Month: May 2025

One of the challenges of similar foods being studied in differing formats and by various research groups is the utility of the data as a combined set. Differences in test materials and experimental designs make integration of data difficult. The proper curation of combined data, whether physiologic, metabolomic, or genomic, is critical to ensure that combined datasets provide synergy, statistical power, and enhanced usefulness.The cardiometabolic benefits from regular consumption of nuts or berries are widely reported and include improved vascular function, reduction of cardiovascular disease risk factors, improved insulin sensitivity, and reduced risk of type 2 diabetes mellitus. Metabolic outcomes may be context-specific and related to the physiologic state of the individual and host microbiome composition, among other factors. Examples include findings of ellagitannin and ellagic acid rich foods resulting in differential responses in healthy individuals compared to those with prediabetes, who are dependent on gut microbial-derived metabolite profiles. Many factors contribute to interindividual variability in response to diet that can extend to context-specific aspects influencing the magnitude of health benefits and reinforces the importance for further research aimed at advancing discoveries in precision nutrition. Additional health outcomes related to nut or berry intake are outlined below.Adding nuts or berries to the daily diet may be advantageous for weight management for several physiological reasons. One is that these foods produce feelings of satiety, helping to reduce the desire to consume calorie-rich snacks that are low in vitamins, minerals, and fibers, dutch buckets ultimately improving body composition over time. A second possibility is due to urolithins, secondary metabolites produced from ellagitannins in nuts and berries.

Urolithins increase the activation of the adenosine monophosphate-activated protein kinase pathway, resulting in anti-obesogenic properties in vitro and in animal models. AMPK increases fatty acid oxidation and decreases triglyceride accumulation. Phosphorylation of AMPK may also decrease cholesterol synthesis and lipogenesis by downregulating 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol regulatory-element binding protein expression. In clinical studies exploring the relationship between food and body composition, the incorporation of nuts and berries into the diet was associated with weight loss or maintenance.Regular consumption of nuts or berries has been reported to support brain health and cognitive function, motor control, mood, and executive function at physiologically relevant intakes. Middle-aged and older adults experienced improvements in balance, gait, and memory, and children experienced higher executive function and positive affect after acute and regular intake of both strawberries and blueberries. These beneficial effects may be the result of direct effects on brain signaling or indirect effects through oxidant defense and anti-inflammatory properties of polyphenols and other bio-active compounds in nuts and berry foods. The gut-brain axis is an emerging area of research. Most studies are preclinical in nature using animal models but are suggestive of a significant role of gut microbial-derived ellagitannin metabolites on brain health and neuroprotection.The influence of nuts and berries on skin health and appearance is an emerging area of research. Regular intake of almonds, a good source of fatty acids and polyphenols, has been associated with a significant decrease in facial hyperpigmentation and wrinkle severity. A walnut protein hydrolysate administered to rats exposed to ultraviolet radiation significantly reduced skin photoaging and enhanced skin elasticity. Supplementation with ellagic acid, a compound found in many berries, prevented ultraviolet B -related inflammation and collagen degradation related to skin wrinkling and aging in a murine model.

More human studies, using objective measures of skin wrinkles, skin elasticity and response to low-dose UVB radiation exposure are warranted. Monitoring skin responses to a UVB radiation challenge has been used as a marker of whole-body antioxidant status in response to almond consumption. The response to a UVB challenge has also been used to monitor oxidant defenses and changes in skin microbiome following the intake of pomegranate juice.Age-related macular degeneration is the third leading cause of vision loss worldwide. Anthocyanins, carotenoids, flavonoids, and vitamins C and E, found in many berries, have been shown to reduce risk of eye-related diseases. Goji berries, containing the highest amount of zeaxanthin of any known food, hold particular promise since this compound binds to receptors in the macula to offer protection from blue and ultraviolet light. Regular supplementation with 28 g/d of goji berries for 3 mo increased macular pigment optical density, a biomarker for AMD, as well as the skin carotenoid index. Nuts may also be protective against AMD since they are a rich source of vitamin E and essential fatty acids. Regular intake of nuts has been associated with a reduced risk and slower progression of AMD in 2 epidemiological studies, thought to be due to the beneficial role of polyunsaturated fatty acids.Identification of new cultivars with traits desirable for growers, processors, and consumers is a continuous effort. As researchers continue to produce new varieties by both conventional and molecular-driven approaches, assessing these varieties for nutritional value is a challenge. A combination of broad targeted and untargeted metabolomic approaches, along with defined functional phenotyping could be used for rapid screening and defining of mechanistic pathways associated with health. However, consumer preferences for new cultivars are often driven by size and appearance of the berry or nut and flavor, rather than its nutritional value. This would further confirm the need to balance improvements to nutritional profiles with enhancement of consumer-driven traits, maintaining the marketable nature of the berries and nuts.Biomedical research, particularly for clinical studies, is expensive and resource intensive. Although the USDA competitive grants program offers funding for outstanding research projects, budget limitations favor animal or in vitro study proposals. Compelling pilot data is needed to be competitive for clinical studies funded by the USDA or NIH, so many researchers submit their initial ideas to commodity groups representing specific nuts or berries. Commodity groups represent farmers, processors, and distributors and have been instrumental in supporting fundamental and applied research focused on their specific berry or nut. The perception that studies funded by nut and berry commodity groups are inherently biased in favor of the test food is an issue sometimes raised by critics, journalists, and the general public. As in all nutrition research, ethical considerations regarding the structure of research questions, hypotheses, study design, outcome measures, interpretation of data, and conclusions must be rigorously considered. The food and beverage industries have played a key role in providing funds and supporting nutrition research on individual foods and beverages, including berries and nuts. Although this draws scrutiny regarding scientific integrity and data reporting, collaboration between academia and industry compared to exclusive corporate funding may help offset some of these concerns. For example, in multiple reported studies, matching funds were also provided by nonindustry sources, including institutional and federal agencies. In other cases, while the food industry provided the test agents, key research personnel and staff were not supported by the same funding source.

The academia-industry collaboration has also led to the formation of scientific advisory committees that evaluate and recommend proposals for funding, a peer review process that helps ensure rigorous study designs, data reporting, and dissemination of results. Human studies of sufficient statistical power are expensive, labor-intensive efforts requiring sophisticated and costly laboratory equipment and supplies. In order for research proposals to be competitive for funding from the USDA or NIH, pilot data is required, grow bucket and for nuts and berries, the only realistic source of funding for these exploratory trials is from industry sources. Critics of industry support for nutrition research have yet to propose realistic alternatives for funding needed to generate initial data. Further, ongoing industry funding of nuts and berries research has yielded important insights into the molecular and physiological understanding of mechanisms of action. Without industry support, provided in an ethical and transparent manner, advances in our understanding of the role of nuts and berries in a healthy dietary pattern would be limited. A risk-of-bias study of 5675 journal articles used in systematic reviews published between 1930 and 2015, representing a wide variety of nutrition topics, concluded that ROB domains started to significantly decrease after 1990, and particularly after 2000. Another study examined the incidence of favorable outcomes reported in studies funded by the food industry in the 10 most-cited nutrition and dietetics journals in 2018. Of the 1461 articles included in the analysis, 196 reported industry support, with processed food and dietary supplement manufacturers supporting 68% of the studies included. Studies supported by any nut or berry commodity group were not considered due to an incidence lower than 3% of qualifying articles. Studies with food industry support reported favorable results in 56% of their articles, compared to 10% of articles with no industry involvement. The authors offer a number of suggestions to help minimize real or perceived bias, calling on research institutions to enforce strict, regularly updated, and transparent oversight of all research projects involving industry. Suggestions in support of research transparency and integrity have also been advanced from guidelines adapted from the International Life Sciences Institute North America. This served as the basis for the development of consensus guiding principles for public-private partnerships developed by a group of representatives from academia, scientific societies and organizations, industry scientists, and the USDA, NIH, US Centers for Disease Control, and the US Food and Drug Administration. These provisions include full disclosure of funding and confirmation of no direct industry involvement in the study design, data and statistical analyses, and interpretation of the results and only minimal, if any, involvement of industry coauthor, often given as a courtesy to acknowledge funding and logistical support by the investigators with no intellectual involvement by the study sponsor. This is in contrast to industry-initiated research, where the industry office or commodity group sets predetermined research objectives, provides intellectual collaboration, and often has input on the study design, interpretation of results, and decisions regarding publication. Although some critics may argue that repeated industry funding in support of research groups that report favorable results on a particular nut or berry shows a bias toward positive outcomes, other interpretations are also possible. First, few labs have the infrastructure, detailed methodology and analytical equipment, and trained personnel to conduct clinical studies in an efficient and timely manner. Industry funded studies conducted at major universities have layers of review and accountability within their organizations to guard against malfeasance, and while these layers may not focus directly on precise elements of research design and interpretation of results, faculty members at such institutions generally have a level of integrity and accountability, knowing that administrative review exists. Calls for industry-funded research are often broad in scope, which allows researchers to generate proposals, research questions, and hypotheses that do not have preconceived outcomes. A third consideration is that the nuts or berries under study may simply have sufficient bio-activity to produce favorable outcomes, independent of potential researcher bias.With the depletion of natural resources and growing populations, there is a great motivation for recovering valuable compounds from fruit and vegetable wastes and converting them into functional ingredients as a part of sustainable agricultural practices. Pomegranate is an important California commodity and pomegranate by-products are a large portion of the process. These by-products could be repurposed into valuable compounds, but they are not unutilized or underutilized, ending up in landfills or animal feeds. In this dissertation, physiochemical properties and extraction methods of bio-active compounds in pomegranate peel were comprehensively studied to determine the feasibility of converting these pomaces into healthful food ingredients. The research objectives were to identify the physiochemical characteristics of pomegranate peel and assess different size reduction methods to obtain suitable peel particles for effective extraction by using water as a solvent; evaluate the effects of extraction temperature, time, and water usage on the phenolic yield, phenolic composition, and antioxidant activity to optimize the extraction conditions; study the bio-active properties and nutritional benefits of pomegranate peel powder and extract through animal models; investigate the characteristics of food products developed with pomegranate peel powder and extract; and study the co-extraction mechanism of pectin and polyphenol to improve extract yield and phenolic stability. There are 6 chapters in this dissertation. Chapter 1 includes an introduction, research objectives, and literature review on the processing and waste utilization of pomegranate fruits, with a focus on the current waste management practices and effects of punicalagin, which is the most unique polyphenol in pomegranate.In Chapter 2, the physicochemical properties of pomegranate fruits were investigated to explore the potential of recovering bio-active food ingredients from the inedible fruit peel.

There is also evidence that CRISPR-Cas9 modifications to upstream open reading frames can impact metabolite regulation across species, as vitamin C concentrations increased by 150% in Arabidopsis and lettuce by removing the regulatory effects of the associated uORF . While altering the levels of endogenously produced chemicals is effective, this approach is limited in the diversity of nutrients it can deliver. However, plant synthetic biology offers a means to bypass this limitation through the introduction of heterologous biosynthetic pathways. One of the first successes in this came with the production of golden rice, a ß-carotene enhanced rice grain that was generated through the addition of three genes . Since then, replacement of the daffodil phytoene synthase with a maize phytoene synthase has increased the titers of ß- carotene in golden rice 2 , providing greater nutritional benefits . Furthermore, the authors were able to not only add ß-carotene to rice grains but also increase the levels of iron and zinc with a single genetic insertion. This shows the potential of a biofortified staple crop to have an enhanced nutritive capacity suitable to meet multiple minimal daily requirements. Additional work has shown that the added ß- carotene in the rice grain can be further modified into astaxanthin and canthaxanthin, two antioxidants of interest, with one or two additional genes . Iron deficiency is one of the world’s leading micronutrient deficiencies and is responsible for around one million deaths annually. As such, hydroponic nft channel iron has been a target of biofortification efforts over the last few decades.

Early work showed that the overexpression of a root zinc transporter from Arabidopsis could not only increase the zinc concentration in grains, another significant deficiency, but it also increased the iron content for Hordeumvulgare . The addition of genes increasing nicotianamine, the nicotianamine-iron transporter and the iron storage protein ferritin was shown to have a concerted effect on rice seed iron concentration of more than fourfold . Perhaps, with the combination of nutrient transport and sequestration mechanisms in the grain or fruit, levels of many micronutrients could be enhanced. Other vitamin deficiencies also pose major concerns to human health and plant synthetic biology efforts can alleviate some of these nutrient shortcomings. Inadequate folate consumption is linked to cardiovascular disease, Alzheimer’s disease, and cancer, but it is especially important for pregnant women, as deficiencies can cause neural tube defects or megaloblastic anemia . Folate content in rice has been improved by 150-fold through the expression of a folate binding protein and a folylpolyglutamate synthase , offering a means to increase folate consumption without dietary supplementation. This work also showed that the binding protein provided stability to folate so that it was maintained at a similar level even after months of storage, which is a concern for any crop that does not get eaten immediately after harvest.

Vitamin B6 deficiency has been shown to be linked to cardiovascular disease, diabetes, neurological diseases, as well as nodding syndrome . Efforts to biofortify cassava, a staple vegetable crop in many parts of Africa, have been successful with the expression of the Arabidopsis thaliana genes PDX1.1 and PDX2, which are involved in de novo production of vitamin B6. Root-specific expression of PDX1.1 and PDX2 increased vitamin B6 and B6-glycoside concentrations by 4- and 14-fold, respectively, even after cooking . While biofortification of crops has taken great strides forward, there remain several considerations for effective implementation of this synthetic biology strategy. As more compounds are engineered into crops, it will be important that metabolic flux and regulation are not drastically altered, as this can have detrimental pleiotropic effects on the plant’s health. For example, the overproduction of γ-aminobutyric acid in tomatoes causes gross developmental defects . Moreover, there will be clear challenges in transferring metabolic engineering strategies from one crop to another. There has been moderate success in this, such as with enhancing ß-carotene in golden bananas , but each crop is unique and requires experimentation to optimize production. As we continue to understand the intricacies of plant metabolism, these issues will be more easily addressed.Nutraceuticals are defined as consumed bio-active compounds that are not essential for nutrition but offer other health benefits. These molecules are distinct from pharmacological compounds—though in some cases they do overlap—and do not require extraction and purification or semi-synthetic modifications before final dosage and distribution, as the source of nutraceuticals are the foods themselves.

While these compounds are not essential for growth and development and many of their health benefits are still under investigation, their presence in our diet has the potential to improve human health. One nutraceutical compound that has been of great interest is resveratrol. This naturally occurring compound, found in wine and chocolate among other plants, has been studied since the 1990s for its health benefits due to its association with the “French paradox,” the quandary of a low incidence of heart disease in a population that eats a high-fat diet. Many health benefits, including prevention of cardiovascular disease and treatment of diabetes, have been attributed to resveratrol and validated clinically, but its low concentration in native plants poses a challenge to extraction for medicinal use . Additionally, consumption of purified resveratrol is mostly metabolized, greatly reducing its beneficial effect, but ingestion alongside other food-based phenolic compounds increases resveratrol levels found in blood serum. Therefore, the addition of resveratrol to foods could provide a more efficient means for delivery than purified supplements. Since all plants contain the precursor molecules for resveratrol, 4-coumaroyl-CoA and malonyl-CoA, only a single gene, stilbene synthase, is required to produce minor amounts of the metabolite in any plant. Optimization of precursor pathways would also be necessary to enhance nutraceutical levels in any given food . Glucosinolates, and their hydrolysis products, isothiocyanates, are another family of compounds with great health potential that have been studied thoroughly . A recent review examined the vast diversity of these compounds, totaling 156 unique entries, emphasizing their identification and synthesis in plants . This diversity can be broken down into three types of glucosinolates based on the amino acid precursor: aliphatic, indolic, or benzenic, with the vast diversity derived from secondary modifications to these basal categories. Over decades of study, biosynthetic pathways for each type have been elucidated . Glucoraphanin is a glucosinolate found in high concentration in broccoli and has been a subject of intensive study due to the health benefits of it and its isothiocyanate, sulforaphane . Various studies have displayed the promise of sulforaphane in treating diseases such as neurological disorders, diabetes, cancer, and cardiovascular disease with variable efficacy observed over a vast range of dosages and sources of the metabolite; however, further clinical validation is needed to verify these health benefits . Medicinal interest in glucoraphanin led to its successful production in Nicotiana benthamiana through transient expression, though only small amounts were produced . This was one of the first examples of a complex pathway being heterologously expressed in plants, and with some optimization, nft growing system it could be stably introduced into a crop species for consumption. The use of breeding practices that select plants for favorable agricultural traits can remove important nutrients from cultivated crops. This can also reduce some of the important nutraceutical compounds found in the foods that we eat. For example, there is a complete lack of iridoids, a class of monoterpenes suggested to benefit human health, in most cultivated varieties of blueberry on the market, while every wild species tested was abundant with them . While this was an inadvertent consequence of breeding, it illustrates the need to investigate wild relatives of food crops to identify nutraceuticals lost during domestication.

Using the tools of plant synthetic biology, we should be able to quickly and easily reintegrate the pathways lost into commercial cultivars without the need for the lengthy breeding process. As we move forward in our efforts to produce advanced nutritive foods, it is important to examine the past uses of nutraceuticals in the prevention and treatment of disease. Traditional medicinal plants have seen renewed interest as sources for undiscovered bio-active compounds . This highlights the need for additional compound and pathway discovery efforts. There are many other reviews that discuss the different types of specialized metabolites found in medicinal plant species . Historically, medicinal plant extracts have been used as food additives, but introduction of their biosynthetic pathways into common food crops offers a means of increasing the availability of nutraceuticals to a broader market. In some cases, the biosynthetic pathways are known and efforts are being made to sequence more medicinal plant genomes in order to accelerate enzyme discovery in candidate organisms . Nevertheless, the major limiting step hampering efforts to engineer plant nutraceuticals is still the discovery of the enzymes involved in the biosynthesis of target plant natural products. Thus, new tools to streamline such research is of great interest to the larger plant synthetic biology field.Plants have many attributes that make them an ideal platform for the production of plant natural products. They are autotrophic, can be grown at a large scale, contain various intracellular compartments and tissue types, are used as a nutrient delivery system, and retain similar cellular features to the productproducing plant that may be required for efficient biosynthesis of compounds. Together, this enables countless strategies for the production and administration of plant natural products . However, the state of plant biotechnology has imposed limitations on the utilization of plants. In this section, we focus on the strengths of plants as a production platform and the technologies needed to improve their efficacy.Perhaps one of the greatest strengths of plants as a synthetic biology platform is their scalability. Large-scale plant production has been a focus of human society and technology since the first crops were cultivated. In addition to a long history of scalable plant cultivation for consumption, agriculture also offers a unique platform for the production and extraction of valuable therapeutic plant natural products. Opioids from Papaver somniferum, taxol from Taxus brevifolia, and QS-21 adjuvants from Quillaja saponaria, are several notable examples of therapeutic molecules that are produced and extracted from their native plant host . While many therapeutic chemicals can be extracted from their native producer, many species are difficult to cultivate or suffer from limited yields . Past efforts have focused on the production of nutrients and phytochemicals in microbes or with the use of chemical synthesis, but plants are an ideal platform for the production of small molecules at a large scale, as they are autotrophic and can be grown in non-axenic conditions like an open field. There are two main approaches for the heterologous expression of a specialized metabolic pathway in planta: transient expression or expression in stable plant lines. Transient expression typically utilizes Agrobacterium tumefaciens to insert genes of interest into a host plant. This process allows for the expression and characterization of genes in 3–6 days as opposed to the months required to make stable plant lines . Additionally, multiple strains of Agrobacterium harboring different genes can be co-infiltrated into host tissue, limiting the need for creating multi-gene constructs . These attributes have made it a valuable tool for in planta pathway discovery and protein production. Transient expression is usually conducted in a species of the genus Nicotiana but is typically only applied at small scales, limiting its viability as a production platform. Recent improvements by Reed and Osbourn have demonstrated the scalability and viability of transient expression for the production of phytochemicals. Through the use of full plant transient expression via vacuum infiltration, gram-scale quantities of various triterpenes were able to be produced and purified from Nicotiana benthamiana . As well as being a production platform, transient expression permits the testing of gene construct efficacy prior to stable line generation; however, the efficiency of Agrobacterium-mediated transient expression is variable in different hosts . This poses a limitation of Agrobacterium as a tool to screen genes in host plants. Additionally, transiently expressed pathways in Nicotiana benthamiana do not always function as expected in the desired host, limiting its use for screening constructs before generating stable lines . One method to circumvent these issues is the use of protoplasts, calli, and cell suspension cultures. These cell types are suitable for the use of direct transformation methods such as electroporation, gene gun bombardment, and microinjection. However, generation of these cell lines is often inefficient depending on the species used, produces fragile cells, and may not behave as an intact plant would .

Counts of uniquely mapping reads were generated through HTSeq for all 35 RNA-seq datasets . Multimapping reads were excluded from the analysis except for the tandem gene expression analysis. Differential gene expression analysis was performed using the DESeq2 pipeline across fruit developmental stages with three biological replicates per developmental stage . Gene expression values were derived by calculating the fragments per kilobase per million reads mapped values using the standard formula for FPKM /gene length in kilobases. To construct the gene co-expression network, genes that were not expressed or very weakly expressed in 30 or more conditions were first excluded from the analysis. The count data was then transformed into variance stabilized values using the variance stabilizing transformation function in DEseq. Pairwise correlations of gene expression were calculated using Pearson correlation coefficient and mutual rank using scripts available for download from the project’s data repository. MR scores were transformed to network edge weights using geometric decay function e−; five different co-expression networks were constructed with x set to 5, 10, 25, 50, and 100, respectively. Edges with PCC <0.6 or edge weight <0.01 were excluded. For each network, modules of coexpressed genes were detected using ClusterONE v1.0 using default parameters, blueberry grow pot and modules with P value > 0.1 or quality score <0.2 were excluded. The results from all co-expression networks were then combined by collapsing modules into meta modules of non-overlapping gene sets.

Total antioxidant capacity of tissues from the fruit developmental panel was analyzed using the ORAC assay. Briefly, ∼20–30 mg of frozen ground fruit tissue was measured for tissue samples prior to extraction. Sample extractions were performed on ground tissue using 1.8 mL of ice cold 50% acetone. Samples were vortexed and then put on a shaker for 5 minutes at room temperature. Samples were then centrifuged at 4◦C for 15 minutes . The ORAC assay was performed in a 96-well black microplate using the FLUOstar OPTIMA microplate reader . Each reaction well contained 150 μL of 0.08 μM fluorescein and 25 μL of 75 mM phosphate buffer , Trolox standards , or diluted sample extracts. For blueberry tissue samples, 1:80–1:20 dilutions were used. Upon loading all appropriate wells, the 96-well microplate was put into the microplate reader and incubated for 10 minutes at 37◦C. Following incubation, 25 μL of 150 mM AAPH was added to each well, and fluorescence measurements began immediately. Fluorescence measurements were taken for 90 seconds per cycle for 70 cycles until the fluorescent probe signal was completely quenched. The area under the fluorescence decay curve was calculated for each well. The total antioxidant capacity of a sample was calculated by subtracting the AUC from the blank curve from the AUC of the sample curve to obtain the net AUC. Using Trolox of a known concentration, a standard curve was generated , and the total antioxidant capacity of each sample was calculated as Trolox equivalents. Each sample was run twice for two technical replicates. The coefficient of variation between technical replicates was required to be less than 0.20. Biological replicates were run for all tissues in the fruit developmental series.Berries from ”Draper” were collected as described above.

Approximately 100 mg of each frozen ground sample was resuspended in extraction solvent in a 2 mL tube . Ground tissue was immediately mixed thoroughly to prevent thawing during extraction and to prevent metabolism of analytes by enzymes in the samples. All tubes were spun down for 10 minutes at 13,000 × g to pellet protein and other insoluble material. Then, 1 mL of supernatant was transferred to an autosampler vial. Anthocyanin content was evaluated by LC-MS as follows: 5 uL of sample extract were separated using a 10 minute gradient on a Waters Acquity HSS-T3 UPLC column on a Waters Acquity UPLC system interfaced with a Waters Xevo G2-XS quadrupole time-of-flight mass spectrometer . Column temperature was maintained at 40◦C, and the flow rate was 0.3 mL/min with starting conditions of 100% solvent A and 0% solvent B . The gradient was as follows: hold at 100% A for 0.5 minutes, ramp to 50% B at 6 minutes, then ramp to 99% B at 6.5 minutes, hold at 99% B to 8.5 minutes, return to 100% A at 8.51 minutes, and hold at 100% A until 10 minutes. Mass spectra were acquired in positive ion mode electrospray ionization over m/z 50–1500 in continuum mode using a data-independent MSE method that acquires data under both low and high collision energy conditions with the high collision energy setting using a ramp from 20–80 V. Capillary voltage was 3 kV, desolvation temperature was 350◦C, source temperature was 100◦C, cone gas flow was 25 L/hr, and desolvation gas flow was 600 L/hr. Correction for mass drift was performed using continuous infusion of the lock mass compound leucine encephalin. Anthocyanins and other related flavonoids were identified based on accurate mass and fragmentation pattern. Peak areas were determined using Quanlynx within the Masslynx software package . Relative anthocyanin content was calculated for each sample using the formula: reported peak area of the compound/peak area of internal standard/weight of extracted tissue .Insect repellents have been used since antiquity to fend off disease-transmitting mosquitoes and other arthropods. They developed gradually from smoke generated by burning plants and topical applications of essential oils into repellent substances, including those isolated from plants and a broad-spectrum synthetic repellent DEET , which was discovered in the early 1940s from a screening of more than 7000 compounds . Thereafter, other synthetic repellents have been developed, including IR3535, aminopropionate and picaridin piperidine-1-carboxylate, but DEET remains the most widely used repellent substance worldwide , particularly in the United States of America. Repellents work primarily as spatial and contact repellents. Mosquitoes attracted to and flying towards vertebrate hosts may make oriented movements away from the source upon approaching chemically treated skin surfaces. In this case, the chemical is a repellent sensu stricto . Because the repellent is acting from a distance, it may be referred to as a spatial repellent . When mosquitoes land on a chemically treated skin thus making contact before starting increasing locomotion activity or taking off, the chemical is called a contact repellent, which is sometimes referred to as excitorepellent, irritant, or contact irritant . From a strict mechanistic viewpoint, these two groups of compounds should be named non-contact and contact disengagent for spatial and contact repellents, respectively. It is now known that at least Culex and Aedes mosquitoes smell DEET . More importantly, it has been demonstrated that an odorant receptor from the Southern house mosquito, Cx. quinquefasciatus, CquiOR136, is essential for reception of DEET as a noncontact disengagent . Recently, it has been demonstrated that as a contact disengagent, DEET is detected by sensilla on the tarsal segments of the legs of the yellow fever mosquito Aedes aegypti , but the receptors remain elusive. Lastly, it has been suggested that DEET merely masks the reception of human emanations by Anopheles coluzzii , thus reducing the attractiveness of the host. Although its modes of action remain a matter of considerable debate, DEET is a gold standard repellent. It also has many “off-label” properties that do not directly affect human mosquito interactions. For example, DEET is a feeding deterrent , hydroponic bucket but if this were the primary mode of action, it would have little value in epidemiology.

The great value of repellents is that they reduce biting rates, which represents a second order parameter in vector capacity . Another property that may have a value in epidemiology, albeit not by decreasing vector capacity, is the deterrent effect of DEET on oviposition, as first observed for Ae. aegypti . While using the Xenopus oocyte recording system to deorphanize odorant receptors involved in the reception of oviposition attractants, we observed that DEET elicited outward currents in our preparations, in contrast to oviposition attractants and other compounds that generated inward currents. We have now systematically investigated this phenomenon using different ORs from three different species of mosquitoes. Here, we report that DEET, IR3535, and picaridin elicit outward currents on OR involved in the reception of mosquito oviposition attractants in the Southern house mosquito, Cx. quinquefasciatus, and orthologues from the yellow fever and malaria mosquitoes. Dose dependent outward currents were also observed with compounds in a panel that included plant-derived and plant-inspired repellents. Like DEET, IR3535 and picaridin , plant-inspired compounds, elicited robust inward current in the DEET receptor, CquiOR136, and showed repellency activity.In vitro transcription, oocytes microinjection, and electrophysiology were performed as previously described . Briefly, in vitro transcription of cRNAs was performed by using an mMESSAGE mMACHINE T7 kit , according to the manufacturer’s protocol. Plasmids were linearized with NheI, SphI, or PstI, and capped cRNAs were transcribed using T7 or SP6 RNA polymerase. cRNA samples were purified with LiCl precipitation solution and resuspended in nuclease-free water at a concentration of 200 μg/mL and stored at −80°C in aliquots. RNA concentrations were determined by UV spectrophotometry. cRNA samples were microinjected into stage V or VI Xenopus laevis oocytes . A two-electrode voltage clamp was used to detect currents. Oocytes were placed in a perfusion chamber and challenged with test compounds. Odorant-induced currents were amplified with an OC-725C amplifier , the voltage held at −80 mV, low-pass filtered at 50 Hz and digitized at 1 kHz. Data acquisition and analysis were carried out with Digidata 1440A and pClamp10 software .In short, two 50-mL Dudley bubbling tubes painted internally with a black hobby and craft enamel were held in a wooden board , 17 cm apart from each end and 15 cm from the bottom. The board was attached to the frame of an aluminum collapsible field cage . Two small openings were made 1 cm above each Dudley tube to hold two syringe needles to deliver CO2. To minimize handling of mosquitoes, test females had been kept inside collapsible field cages since the latest pupal stage. These female cages had their cover premodified for behavioral studies. A red cardstock was placed internally at one face of the cage, openings were made in the cardboard and in the cage cover so the cage could be attached to the wooden board with the two Dudley tubes and CO2 needles projecting inside the mosquito cage 6 and 3 cm, respectively. Additionally, windows were made on the top and the opposite end of the red cardstock for manipulations during the assays and a video camera connection, respectively. The two cages were connected at least 2 h prior to bioassays. At least 10 min before the assays, water at 28°C started to be circulated with a Lauda’s Ecoline water bath, and CO2 at 50 mL/min was delivered from a gas tank just at the time of the behavioral observations. Sample rings were prepared from strips of filter papers 25 cm-long and 4-cm wide and hung on the cardstock wall by insect pins to make a circle around the Dudley tubes. Cotton rolls were loaded with 100 μl of defibrillated sheep blood purchased from UC Davis VetMed shop and placed between a Dudley tube and CO2 needle. For each run, one paper ring was loaded with 200 μL of hexane and the other with 200 μL of test repellent in hexane. The solvent was evaporated for 1-2 min, blood-impregnated cotton plugs and filter paper rings were placed on the arena, CO2 was started, and the assays recorded with an infrared camera . During the assay, the arena was inspected with a flashlight with a red filter. After 5 min, the number of females that have landed and continued to feed on each side of the arena was recorded. Insects were gently removed from the cotton rolls and the assays re-initiated after rotation of sample and control. Thus, repellence for each set of test mosquitoes was measured with the filter paper impregnated with the same sample at least once on the left and once on the right side of the arena.To revisit our earlier observation of repellent-induced outward currents on OR sensitive to oviposition attractants, we challenged CquiOR21/CquiOrco-expressing oocytes with DEET and then skatole. CquiOR21, formerly known as CquiOR10 , is narrowly tuned to the oviposition attractant skatole . CquiOR21/CquiOrcoexpressing oocytes generated robust inward currents when stimulated with 10 μM skatole, whereas 1 mM DEET elicited outward currents . These outward currents were dose-dependent and were not observed when oocytes were injected only with CquiOrco cRNA or CquiOR21 cRNA .

Briefly, for PD3 and PW with and without BSA, media plates solidified with agar or Gelrite were divided into two halves, 10 to 12 spots of each mutant strain were made using a sterile toothpick, and plates were incubated at 28°C for 4 to 5 days. For XFM with and without BSA, plates solidified with agar were used and incubated for 10 to 12 days before measurements were recorded. Colony peripheral fringes were observed under 10 magnification using a Nikon Eclipse Ti inverted microscope , and fringe widths were measured for six colonies per plate per strain, with at least seven measurements per colony using a Nikon DS-Q1 digital camera connected to a Nikon Eclipse Ti inverted microscope and controlled by NIS-Elements imaging software version 3.0. Twitching experiments were performed at least three times independently for PD3 and PW with and without BSA and once for XFM with and without BSA. Three growth conditions were used: solid agar plates , liquid culture tubes , and continuous liquid flow . PD3 without antibiotics was the medium used, and the initial inocula of the NS1-CmR and pglA-KmR mutants were prepared as described above. Competence in tubes. Twenty-five-milliliter glass test tubes containing 3 ml of PD3 were inoculated with 100 l of each of the OD adjusted strain suspensions as donor and recipient cells . Tubes containing single strain inoculations were included as control treatments.

Tubes were then incubated with shaking . After 3 days, the tubes were vortexed well to mix the biofilm formed on the air-liquid interface with the rest of the suspension and serially diluted and plated as described above. Three independent experiments were performed, nft hydroponic system and three replications were included in each experiment . Competence in MCs. MCs were prepared as previously described . Briefly, two parallel channels with separate inlets for bacterial cells and growing media were etched on a silicon wafer. The channels were modeled into polydimethylsiloxane and sandwiched between the PDMS layer and a glass cover slide. The inlets and outlets were then connected to tubings that were connected to syringes . The syringes were connected to pumps which control the media flow rate in the MC. The MC was mounted onto a Nikon Eclipse Ti inverted microscope to observe cell attachment and biofilm formation using phase-contrast and Nomarski differential interference contrast optics. Time-lapse video was taken using a Nikon DS-Q1 digital camera connected to the microscope and controlled by NIS-Elements imaging software version 3.0. For preparing the inocula for MCs, equal volumes of the strain pairs were mixed and inoculated into the cell inlet syringes, and growing medium was injected in the media syringes. MCs were run for 5 to 7 days with a media flow rate of 0.25 l min 1 until abundant growth of biofilm was observed. At the end of the experiment, the fraction of cells collected in the outlet syringe was harvested, and the fraction formed inside the channels was detached and pushed to the outlet collection syringe by increasing the flow rate to 30 to 40 l min 1 . Serial dilution, plating, CFU counts, and the frequency of recombination calculations were done as described above. Four independent experiments were performed with seven replicates in total . 

Competence in MCs with grapevine sap. Grapevine sap was collected from a X. fastidiosa-susceptible variety in Dahlonega, GA, and a tolerant variety in Tallahassee, FL, at the end of the dormant season . A new season cane was pruned, and sap was collected in a 50-ml conical tube, which was stored in ice until it was brought back to the lab. Xylem sap was sterilized by filtering with a 0.22- m vacuum filter and stored at 80°C until used. Sap experiments were performed in the MCs with both pure sap and 50% sap mixed in PD3 . Natural competence assays were same as those for the MC experiment with PD3. Experiments were repeated at least three times for both sap types. Natural competence with heat-killed donor cells and confirmation of homologous recombination. Confirmation of homologous recombination occurring via natural competence was performed by using heat killed donor cells in the solid agar plates. Suspensions of the donor cells were incubated at 90°C for 15 min for heat killing. Complete killing was confirmed by plating an aliquot onto PW plates. The heat-killed donor and live recipients were then spotted on PD3 plates as described above. For confirmation of homologous recombination at the desired genome region, randomly selected recombinant CFU were restreaked onto new double-antibiotic PW plates, and colony PCR was performed using the primers targeting the flanking region of the construct used to generate the mutants according to Kung et al. . Sequences of the flanking regions of antibiotic cassette insertion sites between the parent strains were compared using the muscle pairwise alignment algorithm within the Geneious 9.0.3 platform . Statistical analysis. The number of recombinants, total CFU, and recombination frequency data were analyzed in PROC GLIMMIX , which fits statistical models to data with nonnormal distribution and nonconstant variability.

For the analysis of frequency, the response distribution was used as the binomial distribution of number of recombinants/ total cells. Least-squares differences of means among the treatments were separated by Tukey’s honestly significance difference test at the significance level P 0.05. For the repeated experiments, time factor was used as a random variable. The fringe widths of bacterial colonies among different media also were compared using PROC GLIMMIX in SAS. Accession number. Sequences of the flanking regions of antibiotic cassette insertion sites between the parent strains were deposited in NCBI under accession numbers KU873007 to KU873014.Several hypotheses have been proposed to explain the existence of natural competence in bacteria. One explanation is that starvation signals induce competence, and the incoming DNA serves as a nutrient source under poor nutrient conditions as demonstrated in H. influenzae , Pseudomonas stutzeri , and R. solanacearum . Based on the results with a minimal medium and a rich undefined medium , a previous study speculated that growth in a low-nutrient medium favors natural competence inX. fastidiosa. However, the results of this study with these two media and PD3, another undefined rich medium, demonstrated that growth in PD3 significantly increases the recombination frequency. This suggests that starvation is not necessary to induce competence in X. fastidiosa. Further investigations of the differences between PD3 and PW were performed by either removing or adding these components to/from one another. Initial screening with the components showed a pronounced effect of BSA on the number of recombinants recovered. Additional experiments confirmed that BSA significantly reduces the recombination frequency when present in PD3, PW, and XFM. Since both XFM and PW contain BSA, this may explain the lower recombination frequencies in these media. In a previous study, BSA had been found to reduce the surface attachment and twitching motility of X. fastidiosa . In fact, natural competence and twitching motility are dependent on the activity of type IV pili in X. fastidiosa . Therefore, in this study the correlation between twitching movement and natural competence in different media was investigated. Interestingly, PD3 allowed the highest fringe width, and the presence of BSA significantly reduced twitching motility in all three media. Twitching motility in XFM was lower than in either PD3 or PW as poor growth in XFM resulted in smaller colony sizes. Still the fringe widths of colonies in XFM without BSA were bigger than in XFM with BSA.

Most of the colonies spotted in XFM and XFM-BSA showed very little or no visible growth. This can be expected as XFM is a nutrient-limited minimal medium. Moreover, the pglA-KmR mutant that did not show twitching movement was not competent when tested with heat-killed NS1-CmR mutant and plasmid DNA as the donor. These results of concomitant decreases in natural competence and twitching motility in BSA-supplemented media and non-competency of twitch minus strain suggest that twitching motility is correlated with natural competence in X. fastidiosa. Natural competence in other Gramnegative bacteria is mediated by type IV pili-like structures . In light of the effect of BSA on twitching, hydroponic nft system it remains to be determined if BSA only alters movement or bio-genesis of type IV pili. Our results with different growth settings showed that the recombination frequency is significantly higher in the MC_in fraction than in the MC_out fraction. The MC_in environment closely mimics xylem vessels and the insect foregut with respect to continuous liquid flow, adhesion of cells on channel walls in a fashion similar to adhesion of cells on xylem vessels and the insect foregut, and formation of biofilms. This environment is conducive for both biofilm formation and twitching motility as demonstrated in previous studies . Moreover, expression levels of some of the type IV pili genes were shown to be increased in the MC_in environment compared to those under the other growth conditions , implying that activity of type IV pili is increased in this system, which may explain the higher rates of recombination in the MC_in fraction. The MC_out environment, on the other hand, consists mostly of planktonic cells and some detached biofilm fraction from MC_in, which is washed away with the liquid flow. The differences in recombination frequencies in these two environments suggest that the continuous media flow condition of the xylem vessels and growth in biofilm may increase the chances of recombination. Batch cultures in tubes also allowed recombination but at a lower rate than the continuous flow environment of MC_in and surface-attached condition of solid agar plates. A previous study also showed that growth in solid plates increases recombination compared to the growth in the liquid culture tubes .Recombinantsin theMC_outfraction were recovered when profuse biofilm growth was observed in the MC_in fraction with many recombinants formed. It is possible that the recombinants recovered in the MC_out fraction are due to detachment and washing away of portions of biofilms from the MC_in fractions, supporting the proposition that biofilm formation induces competence. Biofilm formation and quorum sensing signals have been shown to induce natural competence in other naturally competent bacteria such as Vibrio cholerae, Acinetobacter sp. , and Streptococcus mutans . Biofilms, in addition to having dense populations of cells, contain elevated amounts of extracellular DNA , which can be used for transformation by competent cells. Extracellular DNA has been shown to enhance biofilm formation in X. fastidiosa . Moreover, Kung et al. also showed that a knockout mutant on a biosynthetic gene for diffusible signaling factor , a cell-cell communication signal in X. fastidiosa, had a reduced rate of recombination, implying that a cell-cell communication signal also may be involved in regulating natural competence in X. fastidiosa. MC experiments with grapevine sap provide a closer resemblance to the natural habitat than MCs with the artificial culture medium. Previously, we have shown that the biofilm structure in grapevine sap is more similar to the natural biofilm than are the aggregates observed in synthetic medium inside MCs . The experiments with amendments of sap in the MCs detected natural competence, providing an indication that natural competence occurs in the xylem vessels of host plants and possibly in the insect vectors. Although the results with pure sap experiments were not reproducible due to inconsistent growth of one of the strains used, recombinants were recovered once with pure Chardonnay sap as the medium. Recombinants were readily recovered with the 50% sap in PD3 for both tolerant and susceptible varieties. Maintenance of competence with the addition of xylem sap indicates that sap components support DNA acquisition and transformation. Natural competence occurring in environments resembling natural habitats also have been demonstrated in other naturally competent bacteria such as P. stutzeri and V.cholerae, in which artificial medium resembling natural soil extract and natural growth substrate , respectively, induced competence. In R. solanacearum, another xylem-colonizing plant pathogen, natural competence has been demonstrated in planta , and the recipient strains were shown to have increased virulence, acquiring DNA regions as long as 40 kb from donor strains. Findings from competence experiments with grapevine sap and the MCs suggest that when two different strains are established together in the xylem vessels or in the vector foregut, recombination is possible. Noteworthy is the fact that in the experiments reported here, recombination was higher with sap from a tolerant grapevine variety, where infection by X. fastidiosa is symptomless.