent functional MRI scanning on the same day. Memory tests administered were the Buschke-Fuld selective reminding task, from which we calculated two performance measures, total items recalled and consistent long term retrieval; an experimental unrelated word pair associates learning task, based on the Wechsler memory scale paired associates learning in which we used identical procedures to that used in the fMRI scan but with different stimuli. We also obtained behavioral data from the MRI tasks administered in the scanner .

Of the 28 subjects completing the study, 26 had behavioral data on both occasions . Subjects were administered one of two alternate forms of each task, with the order of forms counterbalanced across subjects, to avoid practice effects. To assess changes in verbal memory, we compared performance on total number of items recalled on the Buschke-Fuld selective reminding task and on the consistent long term retrieval score . The TR score emphasizes immediate recall, while CLTR depends upon memory consolidation processes. Because memory performance in this age range is highly variable, we used time 2 minus time 1 difference scores as our dependent variables. At baseline, there were no differences between groups on either TR performance or CLTR . The spatial navigation task involved learning and recalling routes on a virtual map, while subjects play the role of a “taxi driver.” During the encoding phase, subjects watched and rewatched two sets of video clips. In each video, subjects first saw the name of the imaginary town, such as “Johnsberg,” then for 15 second blocks they saw a first-person view of “driving” to a virtual town laid out in 5×5 block grids, to a storefront destination with unique characteristics for specific storefronts, stopping for 3 seconds before proceeding to a new store within the same town starting from a different location. After each city, subjects performed a 30-second distractor task where they pressed a left or right button in response to a probe. This was repeated while subjects learned 6 cities in total. The fMRI data were then collected during memory retrieval in three conditions. Subjects were asked to remember one of three characteristics about each storefront for the separate tasks. Two conditions focused on the physical features of the environment . In condition 1, participants were asked to identify an appearance change of the store , such as signs, colors, and doors. In condition 2, they were asked to identify the spatial location change of the store within the town, relative to other buildings , respectively. Condition 3 required subjects to identify the store for a specific item that might be purchase there, requiring the participant to encode the association between an item and a spatial location. For this source task, subjects had to recall whether the store was in the first or second town . Only the latter condition tapped into the associative memory processes. The order of the three conditions was counterbalanced across subjects.All functional data were analyzed using FSL version 4.1.4 . Preprocessing included motion correction to the mean image, spatial smoothing , and high-pass temporal filtering .Twenty three subjects were able to complete the visual memory fMRI task without significant head motion, while for the verbal memory task, eight pomegranate juice and nine placebo subjects were able to complete both scanning sessions with high quality images. Each functional scan was registered to its corresponding coplanar high-resolution image with rigid body transformations and to the MNI152 standard brain using nonlinear transformation . Additionally, a high-pass temporal filtering of 100 sec was applied to the functional images. Individual preprocessing and statistical analysis of each subject’s functional scan was run using FSL FMRI Expert Analysis Tool ; FEAT was then used to analyze data at the group level.

Given the previous epochs from 1 to 8, has values for their F1 Score and Accuracy. It shows that there is possible room to improve the model with more fine-tuning and possibly an early stop in training to not have the model over-train to get the result of 1.0 for F1 Score and Accuracy. Another possible reason for the result could be the large imbalance of images between classification labels and that the Plant Village data set is considered to be a small data set. These types of results are good but not realistic for model prediction power. Even though these values are incredibly high, the training and validation loss is still fairly low and slowly converging to 0. This means that the model is learning over time, which is a good sign and shows room for improvement. A possible future improvement for this project would be to find another data set with more specific plant disease information to train the model on. Efforts will be made to look for data that includes more plant pests. That variation would be beneficial because the data set for this project mainly contains crop disease images. The disease Spider Mite, is labeled as a disease in this data set but in reality, is not a disease. It is the only classification label that has pest-inflected crop images in this data set. Having more data on crop pests would be beneficial because crop pests also cause a significant amount of crop loss and damage as well. Technical improvements for this project include developing a stronger data augmentation technique. Instead of using the set transform method which artificially augments the data set,garden grow bags using another package that can actually create the separate images and add them to the data set instead would be interesting to see how it would perform. More Hyper-parameter fine-tuning could be done such as exploring the learning rate and adding an optimizer. More Epochs should be tested in order to see how the model will perform over a greater period of time.

Fewer epochs will also be tested to see how early-stopping the model from training will perform. Another idea is to explore more the Training-Validation-Testing splits chosen for the data. Exploring the performance between different splits could show how to better improve the classification model. To achieve our expected agricultural need of feeding 10 billion people by 2050, we must prioritize minimizing crop loss wherever possible. More research is needed to help develop more tools to assist crop growers with preventing crop loss. The study “Mobile phone use is associated with higher smallholder agricultural productivity in Tanzania, East Africa” by Amy Quandt et al. looks into the relationship crop growers have with their cell phones as agricultural tools to help increase crop yields. “A key result is the positive association between phone use for agricultural activities and self-reported agricultural yields”. Cell phones are increasing accessibility to technological tools that help with agriculture. These technologies for assisting with crop loss will not only be utilized by commercial crop growers or the average hobbyist and enthusiast as well. Whether crop growers use a ViT image classification model or a convolutional neural network image classification model, or another type of machine learning architecture is used, more research is needed to help develop tools to assist crop growers worldwide in eradicating crop loss everywhere! The original Specialty Coffee Asscn. of America Coffee Taster’s Flavor Wheel was developed in 1995 by Ted Lingle, before many advances and methods in sensory science had been developed . To revise this longstanding industry tool, sensory science and statistical methods were applied as novel flavor wheel construction techniques. Even today, across food and beverage industries, very few flavor wheels exist that were created using a scientific approach and a sensory lexicon. A lexicon is a list of vocabulary developed using sensory descriptive analysis methods used to describe a product, along with descriptions of each attribute and reference preparation instructions .

Some notable existing flavor wheels have been created using sensory lexicons, for products such as beer, wine, tea, spices, and even drinking water, but the flavor wheel construction methods differed from those used in the current study Suffet and others 1999; Gawel and others 2000; Koch and others 2012; Lawless and others 2012. Lawless and others used similar statistical techniques to those used in this study to develop the McCormick Spice Flavor Wheel; however, the data used were simply a subset of descriptive analysis data gathered from lexicon development, with no sorting task. In the development of a tea flavor wheel, Koch and others used all descriptive analysis data to perform principal component analysis to determine the positioning in the flavor wheel, but no clustering techniques or sorting exercises were utilized. Gawel and others did use a sorting task, as in this study, for mouthfeel attributes in wine, but slightly different clustering statistical techniques were used, and only 9 experts participated in the sorting exercise. Prior to this project, SCAA and World Coffee Research worked with sensory scientists, industry representatives, and trained panels of judges at the Sensory Analysis Center at Kansas State Univ. and Texas A&M Univ. to develop a lexicon of about 110 attributes to describe flavor , texture/mouthfeel, and amplitude . The WCR Sensory Lexicon was then sent to UC Davis to be sorted into categories and levels to be converted into an updated Coffee Taster’s Flavor Wheel. The words in a flavor wheel serve to standardize training and aid in education and discussion. The original Coffee Taster’s Flavor Wheel has served as a communication tool about coffee products among all components of the industry, including tasters, plants, retailers, exporters and importers, producers, baristas, and consumers. The new Coffee Taster’s Flavor Wheel will serve as an improved communication tool, as it is an organized visualization of the WCR Sensory Lexicon . This tool is the 1st step toward enabling the coffee industry to identify and characterize specific flavor changes and relate these changes to specific variables in the coffee process, which brings us one step closer to understanding which factors drive coffee flavor.Although this type of scientific conversion from lexicon to flavor wheel was unprecedented, existing sensory and statistical methods were also applied for the purposes of this study.

A rapid sensory profiling method sans tasting, called single free sorting, was utilized to determine the similarities and dissimilarities among the 99 coffee flavor attributes. Once the data from the individual sorting tasks were collected and summarized, 2 multivariate statistical techniques were applied. First, to determine the major categories, subcategories, and levels, agglomerative hierarchical clustering was used. Conjointly, to determine the arrangement of these categories and subcategories in the wheel structure, multidimensional scaling was used. AHC and MDS are both techniques used in sensory science to observe and visualize the similarities between different products, consumers, or attributes . In this way, existing sensory and statistical methods were adapted to create a novel method for constructing a flavor wheel from a defined lexicon.Twenty-nine trained descriptive analysis panelists were contacted and recruited from other descriptive studies already in progress at UC Davis. These panelists were not required to be trained specifically on coffee, but they were required to be regular coffee drinkers, had all participated in descriptive analysis on products with complex flavors, and had worked with and been exposed to most of the flavor attributes in the WCR Sensory Lexicon. Panelists were not further trained for this experiment,tomato grow bags because group discussions may have allowed the more opinionated panelists to influence the decisions of the quieter panelists. For this experiment, it was decided to simply allow panelists to draw on their individual experiences and subsequently compile and average all the data, rather than holding group discussions and coming to a group consensus. Recruitment, screening, and scheduling were done via email. Once accepted into the study, participants were sent written instructions to perform the sorting task on the web app remotely from their personal computers. The entire process was online and remote. In order to accurately reflect the coffee industry needs, create an additional set of data, and add more statistical power, 43 coffee industry experts recruited by SCAA performed the same online procedure as the UC Davis panelists. The industry panelists came from all areas of the coffee industry and they all had experience as coffee tasters, but not all of them had experience in sensory descriptive tests.Before the sorting task began, the WCR Sensory Lexicon was reduced to 99 flavor attributes, removing all attributes not exclusively referring to flavors. Specifically, the attribute “astringent” and all attributes in the Texture/Mouthfeel and Amplitude sections were removed. The word sort procedure was originally done in a Steinberg study , to be used as a tool for semantic analysis, particularly regarding connotations. This sorting method has since been adapted to food samples for sensory analysis .

Traditionally, panelists are asked to sort food products or other samples into as many clusters or groups as they choose, in a way that makes sense to them . In free multiple sorting, a rapid sensory descriptive method, panelists repeat this procedure until they feel they have exhausted the sorting possibilities, and then they are asked to provide descriptions for each group of samples . In a study comparing single sorting to multiple sorting, Rosenberg and Kim found that multiple sorting was superior in representing all possible dimensions of categorization of the data. Additionally, one drawback to using single sorting is that the individual data need to be summed together in order to analyze it, so individual data are lost . In this experiment, instead of sorting food samples themselves, panelists were asked to sort the attributes into categories and subcategories without tasting samples and therefore based on their experience and expectations of these flavor descriptors. Thus, this sorting exercise was similar to the original word sort procedure performed by Steinberg in 1967. Sorting the words themselves was appropriate in this case, due to the ultimate goal of using the Coffee Taster’s Flavor Wheel as a tool for coffee industry professionals. Since there was no tasting, fatigue, adaptation, and carryover effects did not bias the data . Additionally, as there were 99 attributes, to avoid fatigue, instead of repeating the procedure multiple times, the panelists each only sorted the lexicon once. A user-friendly web interface was created using AngularJS to allow for simple, efficient sorting of the 99 flavor attributes. This helped to minimize the clutter of index cards and catalyze the data collection process, because data could be stored immediately in Firebase , a free database and web application hosting service. The website had a welcome page and the participants would log in and be greeted with brief instructions and a “begin” button. The users would then see further instructions and the list of attributes, each with Table 3–RV-coefficients from multiple factor analysis comparing UC Davis and coffee industry participants. Industry UCD MFA Industry 1 0.414 0.832 UCD 0.414 1 0.850 MFA 0.832 0.850 1 a question mark to the far right with a scroll-over pop-up with the WCR definition of that attribute. If a user was unclear about the meaning of one of the words of the lexicon, he/she could scroll over the information bubble to access the definition. The participant was able to click and drag the attributes into categories and subcategories, for as many hierarchical levels as they deemed necessary. Once the user felt the attributes were adequately sorted into categories and subcategories, they would press “submit” and the results were immediately stored in the Firebase database.The methods of AHC and MDS were used to represent attribute–attribute relations instead of product–attribute relations, because there were no coffee samples, only attributes. To organize the raw data, a program was written in Ruby to translate the sorting data into matrices that could be used for analysis. For both of these methods, 1st, 2 binary matrices were created for each participant , one for “sibling–sibling” relationships, in which the attributes appeared in the same subcategory, and one for “parent–child” relationships, in which one attribute appeared in a subcategory under another attribute.

In summary, DP completely prevented cancellous bone loss caused by irradiation over this short duration study . Our studies demonstrated that DP diet supplementation was equally effective at preventing the skeletal responses to both low-LET gamma radiation, at a dose equivalent to a single fraction of radiotherapy, or combined proton and HZE ions, simulating space radiation. Therefore DP or its components may provide effective interventions for loss of structural integrity caused by radiotherapy or unavoidable exposure to space radiation incurred over long duration spaceflight.Animals. Male C57BL/6J mice experiments at 16 weeks of age were randomized by weight, individually housed, and assigned to groups . Food and water were made available ad libitum and mice were housed on a 12 hours light/dark cycle. Body weights and food consumption were measured throughout the experiments to monitor animal health . The NASA Ames Research Center and the Brookhaven National Laboratory Institutional Animal Care and Use Committee approved all procedures, and studies were conducted in accordance to the IACUC health and ethical standards. Diets. Diet compositions are shown in Fig. 7. The control diets included the following: Control Diet 1 was LabDiet 5001; Control Diet 2 was purified AIN93G and was used as a control for the AOX-supplemented diet of A. Kennedy and colleagues. Control Diet 3 was AIN93M and served as the control for the DP-supplemented diet. The custom antioxidant diet , was prepared by a commercial vendor based on a previously reported diet composition,plastic flower pots with the base AIN93G diet supplemented with five antioxidants: ascorbic acid , N-acetyl cysteine , L-selenomethionine , dihydrolipoic acid , and vitamin E . All antioxidants were obtained from Sigma .

The DP diet was composed of 25% by weight powdered dried plum and was prepared by Teklad as reported by Halloran et al.40. Analysis of custom diets. The candidate interventions were selected in part on the basis of their antioxidant properties. Antioxidant capacity of the custom diets was measured using a Total Antioxidant Capacity measurement assay. In this assay, levels of Trolox equivalents are positively correlated with antioxidant content. DP and DHLA diets had significantly higher antioxidant capacities compared to their respective controls. Specifically, Control diet 1 contained 0.05mM Trolox equivalent per mg of protein. Control diets 2 and 3, CD2 ; and CD3 had lower TAC compared to CD1, 56% and 41% respectively. Both the antioxidant cocktail and dried DP diets had 84% and 74% increased TAC compared to CD1, and their respective controls at 323% and 196% TAC. The AOX and DP diets had comparable TAC’s . Feeding and injection protocols. Mice were separated into groups and fed various diets or injected with DHLA , Ibuprofen . Mice were pre-fed 7 days , 17 days or 21 days with the diets , AOX or DP prior to total body irradiation . The differences in pre-feeding periods were based on previous published findings showing effective prevention of radio damage by AOX diet or bone loss prevention by DP. Feeding with the corresponding diets was continued until euthanasia. For the injection protocols, DHLA or Ibuprofen was delivered via subcutaneous injection twice a day, starting one day prior to TBI as previously reported. In all experiments, mice were irradiated at 16 weeks of age and tissues harvested 1 day later for the gene expression analysis, and 11 days later for the microCT analysis .Radiation exposure. Conscious mice were exposed at 16 weeks of age to 2 Gy Gamma or with 1 Gy of protons and 56Fe ions delivered sequentially to simulate space radiation. The sequential ion exposure was comprised of an initial exposure to 25cGy of 1 H , then 50cGy of 56Fe , and, finally, 25cGy of 1 H; for a total dose of 1Gy, and was performed at the NASA Space Radiation Laboratory at Brookhaven National Laboratory , NY. This exposure regimen was designed to simulate space radiation combining both low-LET and high-LET particles.

Controls were handled identically to the irradiated animals with the exception of exposure to the radiation source, and are referred to as ‘sham-irradiated’. Gene expression. Flushed bone marrow cells from the femora and tibiae were pelleted and lysed in RLT buffer with 1% beta-mercaptoethanol and stored at −80C. Total RNA was extracted using QIAshredder, and RNeasy mini kit . For each sample, RNA was treated with RNase-free DNase Set . RNA quantity was determined using a spectrophotometer and quality confirmed by 2100 Bioanalyzer . Equal amounts of RNA were reversed transcribed, followed by qPCR using GoTaq® RT-qPCR System . Taqman gene expression assays were used : nuclear factor, erythroid 2-like 2 , receptor activator of nuclear factor kappa-B ligand , osteoprotegerin , tumor necrosis factor alpha and monocyte chemotactic protein-1 . The relative gene expression was quantified using the comparative threshold cycle method with normalization to expression levels of glyceraldehyde 3-phosphate dehydrogenase . The reactions were performed in a 7300 RT-PCR System as described previously. Microcomputed tomography . Tibiae were dissected, cut distal to the TFJ to allow PFA infiltration and fixed for 24 hours at 4 °C, followed by storage in 70% Ethanol. The bones were transferred to phosphate-buffered saline and then the proximal metaphysis scanned and analyzed as previously described with a 6.7 μm/voxel resolution using a SkyScan 1174 microCT scanner . For cancellous bone, a 1.0mm thick region located 0.24mm distal to the proximal growth plate of the tibia was selected and semi-autonomously contoured to include cancellous tissue. To assess cancellous bone loss, the bone volume to total volume fraction , trabecular thickness , trabecular number , and trabecular separation were calculated and reported following conventional guidelines. To measure possible changes in cortical features that contribute to whole bone mechanical properties, bones were scanned at 14.6μm/voxel resolution starting at midshaft 2mm proximal to the tibia–fibula junction over a 0.3mm height. Parameters reported include cortical bone cross-sectional area , bone periosteal perimeter , cortical thickness and mean polar moment of inertia .

Tissue mineral density was calculated using the linear attenuation coefficient and calibrated phantoms for diaphyseal cortical bone. Statistics A one-way or two-way analysis of variance was performed as indicated in the legends, with treatment and irradiation as main effects. Where the main effect P< 0.05 by 1-factor ANOVA, or interaction effects by 2-factor ANOVA, differences between groups were analyzed by Dunnett’s post-hoc test comparing experimental groups to the non-irradiated controls, or all pairs Tukey-Kramer . All data are presented as mean and standard deviations.To be labeled as a fruit juice, the US Food and Drug Administration mandates that a product be 100% fruit juice. For juices reconstituted from concentrate, the label must state that the product is reconstituted from concentrate. Any beverage that is less than 100% fruit juice must list the percentage of the product that is fruit juice, and the beverage must include a descriptive term, such as “drink,” “beverage,”or “cocktail.” In general, juice drinks contain between 10% and 99% juice and added sweeteners, flavors, and sometimes fortifiers, such as vitamin C or calcium. These ingredients must be listed on the label according to Food and Drug Administration regulations.Water is the predominant component of fruit juice. Carbohydrates, including sucrose, fructose, glucose, and sorbitol,plastic garden container are the next most prevalent nutrients in juice. The carbohydrate concentration varies from 11 g % to >16 g % . Human milk and standard infant formulas have a carbohydrate concentration of 7 g %. Juice contains a small amount of protein and minerals. Some juices have naturally occurring high contents of potassium, vitamin A, and vitamin C. In addition, some juices and juice drinks are fortified with vitamin C. Juices fortified with calcium have approximately the same calcium content as milk but lack some other nutrients present in milk, such as magnesium and a substantial amount of protein. Many such calcium-fortified juices also are fortified with vitamin D. The vitamin C and flavonoids in juice may have beneficial long-term health effects, such as decreasing the risk of cancer and heart disease.‍ Drinks that contain ascorbic acid consumed simultaneously with food can increase iron absorption by twofold,which may be important for children who consume diets with low iron bio-availability. Juice contains no fat or cholesterol, and unless the pulp is included, it contains no fiber. The fluoride concentration of juice and juice drinks varies. One study found that fluoride ion concentrations in juice ranged from 0.02 to 2.8 ppm.‍The fluoride content of concentrated juice varies with the fluoride content of the water used to reconstitute the juice. Some manufacturers specifically produce juice for infants. These juices do not contain sulfites or added sugars and are more expensive than regular fruit juice. Other forms of fruit juices are frequently consumed. Up to one third of adolescents consume sport drinks, and approximately 10% to 15% consume energy drinks.Pediatricians should inquire about the use of these products as they assess the nutritional intake of their patients.Juices from many fruit contain flavonoids , which can decrease the activity of several enzymes and transport proteins important in drug disposition.‍

Although the ingestion of grapefruit juice has been shown to reduce the activity of intestinal cytochrome P450 3A4 and produce potential drug-nutrient interactions of drugs that are CYP3A4 substrates , recent evidence suggests that grapefruit juice can also inhibit organic acid transporter activity.In addition to grapefruit juice, flavonoids present in oranges and apples have also been shown to reduce the activity of the organic acid transporter OATP2B1.Although the grapefruit juice–CYP3A4 substrate interaction and the potential for producing significant nutrient-drug interactions is the most well characterized, it should be noted that, in addition to inhibiting CYP3A4 activity, cranberry, pomegranate, and blueberry juice can inhibit the activity of CYP2C a cytochrome P450 isoform that catalyzes the bio-transformation of therapeutic drugs such as ibuprofen, flurbiprofen, warfarin, phenytoin, fluvastatin, and amitriptyline. The clinical significance of any of the aforementioned juice-drug interactions is extremely difficult to predict on the basis of a history of coingestion. Substantial variability in the duration and magnitude of resultant interactions is a function of multiple factors, including the following: constitutive expression of the effected enzyme or transport protein, significant genetic polymorphism in the enzyme or transporter, the relative flavonoid composition and potency among different juices, and the amount of juice ingested and its duration of ingestion .In evaluating the potential juice-drug interactions, the coadministration of fruit juice and a drug for which metabolism or transport could be affected by a flavonoid should not be considered immediately as a contraindication for treatment. The amount and type of juice being ingested,9specific information characterizing a given interaction, and whether the drug being taken has a low or high therapeutic index must be considered in the evaluation of a potential interaction. Consultation between the physician and pharmacist can be beneficial in considering the potential clinical significance of a juice-drug interaction.The 4 major sugars in juice are sucrose, glucose, fructose, and sorbitol. Sucrose is a disaccharide that is hydrolyzed into 2 component monosaccharides, glucose and fructose, by sucrase present in the small bowel epithelium. Glucose is then absorbed rapidly via an active-carrier–mediated process in the brush border of the small bowel. Fructose is absorbed by a facilitated transport mechanism via a carrier but not against a concentration gradient. In addition, fructose may be absorbed by a disaccharidase-related transport system, because the absorption of fructose is more efficient in the presence of glucose, with maximal absorption occurring when fructose and glucose are present in equimolar concentrations.‍Clinical studies have shown this process, with more apparent malabsorption when fructose concentration exceeds that of glucose than when the 2 sugars are present in equal concentrations .‍However, when provided in appropriate amounts , these different juices are absorbed equally as well.Sorbitol, found in small amounts in pears, apples, cherries, apricots, and plums and in sugar-free foods and some liquid medications, is absorbed via passive diffusion at slow rates, resulting in much of the ingested sorbitol being unabsorbed.Carbohydrate that is not absorbed in the small intestine is fermented by bacteria in the colon. This bacterial fermentation results in the production of hydrogen, carbon dioxide, methane, and the shortchain fatty acids acetic, propionic, and butyric. Some of these gases and fatty acids are reabsorbed through the colonic epithelium, and in this way, a portion of the malabsorbed carbohydrate can be scavenged.‍Nonabsorbed carbohydrate presents an osmotic load to the gastrointestinal tract, which causes diarrhea.‍ 19

After treated leaf exposure to intense UV light for 120 min, high levels of mite repellency with the spinetoram treatment dropped in persistence from 10 d to 3 d. Mite mortality after treated leaf exposure to UV light was reduced to control levels on abamectin treated leaves by the time of the day 1 bioassay . Because mites trapped in the felt were excluded, percent mite mortality on spinetoram versus spinetoram-UV treated leaves must be interpreted carefully in Fig. 4-7. Based on data from Fig. 4-3 , Fig. 4-8 depicts a more accurate assessment of the results when mites were placed on UV treated spinetoram leaves because it combines the mortality and repellency . There was no effect of the leaf side treated or leaf side the mites were placed on for both treatments and therefore, data were pooled and the new response variable for each treatment became ‘mites deposited on treated side’ versus ‘mites deposited on untreated side’. There was no significant variation observed between the three leaf replicates on any bioassay date and thus, replicate data were pooled. Based upon repeated measures analysis, each response variable showed significant impacts by day , time and location . With spinetoram treatment, there were more mites alive on the untreated side versus the treated side of the leaf on days 1, 3, 7 and 10 . However, on day 14, there was no difference between the numbers of mites on the treated versus the untreated side of the leaf for any of the observation times. The mites in the control treatment were distributed similarly across both sides of the leaf on all days and times . With spinetoram treatment, mite mortality was different from the control on both the treated and untreated sides of the leaves. On the spinetoram-treated side of the leaf,flower bucket mite mortality by day was significantly different from the control treatment for days one through 10 but on day 14 mite mortality was no longer different .

The same pattern was observed for the untreated side of the spinetoram leaves, i.e. mites were dying at higher levels versus control leaves on both the untreated and treated sides of the leaf. In the control treatment, mite mortality was not different between the water treated and untreated sides of the leaf . Mite repellency on the spinetoram treated and untreated sides of the leaf were different than seen on control leaves . On the spinetoram treated side, mite repellency by day was different from the control for days one and three as well as day 7 and day 10 but not for day 14 . Mite repellency on the spinetoram treated side of the leaf by day for each level of time was not different from the control for times 20 min through 10 hours , but was different for each observation interval from 24 hrs through 5 days . On the spinetoram untreated side of the leaf, mite repellency by day was different from the control for days 1, 3, and 7 as well as day 10 but not for day 14 . Mite repellency on the spinetoram treated side of the leaf by day for each level of time was not different from the control for times 20 min through 10 hours , but was different for observation intervals of 24 hrs through 5 days . In our assessment of the four pesticides currently recommended for avocado thrips management , we found that all four products had some negative effects on E. hibisci. Mite exposure to abamectin resulted in relatively high mortality within the first two weeks of the bioassay and dropped sharply; presumably as translaminar movement of the material took place and the ultraviolet light rays broke down surface residues . Fenpropathrin treatment showed the longest and highest amount of activity. Spinetoram was the only material to which the mites exhibited strong repellency and when mites were bioassayed in the Munger cells with spinetoram, mortality was high and consistent with the pattern observed with the repellency over the first two weeks of the bioassays. Mites exposed to sabadilla, a chemical commonly thought to have little non-target effect , showed higher mortality and longer persistence than expected. However, this could be due to the mites feeding on the pesticide-laced sugar on the leaf surface, as sabadilla is formulated with sugar .

Data clearly showed that exposing the treated, field-weathered leaves to UV light increased the survival of the mites on both abamectin and spinetoram-treated leaves. Mite mortality to the UV-treated abamectin leaves was no different than with control leaves on day one of the bioassay , indicating that surface residual activity had been eliminated. With spinetoram treatment, mite repellency and mortality were reduced from 14 d to 10 d. The chief differences in spinetoram from its analog spinosad are: 1) the addition of the 3’-O-ethyl group, which improves potency by altering nicotinic function in the insect nervous system and 2) hydrogenation of the 5,6 double bond, which improves photostability of the molecule and thereby increases residual control . Our data show that these modifications increased the longevity of the material on the leaf surface but with intense UV exposure, that activity was broken down to some degree. The bioassays evaluating mite detection of spinetoram on the leaf surface clearly showed more mites alive on the untreated side of the leaf than the treated side, indicating that the mites were able to detect the material and move away from it. There were fewer mites drowning in the wet felt in the spinetoram detection bioassay on day one than seen in the initial field trial bioassay on day one, indicating that the pesticide free leaf surface provided some sort of refuge for the mites. Spinetoram exposure at days one and three ultimately resulted in some mite mortality or mites drowned in the wet felt, but there were fewer overall mites dying and drowning on both sides of the leaf . Mite repellency was different from the control for both treated and untreated sides of the leaf, but on each subsequent bioassay date, the level of significance dropped until on day 14, there was no statistical separation. Our data suggest that because fewer mites were repelled in the spinetoram detection trial on bioassay days 7 and 10 and because of the pesticide free side of the leaf, more mites were alive, i.e. fewer picked up a toxic dose or drowned. It remains difficult with our bioassay system to precisely separate whether or not mites received a toxic dose when repellency levels were high.

Our studies were conducted with a conservative dilution rate of 2,843 L of water per ha while the majority of California avocados groves are grown on steep hillsides and utilize helicopter application using 468-935 L of applied water per ha. On these hillside groves, speed sprayers cannot be used and relatively few growers use drag hoses because of the high cost of labor in California. Application by helicopter may not provide complete coverage and many of the interior portions of the avocado tree remain untreated. With consideration of the following factors: helicopter application resulting in uneven distribution of pesticide on hillside avocado groves, the conservative dilution rate used in our trial, our containment of mites on the pesticide treated arenas and providing a pesticide treated/ untreated leaf area, our data suggests that in a field setting, mites may not pick up a toxic dose of spinetoram. Those mites that do not pick up a toxic dose will likely be repelled by the spinetoram and this may result in reduced E. hibisci mortality. Growers should be aware of the data presented herein when deciding upon a pesticide rotation management plan, which reduces avocado thrips resistance evolution. Each of the four recommended products have different features with respect to the efficacy of thrips control,square flower bucket concurrent control of avocado mite pests, and persistence of impacts on predaceous mites and other natural enemies . Citrus thrips, Scirtothrips citri , is a significant insect pest of citrus and mango fruits and has been recognized as a major pest of California citrus since the 1890s . In the USA, citrus thrips are known from Arizona, California, Texas and somewhat recently, possibly Florida , whereas in Mexico they are reported only from northern Mexico . Based on its past distribution, several authors have reported that citrus thrips is native to southwestern North America and northwestern Mexico . Citrus thrips is primarily a pest of citrus in California, particularly in the San Joaquin and Coachella valleys. They can have a broad host range, including, but not limited to, alfalfa, rose, grape, laurel, cotton, date, fir, lucerne and various grasses, pecans, and other ornamentals. Citrus thrips have been collected from over 55 different plant species . Their native host plant is hypothesized to be Quercus or more likely Malosmalaurina Abrams which was likely one of citrus thrips more common native host plants in southern California and northwestern Mexico prior to the introduction of citrus. In the SJV of California, S. citri has recently broadened its known host range and become a significant pest of high-bush blueberries . Scirtothrips citri was not known in Florida until 1986 where it was first detected in grape surveys . A collaborator was aware that in Florida, S. citri is not often collected from or abundant in several crops it is notorious for attacking in other regions of the Americas , but it is the most common thrips species he has collected from native vegetation and weeds. Species identifications from slide-mounted specimens can be unreliable or inconsistent and alternative or additional methods of identification may be necessary. Morphological identification suggests that S. citri is present in California, Arizona and Florida, but given that it is not a pest on several crops one might expect in Florida, further investigation is necessary to determine if S. citri is actually a cryptic species complex. The development of molecular genetic techniques , predominantly analysis of mitochondrial DNA , has significantly contributed to an understanding of natural genetic diversity and speciation . Genetic markers offer additional methods of species determination and delineation, especially when coupled with morphological identifications .

These approaches are especially useful in groups that demonstrate a mixture of diverse ecological traits coupled with a conserved morphology. Given the distribution of S. citri in major citrus growing regions of North America and the level of its pest status in those regions, re-evaluation of morphological and molecular identifications was deemed necessary. The goals of this work were to investigate the haplotypic variation among S. citri populations based on phylogenetic analysis of the mitochondrial and ribosomal DNA, and to identify possible cryptic species complexes within the Scirtothrips attacking citrus. The collection records for all specimens used in this study are listed in Table 1. Specimens were collected from various parts of California, Arizona, Texas, Florida , Mexico, Nicaragua and Turkey. Specimens from Turkey were included in this analysis as it is an under-represented area of the world and at the time of collection from citrus, the collector believed the specimens to be citrus thrips. Specimens were collected into 95% ethanol by beating the live thrips onto a white piece of paper, touching a clean 5/0 Princeton paint brush into the ethanol filled collection vial, touching the ethanol imbibed paint brush tip to the live insect so that the insect stuck to the paint brush tip and then depositing it passively into the collection vial. After collection, all specimens were stored at -20°C until analysis. Some of the collections contained Frankliniella occidentalis and Neohydatothrips burungae but these collected groups were not included in our analysis. Thrips were removed from ethanol and allowed to air dry on filter paper for 2 min. Total DNA was isolated using an EDNA HiSpEx Tissue Kit , following the manufacturer’s protocol. This method is non-destructive, allowing slide mounting and morphological examination of the specimen after extraction. After DNA extraction, two separate gene regions were amplified using PCR: the conserved 28S-D2 domain of the large rRNA subunit and the cytochrome c subunit I of mitochondrial DNA . A ~553-bp section of the 28S-D2 domain was amplified in 25-µl reactions containing 2 µl of DNA template , 2.5 × PCR buffer , 1.0 µl of MgCl2, 5 µM dUTP, 0.5 µM each of the primers CF and CR , 2 µl of bovine serum albumin  and 0.2 µl of Taq polymerase .

A personal communication from a collaborator in Florida supplied the idea to investigate the differences seen in citrus thrips in Florida andCalifornia. In Florida, citrus thrips are the most abundantly collected thrips on weeds but is not an agricultural pest in citrus, blueberries and mangos. However, in California citrus thrips is a pest of many agricultural crops especially the three listed crops. Thus, it seems prudent to determine if there are genetic differences between citrus thrips populations within North American citrus growing regions. Citrus thrips is primarily a pest of citrus in California particularly in the San Joaquin and Coachella valleys. They can have a broad host range, including, but not limited to, alfalfa, rose, grape, laurel, cotton, date, fir, Lucerne and various grasses, pecans and other ornamentals. Citrus thrips have been collected from over 55 different plant species . Their native host plant is hypothesized to be Quercusor more likely Malosma laurina . Scirtothrips citri has broadened its known host range and become a significant pest of a relatively new crop to California, blueberries . Thrips feeding on blueberry during the middle and late portions of the season cause distorted, discolored, and stunted flush growth and poor development of fruiting wood for the subsequent crop . Thrips pressure of this magnitude, coupled with repeated pesticide applications of the few effective and registered pesticides, poses a concern regarding pesticide resistance management. Currently, there are no integrated pest management plans available for control of citrus thrips in blueberry. This is primarily due to the recent nature of this crop-pest association. Historically, low-bush varieties of blueberries could only be grown in regions too cold for citrus production. However, the development of heat-tolerant high-bush varieties, which has enabled the development of a blueberry industry in the San Joaquin Valley , has also caused blueberries to be grown in a region where citrus and citrus thrips flourish. This issue is relevant not only to the blueberry industry,black plastic plant pots bulk but also for the 108,665 hectares of California citrus , which has experienced repeated documented cases of pesticide resistance in citrus thrips populations .

It is also important to note that not all varieties of high-bush blueberries are fed on equally by citrus thrips; i.e. there is a distinct varietal preference for some hybrids with similar parentage . Female avocado thrips lay eggs hidden inside the underside of leaves, in young fruit and stems . The first instar is white to pale yellow while the second instar is larger, more robust, and bright yellow . Avocado thrips larvae are typically found along major veins on the underside of younger leaves and anywhere on the surface of young fruit . Although some pupation occurs on the tree in cracks and in crevices, about threefourths of avocado thrips second instars drop from trees to pupate in the upper layer of dry, un-decomposed leaf litter . Propupae and pupae are rarely seen and they do not feed and move little unless disturbed. Adults are 0.7 mm long and have the typical fringed-tipped wings. Adults are orange-yellow with distinct, thin, brown bands between segments of their abdomen and three small red dots on top of the head . Adult avocado thrips resemble citrus thrips to the untrained eye and to an even lesser degree, western flower thrips, which occur on, but do not damage, avocado and citrus. Avocado thrips develop well under cool, humid temperatures . Populations typically begin increasing in late winter and spring, when avocado thrips feed on young leaves and fruit. Population abundance peaks in late spring and early summer, when most fruit are young and after the growth flush when hardening of leaves induces thrips to move from foliage to feed on young fruit. Populations are suppressed by warm, dry conditions, but this weather usually occurs later in the season, when most fruit are larger and no longer susceptible to damage by thrips. Scirtothrips perseae can have 6 or more generations a year. Egg to adult development occurs in about 20 to 30 days when temperatures average 18 to 24°C . Hoddle reported avocado thrips developmental biology and created a developmental degree-day model listing a developmental threshold of 6.9°C, which to our knowledge is the lowest threshold for any insect species. Monitoring temperatures and using degree-day calculations can predict actual development time.

Foliar feeding is usually unimportant, except when very high populations cause premature leaf drop .Avocado thrips adults can feed on over 11 plant species, however, larvae have only been found on avocados in the field in California suggesting that S. perseae has a restricted host range . Although it has little effect on tree health, avocado thrips feed directly on immature fruit , and obvious feeding scars cause severe downgrading or culling damaged fruit . Moreover, severe scarring when fruit are young can slow and stunt fruit growth. As fruit grow, early feeding by avocado thrips becomes apparent as scabby or leathery brown scars that expand across the skin and is sometimes referred to as “alligator skin” . Avocado thrips damage is affected by practices that increase or decrease the abundance of succulent foliage during set and growth of young fruit. Thrips move to young fruit when leaves harden after the growth flush has finished and the most damage occurs when fruit are 5.1 to 15.2 mm long . Although Hass fruit are susceptible to feeding until they reach about 51 mm in length, thrips feeding rarely causes scars on fruit larger than about 19.1 mm. This scarring on young fruit may not become obvious until fruit enlarge. In severe cases, all fruit on a tree can have their entire fruit surface scarred by avocado thrips, causing some packinghouses to sell such fruit with the box marked “papacado.” The California Avocado Commission estimated a $50 million dollar crop lost in the 2006 due to avocado thrips scarring and the costs of control . Sticky card and beating tray sampling are research methods used for these two insects but are rarely used by growers or pest control advisors . Both PCAs and researchers monitor citrus thrips by counting the percent of fruit infested with immature thrips and the number of immature thrips per fruit is also indicative of the severity of the infestation.

Thresholds in use in the San Joaquin Valley are 20% of Valencia oranges or 10% of navel oranges infested with immature thrips until the fruit reaches 20 mm in diameter or more. Thresholds are halved if Euseius tularensis levels are less than 0.2 per leaf . Avocado thripsare monitored by counting the number of immature thrips per leaf prior to fruit set or the number of thrips per fruit. No firm economic threshold has yet been developed for avocado thrips but PCAs typically treat at 3-5 immature thrips per leaf prior to bloom in San Diego County due to restrictions on use of abamectin during bloom. The major documented citrus thrips predator is the phytoseiid mite, E. tularensis ,procona system although Jones and Morse questioned the importance of this predator. Avocado thrips are frequently preyed upon by Franklinothrips orizabensis Johansen and Chrysoperla carnea and is parasitized by the larval parasitoid Ceranisus menes . Franklinothrips vespiformis , black hunter thrips , and several banded-wing thrips also feed on avocado thrips . In many years, natural enemies are unable to suppress avocado and citrus thrips populations below economic thresholds and chemical control is needed to reduce fruit scarring. By the time damage is noticed on ripening fruit, the thrips that caused the injury are often absent from the fruit. A variety of pesticides are registered for thrips control in different cropping systems . After a number of years of use, pesticides like dimethoate , formetante hydrochloride , cyfluthrin , and fenpropathrin resulted in failures in citrus thrips control in some regions, along with an increase in resistance confirmed with both laboratory and field bioassays. Also, these materials are detrimental to natural enemies such as Aphytis melinus DeBach and other biological control agents important to citruspest control. Since it was registered in 1998, spinosad has been the main material used for control of citrus thrips and a related and more effective material, spinetoram , was registered late in 2007 and will soon replace spinosad once MRL issues are resolved with export countries. Resistance to sabadilla has been shown with avocado thrips and a similar pattern of resistance development with abamectin is of concern due to the persistence of this material in leaf tissue. To date, citrus thrips resistance to spinosad has not been documented but there is concern that resistance to it or spinetoram may appear soon. With a limited number of pesticides available for control and the frequency of resistance shown by thrips such as citrus thrips, it is wise to monitor population levels carefully, limit treatments to population levels of concern, and time treatments optimally . Appropriate cultural practices and conservation of natural enemies should be practiced in concert with the use of pesticides only on an as-needed basis. Thus, the search continues for effective biological and chemical controls useful in citrus and avocado thrips management. For both species of thrips, some pupation occurs on the tree in cracks and in crevices, however, about three-fourths of avocado thrips drop as late second instars from trees to pupate in the upper layer of dry leaf litter . Propupae and pupae are rarely seen, move only if disturbed, and do not feed.

This phenomenon of dropping down to the leaf-litter or soil surface for pupation may create the ideal interface for control using the entomopathogenic fungi B. bassiana. Adding coarse organic mulch beneath trees and maintaining a mulch layer may reduce survival of thrips that drop from trees to pupate below the tree, especially in avocados, because this is common practice by many growers as a method of Phytophthora management. The effectiveness of mulching to control thrips is uncertain and labor costs of adding mulch may not be justified solely for thrips control. However, applying coarse organic material such as composted yard waste beneath trees may help control weeds, and thrips reduction might be an additional benefit, particularly for blueberries. The deep mulch layer that is standard practice with blueberry culture in the San Joaquin Valley may also provide an ideal habitat for B. bassiana. It is possible that as citrus thrips are adapted to and evolved in a hot, dry climate, they may be more susceptible to B. bassiana, whereas avocado thrips has adapted to and evolved in a wet and cool climate and may be less susceptible to or even tolerant to B. bassiana. There is increasing pressure in the United States to move away from broadspectrum insecticides and focus on alternative methods of control, e.g., geneticallymodified crop plants expressing Bt toxins, use of entomopathogens, biorational insecticides. Implementation of such methods on avocado and citrus are difficult due to the relatively primitive methods available for thrips sampling, which are labor intensive and rely on experienced and intuitive pest control advisors. The goal of the workdescribed here is to examine alternatives to traditional insecticides such as Bt proteins and entomopathogenic fungi to control avocado and citrus thrips, with the ultimate target of utilizing entomopathogens to aid in field control, evaluate the insecticides registered for avocado thrips management on the native predaceous mite Euseius hibisci, assess citrus thrips oviposition on blueberry varieties, and determine whether citrus thrips is actually a complex of species. Citrus thrips, Scirtothrips citri , is a plant-feeding pest most widely recognized for the damage it causes to citrus and mango fruits and has been recognized as a major pest of California citrus since the 1890s . Recently, its known host range has broadened and they have become a significant pest of a relatively new crop planted in the San Joaquin Valley of California, high bush blueberries . Citrus thrips feed on blueberry foliage during the middle and late portions of the season causing distorted, discolored, and stunted flush growth and poor development of fruiting wood required to obtain the subsequent crop . High numbers of thrips on blueberries , coupled with repeated pesticide applications of the few effective and registered pesticides, poses a concern regarding pesticide resistance management .

A more recent anonymous Internet-based American survey indicated that 46% of dog owners and 38% of cat owners had fed RMBDs to their pets . Additionally, sales of RMBD have increased annually by as much as 15% in recent years . Over the past decade, this feeding practice has continued to increase, and market locations have expanded to include grocery stores, mass merchandisers, pet specialty stores, and veterinary clinics . A 2019 Italian-based survey shed light on dog owners’ motivations for adopting this feeding method . About 80% of respondents reported that they abandoned feeding commercial diets due to distrust in the clarity of ingredients commercial prescription veterinary diet, veterinarians may acquiesce to such requests. However, the reliability of RMBD for this purpose has not been evaluated. The primary aim of this study was to use PCR to test commercially available RMBD for the presence of DNA of animal origin other than that declared on the labels. A secondary objective was to determine the consistency of DNA presence between different batches of the same diets. The hypothesis was that the diets would contain unlisted protein ingredients, and that these unlisted proteins would vary between batches. To the authors’ knowledge, no previous studies have examined these issues. Nine commercial canine and feline RMBD were selected for analysis. All diets were marketed as balanced for complete feeding. The selected diets included a variety of commonly available North American RMBD, some formulated with novel animal source proteins , or limited ingredients , or grain-free diets which may be potentially selected for use as an elimination diet. The diets evaluated were not specifically marketed for feeding as elimination diets, but contained ingredients which may be considered by pet owners for this feeding purpose. Two lot numbers of RMBD representing separately prepared batches of each diet were selected for analysis in order to assess for consistency of any ingredient contamination between the 2 batches. One canine and one feline veterinary prescription extensively hydrolyzed poultry feather-based diet were used as negative controls. Nine species-specific qPCR assays were designed to detect beef, chicken, duck, turkey, salmon, sheep, rabbit, kangaroo, and pig DNA .

Sequences for each species were found in the National Institute of Biotechnology Information database. Two primers and an internal hydrolysis probe , 3 end, quencher dye NFQMGB were designed using Primer Express Software for all species,30 planter pot with the exception of chicken which used a locked nucleic acid probe . A Basic Local Alignment Search Tool of the amplicons confirmed unique species detection. To ensure these assays did not cross-react with DNA of other species, a crossreactivity evaluation was performed by running all assays with control DNA from each species . A housekeeping gene, eukaryotic 18S assay , was run with each sample to confirm successful DNA extraction. All assays were validated for efficiency and sensitivity by running 10-fold standard curves in triplicate from serial dilutions of control DNA. Each assay was 90% to 100% efficient and sensitive enough to detect as few as 10 copies of the target gene. Of the 9 species of animal DNA tested, 8 species, including pork, chicken, duck, rabbit, lamb, beef, salmon, and turkey, were detected in at least 1 sample of the canine and feline RMBD tested. Only kangaroo DNA was not detected in any of the RMBD. The 2 extensively hydrolyzed poultry feather-protein based diets contained either trace amounts of chicken DNA or no detectable DNA . In the canine RMBD, DNA of 1 or more animal species not indicated on the label was identified in 9 out of 9 diets, in either 1 or both of the tested batches . Of the 18 batches tested, 89% tested positive for unlisted animal-source DNA. An average of 4.4 unlisted proteins was detected in each diet. A total of 2 batches were found to contain DNA consistent with the stated label ingredients. The diet with the greatest number of unlisted proteins was a single batch , labeled as containing turkey and sardine. It contained a total of 6 unlisted proteins . The unlisted DNA most frequently detected was lamb . Discrepancy in the unlisted DNA between batches was noted in 78% of batches. In the feline RMBD, DNA of 1 or more animal species not indicated on the label was identified in 7 of 9 diets, in either 1 or both of the tested batches . Of the 18 batches tested, 61% were positive for unlisted animal-source DNA. An average of 2.6 unlisted proteins was detected in each diet. A total of 7 batches were found to contain DNA consistent with the stated label ingredients. The diet with the highest number of unlisted proteins was a single batch , labeled as containing chicken and salmon. It contained 5 unlisted proteins .

The unlisted DNA most frequently detected was turkey . Discrepancy between batches was noted in 56% of batches. All of the canine and feline RMBD included in the analysis were found to contain the proteins listed on their labels. Contamination of one or both batches in all canine RMBD and most of the feline RMBD tested was detected in this study, which supports the hypothesis that cross-contamination would be found in many RMBD. Numerous independent studies have also demonstrated significant discrepancies between label claims and actual contents of dry or canned over-the-counter commercial diets, including those marketed for the management of CAFR . While this finding may have been expected in RMBD due to the prevalence of unlisted DNA detected in other such studies, diet purity could theoretically have been improved in RMBD as they are purported to undergo less processing before distribution, allowing less opportunity for protein contamination. An additional finding of this study was that unlisted animal source proteins varied among batches in most batches tested, including both canine and feline RMBD that were analyzed. Discrepancy among batches has not been previously studied for comparison, but these results showed that differences in unlisted ingredients were common in RMBD. Due to cost limitations restricting the analysis to only 2 batches of each RMBD, a statistically significant batch contamination rate could not be determined. However, the finding of discrepancy among batches represents yet another variable which could impact interpretation of an ED trial in a patient fed a commercially prepared RMBD. While no particular manufacturer’s diets were found to be more likely to contain unlisted proteins, brand F, the producer of feline diets 7 to 9, had the least number of contaminants, as well as the most consistent agreement between each batch. It is possible that this finding is due to the nature of the processing practices of this particular manufacturer, the production of smaller batch sizes to minimize opportunity for contamination, or more limited sourcing of ingredients to restrict the potential for supplier cross-contamination. Overall, the number of animal protein ingredients included in each diet was not a predictor of the number of unlisted proteins isolated in the analysis. Even diets restricted to single proteins were as likely to contain 1 or more sources of unlisted animal DNA as those with multiple animal proteins and batch contamination was unpredictable.

Previous studies have shown that in rare cases,plastic growers pots ingredients listed on product packaging were found to be missing from the analysis . Our study showed that no animal DNA was missing from that declared on the packaging of any RMBD included in the analysis. Due to the target DNA of the qPCR assay, the study was unable to validate the presence of sardine or goose to confirm the inclusion of these ingredients. This was again due to cost limitations precluding the addition of these proteins in the analysis. Some diets also contained animal fat sources such as salmon oil, cod oil, or sardine oil. Through purification processes, fish oils undergo refinement to remove proteins from the oil to render them free of proteins . While cod and sardine were not included in the analysis, our study did evaluate for salmon DNA. Both canine diets 1 and 3 contained salmon oil and tested negative for salmon DNA. Feline diet 5 contained salmon oil, but also contained salmon meat, and tested positive for salmon DNA as would be expected. If extrapolating from the finding that salmon DNA was not found in the diets containing salmon oil, it may be expected that the other diets containing fish sources of oil did not contain cod or sardine DNA as potential allergens. In general, plant- and animal-based oils are not considered allergenic when highly purified . Of additional note was the finding that no diet contained the DNA of kangaroo. Since kangaroo meat is not used by any of the manufacturers in their RMBD, this finding serves as an additional negative control for this study to validate that no DNA of kangaroo origin was detected in the analysis, as would be expected. Analysis of the 2 extensively hydrolyzed poultry featherbased diets revealed no detection of unlisted animal DNA. This is consistent with previous reports that contaminants are detected less commonly and in lower numbers in hydrolyzed diets . The extensive hydrolysis of poultry feather proteins into component amino acids or very short oligopeptides is intended to avoid inducing IgE-mediated mast cell activation that can occur with proteins 10 kDa in size or greater . Extensive hydrolysis to reduce poultry allergenicity has been validated in both serum IgE and feeding trials to show the clinical benefits for CAFR . These negative control diets were selected because of the rigorous quality control methods undertaken by the manufacturer to ensure cross-contamination does not occur before market release . While the canine diet did test positive for chicken DNA, the manufacturer does list the feathers of chicken, turkey, and duck as their sourced raw materials . The target gene of the analysis for chicken DNA, transforming growth factor beta 3, is a protein expressed in chicken feathers . Additionally, the manufacturer is aware that cross-contamination needs to be avoided in a therapeutic diet and has developed and clinically validated calibration curves to prevent contamination . These calibration curves correspond to a known DNA level that was clinically tolerated based on Global Skin Scores in feeding trials in order to set a tolerance level for ancillary proteins known as the NPPI , which is strictly monitored in each diet prior to allowing market release . Based on the manufacturer’s quality control data, 72.3% of these extensively hydrolyzed diets contain DNA below the limit of detection , and 25.7% may have DNA above the LOD but below a safety threshold of 1.2 g/g . However, no diet released to market will exceed the established cut-offs of the NPPI based on the pre-established calibration curves . Therefore, trace copies of chicken DNA may be expected on PCR in some of the diets released to market, as was found in our study. The DNA in the feline extensively hydrolyzed diet in this study was below the LOD, and no animal DNA was detected in the assay. Based on the sensitivity of qPCR, it can be argued that these assays, being sensitive enough to detect as few as 10 copies of the target gene, are of greater sensitivity than that required to detect clinically meaningful contamination that would trigger CAFR. There is no established maximum tolerable level of a contaminating protein that may elicit a pruritic reaction in a food sensitized pet. In humans, soy protein concentration as low as 10 ppm may evoke a reaction in a soy-sensitized individual . Additionally, dose distribution has been demonstrated to vary between different food allergens in sensitized humans, showing that a tolerance range may exist for different food antigens themselves . The additional concern for CAFR pets is that, as opposed to most humans, pets are often fed a specific commercial diet with daily regularity, increasing their risk of chronic re-exposure to a food antigen contained therein. As a result, even small amounts of unknown allergens may lead to a cumulative reaction in a CAFR-affected pet and skew the clinical impression of their response to a particular ED. These reactions may even be sporadic if there is significant variation of the protein constituents of the diet between batches. The need to validate food allergic threshold distributions in canine and feline CAFR is an important area for future research.

The IHR and intermediate types together were considered to define the “recombinant” group of X. fastidiosa subsp. multiplex isolates . Most were observed more than once, and 5 were found in two different U.S. states or districts . The analysis of Nunney et al. was focused on the evolution and host range ofX. fastidiosa subsp. multiplex. For this purpose, it was necessary to identify and exclude isolates whose recent evolution was influenced by inter sub-specific recombination. As such, once the 23 non-IHR STswere identified, there was no further analysis of the remaining recombinant group STs. In particular, no evidence was presented for classifying some alleles as atypical of X. fastidiosa subsp. multiplex beyond the observation that they were never found in the non-IHR group . Nunney et al. did observe one intriguing pattern when they compared their results to those of Parker et al. . Of the 143 isolates, 13 were also used in the study by Parker et al. , in which typing was based on a different set of 9 loci. Unexpectedly, these 13 isolates maintained the same grouping with the IHR and non-IHR types corresponding, respectively, to the clade A and clade B groupings . This highly statistically significant concordance strongly suggested that IHR is not distributed randomly across all X. fastidiosa subsp. multiplex isolates but instead is restricted to a small subset, while the remainder is little influenced by IHR. However, Parker et al. failed to find evidence of inter sub-specific recombination within any of the X. fastidiosa subsp. multiplex isolates, despite applying a series of 9 tests designed to detect recombination contained within the RDP4 program and the PHI program . This result presented a strong argument against our hypothesis that clade A members cluster because they are recombinant types carrying alleles derived from IHR . Here we reexamined the sequence data obtained in their study by using the more sensitive introgression test to determine if their tests missed evidence of IHR and, if so, whether it was confined to clade A.

In particular,plastic planters bulk what could be concluded about the origin of the group given the observation from 2 independent studies that the members appear to form a well-defined cluster of genotypes? Third, we used the sequence data to examine the hypothesis that the introgressed DNA was from X. fastidiosa subsp. fastidiosa, the subspecies that causes Pierce’s disease. X. fastidiosa subsp. fastidiosa is native to Central America, and all known isolates in the United States and northern Mexico can be traced back to a single introduced genotype . IHR would be of limited interest if it simply randomized the genetic differences among the subspecies but had a minimal effect on pathogenesis. For this reason, we were particularly interested in documenting any possible invasion of new plant hosts associated with IHR. The hypothesis is that IHR creates a range of novel genotypes that are far more variable than can arise from a lineage diversifying through point mutations, and this diversity facilitates adaptive evolution of a kind not possible for a clonal lineage. This kind of probabilistic evolutionary hypothesis can rarely be directly proven based on an individual case; however, it makes predictions that, if generally supported, would cause the hypothesis to be accepted. In the case of X. fastidiosa, compelling evidence supporting the hypothesis would be the invasion of a new native host plant that is uniquely associated with IHR. Our data support this hypothesis: in X. fastidiosa subsp. multiplex, IHR is indeed associated with the invasion of at least 2 new native plant hosts, blueberry and blackberry.To investigate inter sub-specific homologous recombination , we analyzed 31 isolates previously identified as IHR-type and 2 isolates previously identified as intermediate-type X. fastidiosa subsp. multiplex , based on sequence of the 7 housekeeping loci used in the MLST scheme defined by Yuan et al. plus a region of the pilUgene. Together, these 33 isolates made up the recombinant group. Details regarding the isolation and typing of the 33 isolates were provided by Nunney et al. , and a summary of salient features is provided in Table S1 in the supplemental material in that article. All sequences used have previously been published and are available both in GenBank and the MLST website . To detect IHR, we employed a modified version of the introgression test developed by Nunney et al. .

In its original form, the test compares a set of target sequences, some of which may have been involved in IHR, to a set of potential donor sequences. Each variable site is classified as F, a fixed difference between the target sequences and the donor sequences, or P, a polymorphic site within the target sequences where at least one variant base is shared with the donor set. A significant shift in the ratio of F to P marks a recombination break point. In the modified version of the test, the targeted introgression test, the target sequence is known a prioriand is compared to two references, the donor group, D , and the ancestral group, A . The minimum number of nucleotide differences between the target and the two references defines a ratio of D to A equivalent to the ratio of F to P and can be tested in the same way . In some cases, there is no break point because the whole locus appears to be an introgressed sequence . Although the signal of introgression across the entire sequenced region may be clear, it is valuable to have a statistical test that documents the strength of the signal. In this case, the null expectation is the ratio that reflects the pairwise differences between the donor and ancestral group versus the pairwise differences within the ancestral group . We used this ratio to define the expectation of the D/A ratio for a chi-square test of complete introgression. Gene diversity and distance trees were calculated using MEGA5 , and the maximum parsimony tree was created using the PARS program in Phylip . Distance trees and the maximum parsimony tree were used rather than other methods, given the known occurrence of inter sub-specific recombination in the data. Clonal Frame was used to provide an independent estimate of the relative importance of recombination versus mutation in the recombinant group.Analysis of the recombinant group ofX. fastidiosa subsp. multiplex showed three important results. First, inter sub-specific recombination was shown to have occurred in 50% of 8 loci scattered throughout the genome that were chosen independently of the data . Second, it was shown that the donor of the introgressed sequence was X. fastidiosa subsp. fastidiosa, a subspecies introduced from Central America into the United States as a single strain .

However,collection pot the introgressed sequence at two of the loci did not come from any of the X. fastidiosa subsp. fastidiosa genotypes that have been found in the United States. This result suggests that another introduction of X. fastidiosa subsp. fastidiosa must have occurred, an introduction that resulted in successful IHR, after which the donor genotype seems to have disappeared. This involvement of an unexpected X. fastidiosa subsp. fastidiosa strain supports the hypothesis that the members of the recombinant group share a single ancestral IHR event. Third, the hypothesis that IHR has facilitated a shift to new hosts is strongly supported by the example of blueberry, where 10 isolates have been typed and potentially supported by the example of blackberry . A link between the shift to a novel plant host and homologous recombination has not been previously identified. Of course, the direct causation of this link can never be proved without knowledge of the genetic changes driving this shift. It can always be argued that the link is fortuitous and that one or more point mutations in the nonrecombined X. fastidiosa subsp. multiplex genome are causal in the host shift. Arguing against this possibility are 2 additional pieces of information. First, both blueberry and blackberry are native to the United States, so if only a simple genetic change was required to infect these species, why did the native nonrecombinant X. fastidiosa subsp. multiplex apparently never acquire these changes? Second, a similar but even more extensive mixing of the genomes of X. fastidiosa subspp. fastidiosa and multiplex is found in the only form of X. fastidiosa that infects another U.S. native plant, mulberry . Furthermore, in other bacterial species, it has been demonstrated that recombination can drive rapid evolution, both in the laboratory and, in the case of Helicobacter pylori, in mice . Similarly, McCarthy et al. concluded that lineages of Campylobacter jejuni in chickens versus cattle and sheep were able to shift host type, because rapid adaptation was facilitated by recombination with the resident host population. In the study by Nunney et al. , it was shown that the recombinant genotypes formed a well-defined group , demonstrating that inter sub-specific homologous recombination was not randomly distributed across the X. fastidiosa subsp. multiplex isolates. This work was based on a survey of 143X. fastidiosa subsp. multiplex isolates using just 8 loci. There were 33 isolates that showed some evidence of IHR in at least 1 locus: all but 2 showed statistically significant evidence in at least 2 loci, while the remaining 110 showed no such evidence . The generality of this discrete group of recombinant forms was supported by our analysis presented here of the sequence data from 9 more loci sequenced by Parker et al. . These loci divided isolates into 2 groups that appeared to correspond to the recombinant and non-IHR groups, respectively , even though Parker et al. found no evidence of IHR. Upon reanalysis, we found statistically significant IHR in 6 of the 9 loci in the clade A data but no evidence of IHR in the clade B data. Clade A included 6 isolates that we had typed in the present study, and each of these showed evidence of IHR in 4 or 5 of the additional 9 loci. Thus, in two independent samplings that together examined 17 loci, there was clear evidence of substantial genome wide IHR in the recombinant group isolates, amounting to 50% of the genes showing IHR across the MLST locis plus the pilU locus .

The average was higher when based on the loci sequenced by Parker et al. ; however, this was probably biased upwards by the manner in which the loci were chosen . None of the IHR events in 6 of the 9 loci identified using the targeted introgression test, or in the case of complete introgression, a chi-square test, were detected by Parker et al. using PHI and the 9 tests implemented in RDP . This failure of the standard tests of recombination to detect IHR was previously noted by Nunney et al. , motivating the development of their introgression test. We examined the hypothesis that the recombinant group STs were derived from a single IHR event involving a X. fastidiosa subsp. multiplex recipient and an X. fastidiosa subsp. fastidiosa donor. The distribution of allelic differences among the recombinant STs was consistent with them all being derived from a single initial event, but a small number of other inter sub-specific and intrasubspecific recombination events would also be needed . More importantly, the genotypes seen in the recombinant group can be accounted for entirely, or very nearly so, based on a single X. fastidiosa subsp. fastidiosa donor genotype. For example, the substantial variation in cysGcan all be accounted for by an ancestral introgression of X. fastidiosa subsp. fastidiosa allele 12 followed by subsequent intrasubspecific recombination of X. fastidiosa subsp. multiplex sequence to form the other two alleles . In contrast, variation at pilU could be accounted for by a second donor contributing the X. fastidiosa subsp. fastidiosa pilU9 allele, but it could also have arisen by a single mutation in pilU1 unique to the recombinant group. A possible single X. fastidiosa subsp. multiplex recipient genotype was also identified . This genotype is consistent with a known ST: setting cysG to allele 3 makes the recipient identical to ST45, which was sampled from the states of California, Kentucky, and Texas . Elsewhere, we consider a slightly different hypothesis regarding the origin of the recombinant group in which the donor and recipient subspecies are reversed—i.e., that it was derived from a single IHR event, but involving an X. fastidiosa subsp. multiplex donor and an X. fastidiosa subsp. fastidiosa recipient; however, apart from the role reversal, the conclusions are unaltered .