Horticulture

As a reduction of water and nutrients increased leaf loss and changed the colour of leaves in the same year when stress was imposed, future studies should investigate plant performance of almond experiencing pollination limitation in subsequent years after stress was imposed and after long-term limitations to plant resources.Worldwide, tephritid fruit flies are among the world’s most economically important crop pests, with at least 200 pest species. In sub-Saharan Africa, several highly polyphagous Africa-native species—belonging to the genera Ceratitis Macleay and Dacus Fabricius have been recognized as economically important pests of several cultivated and wild fruit species, particularly mango , guava , citrus , and several cucurbit and solanaceous vegetables. The traditional problems with tephritid fruit flies have been aggravated in recent years by the invasion of the African continent by the Oriental fruit fly Bactrocera dorsalis , gallon pot which was first detected in coastal Kenya in 2003 and has spread to at least 32 countries in continental Africa and adjacent island countries.

Since the detection of B. dorsalis in Africa, several studies have established the African host range of this species and quantified crop losses due to its infestations. At present, B. dorsalis has been found infesting fruits of 40 host plant species, with mango, guava, citrus, and loquat Lindl. being the major infested cultivated hosts. In addition to causing extensive fruit losses in the field, fruit flies greatly restrict mango and other host fruit exports from Africa, particularly to the European Union , which, for example, intercepted and rejected more than 141 shipments of Cameroonian mango from 2011 to 2018, resulting in substantial financial losses. While several control methods have been developed and deployed across the continent, the large majority of fresh fruit producers in Cameroon and throughout Central Africa continue to experience substantial yield losses caused by fruit flies and they do not yet have the necessary resources and knowledge to successfully use available and new fruit fly control methods. The prevailing agronomic and plant protection practices are of very low or no input type. Apart from the common occasional weeding, pesticide and fertilizer inputs are rare. Knowledge of fruit fly species composition and their respective seasonal abundance using complementary monitoring tools in relation to host plant phenology under different environments is crucial to the understanding of population dynamics of these insects and the subsequent development and implementation of interventions to limit their infestations and damage.

Such knowledge is predicated on proper fruit fly species identification and quantification of the levels of host infestation which are fundamental for establishing the economic status of the pests and ultimately for developing and adopting effective pest control interventions. Two approaches have been traditionally used to provide the aforementioned needed information: effective tools based on food baits and male lures for monitoring and estimating the abundance of adult fruit flies, and systematic fruit samplings to determine host range and quantify the levels and rates of fruit infestations by the various fruit fly species present in the systems. The latter is often complemented with random fruit sampling from areas outside the targeted cultivated fields to determine the fruit fly host range. Ideally, monitoring tools and host fruit infestations should be tested and used over several years and in multiple environments to establish sufficient details of the bio-ecological context where management options will be developed and implemented. Several commercially available male lures and food baits have been developed and used widely for fruit fly monitoring, but their performance has been shown to vary with factors such as climate, fruit fly species, and other factors that affect fruit fly populations. All available studies in Africa are from several agro-ecologies, but none are from the midaltitude, high rainfall agro-ecologies that are prevalent in much of the Congo Basin of Central Africa.

The male lures methyl eugenol and terpinyl acetate are known to, respectively, attract Bactrocera and Ceratitis species, while Culure is known to attract various Dacus species. For principally females, several food baits including Torula yeast, Mazoferm, and Nulure have been developed and used to attract and monitor several fruitfly species. To our knowledge, monitoring the performance of food baits and male lures on fruit flies under the various environments that are prevalent in Central Africa, is lacking. Similarly, compared with other regions of Africa, information on fruit fly species composition, host range, crop losses, and seasonality, as well as various trapping approaches and control measures in Central Africa, are scarce. The Congo Basin of Central Africa harbours a rich humid forest with a high diversity of wild fruit trees that could, at the same time, represent a reservoir for fruit flies and their natural enemies. Central Africa further includes the five key agro-ecologies encountered across the African continent, from desert and arid agro-ecologies to dense high-rainfall and humid forest zones. Preliminary information from fruit collections in Cameroon revealed the presence of several species including B. dorsalis, Ceratitis cosyra , Ceratitis anonae , Ceratitis capitata , Ceratitis quinaria , Dacus punctatifrons Karschand Dacus bivittatus. Continuing to be scarce, however, is multi-year quantitative information on fruit fly’s species composition and their seasonal dynamics, host utilization, and fruit infestation levels, particularly from the principal commercial fruit species mango and guava, and the performance of different monitoring tools in mid-altitude humid and high rainfall agro-ecologies from Central Africa. The broad objective of this study is to establish and validate basic multi-year data necessary for the development of integrated pest management programs of fruit flies across two agro-ecological zones in Cameroon with contrasting climate and farming systems, representing a cross-section of the mid-altitude agro-ecologies of Central Africa. The study has the following specific objectives: determine the diversity of fruit fly species and the level of their infestation of mango, guava and other common fruit hosts; compare the performance of male lures and food baits for monitoring the abundance and seasonality of fruit flies in mango and mixed fruit orchards; and determine the contribution of temperature, relative humidity, and rainfall amount to the variation in fruit fly abundance. The results from this Cameroon study can possibly be extended to the rest of the Congo Basin, gallon nursery pot as the southern half of Cameroon is widely considered agro-ecologically a close representative of much of the rest of Central Africa.The study was conducted over 5–6 years in 2 agro-ecological zones of Cameroon as delimited by the Cameroon Institute of Agricultural Research for Development. The two target AEZs included the western highlands, with a mono-modal rainfall pattern , and humid forest, with bimodal rainfall . Both AEZs differed in their topography, climate characteristics, and cropping systems. The choice of the two zones was based on the richness, diversity and availability of fruit tree species. One experimental site was established in each AEZ for fruit fly trapping using food baits and male lures, and for evaluation of fruit infestations by fruit flies. Because of the long-term nature of the experiments and the need to secure traps for continuous monitoring over a period of 6 years, the traps were installed in the experimental orchard of the IRAD research station in Foumbot, for the WH-MR, and at the International Institute of Tropical Agriculture in Nkolbisson, for the HF-BR . Each orchard was characterized according to the description of the area, climatic conditions, fruit species present and management options . A homogenous hectare of mango was used in Foumbot, while a mixed hectare of fruit species was selected in Nkolbisson .All traps were suspended from tree branches with a galvanized steel wire at ~2 m above ground and at least 20 m apart.

The wire was coated at its middle length with a thick layer of Tanglefoot to prevent cursorial access to the traps by predators, particularly the common weaver ant Oecophylla longinoda . The number of traps varied according to the number of attractants used. For this purpose, 2 and 4 traps of each attractant were installed, respectively, at the Nkolbisson and Foumbot sites, for a total of 10 traps in Nkolbisson and 12 traps in Foumbot. For male lure-based traps, a dental cotton roll soaked with 2 mL of either methyl eugenol or terpinyl acetate was suspended from the centre of the trap lid. Two, 5 cm strips impregnated with 2, 2-Dimethyl dichlorovinyl phosphate  were placed at the bottom of the trap as the killing agent. For food-bait traps, BioLure’s 3 components, packed individually in a sachet containing either ammonium acetate, trimethylamine, or putrescine, were adhered to the inside of the Multilure trap. Torula yeast was used as a liquid bait consisting of 2 pellets dissolved in 350 mL of water per trap. Similarly, the commercial product Mazoferm was diluted in 350 mL of water to obtain a 6% concentration, with 2 g of borax added to the solution as a preservative.Food bait and male lure traps were inspected in both orchards at weekly intervals. Torula yeast and Mazoferm baits were replaced weekly, while BioLure, male lures, DDVP strips, and cotton rolls were renewed monthly. Trap servicing techniques and regular rotation among trees followed those of. All the specimens were transferred and preserved in vials containing 70% ethanol. All samples were brought to the Entomology Laboratory of IITA in Yaoundé for identification. Meteorological data were collected at each location with a Hobo Pro v2 data logger for temperature and RH , and a Tru-Chek® DirectReading rain gauge . Temperature and RH were recorded at hourly intervals and the data were retrieved at monthly intervals, while rain amounts were collected between 7 and 8 am daily throughout the study periods.Fruit sampling was carried out from 2011 to 2015. Systematic random sampling was used in the two AEZs to determine the diversity of fruit flies associated with mango and guava fruits in orchards and home gardens. The mango variety “Camerounaise” and two varieties of guava were available at Nkolbisson orchard. At Foumbot orchard, mango varieties included Ruby, Zill, Irwin, Julie Nyombe, Palmer, and “Camerounaise”, and as in the Nkolbisson orchard, there were local and improved guava varieties of unknown names. Twenty mature fruits each of mango and guava—based on the varieties’ maturity status—were harvested randomly from all sampling sites, and up to 10 fallen mature fruits were collected from the ground at 2-week intervals from five trees of each fruit species. Fruits from other cultivated and wild plants were also collected during their fruiting periods from orchards, home gardens, and natural vegetation within a 70 km radius of each of the two experimental sites in WH-MR and HR-BR to determine the host range of fruit flies and the infestation levels. The number and size of fruit samples from the various plant species were primarily determined by the availability of fruits. Efforts were made to ensure a minimum collection of 20 fruits per sample at each location. Collected fruits were classified by species, known variety, date, and sampling area, then counted and weighed. All fruits were incubated in a screenhouse at the IITA station in Yaoundé. Incubation units consisted of 450 mL plastic containers and 1.5 L circular plastic basins. Owing to their larger size, fruits of mango, papaya, and Annona spp. Were individually incubated in plastic boxes. The other fruit species were incubated in the circular plastic basins, but in groups of 3–5 depending on their size. Fruits were placed on a dome-shaped galvanized steel wire grid which rested on a 2–3 cm layer of moist heat-pasteurized Sanaga river sand as fruit flies pupariating medium. Each incubation unit was then covered with a fine-mesh cloth and secured to prevent larval escape. The incubation units were arranged on metallic shelves. The supports of each shelf were placed inside pint-size containers which were maintained at full capacity with soapy water as barriers against ants and other cursorial insects. Fruit samples were incubated for up to 4 weeks to ensure that all live fruit fly larvae exited the fruits. Sand in each incubation unit was sieved twice at 12 days after the start of incubation, and at the end of incubation for the collection of fruit fly puparia. Collected puparia from each container were placed in 9 cm Petri dishes and transferred to an insectarium maintained at 25 ◦C, 70 ± 5% RH, and photoperiod of 12L:12D for adult emergence. Emergence dishes contained a wet mixture of table sugar and enzymatic yeast as food for full wing development of emerging adults.

However, the underlyinggenetic mechanism has not been revealed for Z. cucurbitae. In fact, nonchemical stimuli, such as color, are associated with vision-related genes that allow the identifcation of different hosts . The genes responsible for color discrimination in Diptera are primarily related to opsin proteins in the photoreceptor cells of the eye . Six types of Rh opsin-expressed genes have been identified as major genes involved in color recognition and photoreception in Diptera insects. The Rh1 and Rh2 opsin genes are associated with motion detection and direction, respectively . Rh3 and Rh4 are UV-sensitive opsin genes, Rh5 is a blue-sensitive gene and Rh6 is a green opsin gene . These opsin genes lead the photoreceptor of eyes to receive various chromophore pigments and then activate a series of visual transduction cascades to launch corresponding color identification behavior. In the genome of polyphagous C. capitata, the long wavelength sensitive genes Rh1, Rh2, and Rh6 and the UV-sensitive genes Rh3 and Rh4 were found, black plastic pots for plants while Rh2-4 and Rh6 were found in the phototransduction pathway of oligophagous B. minax . Moreover, the role of Rh6 in modulating green color discrimination was reported in C. capitata and B. minax .

In B. minax, the function of Rh6, which is responsible for green spectral sensitivity, has been identifed by knockdown of the gene B. minax in female adults, and B. minax flies significantly reduced their preference for green fruit after cutting Rh6 . Absence of a member of the blue sensitive opsin subfamily was found in both tephritid species C. capitata and B. minax, but Rh5 can be specifically expressed in Drosophila . Reports about vision-related genes directly involved in the host expansion of tephritids are still very few.Tephritid fruit fy hosts expand to other new host plants, and the phenology of the new host is another nonchemical stimulus that affects fly adaptation. The phenology of the host plant fruits includes the time of flowering, fruiting, or maturation . Many studies have revealed that dormancy plays a crucial role in assisting insects in responding to various phenological environments, including the phenology of different host fruits . Therefore, genes associated with development are crucial factors that regulate the adaptation of phenology of various hosts. For example, genes related to sensing daylength or photoperiodism and the central nervous system regulate chronic adaptation . Under the regulation of related genes, diapause may involve the deceleration of the developmental progress of tephritids to synchronize the phenological environment . R. pomonella of tephritids is a typical case.

The ancestral host of R. pomonell is the hawthorn Crataegus mollis, but its species host expanded to the domestic apple Malus domestica and subsequently formed a new apple race . Apple fruits ripen earlier than hawthorn. The flies that infest apples and hawthorns must differentially time their life rhythms to match the differences in ripening times of their respective hosts . To realize this process, the flies of the two host races varied their time of overwintering pupal diapause. Under the pressure of different host fruit phenologies, many development-related genes are involved in regulating the adaptation to the different phenologies of two host plant fruits . Functional genes associated with cell/tissue development , metabolism , translation , and cell division are highly enriched . By increasing the expression levels of these genes, the CNS development of apple flies was elevated during their diapausing period compared to that of hawthorn flies. Adult emergence-associated genes, including key hormone signaling genes, the ecdysone receptor partner usp, the ecdysone biosynthesis protein ecd, cell cycling genes Myb and rbf, genes coding Mediator complex proteins, and various genes in the Wnt signaling pathway , etc., were enriched to regulate adult fy eclosion to match their host fruit ripeness .Genes coding for ribosomal proteins are often associated with protein translation by stably expressing ‘housekeeping’ genes. This type of gene is involved in many basic biological processes, such as digestion, detoxification, growth, and development, in most organisms .

Therefore, ribosomal genes may also be involved in the host plant expansion of tephritids after receiving chemical and nonchemical stimuli. As mentioned above, ribosomal genes increased their expression level to regulate the growth of R. pomonella in response to the different phenology of its new host apple . The role of ribosomal genes involved in host expansion and new host adaptation of insects, including tephritid flies, is mainly related to the response of ribosome-inactivating proteins in host plants . RIPs have been found to have insecticidal functions in many insects, including beetles, mosquitoes, and moths . Ribosome genes can help insects such as tephritids realize host shifting by regulating their expression levels to counteract the RIPs of various host plants . In addition, ribosome genes interact with some epigenetic factors, which leads to chromatin remodeling to change gene expression and regulate different biological processes, including host plant adaptation . In response to different secondary chemicals, ribosomal genes were also involved in host detoxification of different species of the R.pomonella complex. R. zephyria and R. pomonella are sister species in the R. pomonella complex that specialize in snowberry and domestic apple plants, respectively . In reciprocal transplant tests of these two Rhagoletis taxa, microarray data indicated signifcant enrichment of mitochondrial ribosomal proteins when the two fly species fed on their new hosts, which contain different complements of phenolic and glycosidic in laboratory studies . Several studies on lepidopteran species revealed the role of ribosomal genes in response to host expansion . For example, ribosomal genes were downregulated in C. suppressalis when extended to the novel host water oats , which may be a more suitable host for C. suppressalis than its native host, rice. In contrast, ribosomal genes were upregulated in H. armigera when shifting to unsuitable novel hosts .The role of genes associated with the oxidative phosphorylation pathway is primarily involved in energy metabolism and provides energy in the form of ATP for most organisms and most biological actions . The OXPHOS pathway is coupled with the mitochondrial electron transport chain, and mitochondria are major sites of reactive oxygen species production in the majority of eukaryotic cells . The level of mitochondrial oxygen fow through the OXPHOS pathway influences ROS homeostasis and regulates the energy supply in different biological processes . OXPHOS genes can take part in many biological activities, and therefore they may also be important in the regulation of the response to host plant expansion of tephritid flies. Research on Bactrocera tau reared on two native cucurbit hosts and a novel host showed a large number of upregulated NADH genes in the OXPHOS pathway in transcript data of B. tau when feeding on banana. These results suggest that OXPHOS genes play an important role in the process of novel host fruit use in B. tau . OXPHOS was also involved in the host expansion of R. pomonella in response to the different phenologies of various hosts, as mentioned above. Certain genes in the fat bodies of tephritids are also involved in the energy supply for many biological processes, including digestion, detoxification, drainage pot development, and immunity . Differentially expressed genes, such as the lipase gene, ATP synthase gene, and alpha-amylase genes , were documented in the tephritids B. dorsalis and P. utilis in response to different secondary chemical environments .The various types of genes summarized above led to multilevel responses in tephritids, including nervous-, behavioral-, chemical-, and physical-level responses, when the flies faced different host environments. These multilevel responses to host expansion result in multilevel adaptations in flies, which lead to successful expansion to a novel host . Adaptation to a novel host is a complex process. Multilevel adaptation in fruit flies results from multigene regulation rather than a single gene or several genes performing various regulatory roles. The transcriptome data revealed that olfactory-, digestion- and detoxifcation-related genes and ribosomal genes were all involved in novel host adaptation in R. pomonella . Laboratory strains of B. tau also had activated OXPHOS genes and digestive and detoxification genes when the fy responded to a novel host environment .

The multiple-gene regulation mechanism during host expansion to a novel host was also documented in other insects. For example, C. suppressalis launched three types of genes simultaneously to regulate adaptation to the new novel host water oat . S. yangi differentially expressed genes related to digestion, detoxification, oxidation–reduction, stress response, water deprivation, and osmoregulation during adaptation to the new host Ephedra lepidosperma . Various genes also regulate the adaptation of tephritids to new hosts via multiple mechanisms. As summarized above, the alteration of gene expression levels, gene family expansion, and the use of various gene types or subfamilies are the major mechanisms involved in novel host adaptation.Many tephritid species attack economically important crops, including vegetables and fruits. The economic losses caused by tephritids reach over US$2 billion annually . Control strategies for tephritids primarily involve chemical use in many countries, which may be harmful to the environment and human health. Therefore, more environmentally friendly control methods should be sought and recommended when possible. RNA interference is an effective method to safely control tephritid flies. RNAi control methods suppress the expression of certain target genes by importing dsRNA . Therefore, selecting the target genes to be ‘silenced’ is a key step in the RNAi control method . Some target genes are associated with functions such as temperature sensitivity and sex determination . For tephritid species, including Anastrepha suspense , Anastrepha fraterculus , B. dorsalis , B. minax , Bactrocera tryoni , and C. capitata , effective RNAi controls have been developed based on the suppression of functional genes associated with eye pigmentation, embryonic segmentation regulation, postembryonic growth/development, reproduction, embryonic temperaturesensitive lethality and sex determination . Based on these target genes, RNAi can be applied in pest control not only for tephritid species but also for some Coleopterans and Lepidoptera insects by foliar spays, ingested dsRNA or sterile insect technique application . However, functional genes related to host plant adaptation are also target genes in RNAi control methods for tephritids. For example, the vision-related gene R6 or gustation gene GR59f of B. minax , digestion-related genes try1, try2, try4, and try5 of B. dorsalis , olfactory Orco gene of B. oleae , CSP2 gene of B. dorsalis , and detoxification genes CYP6A41 and CYP6EK1 of B. dorsalis are associated with host adaptation functional genes, and all of these genes possess an exploitable potential as target genes to control fruit flies. More target genes related to host plant expansion for tephritids need to be identified for their major functions and implemented in pest management. Although RNAi is an effective and tractable genetic tool, other novel gene tools, such as clustered regularly interspaced short palindromic repeats and the CRISPR-associated protein 9 gene editing system, can also provide scalable pest control strategies . Compared with traditional RNAi, CRISPR‒Cas9 can knock down or modify the target gene precisely instead of just suppressing the expression of target gene . The target genes edited by the CRISPR‒Cas9 system can create stable and heritable strains, which can be applied in actual tephritid control. Applying the CRISPR‒Cas9-mediated editing system, some target genes in tephritid flies have been evaluated for their potential for functional application, such as the eye pigmentation gene we , embryonic segmentation gene prd , sex-determination gene Astra-2 , tra2 , and pupae color gene wp . CRISPR/Cas9-mediated precise editing is a process in whichCas9 endonuclease recognizes a specific genomic region under the leading of chimeric single guide RNA . The CRISPR/Cas9 system editing the functional target gene shibire, tsl in B. tryoni and the white pupae gene wt in B. dorsalis, C. capitate, and Z. cucurbitae have been applied in the development of genetic sexing strain application in SIT control. This gene tool also has broad application prospects in tephritid management based on host plant adaptation-related genes in the future. Regulation of host adaptation would be an important mechanism to target because this adaptation allows tephritids to expand in new habitats and change to new biotypes. Therefore, developing suitable novel host adaptation functional genes as target genes in genetic disruption control strategies could help prevent tephritids in an environmentally friendly manner.Fruit trees exhibit two major multiannual reproductive strategies . In the first, the amount of fruit produced allows a sufficient amount of vegetative growth to support production of an ample number of flowers during the following year . Such trees, including fig and some orange and grapefruit cultivars, are defined as regular bearers.

The lemon vacuolar H+-ATPase was purified and characterized by Taiz’s group . They revealed that, in fact, two tonoplast-bound ATPase activities exist, a nitrate-sensitive V-type ATPase that is partially inhibited by vanadate, and a vanadate-sensitive ATPase that is partially inhibited by nitrate . These results should be taken with caution because of the possible cross-contamination of the tonoplast vesicles with other membrane vesicles. Nitrate inhibition seemed to be dependent on the time of tonoplast vesicle preparation; for the same phenological stage, inhibition peaked during the spring and was minimal during the autumn–winter, suggesting an environmental effect resulting in seasonal changes in membrane lipid composition . Moreover, the H+/ATP coupling ratio varied between 1 to 2 as the DpH increased, displaying a pH-dependent slippage, where the hydrolytic activity and the H+ transport are partially uncoupled. Further, pot with drainage holes the fruit V-ATPase reconstituted into artificial proteoliposomes showed a steeper pH gradient than the corresponding reconstituted epicotyl enzyme .

Overall, the following characteristics seem to allow lemon fruit V-ATPase to generate a steep pH gradient: variable coupling, low pH-dependent slip rate, low proton permeability of the membrane, lower H+/ATP stoichiometry, and improved coupling by citrate, the major accumulated organic acid, which also enhance the enzyme’s ability to generate a pH gradient. The pyrophosphatase activity in acid lime fruit was much lower than that of H+-ATPase, suggesting the latter as the major mechanism for proton influx . Tonoplast vesicles isolated from juice cells of ‘Valencia’ oranges displayed similar V-type ATPase and V-PPiase activities, although a steady-state was reached faster with ATP as substrate. At a DpH of 3 units, V-PPiase synthesized PPi in the presence of Pi, indicating that mature orange juice cells acted as a source of PPi, providing a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage . In summary, in light of the possible presence of an additional tonoplastic H+ transport mechanism, P-ATPase, vacuolar proton homeostasis and transport across the tonoplast require further biochemical research. A vacuolar citrate/H+ symporter, CsCit1 , homologous to the Arabidopsis decarboxylate transporter, was characterized in orange fruit; its mRNA and protein levels coincided with the acid-decline stage, suggesting its role in citrate efflux .

Yeast cells expressing the CsCit1 displayed electroneutral coupled citrate–H+ cotransport with a stoichiometry of 1citrate/2H+.Amino acids have been studied in citrus fruit in relation to the nutritional value of the juice provided the motivation, mostly for early workers, to analyze the levels of free amino acids and their patterns of accumulation during fruit development and storage . The exposure of fruit to stress on-the tree and cold or heat treatments during storage was associated with the accumulation of several amino acids. Glycolysis and the tricarboxylic acid cycle are metabolically associated to amino acid metabolism , its relation to citrate decline and the induction of a γ-aminobutyric acid shunt during the second half of fruit development. Moreover, the possible relationships between amino acid accumulation and Huanglongbing resistance/tolerance mechanisms have been recently investigated .In general, all of the amino acids are detected in the juice of mature fruit, with aspartic acid, asparagine, serine, glutamic acid, proline and GABA being the more abundant . A gradual increase in most of the free amino acids was detected during fruit development and toward maturation of Valencia orange .

This increase is associated with citrate decline and it is common to all citrus cultivars . However, different trends were detected in Navel oranges , with most amino acids and their metabolites decreasing from stage II to III of fruit development . A comparative analysis of total amino acid contents among various citrus cultivars showed lemon and mandarin with overall higher contents of essential amino acids than pomelo, grapefruit or sweet orange . Moreover, lemon displayed higher levels of amino acids with bitter taste, such as histidine, phenylalanine and valine, as well as acidic amino acids, aspartic acid and glutamic acid. Following harvest, citrus fruit are usually subjected to relatively long storage periods at low temperatures. However, heat treatments, which vary from 37°C for 24 h to ~50°C for a few minutes, prior to storage, are common to reduce pathogenic agents, as well as to induce resistance to chilling and pathogens. The effects of such treatments on amino acid contents and metabolism were investigated, with conflicting results. In Satsuma mandarins, the contents of most amino acids were reduced or remained unchanged following heat treatment and only ornithine showed a consistent increase following the treatment . On the other hand, Matsumoto and Ikoma found that most Satsuma mandarin amino acids were heat-responsive, showing a remarkable contents increase during postharvest storage at 20°C or 30°C, but not at 5°C or 10°C. However, two amino acids, ornithine and glutamine, were cold-responsive, suggesting active metabolism during postharvest cold storage. Changes in amino acid metabolism during fruit development of various cultivars and in the presence of external stimuli have been studied mostly by transcriptomic and metabolomic analyses. The activation of the GABA shunt, a major route for citrate catabolism , was identified in a transcriptomic analysis and confirmed by proteomics ; these analyses identified an increase in the transcript of glutamate dehydrogenase, aspartate/alanine aminotransferase, glutamate dehydrogenase, glutamine synthase, GABA amino transferase and succinate semialdehyde dehydrogenase during fruit development, and the presence of their corresponding proteins during the declining-citrate stage of fruit development . Moreover, use of an aconitase inhibitor, which induces citrate accumulation, resulted in induced activities of some of the enzymes of the GABA shunt . In addition, proteins of most amino acid-synthesis enzymes were induced either from early stage II to stage II or from stage II to stage III of fruit development, including pathways leading to the synthesis of cysteine, glycine, serine, leucine, valine, asparagine, aspartate, alanine, ornithine and glutamine . Induction of amino acid metabolism was suggested to play a role in the accumulation flavor-associated volatiles . Comparative transcriptomic analysis of high- and low-citrate oranges showed elevated transcript levels of phenylalanine-, arginine-, proline-, cysteine- and methionine-metabolism genes in the high-citrate orange . Cold storage of mandarins resulted in major alterations in amino acid metabolism, including the biosynthesis of proline and arginine, and significant enhancement of the catabolism of branchedchain amino acids . Catabolism of the branched-chain amino acids leucine, isoleucine, and valine releases acetyl-CoA, providing a precursor for amino acid-derived volatiles that are associated with off-flavor development during fruit storage . Water stress also induced alterations in the amino acid metabolism suggested to be involved in defense mechanisms against stress .Citrus HLB, caused by the phloem sap-restricted bacterium Candidatus Liberibacter, is a serious production threat to the citrus industry in various regions of the world. The bacteria are transmitted by phloem sap-piercing citrus psyllids while they feed, mostly on young expanding vegetative shoots. Different citrus cultivars show varied susceptibility/tolerance to HLB. The differential response seems to be associated with psyllid feeding preferences and with plant tolerance to the bacteria. Based on controlled graft-inoculation experiments, cultivars were classified into three major groups, sensitive, moderately tolerant and tolerant, each showing different symptoms, from severe leaf chlorosis, large pot with drainage depressed growth and death in the sensitive cultivars, to fewer and lesser severe symptoms in the tolerant cultivars. The bacteria appeared to be auxotrophic for a few amino acids, supplied by their host. The bacteria were suggested to affect free amino acid availability by altering the expression of amino acid storage proteins, at least in the insect host.

To assess whether amino acid metabolism plays a role in the variable citrus tolerance to HLB, metabolomics analyses were performed in various cultivars on healthy and infected trees. Although most of the analyses were performed with phloem sap, and not the fruit, we include their brief description, as some fruit symptoms might also be associated with changes in amino acid metabolism. In a metabolic survey of phloem sap and leaves of citrus cultivars showing varied sensitivity/tolerance to HLB, the levels of all amino acids were elevated in the tolerant cultivars . Comparative analyses of amino acid contents in the phloem sap of bacterium-permissive and non-permissive hosts showed that seven amino acids, mostly of the glutamate family, were associated with susceptibility, whereas five amino acids, mostly of the serine family, were associated with tolerance/resistance . Moreover, high proline-to-glycine ratios were associated with bacterium-permissive hosts. Overall, the level of consistency in these studies in relation to amino acid composition in sensitive/tolerant plant species was not high. HLB-symptomatic Valencia orange fruits showed an overall increase in the level of most detected amino acids as compared to no symptomatic fruit, possibly due to protein degradation .Disease resistance or susceptibility of a plant depends not only on the specific plant–pathogen combination, but also on the developmental stage of the host tissues. The ripening process of fleshy fruit is an example of a developmental transition that coincides with increased susceptibility to pathogens. Ripening involves a complex network of regulatory and hormone-mediated pathways leading to significant changes in the physiological and biochemical properties of the fruit . Among the ripening events, modifications in cell wall structure and composition, conversion of starch into simple sugars, changes in apoplastic pH and redox state, and decline in the concentration of antimicrobial metabolites contribute to susceptibility of fruit to pathogens . The enhanced susceptibility of ripe fruit to pathogens could be a default outcome of ripening or, alternatively, could be promoted by some, but not all, ripening processes . Fruit pathogens exhibit necrotrophic, biotrophic, or hemibiotrophic lifestyles , categories that reflect different infection strategies . Necrotrophs, such as the ascomycete, Botrytis cinerea, cause necrosis by deploying hydrolytic enzymes , secreting toxins and/or hijacking the plant’s enzymatic machinery . Biotrophs depend on the integrity of plant host tissues and have developed strategies to deceive the host to obtain nutrients without inducing plant defenses or cell death . Hemibiotrophs are those pathogens that switch lifestyles at different developmental phases and/or in certain environmental conditions . Therefore, the infection strategies of different pathogens challenge the competency of the plant host to respond and deploy effective defense mechanisms. Tomato has served as a model organism to study fruit ripening and has emerged as an informative experimental system to characterize the molecular regulation of the ripening-related susceptibility to pathogens, in particular to necrotrophic fungi, such as B. cinerea . B. cinerea fails to develop in unripe tomato fruit, but as fruit start their ripening program and become ripe , concurrently they become more susceptible to infections, which lead to rapid breakdown of host tissues and extensive microbial colonization . The roles of the plant stress hormones, ethylene , salicylic acid , jasmonic acid , and abscisic acid , in the control of plant developmental processes and the initiation of defense mechanisms against necrotrophic, biotrophic, or hemibiotrophic pathogens have been documented mostly for vegetative tissues . However, our understanding of how these hormones influence plant–pathogen interactions in fruit is still limited. The gaseous hormone, ET, is involved in the control of terminal developmental programs, such as organ abscission, leaf and flower senescence, and fleshy fruit ripening . ET also modulates plant resistance and susceptibility to pathogens. Thus, from one point of view, ET controls a variety of immune responses in conjunction with other signaling networks; but from another perspective, it promotes senescence or ripening, processes which facilitate infection by pathogens . JA influences flower development and may be involved in some ripening processes, depending on the plant species . The best-known function of JA is to regulate plant immune responses against insects and pathogens, particularly necrotrophs . JA may also play a role in resistance against abiotic stresses, including mechanical stress, salinity, and UV irradiation . SA is a phenolic compound with hormonal features that is crucial for the establishment of basal defenses, effector-triggered immunity, and both local and systemic acquired resistance . SA is typically involved in the activation of plant defenses against biotrophs and hemibiotrophs, but it also appears to enhance susceptibility to necrotrophs by antagonizing the JA signaling pathway through the regulatory protein NPR1 and by inhibition of auxin signaling . ABA regulates many aspects of plant development, including seed dormancy and germination, and plays a significant role in tolerance to abiotic stress . Negative and positive roles have been described for this hormone depending on the pathosystem, developmental stage of the host, and/or the environmental conditions in which the plant–pathogen interaction occurs .

To evaluate the thermal stability of encapsulated bio-actives, the cells were heat-treated at 90 ◦C for 1, 2, 5, 10, 20, 40, and 60 min in a temperature-controlled water bath. The heating conditions were selected based on prior studies. After the heat treatment, the cell-encapsulated polyphenolics were extracted using the methods described in Section 3.10 of the material and methods section. The total antioxidant concentration of the extract was then measured using the FRAP assay. The control group of cells with encapsulated compounds but without the heat treatment were also extracted using the same approach and used for calculating the retention ratio during the treatment. The results in Figure 4 illustrate the percentage of total antioxidant capacity retained at each time point during the heating process. As observed in Figure 4, the bacterial carrier effectively protected the encapsulated compounds during thermal treatment. Approximately 93% of the antioxidant capacity for the encapsulated MG juice was retained after 1 h of heat treatment , square pot whereas only 74% of the initial antioxidants were preserved without using encapsulation after the heat treatment of juice for 1 h.

These observations indicated that cell carriers can effectively protect encapsulated antioxidant compounds against thermal stress. In addition to the total antioxidant capacity, the retention of anthocyanins was also monitored during the heating process at 90 ◦C. The cells encapsulated with polyphenols from the juice matrix were sampled at 1, 2, 5, 10, 20, 40, and 60 min, and compared to the non-heated polyphenols encapsulated in cells from the juice. As described previously, the anthocyanin content retained in the cells was extracted using methanol and measured using a UV-Vis spectrometer. Figure 5 shows similar patterns of enhanced stability of anthocyanin compounds on cell carriers similar to the results in Figure 4. Despite the fact that the MG juice contains more colored pigments , these compounds seemed to be more susceptible to heat. Only 61% of the anthocyanin pigments in the MG juice were retained after 60 min of heat treatment. In contrast, 90% of the encapsulated anthocyanin pigments were preserved in the cell carriers. These results demonstrated that the bacterial cell carriers effectively protected encapsulated anthocyanins from degradation caused by the thermal treatment.Overall, the results demonstrated that, after 60 min of heat treatment at 90 °C, more than 87% of the total antioxidant capacity and 90% of the anthocyanin content were recovered from the encapsulated MG as compared to the respective juice without encapsulation. The degradation of juice phenolics content including anthocyanin from heating were comparable with previous studies. The thermal stability of the encapsulated active compounds was significantly higher than non-encapsulated MG juice.

This protective effect of microcarriers has been observed in a range of encapsulation systems such as spray-drying particles and emulsions. However, comparable or higher percentages of antioxidant capacity and pigment content retention were observed using the cell carriers compared to the synthetic encapsulation carriers. For instance, more than 20% losses were observed for anthocyanins encapsulated in polymer matrices such as maltodextrin, mixture of maltodextrin and gum arabicadvantage of the cell carriers might be attributed to both the physical cellular structure and its complex chemical composition. As shown in Figure 1, the cell structures persisted through the encapsulation process, and literature has shown that some of the Lactobacillus strains can maintain structural integrity at elevated temperatures around 100–120 ◦C, for 30 to 60 min. The robust structure is essential for protecting encapsulated bio-actives, whereas colloidal encapsulation systems tend to destabilize both physically and chemically during encapsulation or in adverse environmental conditions. Besides the physical structure, the antioxidant property of intracellular content of L. casei has also been reported. Aguilar-Toalá et al. suggested that glutathione and other intracellular lipid and protein components might be involved in the antioxidant activities, which might in turn help stabilize and protect bio-active compounds encapsulated within the cell carrier.

Therefore, cell carriers are an efficient encapsulation material for preserving the bio-active functions of the extracted polyphenolics during thermal processing. In addition, encapsulation using cell carriers exhibits certain advantages in terms of the manufacturing process. In this study, we used L. casei cells to encapsulate a composite profile of polyphenolics with a basic temperature-controlled incubation. Currently, spray drying and freeze drying are the most commonly applied industrial techniques for microencapsulation and stabilization of plant polyphenolics from natural sources. Spray drying is a unit operation where liquid is atomized in a hot gas current to obtain a powder. While spray drying is prevalent with low cost, its limitations have also been extensively discussed. We observed 4 to 5 times higher amounts of anthocyanin content encapsulated in the cell carrier in this study when compared to spray-dried powder. Despite variations in the raw material, loss of heat in sensitive compounds during spray drying might be due to the exposure to oxygen and the thermal treatment . In addition, the drying process may cause the loss of dried material due to wall deposition, low thermal efficiency, broad size distribution, and irregular microstructures. Encapsulation using the preformed cellular structure of probiotic bacteria and passive incubation, on the other hand, significantly simplified the process with more uniform cellular size and microcellular structure.Total antioxidant capacity of juice matrix before and after encapsulation was quantified using the Ferric Reducing Antioxidant Power assay. Antioxidant activity was selected as a representation of the total bio-active compound concentration in the juice. The changes in the antioxidant content of the juice after encapsulation was evaluated to assess the encapsulation efficiency of diverse class of bio-active compounds. The protocol for measuring FRAP activity was adapted from Benzie and Strain. The stock solutions included 300 mM acetate buffer , 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl3·6H2O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3·6H2O solution and then warmed at 37 ◦C before using. Fruit juice before and after encapsulation was allowed to react with 2850 µL of the FRAP solution for 30 min in the dark condition. Change in color of the solution was quantified using a UV-Vis measurement at 593 nm using a spectrometer. The standard curve was generated using a range of Trolox solutions between 25 and 800 µM. Results were expressed in µM T.E./mL fresh juice. The samples were diluted in case the absorbance value measured for the samples was over the linear range of the standard curve.Anthocyanin content in juice before and after encapsulation was also measured using a UV-Vis spectrometry. Grape juice is a significant source of plant anthocyanins and changes in the level of anthocyanins in a juice matrix before and after encapsulation also represent a measure of encapsulation of water-soluble pigments in bacterial cells. The absorbance value of the clarified samples was scanned from 250 nm to 600 nm, and a peak intensity was recorded at 530 nm for all the samples. The samples were diluted accordingly to avoid saturation in the absorbance signal. Standard curves were constructed using different concentrations of keracyanin chloride and anthocyanin content was represented as keracyanin equivalent content.Confocal Laser Scanning Microscopy images of bacterial cells after encapsulation with and without incubation with muscadine juice sample were collected using a Zeiss LSM 510 upright microscope with 40×/1.1 water objective.

Each sample was excited at 405 nm using an argon diode laser. Emission scans were acquired using a 500–550 nm bandpass emission filter. Lambda scans of each sample were collected over a range of 470–670 nm with 20 nm step size. The average intensity of the images acquired at different wavelengths during the lambda scan was measured using ImageJ software and plotted using an Origin 8.0 .Phenolic compounds were extracted by mixing 2 mL of the reconstituted MG juice sample with 13 mL of acidified methanol . After mixing using a vortexer, drainage collection pot the mixture was sonicated using a bath sonicator for 10 min and the extract was separated from the remaining juice solids by centrifugation at 5500 rpm for 5 min. The samples were then diluted 10-fold with milliQ water for HPLC-DAD analysis. To assess encapsulation efficiency and yield in cell-based carriers, phenolic content in the aqueous phase before and after encapsulation process was quantified. Chromatography separation and detection of phenolic compounds were performed on an Agilent 1260 Infinity reverse phase HPLC -D.A.D. system equipped with a thermostatic autosampler, thermostatic column compartment, and a diode array detector according to a method adapted from Plaza et al.. An Agilent PLRP-S 100  column with an Agilent 3 × 5 mm guard column was used at a temperature of 35 ◦C for all the analysis. Mobile phase A: 1.5% phosphoric acid solution. Mobile phase B: acetonitrile solution containing 20% mobile phase A. The gradient protocol for HPLC separation and analysis was as follows: 0 min, 94% solvent A; 73 min, 69% A; 78 min, 38% A; and 90 min, 94% A. The flow rate was 1 mL/min and the injection volume for all samples was 10 µL. Samples were filtered through 0.45 µm type H.A. Millipore filters prior to injection. Absorbance spectra were recorded from 250 nm to 600 nm. The eluted compounds were monitored and identified based on spectral and retention time comparisons with standards at multiple wavelengths, including 280 nm for Flavanol [gallic acid, -catechin and -epicatechin] and polymeric phenols, 320 nm for hydroxycinnamates , and 360 nm for flavonol and derivatives , respectively, using the D.A.D. detector. External calibration curves were constructed for gallic acid, -catechin, -epicatechin, caffeic acid, coutaric acid, quercetin, and myricetin glycosides were used for quantification of the target compounds. Polymeric phenols were quantified as catechin equivalents. Chromatograms were integrated using the Agilent CDSChemStation Software .The thermal stabilities of the encapsulated bio-active compounds were evaluated using a thermostatic water bath at 90 ◦C for up to 60 min. A 1 mL suspension of the cells with encapsulated compounds and 1 mL of juice alone were added to the prewarmed 20 mL glass vials and incubated in the dark for 1, 2, 5, 10, 20, 40, and 60 min. The concentration of the total antioxidant contents in the juice sample and the cell encapsulated sample were maintained the same. After the treatment, 1 mL acidified methanol was added to each vial. Bead-beating at 6.0 m/s for 30 s for 3 times was then carried out to facilitate thorough extraction. Finally, the homogenized samples were sonicated using a bath sonication device for 10 min. The methanolic extract was then centrifuged to remove cell Ecosystems are suffering from the pressures of on-going global change, including climate change and habitat loss. One of the main consequences of ecosystem disturbance is the local extinction of species, yet we have little understanding of the consequences of these extirpations for ecological interactions, community dynamics and ecosystem functions. To predict how ecosystems, which are naturally dynamic, will react to these pressures, we first need to understand how communities react to the natural dynamics that lead to changes in their composition of species, with special emphasis on changes in species interactions and the ability of the community to re-arrange itself and maintain its functioning. Up to now, understanding community-level rearrangements following changes in species composition has proved elusive, given the great complexity of ecological systems, which feature high levels of species diversity, interactions across species and environmental variability. The use of network analyses to represent some of the biotic interactions has allowed us to address part of this complexity. However, many network studies have used temporally and spatially aggregated data of observed interactions representing a snapshot of a community. Aggregating data omits important information regarding the dynamic nature of ecological interactions, and in particular concerning species functional roles, which can change due to competition for resources, the presence of parasites and pathogens or changes in species composition. These changes in species composition have been primarily assessed through studies focusing on species extinctions or invasions. Some of them have used experimental set-ups to explore community level dynamics following species extinctions. For example, Brosi & Briggs temporarily removed the most abundant bumblebee species and analysed how the rest of the pollinator community responded. They found that in manipulated sites, floral fidelity decreased, with consequences for plant reproductive success, and also that the loss of a single pollinator species changed pollination network structure.

The population has phenotypic diversity for each trait. We will compare the QTLs detected in prior studies to the QTLs detected in this study. For the work, a large number of high-quality SNP markers, the use of high-throughput phenotyping, and a parent with blood orange in the pedigree were used. A unique aspect of this research is the possible detection of QTLs associated with red color in the peel derived from the blood orange grandparent.The pollinations were performed on April 11, 2011, using Kiyomi as the female parent and Amoa 8 as the male. 18 pollinations were made. This resulted in 10 fruit that set seed. These 10 fruit produced 232 seeds that were planted in seed cones in early 2012. Approximately 175 seeds germinated and were grafted by Spring 2013. 131 grafted trees were planted in Field 11B on June 24, 2014, vertical tower for strawberries and 39 grafted trees were planted in Field 12C on July 9, 2015. Trees that were planted in 2015 were held back because they were too small to plant the prior year or had to be repropagated. Any grafted trees not planted were likely discarded because they failed to thrive.

Some of the trees in 11B began to set fruit as early as 2016. Both Carrizo and C-35 citranges were used as rootstocks. These trees were about 6 years old when the phenotyping was done.Fruits were collected from 159 Kiyomi x Amoa 8 hybrids at the UCR Citrus fields during the 2019/2020 season in December to identify loci of interest that could be contributing to variation in fruit quality traits for QTL mapping. First, the identities of the trees were written on each bag, and then the fruits were picked according to their tree identities. 50 fruits per tree were collected in the labeled bags for phenotypic evaluation. Before being sent for phenotypic analysis, each bag was washed in detergent water, rinsed, and left to dry under sunlight to reduce the risk of HLB contamination . Each bag was checked for the presence of materials that may carry a contamination risk, such as leaves or stems, which were then removed. All fruit bags were then transportedfor analysis of phenotypic features to the UC Lindcove Research & Extension Center .The other packline trait is the fruit color measured by InVision® software. For each pixel, they assign it to one of ten color categories. They do this for all pixels in the image. They assign each image a value for each of the ten colors. The value is the percentage of pixels that are a certain color. Therefore, each fruit has a value for the color.

Colors were defined and referenced from color sample standards from The Royal Horticultural Society’s Colour Chart. Each color sample was matched to actual fruit colors chosen according to the color chart used by the UC research community.From the 50 fruit per tree analyzed using the packline, 12 fruit were randomly selected for destructive sampling. Then, these twelve fruits per tree were destructively sampled to obtain measurements for the additional traits. The fruits were prepared for destructive data analysis by cutting them in half at the equatorial region. The number of seeds for each fruit of twelve fruits per tree was counted as the number of seeds visible in an equatorial cut. Then, the total number of seeds of these twelve fruits was divided by the number of twelve fruits and the average seeds number was calculated. Fruit peel thickness was determined by measuring the flavedo portion of twelve fruits per tree by a digital caliper and then the values were transferred to the computer automatically. Average peel thickness data was obtained by dividing the total peel thickness value per fruit by the total fruit number. After measuring the seed and peel thickness of the fruits, a hydraulic fruit press was used to extract fruit juice for further measurements.

For each tree, juice from twelve fruit was combined. Then, the juice weight of each juice sample was measured with a precision balance and expressed in grams. The trait specified as the sugar content is the soluble solids content of the fruit. OBrix value was obtained by measuring the juice from twelve fruits per tree using a refractometer . The total OBrix values per tree were divided by the total number of fruits and the sugar ratios of the tree identities were calculated asOBrix.For Packline data analysis, an average of 50 fruits were analyzed. Then, 12 fruits were selected randomly from 50 fruit bags for destructive data analysis, and the destructive analysis was completed. The data of the fruits whose analysis was completed were submitted as two separate files. After taking the averages of each fruit, both data files were merged according to tree identities to create a phenotype file. The data were transformed using the best Normalize package in R Studio Software for each of the fruit characters. The best Normalize package determined which method could be performed to normalize data for each trait. The distribution of the transformed data was examined. In addition, Spearman correlation coefficients were performed to find the relationship among the phenotypic traits of the fruits. The correlation was visualized using the R package corrplot .Linkage analysis was conducted using a pseudo-test cross strategy in the R/qtl package. SNPs were divided into three categories according to their segregation patterns: AB × AA, AA × AB , and AB × AB . Although some individuals were sampled for DNA extraction, they could not be analyzed for phenotype data because they did not have any fruit. For this reason, the individuals were filtered to keep the ones with both phenotype and genotype data. There were 93 samples with complete genotype and phenotype data for QTL analysis. To perform QTL mapping, the R/qtl package was used to build maternal and paternal genetic maps and identify QTL. A total of 4491 markers are informative for mapping QTL derived from Amoa 8 and 7303 markers are informative for mapping QTL derived from Kiyomi . The recommended quality control procedures were followed according to to filter markers prior to QTL mapping. This includes removing distorted markers, identifying markers incorrectly placed, removing duplicated and switched markers, estimating the recombination fraction between them, calculating a LOD score for the test of r = 0.5 for each pair of markers, and counting crossing over numbers. SNPs with any missing data were removed.Fruit quality traits have a significant effect on consumers in the global industry. Identifying the genetic bases of important fruit quality traits is essential for Citrus breeding. Understanding how mandarin fruit quality traits are genetically regulated and correlated is the first step toward improving marker-assisted breeding programs. The distribution of the offspring and the parents of the population in terms of fruit quality characteristics measured was examined as well as the correlation between these features. The fruit quality traits were analyzed for Kiyomi, Amoa 8, and their outcross F1 mandarin progenies. The phenotypic trait distributions of the parents and progenies were shown as non-transformed in Figure A1, A2, and transformed data in Fig. 9, Fig. 10, Fig. 11, and Figure A3. The distributions were transformed using the Best-normalization package in R, container vertical farming and the best transformation method for each phenotypic trait . Trait distributions were typically unimodal with transgressive segregation evident in most of the measured traits. Transformed phenotypic distributions of destructive traits, which were JW, JV, TA, pH, SC, AC, ASN, and APT, in the populations, transgress beyond the two parents . For example, the average sugar content from 12 fruits of Amoa 8 was 14.6 oOBrix while that of Kiyomi was 11.9 OBrix. The minimum and maximum values of hybrids are 10 and 17.8 OBrix, respectively. The value of Amoa 8 was measured as 3.62 mm and Kiyomi was 4.57 mm for APT . The TA, AC, and pH values, which were responsiblefor the acidity of the fruit, and TA, AC, and pH values of the parents were closer to each other. FW, FV, MajorFD, and MinorFD are traits related to fruit size.

Looking at the distribution histograms of these features, it is seen that these features were transgressively segregated beyond two parents . MajorFD is the measurement of the average diameter of the fruits from the equatorial region. Amoa 8’s MajorFD measured 37.9 cm and 68.58 cm for Kiyomi. The min value for MajorFD was measured as 1.77 cm and the max value was 84.21 cm in the population . The distributions of fruit characters, which are ELG, TEX, OVR, FLT, STA, STS, CAS, SMT, RS, and SS that contribute to the difference in fruit shape and size were also examined . In addition, it is seen that the frequency distributions of the hybrids for these fruit characteristics were often substantially broader than those of their parents . The progenies and the parents were also evaluated for fruit color traits. The distribution of ten features, CMR, CR, CRO, CDO, CO, COY, CY, CYG, CG, and CDG, related to fruit color, which is one of the most important fruit quality traits related to the external appearance of the fruit, was shown in Figure 11. Transgressive segregation was observed in all the color traits, but to varying degrees. For CMR, CRO, CG, and CYG distributions, parental values span the range of progeny values with less transgression. Most progeny values were similar to parental values in CMR, CRO, CG, and CYG distributions. On the other hand, parental values are similar with extensive transgression in progenies in the distribution of CR, CDO, COY, and CY traits. Since the value of Kiyomi was very low, it was not seen in the CDG distribution histogram.The first component separated individuals within the population based on their values for the CY and CO color traits. The second component separated individuals within the population based on their values for the CMR and CG color traits. In addition, PC2 separated the parents , which are known to differ in the level of external red fruit color. The same PC plot, but variations in specific colors are highlighted with the yellow-purple shading. In other words, individuals of the population were separated with CY and CMR colors. Phenotypic variation in color was summarized using PC1 and PC2 values and these values were used in all downstream analyses.Fruits were collected from field-grown trees that were subject to local weather conditions. The aim is to understand how robust traits measurements were across years and focused on two traits – SC and MajorFD. To measure the stability of genetic effects across years, according to the measurements for 2020, twenty individuals were selected to represent the phenotypic extremes for two traits – SC and MajorFD. For each trait, 10 individuals with the highest trait values and 10 individuals with the lowest trait value were identified. SC and MajorFD were re-measured in fruit collected in 2021 for these 40 individuals. The measurements for each trait showed consistency between two years for each group, and hence genetic effects across years are stable . However, differences between the high and low groups were smaller in year 2 and during the year 1 when the groups were identified. This is consistent with an environmental contribution to variation in these traits.The recommended quality control procedures to filter markers prior to QTL mapping were followed according to two references , Broman . These procedures included removing distorted markers, identifying markers incorrectly placed, and removing duplicated and switched markers. After completing these procedures, the recombination fraction was estimated and a LOD score for the test of r = 0.5 for each pair of markers was calculated, and then crossing over numbers were counted. According to the plot of estimated pairwise recombination fractions, there were not any problematic markers. It was most probably that markers were placed in their accurate position on chromosomes and. There were no estimated recombination fractions that were r> 0.5, and no large recombination fractions with large LOD scores. There was a distribution between 0-30 crossing overs among the 96 individuals. Although there were some departures from 1:1 segregation on chromosomes 1 and 9, the estimated genetic maps were good to start constructing QTL mapping.In previous QTL studies associated with fruit size, 8 QTLs for fruit weight and 3 QTLs for fruit diameter were determined by Yu et.al . These QTLs on the scaffolds 4, 5, and 8 of the Clementine reference genome explained phenotypic variance from 15.03 % to 24.6%.

Nearly free electron models assume that electrons are free except for at the boundary of the Brillouin zone, where they interact with a potential. As an example, the Kronig-Penney model assumes that there are a periodic array of delta-function potentials.This interaction creates discontinuities and forbidden energies which qualitatively explain the behavior of insulators. Another class of theories of electronic conduction are bound electron models. Tight binding models are a major class of bound electron models. Following Slater, electronic conduction in insulators or near-insulators may be quantitatively modeled by an electron hopping between atomic orbitals to which it is bound. Therefore the energy band structure is a “Linear Combination of Atomic Orbitals,” and electrons move between neighboring atoms. Tight binding models offer superior tunablility relative to free electron models, plastic pots 30 liters and may model topological quantum properties such as the Quantum Hall Effect. A fundamental experiment of electromagnetism is the Hall Effect. A strip with a current running through it under the force of an electric field, in the presence of a normal magnetic field acquires a transverse voltage.

This is expected on the basis of the Lorentz Force Law. With division it may be shown that the transverse conductivity, or “Hall conductivity” is linear in magnetic field strength.These plateaus in resistivity are · h/e2 , for c and integer, to within one part in one trillion. These plateaus are used to define the standard for resistivity. The centers of the plateaus correspond the the Landau Level energy, which is the only energy at which electrons can move at when disorder is present. Electrons at all other energies are localized. This experimental result motivated the introduction of topology into condensed matter to describe such quantized behaviors. Within a magnetic field, charged particles can only occupy certain energy levels. These energy levels are the Landau levels, which for a magnetic field perpendicular to a two dimensional plane describe the cyclotron orbits of electrons. Here, we follow the discussion Electrons in a tight-binding chain of atoms may be modeled in terms of a linear combination of the atomic orbitals of each atom in the chain, modified by additional potentials from the Coulomb interaction of atoms. Here, a model is developed where all orbitals are considered the same at each site, and that “hopping” is only possible between adjacent sites. Here, numerical and analytical solutions are developed to solve for the spectrum of the Hamiltonian of this finite one dimensional lattice. First, the lattice is defined, and the model clarified in terms of base kets corresponding to states on each site, and this formalism is presented with the time-evolution of states in the Schrödinger equation.

Then a model for the energies on each lattice site, and corresponding to “hops” is developed. From this, the Hamiltonian is determined. From the Hamiltonian, the spectrum of energies may either be determined numerically using a numerical diagonalization algorithm, or analytically by developing the “translation operator,” and observing that the Hamiltonian is diagonal in a basis of eigenkets of the translation operator.Electrons may be modeled using the Tight-Binding model. This model assumes a probability to stay on a lattice site , and a probability to hop to a new site Here, a model is developed where all orbitals are identical, and the orbitals are arranged in a 2D square lattice with equal spacing between sites. It is assumed that hopping is only possible between nearest-neighboring sites. An analytical solution is presented, and an expression for the Hamiltonian is developed and numerically solved. The numerical solution agrees within machine precision to the analytical solution. The methods for the two dimensional tight binding model are very similar to methods for the one dimensional tight binding model. The only differences are that the system is a two dimensional square lattice, and that a formalism for the representation of the matrix Hamiltonian is developed which allocates one row per site, and moves through rows and then columns.The detachment of a grape berry from its pedicel generally damages the berry because the vascular tissues and associated parenchyma, collectively known as “the brush”, remain attached to the pedicel and are pulled out of the berry on detachment, leaving an open wound sometimes called a “wet” stem scar on the berry’s stem-end. Berry detachment may also remove pieces of skin or cause the whole berry to rupture. Such mechanical damage can reduce the yield and quality of machine-harvested grapes for wine or raisins. Stem-end picking damage also limits the quality and storage life of stemless table grapes. Certain plant growth regulators known as “abscission agents” activate an abscission zone at the pedicel-fruit boundary .

The activation of this abscission zone reduces fruit detachment force and promotes the development of dry stem scars . Abscission agents could reduce picking damage and thereby serve as harvest aides if treated grapes can be harvested after the abscission zone is activated, but before the fruit abscises. However, once the abscission zone has been activated, development proceeds quickly and may lead to excessive preharvest fruit drop.The first compound tested as an abscission agent for grape was ethephon, a phosphonic-acid compound that decomposes to release the gaseous plant hormone ethylene. Ethephon can induce the abscission of mature grape berries within 7 to 14 days after treatment, but high dosages are needed. The use of ethephon as an abscission agent for grapes would require an application dosage which is higher, and a preharvest interval that is shorter, than those for the current registered use of ethephon on grapes in order to enhance berry color. Such changes could be expected to increase ethephon residues on treated fruit, and it seems unlikely that regulatory agencies would approve a use that could increase ethephon residues on grapes since existing residues are already a concern. However, it should be noted that Ferrara et al. found that effective dosages of ethephon did not result in excessive residues. Jasmonates, including methyl jasmonate, a natural product, have also been shown to induce the abscission of various fleshy fruits, including blueberry , orange , and tomato. Moreover, the Environmental Protection Agency of the United States ruled that MeJA was exempted from the requirement of a tolerance for residues in or on all food commodities when applied pre-harvest, a ruling that could facilitate the development of jasmonates as active ingredients in agrichemicals. A screening trial confirmed that MeJA and coronatine, a jasmonate mimic, also induce abscission in grape. The exogenous application of jasmonates stimulates ethylene production in several fruits, including apple , orange, grape, strawberry , and tomato. The work carried out on orange and grape showed that the application of MeJA stimulated ethylene production by the fruit which was followed by fruit abscission. Malladi et al. provided indirect evidence that MeJA stimulates the abscission of blueberry fruits at least partly via ethylene action, as the co-application of MeJA with aminoethoxyvinylglycine , an ethylene biosynthesis inhibitor, round plastic pots attenuated MeJA effects on abscission. However, MeJA still induced some abscission in blueberry, even when co-applied with AVG which suggests that MeJA may initiate some abscission processes independently of ethylene. Moreover,grape berries treated with MeJA or 1-aminocyclopropane-1-carboxylic acid , the direct precursor of ethylene, produced similar levels of ethylene in the first 2 days after treatment , but thereafter, berries treated with MeJA produced less ethylene than berries treated with ACC, even though the MeJA-treated grapes generally had lower FDF, greater abscission, and a higher proportion of dry stem scars than the grapes treated with ACC. Together, these findings suggest that ethylene and jasmonic acid can promote fruit abscission via independent pathways and interact to promote abscission.

The potential for synergistic effects has sustained interest in research on the coapplication of jasmonates and ethylene-promoting compounds. This is especially important because of the relatively high dosages of MeJA needed for consistent efficacy when applied alone and because MeJA is much more expensive than ethephon. Because of the high rate of ethephon needed for abscission activity, the short time between abscission zone activation and fruit drop, and concerns about ethephon residues, alternatives to ethephon are desired. 1-Aminocyclopropane-1-carboxylic acid is not particularly effective at stimulating abscission on its own, but the co-application of MeJA with ACC improved efficacy in such a way that lower dosages of MeJA could be used. Recent improvements in jasmonic acid biosynthesis have the potential to make JA and its metabolite MeJA more available and affordable than they are now. The effects of JA on the abscission of fleshy fruits has not been tested, and the relative efficacy of JA versus MeJA with respect to grape berry abscission is unknown. Therefore, two studies were conducted to compare the efficacy of JA and MeJA at inducing abscission of Thompson Seedless grapes and to determine if JA interacts with ACC to promote abscission.Methyl jasmonate at 2 mM was ineffective, whereas 4 mM and 8 mM MeJA were equally effective at inducing preharvest abscission, reducing fruit detachment force , and increasing the proportion of detached berries with dry stem scars . Jasmonic acid was as effective or more effective than MeJA at inducing the abscission of Thompson Seedless grapes. Compared with MeJA, JA induced a similar or higher preharvest berry abscission, a similar or lower fruit detachment force, and a similar or higher percentage of detached berries with dry stem scars after treatment with JA, versus MeJA . The most effective treatment overall was 4 mM JA, which induced the highest level of preharvest abscission and percentage of detached berries with dry stem scars . Grapes that were treated with 4 mM JA also measured among the lowest FDF values .MeJA can stimulate the abscission of many fruits and endogenous JA is known to promote abscission of floral organs and fruits, but the data presented here may be the first report of an exogenous application of JA stimulating the abscission of a mature fruit. Moreover, JA appears to be at least as effective, and possibly more effective, than MeJA at stimulating grape berry abscission. Interestingly, this is in contrast with a recent study that showed MeJA was more effective than JA at inducing abscission in lupine flowers. Improved efficacy at lower dosages could facilitate the commercial development of jasmonate-based abscission agents for grapes and other fleshy fruits because these natural products are expensive. In the first study, 4 mM JA was more effective than 8 mM JA; however, the opposite appeared to be the case in the second study. Therefore, additional research is needed to clarify the lowest dosage of JA that is consistently effective. The general range of effective dosages agrees with previous research that used MeJA as the active ingredient. Abscission zone activation reduces FDF and promotes the development of dry stem scars, both of which could help minimize picking damage and possibly improve the quality of destemmed table grapes. However, the final stage of AZ activation is undesirable unless catchment systems can be employed. Previous studies with MeJA suggested that harvest should occur within 3 days after treatment . Jasmonic acid also stimulates rapid abscission zone activation, with 14 to 25% abscission observed 2 days after treatment, and 52% to 60% by 3 DAT. Without catchment systems, it may be necessary to harvest treated fruit within 2 days to avoid excess crop loss. This could be logistically difficult, and data are lacking to determine whether the abscission zone would be sufficiently developed by 1 or 2 DAT to provide the potential quality benefits that are desired from abscission agents. Another outcome that needs to be determined is whether the abscission zone could be activated preharvest and develop during postharvest storage. If so, this could be a way to achieve fruit quality benefits for stemless table grapes while minimizing the risk of preharvest fruit drop. Lavee demonstrated that preharvest applications of plant growth regulators can affect the postharvest abscission of grapes. Specifically, the preharvest application of 1- naphthaleneacetic acid and some other synthetic auxins reduced the postharvest abscission of “Muscat of Alexandria” that were held at room temperature for three days before entering cold storage. However, the abscission of grapes that were placed into cold storage immediately after picking was greatly suppressed, regardless of whether the grapes were pretreated with auxins or not. This suggests that the effect of plant growth regulators on postharvest abscission may depend on postharvest storage conditions.

This fact combined with the application of data analysis techniques such as outlier detection and machine learning using back-end software allow for faster response times to possible contamination events, collection and analysis of large spatio temporal data sets, and diagnostic capabilities for water treatment systems, as just a few of the potential benefits of realtime water quality monitoring. The work presented in this manuscript describes the design, procurement, building, and testing of an IoT-enabled monitoring system prototype for POU applications with these goals in mind. The proposed system consists of a sensor package that includes basic water quality parameters of pH, EC, ORP, Dissolved Oxygen , and temperature. The system uses a Raspberry Pi 4 Model B microprocessor. It includes a software program that enables data transfer from the sensors to an online repository where any collaborators who have access can view the data.

Preliminary algorithms that utilize reference correlations, statistical analysis techniques, hydroponic bucket and physical and chemical constants to calculate and predict other water quality parameters such as corrosivity risk have also been developed.Wireless sensor networks have been applied to an array of environmental monitoring problems. Those focused on water quality monitoring cover a range of applications for environmental waters, drinking water, and wastewater. Some primary questions addressed by previous work include lowering the costs of wireless sensor networks, optimizing power usage for proposed systems, developing software for contamination detection, and improving solutions for data transmission. The works presented utilize an array of programming languages, sensors, circuitry, and communication protocols that all serve to make real-time data monitoring accessible for operators and consumers. Often environmental waters are prone to various perturbances, requiring the need for real-time monitoring. Rivers and marine coasts have been sites for the deployment of smart wireless sensor networks; Adu-Manu et al. employed wireless sensor nodes and used an energy-efficient data transmission schedule to obtain real-time data for pH, conductivity, calcium, temperature, fluoride, and dissolved oxygen.

The author’s system architecture includes a Libelium Waspmote that uses communication modules such as 3G/4G, General Packet Radio Service, long-range 802.12.4/ZigBee, and Wideband Code Division Multiple Access connectivity to transmit data to the cloud. A low-cost Arduino-based sonde was designed in the marine environment that used the Arduino Mega 2560 Mega and ArduinoUno platforms for two design configurations The first design was for a Lagrangian style drifter that Lockridge et al. deployed for 55 hours to measure salinity and temperature using Atlas Scientific K 1.0 Conductivity Probe and the Atlas Scientific ENV-TMP temperature probe. The data collected by the drifter was compared to values from the Dauphin Island Sea Lab Weather Station YSI 6600 sonde. The RMS error was 1.35 ppt for the salinity and was 0.154 °C for the temperature measurements. Salinity and temperature regressions were also performed and found to be highly correlated with R2 = 0.96 for salinity and R2 = 0.99 for temperature. The first data logger demonstrated consistently higher salinity results than the second, whereas temperature tracked similarly for both data loggers over the entire deployment. The system contributed significantly to verifying sensor performance for harsh aquatic environments. However, the work only stored data locally as .csv files and did not transmit data wirelessly or in real-time.

For IoT-enabled systems, some of the most straightforward system configurations are proposed by Vijayakumar & Ramya and Pasika & Gandla . Work by Vijayakumar presents a low-cost system for developing a real-time water quality monitoring system in the IoT environment. The core controller used is the Raspberry Pi B+, and the system has temperature, turbidity, pH, conductivity, and dissolved oxygen sensors. The Raspberry Pi runs on a LINUX kernel and uses an external USR-WIFI232-X-V4.4 module to transfer the data to the internet and is visible on the ioBridge Server. Pasika et al. propose a system consisting of four sensors with the Arduino Mega microcontroller. Their approach is designed in Embedded-C and accomplishes data transmission using the ESP8266 Wi-Fi module. Authorized users can access the data using a User ID and password to log into a Thing Speak server. The information is gathered, stored, analyzed, and transmitted in real-time.Similar systems to those mentioned previously build upon similar design principles but include additional features incorporated into the software designs, such as power-saving capabilities, user alerts, and contamination detection. Power saving capabilities are primarily emphasized for more rugged environments where connectivity issues and power loss can be concerns. One such area is Malawi, the site for a proposed integrated sensor network. The solution was to develop a low power gateway node that reduces energy consumption using a wake-up mechanism that triggers waking and sleeping modes. A gateway solution built upon the ALIX2 embedded Linux board provided a way to interconnect different networks to address connectivity issues. Parra et al. also addressed power-saving algorithms for their fish farm monitoring wireless sensor network, which was achieved by only sending data if the difference between predesignated reference and threshold values were exceeded. Thus, reducing the amount of data moved to the external web server employed in their system and saving computing power. Threshold values can also be employed for systems alerting users of parameter exceedance of WHO or EPA guidelines. This is the case in the IoT-enabled system developed by Geetha & Gouthami which measures conductivity, turbidity, water level, and pH. The hardware design consists of a TI CC3200 single-chip microcontroller with an in-built WIFI module, and ARM Cortex can connect to the nearest Wi-Fi hotspot. The data collected by the sensors is sent to the cloud, and if the values exceed threshold limits obtained from WHO, then an alert is sent to the mobile application developed as part of the proposed system. The programming for the software design that shows the real-time updates was done using ENERGIA IDE, and data are stored in the Ubidots cloud, which includes a real-time dashboard to analyze data, control devices, and shares the data through public links. Lambrou et al. further developed the event detection capabilities of wireless sensor networks by designing and developing a low-cost network embedded system consisting of a central measurement node, a control node, and a notification node. The system contained sensors to measure temperature, turbidity, electrical conductivity, and pH. These three subsystems served to collect water quality measurements from the sensors, implemented contamination event detection algorithms, and stored the measured data in a local database, visualized the data in the form of charts, and sent email/SMS alerts for contamination events. The system architecture utilizes a PIC MCU , an ARM processor, and a ZigBee RF transceiver. The data is posted to the web using the Pachube IoT platform. For the contamination event detection algorithms, the first system was denoted as the Vector Distance Algorithm with a risk indicator function estimated on the Euclidean distance between the normalized sensor signal vector and the normalized control signal vector of clean water. The second was the polygon area algorithm which calculated a separate risk indicator function estimated on the ratio of the polygon area formed by the vector components of each sensor on a two-dimensional spider graph. To validate the event detection algorithms, intentional contamination was performed using E. Coli bacteria and Arsenic, stackable planters which was added to potable water at various concentrations at discrete time intervals. Overall, the PAA had better performance for both contaminants due to fewer false alarms.It is with these developed wireless sensor networks in mind that the prototype in this work has been uniquely designed to combine, improve upon, and fill in gaps from the existing technologies and methods for applications specific to point-of-use water filtration applications.

After investigating the current hardware and software utilized for wireless sensor networks, a key research question to be addressed was to determine the sensors and algorithms that were best suited for smart point-of-use water quality monitoring. Therefore, some of the existing relationships between various sensor parameters and water quality concerns were reviewed to better guide the sensor selection process. Here, we present some of the findings on what water quality information can be related to pH, ORP, DO, and EC. For acute effects from biological contaminants, it is imperative that sufficient disinfection levels are maintained. In drinking water, this is primarily achieved with Chlorine and Chloramine, which previous research has attempted to link to ORP measurements. Experiments have been conducted that investigate the ability of ORP to be a predictor for kill level of organisms including total coliform, E. Coli, and enterococci by measuring the ORP in conjunction with Chlorine dose for wastewater . It was found that the ORP increased with an increase in chlorine added, total chlorine, and free chlorine. However, the regression slopes were relatively low indicating low sensitivity of ORP measurements as a function of each chlorine species. As more chlorine was added, a second increase in ORP was observed. From this point, Monochloramine is oxidized to Dichloramine, which has a higher redox potential and produces H+ leading to a lower solution pH. Once all the Dichloramine was oxidized, free chlorine became available, and the third increase in ORP was observed, indicating the chlorine break point after which viable counting of microorganisms was not observed. The control system implemented could continuously detect the break point and determine the correct chlorine does for inactivating microorganisms in the water. The proposed system ultimately can easily detect the shifting point where dominating chemical species change by monitoring points along with the ORP and pH profiles during chlorination to achieve proper disinfection. ORP measurements have also been explored for monitoring and controlling water disinfection for the produce washing industry . ORP ranges between 600 and 700 show that free-floating decay and spoilage bacteria, as well as pathogenic bacteria such as E. coli or Salmonella species, are killed within 30 seconds. Recent research in commercial and model post harvest water systems has shown that, if necessary, ORP criteria can be relied on to determine microbial kill potential across a broad range of water quality. However, it is important to note the limitation of ORP and pH because the effect of pH on chlorine speciation, one must use caution in not having a false sense of adequate disinfection rates at high pH’s. In general, a ten-fold increase in total or free chlorine concentrations does not result in a corresponding proportional increase in ORP millivolts. Their results showed that good water quality likely results in measurements of 650 to 700 ORP if the water pH is 6.5 to 7. Raising the pH to 8.0 lowers the ORP value, as more hypochlorite ions are present. Maintaining constant pH but adding more chlorine raises the ORP to a plateau of about 950 ??. Finally, Myron L Company describes their correlation for predicting free available chlorine using ORP and pH levels. The correlation was obtained from a series of experiments where an exact amount of chlorine in the form of laboratory-grade bleach was added to DI water in a closed system and measuring pH and ORP to create calibration curves for their Myron L Ultrameter II 6PFCE Water Quality Meter. From some of the listed works, it is evidenced that ORP can be used as an effective indicator for disinfection levels in water and has the potential to protect users from potential biological contamination using realtime measurements of ORP in a wireless sensor network. Other correlations between the variables measured in the wireless sensor network proposed in this work and water quality have been discovered utilizing statistical data analysis methods and machine learning techniques. In 2014, Zhang et al. focused on robust online clustering and modified pixel-based adaptive segmentation and concluded that the MoPBAS method was suitable for detecting anomalous sensor readings and event clustering from salinity and turbidity measurements, which can assist in addressing root environmental causes and significance levels of disturbance events. Forough et al. used an alternative machine learning technique known as the support vector machine model topredict a water quality index using pH, DO, TDS, temperature, Nitrate, phosphate, BOD, turbidity, and fecal coliform data obtained from water samples collected over 11 months. Their SVM model, developed in MATLAB, successfully explained 87% of the variability in total WQI, with Nitrate being the most important attribute influencing the WQI as calculated from the sensitivity ratio.

The Z and L content among dried goji berries ranges from 25 to 152 mg/100g, and 0.3 to 1.9 mg/100g, respectively, based on different cultivars. Additionally, the predominant form of Z in goji berries is a dipalmitate , connecting with a diester linkage, which has shown a significantly higher intestinal absorption than monoester and free Z due to the high efficacy of hydrolysis, mainly by carboxyl ester lipase. According to the National Health and Nutrition Examination Survey, the average L and Z intake among adults in the United States was 1.58 – 1.76 mg/d. Therefore, to increase dietary intake of these two xanthophylls, goji berry is an excellent source, which can complement other food sources such as green leafy vegetables, egg yolk, yellow corn products, and orange bell peppers. Recent clinical research on goji berry shows promising effects in protecting against AMD. Healthy older individuals who consumed 10 mg of Z extract from goji berries daily for 90 days showed no change in macular pigmentation or soft drusen, hydroponic nft system and significantly higher circulating Z levels, compared to a control group, which presented an increase in soft drusen. 

In an uncontrolled trial, individuals with early stage AMD who consume a beverage daily for five months that contained 12 mg of L and 2 mg of Z derived from marigold flower and goji berry, respectively, showed lower interocular pressures, better best-corrected visual acuity scores and higher circulating levels of L and Z compared to their baseline levels. Unfortunately, the study lacked a control group, did not test the effect of Z separately, and did not clarify whether the form of Z extracted from goji berry was the dipalmitate. Another study investigating the effects of an herbal formula among healthy adults with dry eyes noted that those chewing tablets containing L , Z , combined with extracts from blackcurrant, chrysanthemum, and goji berry showed dose-dependent reductions in eye fatigue symptoms and tear secretion, as well as improved macular pigment optical density , a common non-invasive method to measure the total L and Z in the center of the macula, compared to those in a placebo group. The basis of this formula was derived from TCM, so the multi-component formulation could not directly assess the role of any single ingredient.

Another study in patients with early AMD reported that the MPOD was significantly higher in those consuming 25 g/day of goji berries for 90 days, compared to their baseline levels and to a habitual diet control group. The BCVA was also significantly improved in the goji berry group compared to their baseline values. We recently reported that MPOD and skin carotenoid scores were significantly increased in healthy middle-aged individuals consuming 28 g/day of goji berries five times a week for 90 days, compared to a group taking a dietary supplement with 6 mg of L and 4 mg of Z. These results illustrate that MPOD levels can increase in healthy individuals even without early signs of AMD. While these results are encouraging, longer intervention periods with a larger number of participants would be helpful to replicate and extend these initial observations. In addition to AMD, goji berries have also been studied as a therapy for retinitis pigmentosa, an inherited retinal disease. Patients who consume 0.35 g/d of Lycium barbarum polysaccharides for 12 months showed a significant improvement in visual acuity and macular thickness, compared to control subjects who did not consume L or Z. 

Other bioactive compounds found in goji berries include flavonoids, vitamins, minerals, betaine, cerebrosides, phenolic acids, and certain amino acids which may also support the overall health of the eye, particularly when working synergistically. Based on preclinical evidence, potential benefits of goji berry intake on glaucoma and diabetic retinopathy have also been suggested. Goji berry extract ameliorated the high glucose-induced blood-retinal barrier disruption in human retinal pigment epithelial cells. Studies reported that LBP showed significant neuroprotective effects on retinal ganglion cells in male C57BL/6N mice and Sprague-Dawley rats with ocular hypertension. In db/db mice with thin retina, goji berry extract restored the thickness of the retina, the ganglion cell number, and the integrity of retinal pigment epithelium after daily intake for eight weeks.10Traditional uses of food in general, and fruits specifically, provide useful guides for modern research in both nutrition and pharmaceuticals. This literature review illustrates the evidence and certain proposed mechanisms of select fruits with a high phytonutrient profile on cardiovascular and eye health. The consumption of these fruits and bio-active compounds derived from them demonstrate great potential to protect against certain age-related diseases. Therefore, the objectives of this dissertation are i) to investigate the effects of mango consumption on markers of cardiovascular diseases, ii) to examine the effects of goji berry consumption on macular pigment accumulation in human eyes, and iii) to review evidence for L, Z, and goji berries on eye health throughout the lifespan, with an emphasis on clinical studies.Age-related macular degeneration is the leading cause of blindness among seniors in developed countries, and third worldwide after uncorrected refractive errors and cataracts. In early stages, the disease is characterized by small to intermediate drusen with pigmentary changes that may progress rapidly to more advanced forms such as choroidal neovascularization or central geographic atrophy with loss of central vision. Lutein , zeaxanthin , and the isomer meso-zeaxanthin are macular pigments that filter damaging blue light and provide oxidative defense in the macula. These pigments are found in plants as xanthophylls, with increased dietary intake proposed to reduce the development and progression of AMD. The relative concentration of xanthophyll carotenoids in the retina can be measured noninvasively by psychophysical and objective methods, expressed as macular pigment optical density. Numerous epidemiological studies report that individuals with a low MPOD level are at an increased risk of AMD. Dietary L and Z are found in certain fruits and vegetables with red, yellow, or orange color, egg yolk, and in some green leafy vegetables. The dietary intake of Z is lower than L in all age groups and ethnicities in the U.S.. Dietary intakes of L and Z are strongly associated with their serum levels, as well as with MPOD. Previous studies have shown that high intakes of these carotenoids from dietary sources or supplements can increase plasma L and Z, and MPOD. Once early AMD has progressed to the intermediate stage, dietary supplements are indicated, but no clinical evidence yet exists for interventions that can address the prevention of small-intermediate drusen with pigmentary changes, the initial clinical signs of macular disruption.Goji berry , also termed wolfberry or Go Chi Zi, has been used in traditional Chinese medicine for more than 2000 years. The bright red berry contains the highest amount of Z among all known dietary sources and is mainly present in a dipalmitate form. The intake of zeaxanthin dipalmitate extracts from goji berry increases plasma Z to a greater extent than non-esterified Z supplementation. The berries also contain unique carbohydrates that are present as conjugates with peptides or proteins, nft channel which are often referred to L. barbarum polysaccharides . These have shown anti-inflammatory and neuroprotective effects in animal and cell culture studies . The typical adult human eye has approximately 2.4 times more Z than L in the central fovea of the macula, making goji berry intake a prime candidate for increasing MPOD. Nevertheless, there is a paucity of clinical evidence on goji berry and MPOD particularly for the prevention or delay of progression from early to intermediate AMD.

In individuals from China with signs of early AMD, 25 g of daily consumption of goji berries for 90 days significantly increased both serum Z and MPOD. However, this study had a broad age range , some participants smoked, and others had certain pre-existing medical conditions. Additionally, the authors only reported central MPOD values up to 0.5 retinal eccentricity , whereas macular pathology and visual dysfunction in AMD may extend beyond that central region. Therefore, to provide a more complete understanding of the influence of goji berry intake on the progression AMD, data is needed on for different population groups that measures MPOD at eccentricities over the entirety of the macula. In the current study, we prospectively evaluated if the daily intake of 28 g of goji berries or a commercially available supplement providing 6 mg of L and 4 mg for 90 days can improve MPOD and skin carotenoid levels, an index of total carotenoid intake, among healthy middle aged adults, 45 to 65 years old, with no signs of drusen or early AMD.Eighty-eight volunteers, ages from 45 to 65 years old, were recruited from an online website and public advertisements in the area of greater Sacramento, California. Participants provided informed consent and were screened with a questionnaire. Inclusion criteria were being generally healthy , having a normal macular condition as verified by an optometrist, and if relevant, being prescribed the same medication regimen for at least 6 months that was not related to carotenoid metabolism and was approved by the study physician. Exclusion criteria were a dislike of, or allergy to goji berries, diseases of the eye, malabsorption problems, substance or alcohol abuse, smoking, drugs for management of lipids, glucose, or blood pressure, use of dietary supplements other than multivitamins and minerals that provided greater than 100% of the U.S. Dietary Reference Intake, or any supplement containing L or Z. The intervention was registered on ClinicalTrials.gov , with the first posted date of 6 December 2019, complied with the tenets of the Declaration of Helsinki, was approved by the Institutional Review Board of the University of California , Davis and was conducted at the UC Davis Ragle Human Nutrition Research Center.Qualified participants were randomized into a prospective, parallel-arm, unmasked study to consume either 28 g of goji berries or a commercially available supplement of L and Z five days per week for 90 days. Study measurements were collected at baseline , at 45 ± 2 days and 90 ± 2 days after intake. Twenty-eight grams of goji berries is considered a single serving size. The berries in this study were USDA-certified organic goji berries grown in the Ningxia region of northern China and provided by Navitas Organics, Novato CA, USA. The goji berries were portioned into clean, single-serving plastic bags and provided in 45-day allotments. The commercially available supplements were purchased online, contained 6 mg of L and 4 mg of Z per serving and were repackaged into 45-day supplies in clean plastic bottles. Compliance was monitored by a self-administered log. Habitual dietary information was collected with the Automated Self-Administered 24 h dietary assessment webbased tool once between day 0 and 45, and once again between day 45 and 90. The MPOD was assessed by the psychophysical method of customized heterochromatic flicker photometry using a macular densitometer . After participants viewed a 5-minute video detailing the measurement procedures, they were dark adapted for 7 minutes and then began the test. The flicker frequency was selected based on a preliminary test of the participant’s sensitivity. The task was to eliminate or minimize the flicker in the visual field three times by turning a dial that changed the intensity of a 460 nm light. Each participant performed the test while looking directly at the flickering light at 0.25, 0.5, 1, and 1.75 RE degrees, representing the MPOD level from the center to the periphery of the macula.Skin carotenoid content was measured by reflection spectroscopy . After cleaning, the tip of the right index finger was inserted into the spectrophotometer and three measurements were collected. A skin carotenoid score was calculated by the system software. Carotenoids that exist in human plasma, including β-carotene, lycopene, L, Z, and their isomers have been successfully detected in toto and quantified by this device , which has been validated to reflect fruit and vegetable consumption.Sample size was based on a study that assessed the impact of a Z supplement on MPOD in 24 healthy people. Statistical analyses were performed with JMP version 16 . Two-tailed t-tests evaluated potential between-group differences at baseline. The MPOD and skin carotenoid data were analyzed with mixed-effects models using time and treatment as the main factors, with age and sex as the covariates, and participant ID as the random effect. For main effects, student t-tests determined significance within group pairs. pValues of 0.05 or less were considered statistically significant. Correlation coefficients between the outcome measures were determined via Spearman’s method. The mean values of the dietary intake data were compared by two-tailed t-tests, which were log-transformed when necessary, and presented as the mean ± S.E.M. or the back-transformed mean with 95% confidence intervals .

To answer this question, RNA-seq data generated at one ripening timepoint was associated to the changes in polyphenolic levels using a systems biology approach, WGCNA . The module-trait association analysis positively correlated the key flavonoid/PAs biosynthetic genes with the accumulation of tannins, catechin, and quercetin glycosides exclusively in V9-berries. The modulation of the berry’s transcriptomic profile is concomitant with its polyphenols’ composition, which finally disturbs berry quality, including astringency levels.Five-year-old V. vinifera cv. Scarlet Royal grafted on Freedom rootstock was chosen for its berry astringency diversity at two commercial vineyards located in Delano, San Joaquin Valley, California, USA. Vineyards were located at a close distance of 10 km, and the local weather conditions during the two seasons were collected from the Delano CIMIS weather station . Both vineyards were planted at the spacing of 2.44 and 3.66 m in an open gable trellis supporting system with East-West row orientation.

Vines were pruned in a Quadrilateral cordon training with 7–8 spurs left on each cordon during the winter pruning. In addition, nft hydroponic general UC guidelines practices were applied in both vineyard. Random forty vines from different four rows from each vineyard were used in this study. Starting from veraison and until the end of the season, during two consecutive years . During the first year, sampling dates were July 8th , August 1st , August 10th , September 9th , September 15th , and October 19th ; and for the second year, sampling dates were: July 15th , August 10th , August 25th , September 10th , September 29th , and October 21st . Sampling dates varied from the first to the second year due to the vineyard’s accessibility. At each sampling point, two sets of fifty berries were collected periodically. The first set was used to measure the berry weight, and then these berries were macerated in an electric blender, filtered through a paper towel, and an aliquot of juice was used to determine soluble solids , pH, and titratable acidity . Soluble solids were determined using a tabletop Milwaukee MA871-BOX digital refractometer . The TA and pH were determined by titrating a 40 mL aliquot of juice with 0.1 N NaOH to a pH of 8.2 using an automatic titrator Excellence T5 .

Another random 50 berries from each replicate were collected for color, tannins, and phenolic compounds and sent immediately in a cooler to EST laboratories. At harvest, which was during the month of September, an extra set of samples was collected and promptly frozen in liquid nitrogen and stored at −80°C for subsequent analysis, including RNA extraction and gene expression studies. Harvest time was determined by the growers, and the marketable clusters were picked based on the color, and yield was determined from the three harvest dates.Scarlet Royal table grape is one of the major red varieties in California. Despite the premium fruit quality of the variety, in some cases, an undesirable taste was observed under certain unknown circumstances. To gain comprehensive insights into the development of the occasional berry astringency of Scarlet Royal and understand the underlying mechanism of this phenomenon, berries were investigated at two contrasting vineyards , both following the same commercial cultural practices. However,leaf petioles analysis of grapes from both vineyards showed considerable differences in nutrient levels, especially in the primary macronutrients . During both seasons, the amount of nitrogen in the form of nitrate in LP-V9 was roughly 2 to 3 times higher than the normal levels, in contrast to its counterpart in LP-V7, which slightly accumulated more or less N. Similarly, LP-V9 contained higher percentages of phosphorus and potassium compared to LP-V7 . Conversely, the amounts of secondary macronutrients, calcium and magnesium , in LP-V7 were within the normal range but greater than LP-V9, which showed Mg deficiency in the first year only.

Regarding the micronutrients, their levels were mainly within or around the normal range at both vineyards and during both seasons, with some differences . For example, zinc was slightly higher in LP-V9, especially in the first year. On the contrary, manganese and chlorine were roughly 2 times higher in V7 . Similarly, soil analysis shoed a higher level of nitrogen, potassium and magnesium . However, no significant difference was observed in all other soil macro and micronutrients. During the two seasons of the study, we determined the total marketable yield and the number of clusters in both vineyards. Our data revealed a higher yield in V7 compared to V9 in 2016 and 2017, respectively. The lower yield in V9 can likely be attributed to the smaller number of clusters in V9 compared to V7 during 2016 and 2017. To monitor the changes in the biochemical composition of Scarlet Royal berries, V7 and V9 berries were periodically sampled at six time points from veraison until the end of the season . The obtained data showed that berry polyphenols exhibited discernible patterns in both vineyards, most notably during the ripening stage . Of special interest were the tannin compounds, which widely affect organoleptic properties such as astringency and bitterness . Our data showed that berries from both V7 and V9 vineyards maintained lower levels of tannin from veraison up to the middle of August . Subsequently, a significant gradual increment of tannin took place. However, only V9-berries showed consistent accumulation of tannin over the two studied seasons compared to V7-berries, where the significant induction occurred only during the first season. It is worth noting that the levels of tannin were lower in both vineyards during the second year compared to the first season. Nevertheless, they were more pronounced in V9-berries compared to V7-berries, with roughly 2- to 4.5-fold increases by the end of the harvesting time during the two seasons, respectively . The patterns of catechin and quercetin glycosides were inconsistent during both seasons, particularly within V7-berries . During the first year, for instance, the levels of catechin were similar in both vineyards, showing a dramatic increase only by the end of the season . In contrast, during the second year, such induction of catechin was exclusively restricted to V9-berries, starting from time S3 . For quercetin glycosides, V7-berries exhibited significantly higher amounts at early stages during both seasons relative to V9-berries . However, subsequent amounts were comparable in both vineyards during the first season only , but not in the second one, where V7-berries showed a significant drop at the last sample S6 . Interestingly, the levels of quercetin glycosides were roughly equal at the last V9-berries sample between both seasons despite such inconsistency. For total anthocyanins , the levels in early samples were comparable in both vineyards and seasons . Afterwards, their pattern started to vary between V7 and V9 within the same season, as well as from the first season to the second, as the nutrient amounts fluctuated as well . Nevertheless, TAC accumulation was positively correlated with the progress of ripening in V7-berries, but not V9-berries. To further confirm our data, we measured these phenolic compounds for the third time in mid-September of the next year . Overall, the results showed that the patterns of tannins and TAC were reciprocally inverted between V7-berries and V9-berries as ripening advanced. In addition, both catechin and quercetin glycosides most likely followed the pattern of tannins despite their seasonal fluctuations. To further distinguish V7-berries and V9-berries and assess their astringency development, a panel test was performed using samples at three commercial harvest times . A group of 12 nontechnical panelists scored berry astringency on a scale from 1 to 7, where 1 is extremely low and 7 is extremely high. The panelists were trained using samples from contrasting standard varieties, including Flame Seedless and Crimson as non-astringent and Vintage Red known for its astringent taste . The results showed that V7-berries exhibited lower intensity of astringency compared to V9-berries . As ripening proceeded, nft system astringency levels increased in V9-berries, but decreased in V7-berries.

Moreover, we collected samples from clusters with various astringent taste and measured its tannins content. We were able to determine that the threshold level of tannins that causes the Scarlet Royal astringency taste is around 400 mg/L . Taking into account the levels of polyphenol compounds and the taste panel data together , it is evident that astringency development is positively associated with tannins’ accumulation throughout the ripening process of V9-berries. Nevertheless, organoleptic analysis revealed a significant difference in the berries of the two vineyards, particularly in terms of total soluble solids and titratable acidity . Notably, V9 berries exhibited higher titratable acidity and lower total soluble solids, especially in the later stages . To better understand the molecular events associated with the induction of tannins and astringency upon ripening, the berry transcriptome profile was analyzed in both V7-berries and V9- berries at the late commercial harvest date . Following the quality and quantity check, extracted RNA from quadruplicate samples was deeply sequenced . Of the 19.7 to 24.4 million high-quality clean reads per replicate, 61.9% to 66.1% were mapped against the V. vinifera transcriptome . Hierarchical clustering of the RNAseq data showed explicit changes in the berry transcriptome profile between V7- berries and V9-berries . The Principal Component Analysis showed high consistency among biological replicates . Samples were mainly separated along the first component , which was responsible for 97% of the variance, and was definitely associated with the site of cultivation; V7 and V9. In contrast, the second component was trivial, accounting for only 1% of the variance and was probably attributed to experimental error. Such results were expected, as berry samples came from the same cultivar, Scarlet Royal , and the only difference between them was the vineyard locations. To identify the differentially expressed genes in V7- berries and V9-berries at this specific time within the ripening window, the RNAseq data were analyzed using two different Bioconductor packages, DESeq2, and EdgeR . Subsequently, the DEGs with FDR < 0.05 and log2fold change > 1.5 or < –1.5 generated by both pipelines were considered . The pairwise comparison between berry transcriptomes resulted in 2134 DEGs, with 1514 up-regulated and 620 down-regulated . The data manifested the impact of the cultivation site on the transcriptional reprogramming of a large number of genes that ultimately affect berry quality. Most apparently, at the V9 vineyard, where roughly 2.5-fold higher number of berry transcripts were upregulated compared to V7 . Subsequently, the enrichment of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed among the up- and down-regulated DEGs using the Vitis vinifera Ensembl GeneID . Among the significantly enriched GO terms, the up-regulated transcripts in V9-berries exhibited high enrichment in the molecular function GO terms for quercetin 3-O-glucosyltransferase activity and quercetin 7-Oglucosyltransferase activity . Additionally, the V9-berries induced DEGs were highly enriched in the biological process GO terms for the jasmonic acid signaling pathway and cellular response , Lphenylalanine metabolic process , L-phenylalanine biosynthetic process , and nitrogen compound metabolic process . Similarly, these DEGs were highly enriched in the KEGG pathways for the biosynthesis of secondary metabolites and phenylpropanoid biosynthesis . On the other hand, the down-regulated transcripts in V9-berries showed substantial augmentation in the MF GO terms for hormone binding , abscisic acid binding , and potassium ion transmembrane transporter activity . Correspondingly, the BP GO terms for hormone-mediated signaling pathway and response , auxin-activated signaling, cellular response, and homeostasis , abscisic acid-activated signaling, response, and cellular response , response to strigolactone , potassium ion transmembrane transport , and potassium ion transport , as well as the KEGG pathways for plant hormone signal transduction , brassinosteroid biosynthesis , and carotenoid biosynthesis were highly enriched in the down-regulated genes of V9-berries . Overall, the transcriptome analysis pointed out the substantial changes in transcript abundance that coordinate and reflect the observed induction of tannins/astringency during the maturation and ripening of V9-berries compared to the V7-berries .To elucidate which fundamental processes were altered during tannins/astringency induction within berries, the Weighted Gene CoExpression Network Analysis was applied to construct coexpression networks. Forty-two modules were identified based on pairwise correlations among the 17553 non-lowly expressed genes . Subsequently, the biochemical data from both V7-berries and V9-berries were correlated to the WGCNA modules, and only 2 modules, M21 and M30, displayed substantial correlations with berry polyphenols, containing 5349 and 4559 genes, respectively .

The wetting characteristics were assessed by measuring the equilibrium contact angles of the silica sol-gel on flat PVP-coated gold and amidine-functionalized PS substrates . The prehydrolyzed silica sol-gel solution has a very low contact angle of ≈10° and 30° on a gold surface and PS surface, respectively. These contact angles and the sizes of the NPs and PS colloids were used as input for numerical calculations of the equilibrium solid-liquid-vapor contact line shapes that form a stable sol-gel interface between the NP and the PS colloid for different volumes of air trapped around NPs during the infiltration step . Specifically, the calculations allowed accessing the maximum possible air volume that gives a stable silica-air interface . The upper limit is an air volume of ≈1.6 × 102 nm3, yielding a minimum theoretical value for NP immersion into the sol-gel solution of at least 30% of its surface . Full NP immersion occurs when no air is trapped around the NP upon infiltration, and all the intermediate immersion values are possible, as a function of the volume of air trapped around the individual NPs. Upon calcination, the immersed part of the particle will become embedded into the solidified silica matrix , and hence, the NPs in the RCT catalysts are expected to be significantly entrenched, with a broad distribution of embedding values.To verify the theoretical predictions, hydroponic channel the embedding of more than 200 individual Pd4Au96 NPs in silica-supported RCT catalyst was quantified using dual-axis electron tomography .

Previously, electron tomography has successfully been applied to obtain the distribution, size, and shape of metal particles in catalytic materials. Yet, an important obstacle in using electron tomography to investigate metal-support interfaces is so-called missing wedge artifacts. These artifacts stem from the reduced range of orientations in which the sample can be imaged. Ideally, one would rotate the sample over the full 180° range, but in practice, only tilt angles in the range of 140° can be probed. The missing range of angles, called the missing wedge, causes artifacts in the final 3D reconstruction. These artifacts are particularly severe around the metal particle due to diffraction contrast, thereby obscuring the metal-support interface and the local NP environment. Dual-axis tomography allows a significant reduction of missing wedge artifacts and therefore the assessment of the NP-support interfaces . As the direction of the artifacts around the metal particles depends on their orientation with respect to the tilt axis, combining the information of two tilt series recorded over perpendicular axes, renders a more complete picture of the local NP environment. The analysis procedure to achieve a quantitative and statistically relevant characterization of the local environment of the embedded NPs consisted of five steps : i) acquisition and alignment of the tilt series, ii) separate reconstruction of both data sets, iii) recombination of the reconstructions, iv) segmentation into particle, support and pore, and v) quantification of the embedding.

Full details are provided in the Experimental Section and Supporting Information. Both tilt series can be viewed in Movies S1 and S2, Supporting Information. No beam damage during the tomography acquisition occurred, which is crucial when combining the information from both tilt series into one . Each series was individually processed by aligning the images using 12 fiducial markers in the 141 tilt images per data set and reconstructing the 2 tilt series separately . Subsequently, the two 3D models were combined into one reconstruction using the known positions of 8 overlapping fiducial markers in both data sets . The combined reconstruction at full resolution consisted of more than 3 × 1010 voxels, making further analysis computationally challenging. Therefore, regions of interest of 150 × 150 × 150 voxels around the metal particles were carved out, allowing a reduction in the number of voxels by 3 orders of magnitude. To identify the ROIs, the images were downsized twice and a simple thresholding algorithm was applied to locate the NPs’ positions. The coordinates obtained from the lower-resolution images were mapped back to the high-resolution images and the ROIs were extracted around the estimated particles centers . Successively, each ROI was segmented, that is, every voxel was classified as particle, support, or void . The segmentation procedure consisted of i) denoising the raw images, by employing a total variation denoising algorithm; ii) identifying the voxels belonging to the particle, by applying contrast thresholding; iii) identifying the voxels belonging to the support, by employing a watershed algorithm . From the segmented ROI, the particle size and the fraction of particle surface exposed to the pore were calculated. After visual inspection, accurate quantification of the local environment of 204 particles was confirmed . The quantitative analysis revealed that the NPs were indeed largely embedded in the silica support .

The accuracy of our quantification procedure is supported by the excellent agreement between the average particle size as obtained via automatic segmentation and the manually measured particle size distribution based on a set of 2D images taken at different locations in the sample . Both approaches resulted in an average particle size of 7.4 nm. The relative amount of NPs surface area exposed to the macropores as a function of particle size for 204 particles present in the catalysts fragment, showed that 200 particles were more than 50% embedded in the support. The average and median exposed surface areas were 15% and 12%, respectively. However, the distribution of the exposed surface per particle in the right panel reveals that a substantial number of particles have less than 10% of the surface exposed. Furthermore, there are only 2 particles that have 60% of their surface exposed, which is the minimum embedding observed. As an example, two NPs, one moderately embedded and the other significantly embedded , are shown in Figure 3c, where the green and yellow borders indicate the exposed and embedded NP surfaces, respectively. Overall, the distribution in exposed surface area is broad, and no obvious correlation between particle size and the amount of exposed NP surface was found. Both, the broad distribution of the particles exposed surfaces and the smallest observed embedding of 40% are in excellent agreement with the numerical modeling results shown in Figure 1g.The Au96Pd4 NPs exhibited a characteristic penta-twinned crystal structure, irrespective of their size and degree of embedding . Three representative particles of 9, 11, and 14 nm in diameter at different tilt angles are shown in Figure S9, Supporting Information. The diffraction contrast in the 2D images acquired at different tilt angles clearly shows the fivefold symmetry in the crystal structure of the particles. It is likely that this stems from the citrate ligands used in the NP synthesis, hydroponic dutch buckets which are known to give rise to penta-twinned crystal structures in Au NPs. The fivefold symmetry of the free-standing AuNPs remained intact after introducing a small amount of Pd, attaching the NPs to PS and thermally treating the RCT catalyst at 500 °C. This is in line with previous work in which the crystal structure of free-standing AuNPs was studied as a function of temperature. The decahedral shape of 2–10 nm AuNPs was stable up to 550–600 °C, above which surface roughening and ultimately melting occurred. Another indication that the penta-twinned crystal structure was preserved during the RCT synthesis, is the random orientation of the facets with respect to the pore, which is expected, based on the random attachment of the metal NPs to the PS colloids. No evidence for differences in faceting of the exposed and embedded side of NPs was found.The electron tomography data provided quantitative information on the embedding of the metal NPs in the silica matrix and insight into the particle shape, but raised the question of whether the heavily embedded interfaces of the NPs were still chemically accessible. To assess this, a novel and complementary characterization approach was employed, relying on epitaxial overgrowth of the embedded NPs with a second metal, in this case, silver. Ag was chosen as it is known to grow epitaxially on Au due to the negligible lattice mismatch between the two metals . Hence, the epitaxial growth of Ag on Au enabled visualization of the crystal orientation of the Au surface facets exposed to the macropores. Furthermore, Ag has a significantly lower Z-contrast compared to Au, ensuring a clear difference between the Ag-shell and AuNPs in the electron microscopy visualization.

The high-resolution high-angle annular dark-field scanning transmission electron microscopy image shows a smooth, epitaxial spherical Ag-shell around the free-standing AuNPs with roughly the same 1.5 nm shell thickness at all sides, whereas clear anisotropic Ag-shell growth was observed on the embedded AuNPs . The difference in Ag-shell morphology is also evident from the UV–vis spectra . The localized surface plasmon resonance of the free-standing AuNPs blue-shifted from 517 to 500 nm and increased in intensity upon Ag growth, which is in line with previously reported theoretical and experimental work, where the increase in intensity of the LSPR peak can be ascribed to both the stronger plasmonic properties of Ag andincrease in particle volume. Contrarily, the LSPR peak of the embedded AuNPs split up into 2 separate peaks upon Ag-shell growth: a transverse and longitudinal LSPR peak, at high and low energy, respectively, similar to the plasmonic properties of Ag nanorods. The red-shift of the longitudinal LSPR peak from 521 to 565 nm indicates the growth of a more elongated particle, and hence matches well with the particle Janus-like shape observed in the HAADF-STEM images. In both cases, the shell thickness was controlled via the AgNO3 concentration in the reaction mixture. Although the Ag-shell preferentially grew on the NP surface exposed to the macropores, Energy-dispersive X-ray spectroscopy analysis revealed that Ag also grew on the embedded side of the NPs. This is further confirmed by the high-resolution images in Figure 5, where a continuous shell of approximately three atomic Ag layers was observed on the embedded side of the NPs . Furthermore, the crystal structure of the Ag-shell provided insight into the type and number of surface facets of the AuNPs facing the macropores. By “freezing in” the surface structure of the NPs in the Ag-shell, imaging at high resolution was possible without beam-induced restructuring. The high-resolution images in Figure 5 reveal that depending on the type of facets exposed to the pore, either single-crystalline or twinned Ag-shell formed. In line with our findings shown in Figure 4, no evidence for preferential orientation of specific surface facets towards the macropores was observed. The embedded metal surface area could be available to the reactants through the 0.7 nm gap, but its accessibility might also be related to the porosity of the silica support itself. As the micropores cannot easily be characterized using electron microscopy, nitrogen physisorption was employed to investigate the effect of the latter. The physisorption data confirm that the silica support is indeed microporous , thereby allowing mass-transport to the embedded part of the AuNPs. Furthermore, the physisorption data show that the porosity of the matrix can be tuned via thermal treatment, where repetitive thermal treatment at elevated temperature reduces the microporosity of the silica matrix.The numerical calculation, tomography, and overgrowth results concur in showing that the NPs in RCT catalysts are substantially embedded in the silica support. 200 out of 204 particles were more than 50% embedded in the silica support, and the majority of particles even more than 90%. NP embedding is known to prevent both Oswald ripening and particle sintering, the two mechanisms for particle growth. The large degree of embedding, and in particular the fact that the particles are embedded up to or above their waste line, prevents particle dislodgement and migration, explaining the absence of NP growth even upon thermal treatment at 800 and 950 °C, and during catalysis at elevated temperatures. The thermal stability is substantially enhanced compared to gold based catalysts with NPs lying on top of the support surface, which start to grow to larger particle sizes below 800 °C, and is comparable to fully encapsulated NPs. Hence, the RCT preparation approach endows a robust, porous 3D architecture with all catalytic NPs residing on the pore surfaces and available to the reactants, but significantly embedded into and thus stabilized within the support. The macropores ensure facile mass transport throughout the catalysts, preventing mass-transport limitations in the catalyst bed, whereas the micropores of the matrix enable local diffusion to the embedded side of the NP surface.