Month: June 2024

Some of the pitfalls of this assumption are highlighted in Figure 5.2a. Consider a scenario where we want to compare samples 1-3. An analysis schema that does not account for the chemical relationships among the molecules in these samples , will assume that the sugars in samples 2 and 3 are as chemically related to the lipids in sample 1 as they are to each other. This would lead to the naive conclusion that samples 1 and 2, and samples 2 and 3 are equally distinct, yet they are not from a chemical perspective. On the other hand, if we account for the fact that sugar molecules are more chemically related to one another than they are to lipids, we can obtain a chemically-informed sample-to-sample comparison. Sedio and coworkers developed the chemical structural compositional similarity metric to account for relationships between molecules based on the similarity of their fragmentation spectra. While CSCS compares samples based on modified cosine scores obtained from molecular networking, we calculate chemical relationships based on structurally-informed molecular fingerprints. We express these relationships in the form of a hierarchy which enables the use of other tree-based tools for downstream data analyses. For example, in Figure 5.2a, we show that by using a tree of structural relationships between molecules in samples 1-3, we can apply UniFrac , a tree-informed distance metric and demonstrate that the composition of sample 1 is distinct from samples 2 and 3.The importance of comparing samples by accounting for their molecular relatedness is highlighted when we contrast the results from ignoring the tree structure to those which integrate it . With the structural context provided by Qemistree,square planter pots the differences between replicates across batches are comparable to the within-batch differences .

The retention time shift in this dataset leads to a strong technical signal that obscures the biological relationships among the samples pseudo-F=120.75, p=0.001 vs. tree informed pseudo-F=18.2239, p=0.001. We observed and remediated a similar pattern originating from plate-to-plate variation in a recently published study investigating the metabolome and microbiome of captive cheetahs . In this study, placing the molecules in a tree using Qemistree reduced the observed technical variation , and highlighted the dietary effect that was expected . These results show how systematic and spurious molecular differences can be mitigated in an unsupervised manner using chemically-informed distance measures based on a tree structure. As a case study, we used Qemistree to explore chemical diversity in a set of food samples collected as a part of the Global FoodOmics initiative . We selected a diverse range of food ingredients to represent animal, plant, and fungal groupings. We first performed feature-based molecular networking using MZmine to obtain spectral library matches for a subset of the chemical features . Understanding the chemical relationships between different foods is challenging because most molecules within foods are unannotated. Using Qemistree, we collated GNPS spectral library matches and in silico predictions from CSI:FingerID to annotate ~91% of the chemical features with molecular structures. Using ClassyFire , we assigned a chemical taxonomy to 60% of these structures; the remaining 40% returned no ClassyFire taxonomy. Labeling annotations allowed us to retrieve subtrees of distinct chemical classes such as flavonoids, alkaloids, phospholipids, acyl-carnitines, and O-glycosyl compounds in food products. We propagated ClassyFire annotations of chemical features to each internal node of the tree and labeled the nodes by pie charts depicting the distribution in chemical superclasses and classes of its tips. The molecular fingerprint-based hierarchy of chemical features agreed well with ClassyFire taxonomy assignment, further demonstrating that molecular fingerprints can meaningfully capture structural relationships among molecules in a hierarchical manner.

Furthermore, Qemistree coupled the chemical tree to sample metadata, revealing distinct chemical classes expected for each sample type. In contrast, honey, although categorized as an animal product, shared most of its chemical space with plant products, reflective of the plant nectar and pollen-based diet of honey bees. We observed a clade of flavonoids in both plant products and honey , but no other animal-based foods.While it is expected that a complex food such as blueberry kefir contains molecules from both blueberries and dairy, we can now visualize how individual ingredients and food preparation contribute to the chemical composition of complex foods. We noted that metabolite signatures that stem directly from particular ingredients, such as phosphoethanolamine from eggs, are present in egg scramble , but not in the other two foods highlighted . We can also observe the addition of ingredients in foods that were not listed as present in the initial set of ingredients. We were able to retrieve that there is black pepper in the egg scramble with chorizo and orange chicken, but that this signal is absent from the blueberry kefir . We show that our tree-based approach coherently captures chemical ontologies and relationships among molecules and samples in various publicly available datasets. Qemistree depends on representing chemical features as molecular fingerprints, and shares limitations with the underlying fingerprint prediction tool CSI:FingerID. For example, fingerprint prediction depends on the quality and coverage of MS/MS spectral databases available for training the predictive models, and these will improve as databases are enriched with more compound classes. Qemistree is also applicable in negative ionization mode; however, less molecular fingerprints can be confidently predicted due to less publicly available reference spectra, resulting in less extensive trees. In summary, we introduce a new tree-based approach for computing and representing chemical features detected in untargeted metabolomics studies. A hierarchy enables us to leverage existing tree-based tools, and can be augmented with structural and environmental annotations, greatly facilitating analysis and interpretation.

We anticipate that Qemistree, as a data organization strategy, will be broadly applicable across fields that perform global chemical analysis, from medicine to environmental microbiology to food science, and well beyond the examples shown here.We use SIRIUS , ZODIAC and CSI:FingerID to predict molecular substructures within mass spectrometry features in the MGF files imported as Mass Spectrometry Features. SIRIUS computes fragmentation trees for each molecular formula candidate of a feature and ranks these by score. SIRIUS uses MS1 spectrum in the MGF file to determine the candidate ion adduct to be used for the fragmentation tree computation of each feature. ZODIAC takes the top SIRIUS candidates as input and re-ranks molecular formula candidates considering reciprocal compound similarities in the dataset to increase correct molecular formula assignments. Subsequently, CSI:FingerID predicts molecular fingerprints for each feature based on the molecular formula with the highest ZODIAC score. Note that all spectra provided to the Qemistree pipeline do not necessarily produce a fingerprint. Indeed, SIRIUS does not compute fragmentation trees for multiply charged compounds and CSI:FingerID does not predict molecular fingerprints from spectra with less than 3 explained peaks. To ensure that high confidence molecular formulas are used in Qemistree, we only consider compounds with a ZODIAC score above 0.98 .Samples were analyzed using ultra high performance liquid chromatography coupled to a quadrupole-Orbitrap mass spectrometer . The quadrupole Orbitrap mass spectrometer was fitted with an electrospray source operating in positive ionisation mode. The source used the following parameters: spray voltage, +3500 V; heater temperature, 437.5°C; capillary temperature, 268.75°C; S-lens RF, 50 ; sheath gas flow rate, 52.5 ; and auxiliary gas flow rate, 13.75 . The samples were acquired in non-targeted MS2 acquisition mode,growing blackberries in containers with up to four MS2 scans of the most abundant ions per MS1 scan. The spectra were recorded from 0.48 to 17 min. The following parameters were used for full MS scan: resolution , Automatic Gain Control target , maximum injection time , scan range . For the datadependent in MS2 , the following parameters were used: resolution , AGC target , maximum injection time , loop count , isolation window fixed first mass . and up to four MS/MS scans of the most abundant ions per duty cycle. Higher energy collision induced dissociation was performed with a normalized collision energy of 30 . The data-dependent settings were set as follows: minimum AGC , apex trigger 3 to 15 s, charge exclusion 3-8 and > 8, exclude isotopes , dynamic exclusion .

Samples were collected, extracted, and MS data were acquired as a part of the Global FoodOmics project according to the sampling and data acquisition protocols described in Gauglitz et al., 2020 Food Chemistry. Briefly, 126 food samples were selected from the Global FoodOmics dataset. 119 simple food samples were selected to cover a broad spectrum of fruits, vegetables, meat and fungi. Each food was represented in at least triplicate in the data subset. Additionally 7 complex samples were selected that contained simple foods from the simple food subset in their ingredient lists. The complex foods were from two separate meals of orange chicken, a cooked cucumber and the sauce from a meal , sour cream, blueberry kefir, and egg scramble with chorizo. Sample metadata describes the food samples based on a food hierarchy beginning with plant vs. animal vs. fungus and increasing in detail down to persian cucumber vs. cherry tomato etc. . Briefly, samples were extracted in 95% LC-MS grade Ethanol; 5% LC-MS grade water. Samples were analyzed using the same LC-MS/MS setup and software as described above for the maXis II QTOF mass spectrometer , using a Phenomenex Kinetex C18 1.7 µm 100 x 2.1 column equipped with a guard cartridge . The instrument tuning and internal calibrant remained the same as described above. MS spectra were acquired in a positive ion mode in the range m/z 50–1,500. The mobile phases consisted of A and B , and the flow rate was set to 0.5 µL/min throughout the experiment, and the column maintained at 40℃.AT conceived the concept and managed the project. AT and YVB developed the algorithm and wrote the code for Qemistree. AT and YVB contributed equally to the work. LFN, RK, PCD supervised method implementation. KD, MW, JJJvdH, ME, DM, and AG tested and provided suggestions on how to improve the method. MW managed the deployment of Qemistree on GNPS. AT and MW developed the GNPS-Qemistree Dashboard. DA and AT wrote the documentation for the GNPS-Qemistree workflow. YVB, QZ, and AT developed Qemistree-iTOL visualization. LFN and MNE performed the mass-spectrometry for the evaluation dataset. AT, YVB, and LFN analyzed and interpreted the evaluation data. JMG performed mass spectrometry of the Global Foodomics samples. AT, JMG analyzed and interpreted the Global Foodomics data. KD, MF, ML, and SB supported the integration of SIRIUS, Zodiac, and CSI:FingerID. AT, YVB, PCD, and RK wrote the manuscript. LFN, JMG, MNE, JJJvdH, ME, KD, QZ, DM, AG, JH, MF, ML, SB, and RK improved the manuscript. The co-authors listed above supervised or provided support for the research and have given permission for the inclusion of the work in this dissertation.Flavor chemicals in electronic cigarette fluids , which may negatively impact human health, have been studied in a limited number of countries/locations. To gain an understanding of how the composition and concentrations of flavor chemicals in ECs are influenced by product sale location, we evaluated refill fluids manufactured by one company and purchased worldwide. Flavor chemicals were identified and quantified using gas chromatography-mass spectrometry . We then screened the fluids for their effects on cytotoxicity and proliferation and tested authentic standards of specific flavor chemicals to identify those that were cytotoxic at concentrations found in refill fluids. One hundred twenty-six flavor chemicals were detected in 103 bottles of refill fluid, and their number per/bottle ranged from 1 – 50 based on our target list. Two products had none of the flavor chemicals on our target list, nor did they have any non-targeted flavor chemicals. Twenty-eight flavor chemicals were present at concentrations ≥ 1 mg/mL in at least one product, and 6 of these were present at concentrations ≥ 10 mg/mL. The total flavor chemical concentration was ≥ 1 mg/mL in 70% of the refill fluids and ≥ 10 mg/mL in 26%. For sub-brand duplicate bottles purchased in different countries, flavor chemical concentrations were similar and induced similar responses in the in vitro assays . The levels of furaneol, benzyl alcohol, ethyl maltol, ethyl vanillin, corylone, and vanillin were significantly correlated with cytotoxicity. The margin of exposure calculations showed that pulegone and estragole levels were high enough in some products to present a non-trivial calculated risk for cancer.

Much of our knowledge of the human microbiome comes from association studies that use either a cross-sectional or case–control design. Well-designed case–control studies are critical to demonstrate a potential relationship between microbes and a disease of interest. However, these studies cannot establish causality, and are often subject to confounding variables such as differences in diet or medication between cases and controls. Most studies are conducted at a single time point in a population with the disease, and no long-term follow-up is performed. Consequently, these studies can only identify microbes that differentiate individuals with the disease and the control population. Although these microbes identified might have been causative agents, it is nearly impossible to separate this association from secondary effects associated with the condition. For example, medication plays a major partin shaping the microbiome; a study of patients with type II diabetes mellitus found that treatment with metformin had a larger effect on the microbiome than the disease . Similarly, we hypothesize the physiology of the disease might also contribute to changes in community structure. Association studies are also often confounded by the selection of poor controls. The microbiome is dynamic , and cumulative exposures over an individual’s life, shaped by their diet , lifestyle , medical history , genetics and other factors create a unique community. Thus, if cases and controls are not correctly selected,blueberry container association studies might detect differences due to confounding factors. Matching cases and controls based on age and sex is often not sufficient. In cases in which this matching to control for confounding variables is not possible, it is critically important to collect information about potential confounding factors.

Comparisons across current cross-sectional studies are also challenging due to large effects caused by inter-study differences in technical parameters, including sample collection, storage, primer selection and analysis techniques . Differences across studies increase the challenge of meta-analysis and make identifying causative clades more difficult . Some of these problems can be ameliorated by using consistent methodology between . Efforts like the Microbiome Quality Control Project are exploring sources of technical variation , while analysis platforms like Qiita provide a database of consistently annotated studies for comparison.Twin studies provide a potential antidote to some of the problems with association studies. Twin pairs are naturally controlled for age and some early life exposures. Monozygotic twin pairs also share the same genetic background, further limiting potential confounders . Twin studies can be leveraged in two ways. First, identifying differences between discordant and concordant twin pairs represent more powerful association studies, due to the partial internal control. Although these studies are particularly useful in young children due to shared environment, the approach can also be used with adults . Second, twin studies are critical to examine genetic control of the microbiome. A study published in 2016 of the UK Twins cohort suggested strong association of the microbiome and genes, including those associated with dietary preference and serum lipids . Twin studies provide a unique opportunity to assess if the familial risk factors are either genetic or environmental in nature. These studies have been applied to study heritability for studying hepatic steatosis and fibrosis now that advanced magnetic resonance imaging based assessment can be used to phenotype individuals . However, the sample size requirements for microbiome assessment in twins is large compared to the sample sizes heeded to study heritability may making recruitment for such a study challenging .

Twin studies may not be appropriate for other rare causes of liver diseases e.g. alpha-1 antitrypsin deficiency, cirrhosis, primary biliary cholangitis, and for such low prevalence diseases a trio family design would be more appropriate and would provide the highest power with the most efficient study design to detect association of a trait such as the role of microbiome on the risk of liver diseases .As the cost of microbiome analysis decreases, longitudinal studies are becoming more common. Understanding temporal fluctuation in the microbiome, and the role of microbes in contributing to disease etiology, will rely on studies over time. Work suggests that community instability might, in and of itself, be a characteristic of an unhealthy ecosystem . Prospective studies, such as an investigation examining death from HCC in individuals with NAFLD, have helped identify the role of exposures and etiological factors in contributing to disease outcomes . Currently, the appropriate sampling frequency for understanding the microbiome in prospective studies is unknown, in part due to an overall lack of long term follow up with microbiome studies. Initially, sample collection during standard clinic visits may provide information about the population-scale changes in the population. However, incorporating microbiome samples into these long-term studies will help examine the role of microbial communities—either at a single time point or the community dynamics—as a contributing factor to complex conditions .Model animals also have an important role in shaping our understanding of the microbiome in disease . Although rodent microbial communities are distinct from the human microbiota, there are some shared physiological and microbial traits . Both rodent and human communities are dominated by the same set of bacterial phyla, although a smaller percentage of genera are shared .

As such, experimental findings implicating individual organisms or genera in rodents should be taken with caution until they are validated in humans. Instead, rodent models can show phenotypic consequences of microbiome manipulation. This aspect makes rodent models a useful model system to investigate causality, explore interactions and test early interventions. Both antibiotics and probiotics have been used to study the effect of changing the conventional mouse microbiome on a phenotypic outcome. Broad spectrum antibiotics decrease the total bacterial load, as well as causing major perturbations in the microbial communities . In some cases, such as in liver disease models, this approach can demonstrate the role of bacterial products like LPS in modulating inflammation . In other cases, like a reported addiction model, it can be used to demonstrate the importance of an intact microbiome in regulating behavior . Probiotics can also be used to investigate the effect of a specific bacteria or bacterial cocktail within a controlled environment. A study of alcoholic fatty liver disease demonstrated an attenuation of the microbiome-mediated inflammation when a probiotic was used . Gnotobiotic or germ-free mice can be used in multiple contexts. Comparisons of specific pathogen-free laboratory mice and germ-free mice can be used to examine the role of the microbiome in modulating an expressed or induced phenotype . More importantly, gnotobiotic mice can be humanized with a donor’s stool. This approach creates a system in which an individual’s microbiota can be tested, either for its ability to modulate a disease phenotype or as a target for intervention . For instance, in a small study, mice received their microbiome from a donor with either severe alcoholic hepatitis or no liver disease. Following alcohol treatment, the mice with the microbiome from the patient with alcoholic hepatitis showed greater liver damage than mice that received stool from the healthy donor . Well-designed mouse models that combine our current understanding of liver disease with humanized microbiomes offer some of the greatest potential for preclinical interventions. Avatar, or sometimes called patient-derived xenograft mice, are widely used in the cancer community to test the efficacy of chemotherapeutics for individual tumors, including HCC . This model better re-capitulates the complexity of a tumor than cell culture. Avatar mice can be further personalized by introducing a human immune system into an immuno compromised mouse, along with the tumor . Generating this model in germ-free mice with a humanized microbiome and immune system expands our capacity to understand the role of the microbiome in modulating cancer. For example,growing blueberries in containers this model could be used to study whether the microbiome of a patient with ALD leads to more tumor growth than the microbiome from a healthy individual as control. An accumulating body of research suggests that the disparate observations in liver-disease related studies can be unified and explained by the microbiome. It is now widely accepted that liver damage can result from extensive interplay between gut microbiota via specialized molecules and host-immune system via Kupffer-cell-mediated liver inflammation. However, a comprehensive understanding of the interactions between the microbiome and the liver still evades us. Animal models, particularly rodents, have been instrumental in elucidating many important mechanistic pathways in liver disease etiology. The introduction of the microbiome into these models will provide a more complete view of the cancer ecosystem. Because microbiome research is sensitive to technical variability that often masks underlying biological signals, there is a need for consistency in technical platforms and standardized protocols, so that findings from different laboratories can be replicated and validated. Additionally, it is critical to use an animal model that mimics human disease as closely as possible in all its physiological and metabolic manifestations. We are slowly advancing from observation-based studies in humans as research establishes grounds for microbiome-based therapeutic modalities such as FMT and probiotic interventions. However, effectively translating and applying findings accrued through animal models to humans requires well-designed, large-scale clinical trials spanning multiple disease etiologies and patient characteristics.

As the role of microbiota in liver disease development, prognosis and treatment is increasingly recognized, we emphasize the need for focused, microbiome-aware efforts to efficiently tackle the socioeconomic burden of this spectrum of liver diseases. The authors would like to thank D. McDonald, T. Kościółek, Z. Xu and A. Plymoth for their helpful discussions. M.K. is supported by the NIH grants R01 AI043477 and R01 CA118165. R.L. is supported in part by the grant R01-DK106419-03. Research reported in this publication was supported in part by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number P42ES010337. B.S. is supported by NIH grants R01 AA020703, U01 AA021856, U01AA24726, and by Award Number I01BX002213 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development. J.D. is supported by the Robert Wood Johnson Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Primary biliary cholangitis is characterized by inflammation-mediated damage to the small bile ducts inside the liver, which gradually progresses to liver fibrosis and cirrhosis . Previously considered a typical autoimmune disorder, the modified etiological understanding of PBC considers proinflammatory changes in the gut-microbiota, intestinal bile acid disruptions and gut-barrier dysfunction . Consequently, microbe-associated molecular patterns ascend the biliary duct, perpetuating infection. An immune attack against the biliary epithelial cells is mediated by antibodies that recognize E2 subunit of pyruvate dehydrogenase complex due to cross-reactivity with conserved proteins in Escherichia coli, Lactobacillus delbrueckiiand Novosphingobium aromaticivoransIn fact, genetically susceptible mouse strains developed liver lesions mimicking PBC when infected with Novosphingobium aromaticivoransm, which further implicates a role for the microbiome in this disease . Ursodeoxycholic acid, a tertiary bile acid produced by Ruminococcus, has been approved for PBC treatment . Thus, microbiome-based treatment modalities hold promise for managing PBC and should be studied further. Primary sclerosing cholangitis is also an immune-mediated disease of the bile ducts . However, unlike PBC, PSC can affect bile ducts, both inside and outside of the liver. Gut dysbiosis-mediated bile dysregulation, intestinal permeability and translocation of proinflammatory molecules in the portal vein characterizes PSC . The immune reaction in PSC is mediated by autoantibodies, including perinuclear antineutrophil cytoplasmic antibody, that recognize the ubiquitously expressed bacterial antigen FtsZ . Furthermore, increase in microbe-associated Toll-like receptor expression and T helper type 17 cells has been reported in PSC, which strongly suggests microbiome involvement in disease pathogenesis . PSC is closely associated with IBD , in particular ulcerative colitis and shares some of its characteristic features . Thus, a common disease mechanism might be at play, and novel treatment avenues by targeting microbe associated immune pathways can be explored.Limited data exist concerning the diagnostic accuracy of gut-microbiome-derived signatures for detecting NAFLD-cirrhosis. Here we report 16S gut-microbiome compositions of 203 uniquely well-characterized participants from a prospective twin and family cohort, including 98 probands encompassing the entire spectrum of NAFLD and 105 of their first-degree relatives , assessed by advanced magnetic-resonanceimaging . We show strong familial correlation of gut-microbiome profiles, driven by shared housing.

Table 3.1 summarized the diet composition. During 4 weeks of feeding, fresh food was provided to hamsters weekly. Their body weight and food intake were recorded weekly. Food efficacy, known as the weight gain per gram of diet consumption was calculated. At the end of the 4th week, hamsters were fasted for 14 h, anesthetized using isoflurane , and sacrificed based on the procedures in the previous study . 5 mL blood was drained by cardiac puncture into EDTA rinsed syringes, then centrifuged at 2000 g, 4 °C for 15 mins to collect plasma and stored at -80 ⁰C. The liver, kidney, and epididymal adipose were excised, weighed, and frozen in liquid nitrogen then stored at -80⁰C. Feces were collected during the last day of feeding and stored at -20 ⁰C. Plasma lipoproteins, including very-low-density lipoprotein -, low-density lipoprotein -, and high-density lipoprotein – cholesterol, were determined using size-exclusion chromatography as described . Specifically, A cholesterol reagent was delivered using Hewlett-Packard HPLC pump 79851-A at a flow rate of 0.2 mL/min. Lipoproteins were separated by injecting 15 μL of plasma onto the Superose 6HR HPLC column in Agilent 1100 HPLC chromatography system. Then the lipoproteins were eluted with a solution containing 0.15 M NaCl and 0.02% sodium azide at a flow rate of 0.5 mL/min. Base signals of the peak areas were calibrated by applying bovine cholesterol lipoprotein standards . Total cholesterols in plasma were counted as the sum of the VLDL-, LDL-, and HDL-cholesterol concentrations, interpolating from the standard curve. Plasma triglyceride was quantified using an enzyme colorimetric assay kit based on the absorbance at 505 nm . The liver and feces samples were ground in mortar and pestle, snap clamps for greenhouse then the lipid was extracted with hexane/isopropanol at 60 °C using Dionex ASE 350 accelerated solvent extractor . The lipid extracts were evaporated to dryness at 37 °C under nitrogen.

Total hepatic and fecal lipid contents were calculated based on the weighed extracted lipid over liver weight. Hepatic lipids were re-dissolved in hexane/isopropanol solvent and mixed with Triton X-100. After drying and diluting the mixture in DI water, total cholesterol and free cholesterol were determined with enzymatic kits , then esterified cholesterol was calculated based on the difference between TC and FC. Liver TG was measured using the same method for plasma TG.The body weight gains over 4-weeks of feeding were shown in Figure 3.1. All the diets elevated the total weight gain ranging from 18.14 to 33.82g, in order of HP, HF, HE, LP, and LE . The corresponding feed efficacy ratio was from 0.11 to 0.18 g/g. In both total weight gain and feed efficacy ratio, the HP diet was significantly lower while the LE diet was significantly higher compared to HF and HP diets . Possible reasons could be that the high fiber content in peel promoted earlier satiety , reduced the digestion and absorption of lipid content. At the same time, liquid extract in LE and HE diets tended to dissolve more reducing sugars from the pomegranate peel. Therefore, it promoted absorption efficiency and further weight gain. Sangüesa et al investigated the different adverse effects of glucose and fructose on the metabolism of female rats and observed that fructose had a greater impact on metabolic dysfunction . The carbohydrate composition of the extract and peel could be examined in the future for a better understanding of the mechanisms. As unusual LDL-elevating effects in plasma after PPP and PPE ingestion were observed, real-time qPCR was applied to investigate the potential mechanisms of diets modulating the lipid and cholesterol metabolism. As shown in Figure 3.4, the expression level of CYP7A1 , a gene that controls bile acid synthesis rate from cholesterol, was increased by 2.1-folds in the HP diet, while that was reduced to 0.86-, 0.91- and 0.90- fold in LP, LE, and HE diets. HMG-CoAR and Cyp51 are two important genes in cholesterol biosynthesis. Compared with the control diet, LP, HP, LE, HE demonstrated the same up-regulating pattern in these two genes — 1.03-, 1.51-, 1.11- and 1.18-folds for HMG-CoAR , as well as 1.18-, 1.72-, 1.25- and 1.22-folds for Cyp51 , respectively. LDL receptor facilitates the hepatic LDL uptake from circulation.

In the present study, LP and HP diets up-regulated LDLR expression by 1.18- and 1.38-folds , while that was slightly down-regulated by 0.95-fold in both LE and HE diets. These findings were in line with hepatic LDL cholesterol — all the reformulated diets prompted hepatic cholesterol synthesis. HP diet increased bile acid synthesis while other diets decreased it. Peel-formulated diets elevated LDL uptake and extract-formulated ones alleviated it. For peel-formulated diets, bile acid synthesis was dominating in lowering hepatic cholesterol, whereas for extract formulated diets it lowered LDL uptake. PPARα was an essential transcription factor regulating fatty acid β-oxidation. It was up-regulated in LP and HP diet-fed hamsters by 1.04- and 1.61- folds. In contrast, a lower expression of 0.90- and 0.93-fold of PPARα was observed in LE and HE diets . SREBP-1c is a gene encoding transcription factor for fatty acid synthesis . It targets SCD-1 to catalyze the synthesis of monounsaturated fatty acids, which is a substrate for TG synthesis and storage. LP diet slightly up-regulated the expression of SREBP-1c by 1.07-folds, while HP, LE, and HE diet down regulated that by 0.74-, 0.85- and 0.92-fold . All the diets significantly reduced SCD-1 expression level by 0.42-, 0.24-, 0.73- and 0.61-fold . These results indicated that all the formulated diets induced lower uptake of fatty acids. Peel-enhanced diet slightly promoted fatty acid β-oxidation in a dose-dependent manner, while extract-enhanced diet mitigated that, which was aligned with liver TG levels and hepatic lipid contents. To identify the microbiota-changing effects of different diets on lipid metabolism, relative abundance was assessed at the phylum level . Human gut microbiota is generally dominated by the bacterial phyla Bacteroidetes and Firmicutes . Besides these, Proteobacteria is considered correlated for the variation of the functionality of gut microbiota . Compared to the control HF diet, HE and HP diets reduced the relative abundance of Firmicutes by 9.33% and 18.3% while increasing the RA of Bacteroidetes by 43.1% and 41.9% and nearly triplicated the RA of Proteobacteria.

The corresponding ratio of Firmicutes/ Bacteroidetes dropped by 39.4% and 42.4%, with an increase of Proteobacteria/ Bacteroidetes ratio by 89.6% and 105.1%, indicating that a shift of fecal microbiota towards leaner phenotypes. Verrucomicrobia was boosted to 4.0% and 8.5% for HP and HE diets, which was non-detectable for the HF diet. Similar observations of Verrucomicrobia increase were found in formulated diets with PPE , cranberry , and black raspberry , which could be attributed to the abundant polyphenol content. At the same time, Cyanobacteria was elevated from 0.3% in the HF diet to 2.3% in HE and shown of associated with improved gut health . To the best of our knowledge, no other research reported the change of Verrucomicrobia and Cyanobacteria after PPP incorporation in hypolipidemic diets. Further metagenomics studies are required to understand how Verrucomicrobia and Cyanobacteria modulate the lipid metabolism pathways. As shown in Figure 3.6, the expression of hepatic HMG-CoAR was significantly correlated with obesity-related indices, with a positive correlation with plasma concentrations of the total- and LDL-cholesterol , and a negative correlation with liver , adipose and body weight . The expression of LDLR also exhibited a significant positive correlation with total cholesterol. This was consistent with previous research from Teh et al. , who concluded that HMG-GoAR and LDLR were the two major regulating factors in meditating hypo cholesterol effects of hamsters fed with fruit and vegetable seed meals. As for types of bacteria, total plasma cholesterol exhibited a significant positive correlation with the phylum Bacteroidetes,greenhouse snap on clamp and a significant negative correlation with the F/B ratio in response, suggesting that the increase of Bacteroidetes attributed to the decrease of F/B ratio and played a role in elevating total cholesterol level. Pomegranate peel, a commonly underutilized by-product with high phenolic and fiber content, was incorporated into the hypolipidemic diet in the form of powder and extract to investigate its hypoalipidemic potential. PPP and PPE containing a rich mixture of phytonutrients demonstrated sufficient effects to suppress weight gain, hepatic lipid profile and ameliorate the symptoms of metabolic syndrome in Golden Lakeview Golden Syrian hamsters with HF diet-induced obesity. These observations can be at least partially explained by hepatic metabolism changes and changes in gut microbiota composition. In this study, PPP and PPE lowered Firmicutes and boosted Bacteroidetes, Verrucomicrobia, and Cyanobacteria to lower the F/B ratio, as well as increased microbiota diversity. These indices were significantly correlated with obesity-related indices, indicating that microbiota might play an important role in the hypolipidemic effects of PPP and PPE. 2 hepatic genes were closely related to modulating the plasma and lipid profile, suggesting the ingested cholesterol and LDL uptake level were crucial metabolic changes. However, adverse plasma LDL-elevating effects were observed in a higher dose of PPP and PPE intake, which required further study on the potential toxicity.

Since the 21st century, society has an increasing awareness of health and is switching to healthier lifestyles and eating habits . Several sectors for product development are responsible for the change, such as food industries, researchers, health professionals, and regulatory authorities . In this context, functional foods have great potential. Functional foods represent the portion of the human diet that could provide health benefits and reduce the risk of chronic diseases beyond nutrition. Polyphenol-containing products are a common type of functional food with proven health benefits, such as protecting against certain cancers, cardiovascular diseases, type 2 diabetes, osteoporosis, pancreatitis, gastrointestinal problems, lung damage, and neurodegenerative diseases . According to Scalbert & Williamson , 1 g of daily consumption of polyphenols in long term is suggested to fulfill all the aforementioned health benefits of polyphenols. U.S. dietary guidelines recommended daily food intake to satisfy certain nutrient needs. However, polyphenols are not included and only 552 mg of polyphenol is satisfied through the recommended diet based on our calculation . Yogurt is a popular fermented dairy product known for its high nutritional value, especially the significant content of proteins and essential minerals, such as calcium. Greek Style Yogurt is a type of nutrient-dense yogurt with increasing popularity among consumers. According to Statista , from 2015 to 2020, the consumption of GSY in the U.S. significantly improved 50%, worth $3.7B and accounting for 52% of the U.S. yogurt market share. Compared to regular yogurts, GSY contains a higher solids content and is often perceived as being less acidic. The nutritional information commonly claims “twice the amount of protein as in regular yogurt” . However, they are never considered a source of functional foods. Therefore, it is of great interest to investigate the GSY fortified with polyphenol ingredients for improved polyphenol intake and increased consumption of dairy foods . El-Said et al., evaluated the antioxidant activities and physical properties of stirred yogurt fortified with the extracts of oven-dried and solar-dried pomegranate peel. Results showed that increasing the percentage of the added pomegranate peel extract statistically increased antioxidant activities and reduced the viscosity without significantly affecting the sensory attributes. However, since stirred yogurt has lower protein content and viscosity than GSY, a modified manufacturing process is needed for GSY. Kharchoufi et al. investigated quality changes of GSY fortified with pomegranate juice and arils during 18 days of storage. Researchers directly incorporated PJ and PA into GSY and quantified the total phenolic content, antioxidant activity, and sensory 1, 6, 12, and 18 days. This research demonstrated that the addition of PJ and PA in fermented GSY could significantly increase the antioxidant content and activity without negatively altering the sensory quality. However, incorporation of PPE in GSY prior to fermentation was not discussed, which could provide unique product characteristics and health benefits, as research has shown that bacteria could transform polyphenolic compounds into smaller units for increased extractability and stability . According to Ozcan , several main processing parameters influence the yogurt texture, including . fortification level and materials used, . stabilizers type and usage levels, . fat content and homogenization conditions, . milk heat treatment conditions, . starter culture , . incubation temperature , . pH at breaking, . cooling conditions and . handling of product post manufacture . Based on these considerations, this study aimed to investigate the effects of different contents of protein and PPE on the sensory, nutritional, and functional attributes of GSY.

Breeders increasingly are focused on meeting the needs of consumers, but genetic improvement of flavor is challenging as a consequence of the chemical and genetic complexities of the flavor phenotype . These challenges are accentuated in heterozygous, polyploid species. For example, fewer significant single nucleotide polymorphisms were detected in genome-wide association study of tetraploid blueberry when diploid models were applied ; in octoploid strawberry, structural variation underlying a locus affecting volatile production was difficult to resolve using a single reference genome . Recent advances have been made via chemical–sensory studies to identified specific volatiles associated with consumer preference . Although important volatile compounds in fruit crops are being identified, too little is known about the metabolomic and genetic diversity within species and breeding populations. Some volatiles have been lost during domestication and breeding as a combined result of negative selection and linkage drag in tomato and watermelon . Likewise, gain and loss of terpene compounds during strawberry domestication and its genetic causes have been investigated . Recent advances in sequencing technology and analytical approaches have opened new opportunities to understand the chemistry and genetics of fruit flavor. Meanwhile, genomes-wide expression quantitative trait loci studies have the capability to bridge the gaps between GWAS signals and their underlying causative genes. Integration of GWAS and eQTL studies has led to discovery of a master metabolite regulator in tomato and a flesh-color-determining gene in melon .

Long-read sequencing now allows assembly of genomes with high contiguity, and when coupled with parental short-read data ,large pot with drainage the two haplotypes of a heterozygous individual can be fully resolved. Phased assemblies have improved variant discovery, especially for large structural variants . The extent, diversity and impact of SVs increasingly are being studied in horticultural crops and have been shown to alter fruit flavor, fruit shape and sex determination . Great opportunity exists to coherently integrate these multi-omics resources for the discovery of flavor genes. Garden strawberry is an allo-octoploid species with highly palatable non-climacteric fruit . It increasingly has been utilized as a model for Rosaceae fruit crops genomics and flavor research as a result of its short generation time, wide cultivation and high value. Through exploration of spatiotemporal changes in gene expression and homolog search, several flavor genes have been cloned and validated, including an alcohol dehydrogenase and several alcohol acyltransferases for esters, a nerolidol synthase 1 for terpenes and a quinone oxidoreductase for furaneol. Recently, QTL studies and transcriptome data analyses for strawberry volatiles using biparental crosses have detected QTL and causative genes for mesifurane and gamma-decalactone . Nevertheless, low mapping resolution and a lack of subgenome-specific markers have hampered further characterization of causal genes underlying other QTL. This problem recently was addressed by the development of 50K Fana SNP array using probe DNA sequences physically anchored to the octoploid ‘Camarosa’ genome . High heterozygosity combined with an allopolyploid genome presents difficulties for resolving causative genes and their haplotypes. To further the goal of discovering causative genes affecting flavor in strawberry, association studies with larger sample sizes and additional genetic resources such as eQTL and additional genomes are required. Furthermore, these resources must span the breadth of natural variation in breeding germplasm.

Here we present multi-omics resources consisting of an eQTL study representing the genetic diversity of strawberry breeding programs in the US, phased genome assemblies of a highly- flavored University of Florida breeding selection, a structural variation map in octoploid strawberry and a volatile GWAS of 305 individuals. These are combined to leverage the extensive metabolomic, genomic and regulatory complexity in strawberry for the discovery of natural variation in genes affecting flavor. Ultimately, the functional alleles identified will be selected in breeding to achieve superior flavor.The eQTL population consisted of 196 genotypes including 133 newly sequenced accessions . The University of Florida genotypes were grown at GCREC and collected in the spring of 2020 and 2021. The University of California-Davis collection of diverse selections from multiple breeding programs were grown at either Santa Maria CA or Oxnard CA, for day-neutral and short-day accessions, respectively, and collected in the spring of 2021. Four UC genotypes were collected at both sites to ensure sequencing and SNP quality. Total RNA was extracted from a bulk of three fully ripe fruits using a Spectrum™ Plant Total RNA Kit , after flash freezing in liquid nitrogen. Illumina 150-bp pair-end sequencing was performed on the Illumina NovoSeq platform by Novogene Co. . On average, 6.9 Gb of sequence data were obtained for each sample. Raw RNA-Seq data of 63 samples from previous published studies were retrieved from the NCBI SRA database . In order to quantify gene expression, short reads were trimmed for adapter sequences and low-quality reads with TRIMMOMATIC v.0.39 and aligned against the reference genome using STAR v.2.7.6a in the two-pass mode . Only unique aligned reads were scored by HTSEQ v.0.11.2 in the union mode with the ‘–nonunique none’ flag supplied with the latest Fragaria_ananassa_v1.0.a2 annotation . All count files were compiled in R and normalized with the DESEQ package . To generate the marker dataset for eQTL mapping, SNPs and InDels were called using the mpileup and call commands. Markers were further hard-filtered using BCFTOOLS with the following steps: individual calls with lower than sequencing depth of three were set to missing using + setGT plugin; marker sites with quality < 30, missing rate > 0.3, heterozygous call rate > 0.98, minor allele frequency < 0.05, or number of alternative alleles > 1 were purged; the filtered markers were imported and analyzed in R, and only markers showing more than three matched calls in four duplicated sample pairs were retained.

A total of 491 896 markers passed the three stages of filtering. The missing calls were imputed, and all calls were phased using BEAGLE v.5.2 using the default settings . The eQTL mapping was performed for 62 181 fruit expressed genes using the filtered markers. Linear mixed models implemented in GEMMA were used for association analysis . The relationship matrix was computed in GEMMA and supplied to explain relationship within populations, and the top five principal components with a total of 25.0% variance explained were imported as covariates to reduce effects from population stratification to signify the genetic variance underlying the target traits. The Bonferroni corrected 5% significance threshold was used, determined the by number of LD-pruned markers . The approach to define an eQTL was similar to that used in previous studies . Briefly, we first clustered all significant markers with distance < 100 kb and purged clusters with fewer than three markers. The lead marker with lowest P-value was used to identify the eQTL, and boundaries of eQTL were defined as the furthest flanking significant markers. Clusters in LD were merged and boundaries were updated. The longest distance between cis-eQTL boundaries and eGene boundaries was limitedto 500 kb. Trans-eQTL hotspots were searched using the density function in R .In this study we leveraged eQTL, GWAS and haplotype-resolved genome assemblies of a heterozygous octoploid to identify allelic variation in flavor genes and their regulatory elements. Fine tuning of metabolomic traits such as amylose content in rice and sugar content in wild strawberry recently were made possible via CRISPR-Cas9 gene-editing technology. Similar approaches can be taken in cultivated strawberry for flavor improvement, but not before the biosynthetic genes responsible for metabolites production and their regulatory elements are identified. Our pipeline has proven to be effective in identification of novel causal mutations for flavor genes responsible for natural variation in volatile content and can be further applied to various metabolomic and morphological aspects of strawberry fruit such as anthocyanin biosynthesis , sugar content and fruit firmness. These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Strawberry also shares common volatiles with a variety of fruit crops. Specific esters are shared with apple ,drainage collection pot certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and subgenome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication .

Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, but rather is concentrated at a few hotspots controlled by putative master regulators . The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species . In cotton, physical interactions among chromatins from different subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes and limited length of Illumina reads, false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals. Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits. Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.The gastrointestinal tract, especially the large intestine, houses the most abundant and complex microbiota in humans. Most of intestinal bacteria belong to the phylum Firmicutesand Bacteroidetes , which make up more than 90% of known phylogenetic categories and dominate the distal gut microbiota. Other lower abundance bacteria include Actinobacteria, Fusobacteria, Proteobacteria, and Verrucomicrobia. Diet is one of the important factors contributing to the gut microbial composition that ultimately affects human health. Obesity and associated metabolic diseases, including type 2 diabetes, are intimately linked to diet . A number of recent in vitro, in vivo, and human studies showed that polyphenols or polyphenol-rich dietary sources, particularly tea, wine, cocoa, fruits, and fruit juices, influence the relative abundance of different bacterial groups within the gut microbiota byreducing the numbers of potential pathogens and certain gramnegative Bacteroides spp. and enhance beneficial bifidobacteria and lactobacilli . Spices are derived from bark, fruit, seeds, or leaves of plants and often contain spice-specific phytochemicals. Spices have been used not only for seasoning of foods but also for medicinal purposes, and have a number of demonstrated disease preventive functions such as antimicrobial, antiinflammatory, antimutagenic activities, and are known to reduce the risk of cancer, heart disease, and diabetes . They are best known for their strong antioxidant properties that exceed most foods. It was reported that of the 50 food products highest in antioxidant concentrations among 1113 U.S. food samples, 13 were spices. Among them, oregano, ginger, cinnamon, and turmeric ranked #2, 3, 4, and 5, respectively .

Familial CRC cases reporting regular wine consumption were noted to have earlier stage at presentation, and the major OS benefit from regular wine consumption was observed for local stage cases . However, this survival improvement for regular wine consumption among familial CRC cases was independent of stage, age, and other clinical variables in an adjusted analysis as shown in Table 4. Local and regional stage CRC patients represent those with potentially curable disease after surgical resection and adjuvant chemotherapy. The cause of death analysis reveals that most CRC cases died from their cancer among familial and sporadic CRC cases. Thus, any protective benefit of wine consumption among familial CRC cases is not likely to be due to a specific decreased mortality risk from death by other causes when compared to sporadic CRC cases. It is not known whether the cases in our study continued with the same wine consumption habits after their diagnosis compared to before diagnosis, which remains a limitation of this study. Nonetheless, potentially operative gene-environment survival effects among wine-consuming early stage, familial CRC cases could be investigated further. Among the numerous compounds found in wine, resveratrol and anthocyanin have been reported to have anticancer activity in vitro. Experimental studies suggest that resveratrol, a naturally occurring compound found in grape skin and wine , has been shown to inhibit tumor initiation, promotion, and progression and is reported to have anti-proliferative effects in colon cancer cells and in experimental murine models . Several mechanisms of action have been proposed for the antitumorigenic effects of resveratrol including inhibition of polyamine synthesis, cyclooxegenase inhibition,round plastic planter and inhibition of NF-kappa-B signaling, among others. Abnormalities in the control of polyamine metabolism result in increased polyamine levels that can promote tumorigenesis .

Resveratrol has been shown to inhibit ornithine decarboxylase , which is the rate-limiting step in polyamine biosynthesis . The polyamine catabolic enzyme spermine spermidine acetyltranferase is upregulated by resveratrol in colon cancer cell lines , promoting polyamine efflux. Thus, resveratrol, through its interactions with ODC and SSAT, affects both the biosynthetic and catabolic pathways involved in polyamine regulation to decrease polyamine levels. Resveratrol was reported to inhibit cyclooxegenase-1 and tumor initiation . Additionally, resveratrol has been shown to inhibit cyclooxygenase-2 and I-kappa-B, thereby inhibiting NF-kappa-B dependent signaling . Preliminary data from our laboratory indicate that resveratrol down regulates signaling through the Wnt pathway , a pathway that is activated in over 85% of CRC cases . Recently, it was demonstrated that resveratrol improves health and survival in obese mice that had been fed a high-caloric diet—although the amounts of resveratrol used in that study were far beyond what could be obtained through wine consumption in humans . The flavonoid extract anthocyanin from red wine showed a suppressive effect on human colon and gastric cancer cells in vitro . White wine was not found to have anthocyanins, and yet the nonanthocyanic extractions from red wine and white wine still suppressed cell growth in the aforementioned study but at a reduced rate compared to anthocyanin . Phenolic acid and anthocyanin extracts from certain blueberries and grapes have been shown to inhibit viability of colon cancer cells, with increased apoptosis noted after anthocyanin treatment. Thus, a variety of compounds in wine may contribute to the observed survival benefit noted in our study through effects on multiple signaling pathways. The observed survival differences for familial CRC cases in our study may reflect other unique differences between regular wine consumers and infrequent wine users. For example, Johansen et al. conducted a cross-sectional study of dietary habits among wine drinkers and beer drinkers and found that people who purchase wine at grocery stores in Denmark also have a healthier selection of other foods.

Wine buyers purchased more olives, chicken, fruits, vegetables, milk, and meat than beer buyers in that study. People buying beer purchased more ready cooked dishes, sugar, chips, and sausages than wine buyers. Although dietary fiber, total daily energy intake, fruit and vegetable consumption, and BMI were not associated with survival in this study, it should be recognized that other dietary factors, lifestyle factors, or habits may contribute to the observed survival effects of wine consumption among familial CRC cases. Importantly, wine consumption has been associated with higher SES compared to beer drinkers , but SES was not a major determinant of survival in our epidemiologic study. This is consistent with other studies on CRC cases that have shown no effect of SES on survival . In contrast, higher SES has been associated with a higher incidence of breast cancer , among others, and has been associated with improved survival in aggregate data for all U.S. cancer patients . High SES is generally associated with improved access to care and insurance coverage—factors that affect screening practices and thus stage at presentation. In our study, there was an association with wine consumption and SES; however, there was not a statistically significant association with SES and stage at presentation among familial or sporadic CRC cases. Larger epidemiologic studies are needed to investigate SES as a potential confounder of the effects of wine consumption on stage at diagnosis among familial CRC cases. It is important to note that the proportion of cases reporting regular wine consumption in this study is low compared to what has been reported in other major studies on alcohol consumption and mortality or risk of CRC . Even among the regular wine consumers in this study, reported consumption appeared quite moderate . This may reflect population differences between otherwise “healthy” cohort study subjects and our population, which was comprised purely of CRC cases. This epidemiologic study provides important hypothesisgenerating results related to wine consumption among familial CRC cases. Some of the genes and gene variants involved in multi-factorial inherited susceptibility to colorectal adenomas have now been described .

However, currently, there is an incomplete understanding of the genes involved, and even less is known about how these genes interact with environmental exposures to affect survival. Notwithstanding available investigations of wine consumption on CRC incidence, this study represents the first large, population based study addressing outcomes for CRC patients based on reported wine consumption. Further investigations aimed at elucidating the mechanisms for the observed benefits of wine consumption in familial CRC patients are needed.Survival of humans will depend on increased agricultural productivity. Agricultural productivity is not only more yield per area, but also higher nutritional content, less dependence on fertilizers, and more resilience against environmental hazards. All of these traits impinge upon plant metabolism. Plants carry out a myriad of metabolic reactions that are intricately connected into complex networks. To understand and engineer plant metabolism, it is important that metabolic complements of plant genomes are accurately and consistently annotated across species. To provide the research community with comprehensive information about plant small-molecule metabolism, we previously introduced the Plant Metabolic Network , a plant-specific online resource of metabolic databases . PMN consists of PlantCyc, a database of all experimentally-supported information found in the literature from any plant species, as well as 22 single-species databases with a mix of experimentally-supported and computationally-predicted information,round plastic plant pot which allow researchers to explore each species’ unique metabolism. Here we describe the substantial expansion of PMN in both quantity and quality, which includes 126 single-species databases. We demonstrate the utility of the PMN resource by applying recently published omics data to gain insights into plant physiology and cellular level metabolism. Additionally, we systematically compare 126 species in the context of metabolism to identify metabolic domains and pathways that distinguish plant families. Finally, we present new website tools for viewing and analyzing metabolic data including a CoExpression Viewer and subcellular boundaries for metabolic pathways.PMN is a compendium of databases for plant metabolism with a substantial amount of experimentally supported information. The latest release contains 126 databases of organism-specific genome-scale information of small-molecule metabolism alongside the pan-plant reference database PlantCyc . Together, these databases include 1,280 pathways, of which 1,163 have direct experimental evidence of presence in at least one plant species. In addition, PMN 15 includes 1,167,691 proteins encoding metabolic enzymes and transporters where 3,436 have direct experimental evidence for at least one assigned enzymatic function. There are 9,129 reactions , and 7,316 compounds. Compared to the PMN 10 release described in Schläpfer et al. , PMN 15 increases the number of species 4.7-fold and proteins 8-fold, and adds 2,929 more reactions, 2,178 more compounds, 66 more pathways, and 3,229 more references . Data in the PMN databases are represented using structured ontologies consisting of hierarchical classes to which pathways and compounds are assigned by PMN curators, which makes statistical enrichment analyses possible. The pathway and compound ontology classes, alongside the phylogeny of the included species, illustrate the breadth of metabolic information and species included in the database . Prominent specialized metabolism classes such as terpenoids and phenylpropanoids are highly represented in the databases. This large volume of metabolic information makes PMN a unique resource for plant metabolism. The reference database, PlantCyc, is a comprehensive plant metabolic pathway database. PlantCyc 15.0.1 contains experimentally supported metabolic information from 515 species. Most of the data come from a few model and crop species .

For example, Arabidopsis thaliana contributes to 43.4% of experimentally supported enzyme information in PlantCyc, followed by 7.46% from Chlamydomonas reinhardtii and 3.37% from Zea mays. Compared to other metabolic pathway databases such as KEGG and Plant Reactome , PlantCychas substantially higher numbers of experimentally supported reaction and pathway data . PlantCyc 15 includes 3,077 experimentally validated reactions with at least one curated enzyme and 1,163 curated pathways . Plant Reactome includes 1,887 and 320 curated reactions and pathways, with 677 reactions and 266 pathways predicted to occur in A. thaliana , while KEGG includes 543 experimentally supported pathways as of February, 2021, with 136 occurring in A. thaliana. Data on the number of reactions in KEGG that were experimentally validated were not available at the time of publication. The reference information in PlantCyc is incorporated into MetaCyc, which also includes experimentally supported metabolic information from non-plant organisms and is used to predict species-specific pathway databases . In addition to the reference database PlantCyc, PMN 15 contains 126 organism-specific metabolism databases . These databases range widely in the plant lineage including several green algae and nonvascular plants. The majority of the plants are angiosperms with the Poaceae family most highly represented with 25 organisms. There are also 8 pairs of wild and domesticated plants, including rice, wheat, tomato, switch grass, millet, rose, cabbage, and banana, alongside their wild relatives . Finally, PMN 15 includes 6 medicinal plants : Camptotheca acuminata, Cannabis sativa, Catharanthus roseus, Ginkgo biloba, Salvia miltiorrhiza, and Senna tora. The newest addition to the list of the medicinal plants is Senna tora, which is a rich source for anthraquinones and whose recent genome sequencing and metabolic complement annotation helped discover the first plant gene encoding a type III polyketide synthase catalyzing the first committed step in anthraquinone biosynthesis . This rich collection of species-specific metabolic pathway databases enables a wide range of analyses and comparisons. To promote interoperability and cross-referencing with other databases, PMN databases contain links to several compound databases such as ChEBI , PubChem , and KNApSAcK . PubChem containins over 270 million chemical entries as of March 2021, and 95% of PMN compounds link to it. ChEBI release 197 has 58,829 entries and serves as a primary source of compound structural information during curation into PMN databases. Within PMN, 65% of compounds link to ChEBI. Examining 50 randomly chosen compounds that are not mapped to ChEBI suggest that the majority of the remaining 35% compounds do not yet exist in ChEBI . KNApSAcK links are comparatively rare, as only 1.7% of compounds have had a KNApSAcK link added by curators. Linking to these chemical databases provides a more in-depth source of information on the compounds and their physical and chemical properties. In summary, PMN is a broad resource for plant metabolism and continues to be under active development and expansion. The single-species databases were created using a computational pipeline and Methods. The large number of predicted databases in PMN 15 allows us to evaluate the quality of the predictions quantitatively.

During the mid-Pliocene, the Canadian High Arctic would have been forested, and the latitudinal gradient was much less than modern, so that although global temperatures were 3-4 degrees warmer than modern, the mean annual temperature of the terrestrial High Arctic was ~22 °C warmer . The Beaver Pond site comprises the remains of a Pliocene forest wetland community that was dominated by larch , and also supported alder and birch , spruce , pine and cedar . Multiple proxies consistently suggest a Pliocene mean annual temperature at the Beaver Pond site of slightly above freezing, with plant community composition indicating a warmest summer air-temperatures of ~20°C. Coldest winter temperatures have been recently estimated from vegetation to be ~−12°C, though a prior estimation from beetle fauna suggest −27 °C. Modern Mean Annual precipitation in the area is 104mm/year, whereas in the Pliocene the plant community implies precipitation to have been ~550mm/year . In fossil vertebrates, the Beaver Pond site, in combination with the nearby Fyles Leaf Bed fossil site, has produced four native North American mammals: a castoroidine beaver Dipoides sp., an archaeolagine rabbit Hypolagus cf. H. vetus, a small canine dog Eucyon, and a cameline camel. Of these, Eucyonand Paracamelus had arrived at Eurasia near the Mio-Pliocene boundary,pots with drainage holes and they may be closely related to the ancestral stock that gave rise to the Eurasian forms.

The rest of the faunal components include a frog, a percid fish, Sander teneri, of Eurasian origin, and ten mammal taxa which share considerable similarity to equivalent-aged faunal assemblages in East Asia, including a neomyine shrew Arctisorex polaris, a microtine-like cricetid similar to Microtodon or Promimomys, a large wolverine , a fisher -like carnivore, a marten-like carnivore Martes cf. M. americana, a weasel Mustela sp., a meline badger Arctomeles sotnikovae, a three-toed horse Plesiohipparion, a possible cervoid Boreameryx braskerudi of unknown origin, plus an ursine bear “Ursus abstrusus” described herein. Te third author has also identified a duck closest to the Greater Scaup .Skeletal remains of the fossil bear were collected in different years from the Beaver Pond site . Te skull specimen, with upper teeth appears to be a young adult . Te exoccipital-basioccipital, exoccipital-supraoccipital, and premaxilla-maxilla sutures are largely fused, whereas the internasal and interfrontal elements are unfused. In the modern black bear this degree of fusion of the cranial elements suggests the individual is between five and seven years old. Te upper teeth, particularly the premolar and molar cheek teeth, are essentially pristine and show wear only on the tip of the upper right canine, incisors, and anterior edge of M1, which also suggest a relatively young individual . In contrast, there is extensive wear on the lower teeth , indicating the mandibles are from an individual much older than that of the cranium . Te symphysial sutures of the leftt and right dentaries occlude perfectly, and the wear patterns on the lower teeth on either side are comparable, indicating a single individual for the lower jaws. There are thus a minimum of two individuals. Judging by the lack of fusion between epiphysis and diaphysis, the postcranial elements may belong to the younger individual represented by the skull .A phylogenetic analysis was conducted using 24 taxa and 59 morphological characters and all seven living ursines . A new single shortest tree was found by New Technology search in TnT with a tree length of 145, consistency index of 0.51, and retention index of 0.70 .

The topology of modern taxa was constrained using nuclear DNA evidence of Kutschera et al. and the whole genome analysis of Kumar et al. . Assuming the molecular relationship is correct, six extra morphological steps are required to account for this new relationship. Protarctos abstrusus appears basal to all modern bears, including Tremarctos, the spectacle bear of South America. Moreover, its phylogenetic position suggests aEurasian origin for this lineage. Asia appears to be of vital importance in the early diversifcation of ursines: Not only is Asia home to all basal ursines still alive today but the most advanced stem form, Ursavus tedfordi, leading to the ursines is also found in east Asia, as well as early ursines such as Ursus yinanensis .Judging from dental wear, the partial skull and mandibles from the Beaver Pond site represent two individuals of Protarctos abstrusus. Both show evidence of dental caries, particularly on teeth that have sustained the most wear; a pit usually develops on the exposed dentine surface . We used microCT scanning to investigate features of the left upper second left molar , and the right side lower first and second molars of the mandibular specimen, CMN 52078-A. The M2 has deep occlusal and proximal surface lesions. Scans show that both lesions are characterized by a thin zone of demineralization at the cavity boundary and deeper sclerosis of the dentinal tubules. There is also evidence of mild formation of reparative dentin formation in the adjacent pulp associated with each lesion. The lower carnassial, m1, revealed five structures of interest . Feature m1.1 is a fragment of dentin that is slightly elevated from a worn surface because of cracks in the desiccated dentin. Feature m1.2 represents a series of carious lesions extending apically to the worn surface . Tree small pit-like lesions and one large lesion are identified. MicroCT scans reveal that lesions undercut the worn surface and show slight demineralization of their margins.

Demineralization of dentinal tubules is a reaction to actively spreading caries while dentinal sclerosis and formation of reparative dentin are evidence of protective responses. Feature m1.3 demonstrates subsurface demineralization extending about 0.1mm from the margins of the cavity. Features m1.4 and m1.5 are early carious lesions that clearly extend below the worn surface. Seven features of interest were identified in the occlusal surface of m2 . Five early carious lesions are identified under the scale bars of m2.1, m2.3, m2.4, m2.5 and m2.6. Feature m2.2 shows demineralization of the pulpal surface of the lesion. There is also evidence of demineralization of the dentinal tracts between the depth of the lesion and the pulp as well as a mildly sclerotic peripheral zone. Further, it is most likely that some reparative dentin has formed in the region of the pulp adjacent to the demineralized dentinal tracts. Feature 2.7 also shows slight demineralization of the lesion surface as well as slight demineralization of dentinal tracks just pulpal to the lesion as well as deeper sclerotic changes. For comparative purposes, we assessed the prevalence of caries in modern American black bear populations using museum collections, as well as published data from museum collections and a living population. Dental caries in extant black bears are seen in both museum specimens and in vivo bears , in contrast to their general absence in other carnivores. Moreover, examination of northern boreal forest black bears from Canadian Museum of Nature collections revealed prevalence of caries increasing with age .Analysis of new fossil material of Protarctos abstrusus from the North America High Arctic shows that, although ecomorphologically similar to the modern North American black bear , P. abstrusus represents a basal ursine. The most prominent cranial features of P. abstrusus are its relatively short rostrum,drainage pot fat forehead above the orbit, and high sagittal crest that extends posteriorly and overhangs the occipital condyles , characters that generally signal primitive status within Ursinae. P. abstrusus appears to have been an isolated immigration event from Eurasia to North America, separate from Ursus, representing a time of Asian-North American high latitude foral and faunal interchange, when the high-latitude forests of Asia and North America were connected across the Beringian isthmus.Te American black bear, by contrast, appears in the North America fossil record in the Early Pleistocene as a result of an independent dispersal event from Eurasia. Fossil records of true American black bear, Ursus americanus Pallas, range from Irvingtonian to late Rancholabrean. From the Irvingtonian age, Brown described abundant materials from the Conard Fissure, Arkansas, which he referred to U. americanus. Gidley named Ursus vitabilis from the Cumberland Cave, Maryland, which was later referred to Euarctos vitabilis. By late Rancholabrean black bears appear widespread throughout North America. Several species or subspecies of late Pleistocene black bears have been named, which were sometimes confused with brown bears because of overlap in size and pronounced sexual dimorphism, such as Ursus optimus from late Pleistocene McKittrick brea deposits of southern California, which was determined by Graham to be a brown bear. Grahamconcluded that only one species, Ursus americanus, is valid throughout the Pleistocene with late Pleistocene fossil forms being larger than their living descendants, suggesting continuity of the black bear linage in North America, as was also pointed out earlier by Kurtén. 

Within Ursinae, P. abstrusus represents a stage of dental evolution that is intermediate in its specialization for ingesting plants, and significantly less than modern bears – polar bears being an exception, showing evolutionary reversal toward increased carnivory. Te evolutionary history of ursines is generally characterized by a shift in dental specialization from carnivory to increased omnivory, with the posteriormost molars of more recent forms being more elongate, and wrinkled, allowing for more crushing surface . Although, morphologically, P. abstrusus is less specialized than modern bears, the presence of dental caries suggests the diet of this 3.5 million-year-old transitional form already included a significant carbohydrate component. Dental evidence from the beaver pond site P. abstrusus appears to be from two individuals, including an apparent young adult, and both show dental caries, suggesting their diets included high amounts of fermentable carbohydrates early in their lives. Simple sugars, such as glucose and fructose, are readily metabolized by many bacteria found in the oral bio-film into various acids. These acids demineralize enamel and dentin and may lead to dental caries. Cariogenicity is highly correlated to the amount and frequency of sugar intakes. Te type of sugar consumed and associated dental caries are also found to differ. Despite their high sugar content, raw fruits by themselves are not always implicated for cariogenicity, although high frequency may induce caries. In humans, there is convincing evidence that free-sugar consumption of more than four times a day or more than 6–10% energy intake will increase incidents of dental caries. Historically in humans, increase in prevalence of dental caries has generally been associated with dietary shifts, linked with a reduction of nomadic lifestyles, the development of agriculture in Neolithic populations, and even more so with industrialization. In bears, carbohydrate intake may account for the appearance of dental caries , and may also be related to sedentary behavior, particularly for northern bears which hibernate. Northern black bears hibernate five to seven months, and survive better if they have high fat reserves. In bears, the optimal diet for production of fat reserves appears to be one of high-energy carbohydrates and low in protein. High-latitude berries often have a wide, circumpolar distribution and can be found in a variety of northern habitats including forest, woodland, wetland and tundra habitats. Black bears and grizzly bears in boreal forest eat berry fruits in the autumn, but some fruits, such as cranberry and bearberry, frequently remain on the vine over winter and are important to bears coming out of hibernation in the early spring. Bearberry fruits are relished and highly important to black bear in Pelly River Valley of Yukon Territory. Berries are found in nearly 80% of bear scats collected during the fall period and consistently represent a large component of black bear diet in Alaska, with blueberries being the most common. However, fruit intake may be mitigated by factors such as fruit abundance and body size. For example, larger bodied bears appear to tend toward carnivory, as they are less efficient than smaller bears at exploiting small fruits. These factors may underlie the high variation observed in caries prevalence seen among populations of modern black bears . Floral macrofossils from the Beaver Pond shows a diversity of berries would have been available to U. abstrusus, including Empetrum nigrum , Vaccinium sp. , Rubus idaeus , and their abundance may have been enhanced following forest fires, which is evident at this site. Therefore berries may have constituted a component of the Beaver Pond bear’s diet, particularly during the peak seasons, and their high sugar and acid contents could have resulted in the observed pronounced dental caries.

When humans lose weight, either in outer space or on Earth, this weight is in the form of both lean body mass and fat mass . In non-astronaut populations, the goal of weight loss is usually to lose more fat mass than lean body mass. Indeed, when healthy, ambulatory humans lose weight on Earth, most of this weight is in the form of fat mass, with a lesser percentage of the lost weight coming from lean body mass . However, when humans are not ambulatory, as is the case with astronauts during transport, a larger percentage of the lost weight can come from lean body mass. In one crossover study – a type of study in which the same participants are exposed to all conditions – Biolo and colleagues tested the consequences of a weight-loss diet versus a weight-maintenance diet during either ambulatory or non-ambulatory conditions. When participants were on the weight-loss diet and ambulatory, most of their weight loss was in the form of lost fat mass rather than lean body mass . However, when participants were on the weight-loss diet and bed rest, their weight loss was in the form of similar amounts of lost fat mass and lean body mass . In other words, weight loss coupled with inactivity leads to greater loss of lean body mass. The loss of lean body mass is already a phenomenon that occurs in human and nonhuman animals living in micro-gravity . Therefore, weight loss could exacerbate this loss of lean body mass in astronauts, which could lead to the loss of muscle volume and the loss of bone mineral content . These outcomes could lead to increased risk for soft tissue injury , bone fracture, and decreased stamina , which could increase astronauts’ risk of being injured during extravehicular activities and of having difficulty readjusting to gravity on Earth or other planets. In summary,vertical agriculture when astronauts are in a micro-gravity environment, and especially when they are physically inactive, such as they will be in the Crew Transportation Vehicle, weight loss could result in the loss of lean body mass.

Consequently, assessing the impact of space food on weight loss will be important. Sleep difficulties are also common in astronauts. Astronauts sleep significantly less during ISS missions, as compared to after, and 75% of ISS crew members report using medications to promote sleep . The most frequently used sleep medication during spaceflight is zolpidem , a medication with side effects that can include difficulty with balance, unusual dreams, headache, gastrointestinal problems, and feeling “drugged” . Despite the common use of this and other sleep-promoting medications among astronauts, and despite a NASA mandate that astronauts have 8.5 hours for sleep per night, ISS astronauts average only 6.09 hours of sleep a night . Sleeping 6 to 6.9 hours a night was shown in one epidemiological study of over 175,000 people to significantly increase the risk for work-related injury . Sleep deprivation has also been linked to psychological impairments such as negatively biased mood, difficulty using one’s emotions to make decisions, impaired frustration tolerance, and reduced attention, vigilance, and memory . Even when alertness is restored with stimulants such as caffeine, many of these impairments remain . Astronauts have access to coffee, caffeine pills, and stimulant pills, such as modafinil . Yet their journals still report numerous instances of sleep deprivation leading to impaired functioning, such as “I fell asleep while typing” and “The fatigue was evident when a couple of minor mistakes were made today on some payload activities . . . it is an obvious indicator of fatigue” . Astronauts’ sleep deprivation could pose a serious threat to both themselves and to the success of space missions . Factors that can exacerbate sleep difficulties should therefore be avoided . Food is one such factor that could exacerbate sleep difficulties. In a nationally representative study of 5,587 U.S. adults, eating a reduced-variety diet, consuming less salt, and consuming a low number of calories was associated with self-reports of sleeping less than five hours a night, as compared to self-reports of sleeping 7 to 8 hours a night . The researchers also found significant associations between sleep duration and the intake of numerous nutrients, including intake of carbohydrates, proteins, vitamins, and minerals . In summary, aspects of food intake have been associated with impaired sleep, and because astronauts already have sleep difficulties, it is important to evaluate how new space food diets impact sleep.

Unlike sleep difficulties and weight loss, poor self-reported health is not often emphasized by NASA as a potential health risk. However, asking participants to rate their own health as excellent, very good, good, fair, or poor is well recognized in health fields as a robust, reliable, and valid way to assess overall health status . Self-reported health, also called self-rated health or perceived health, predicts functional ability and mortality . Self-reported health predicts mortality even after controlling for numerous potential confounds such as age, sex, income, education, and health practices . Asking participants to rate their own health is a robust predictor of health outcomes, even when the wording of the question varies . Self-reported health is usually used as an indicator of health over long time periods, but researchers have found that participants’ ratings can also change over relatively short periods of time, indicating that self-reported health could be used as a marker of short-term changes in health. For instance, when 9,235 participants in the 2005-2008 National Health and Nutrition Examination Survey were surveyed at two time points one month apart, nearly 40% of participants changed their response to the question, “In general, would you say your health is excellent, very good, good, fair, or poor?” . Additionally, when a variant of the self-reported health question, “Overall, how satisfied were you with your health today?” was used in a 56-day survey study, researchers found significant day-to-day, within person variability . Greater day-to-day health satisfaction was also associated with fewer health events . Consequently, although self reported health is traditionally used as an indicator of health over long time periods, it can likely also be used to assess variation in self-reported health over days or weeks. Self-reported health could therefore be used to assess how new space food diets impact health over time.To ensure participants were representative of future commercial astronauts, all participants had to express an interest in traveling to outer space. Participants also had to be willing and able to eat breakfast, lunch, and dinner at work Monday through Thursday for two weeks. Additionally, participants had to be willing and able to eat only the food provided during the experimental condition and to eat from a restricted number of food venues at the workplace during the control condition. These eligibility requirements were anticipated to increase study adherence. Participants also had to report no endocrine or metabolic disorder, as eating the processed, ready-to-eat food could have exacerbated these disorders. Finally, participants had to report no current dieting and no history of an eating disorder, as these factors could have adversely impacted participants’ eating or rating of the foods. UCLA Institutional Review Board approval was obtained prior to conducting study procedures.

A parallel crossover design was used over the course of two weeks. The parallel crossover design was selected because this type of design achieves greater statistical power and precision with fewer participants, as compared to a parallel design , and recruiting participants from the pool of commercial aerospace employees was anticipated to be difficult. For the parallel crossover design to be most effective, a “washout” period between conditions is required . Therefore, this study involved food manipulation Monday through Thursday with a three-day “washout” period Friday through Sunday. During this washout period, participants ate as normal without recording their food intake. During week 1, half the participants were randomly assigned to the experimental condition, while the other half were in the control condition. During week 2, this arrangement flipped,vertical farming aeroponics with the participants who were previously in the experimental condition switching to the control condition, and vice versa. Both the experimental and control conditions lasted 4 days in Week 1 and 4 days in Week 2 . All participants completed the study during the same two-week period. The procedure is shown in Table 3. Each week of the study required approximately 107 minutes of the participant’s time. Participants also completed a brief qualitative assessment at the very end of the study, which took approximately 10 minutes. This brought a participant’s total time commitment to 224 minutes, or approximately 3.75 hours, for the entire study.Regardless of the condition to which they were assigned, participants met me in a workplace conference room between 8:00am and 10:00am on Monday morning. At that time, they received their condition instructions , along with a bullet-point list of foods they could and could not week eat that week. All participants were reminded they would be weighed right then and on Friday morning, before eating. They were also reminded they would receive an email with a link to an online survey at 5:00pm on Monday, Tuesday, Wednesday, and Thursday of both weeks. Finally, all participants were told they would receive text message reminders to complete the survey at 6:00pm and 7:00pm each night, and they were expected to complete the survey before 8:00pm. Participants were asked to complete the survey on a computer, rather than on a smartphone, so that all questions would display in correct formatting. The time frame of 5:00 to 8:00pm was chosen for the evening survey because participants often remained at work until 7:00pm or later, and I expected participants would be more likely to complete the survey if they completed it before leaving work. However, data were not discarded for being submitted after 8:00pm.When participants met me on Monday morning, they received their tote of experimental condition food. The tote included four gallon-sized plastic bags, with each bag containing one day’s worth of food. The bags were labeled Day 1, Day 2, Day 3, and Day 4. Each gallon-sized bag contained four smaller plastic bags in which the food was actually stored. These bags were labeled Breakfast, Lunch, Dinner, and Snack. Participants were also provided with a binder that showed the breakfast, lunch, dinner, and snack options that were provided for each day. Participants were informed that the meal categories were recommendations only and that they could eat anything from the tote at any time. Because the food was designed to be lightweight and portable, participants were able to carry the tote with them everywhere they went. All totes contained the same exact food . This food was designed to provide sufficient calories for all participants. All totes also included eight empty two-gallon-sized plastic zip bags for storing leftover food. When participants met me on Monday morning, they received vouchers for four days of meals at work, the control condition instructions , and four Food Rating Logs . The control condition instructions outlined that, for the next four days, they would be allowed to eat from the workplace food outlets that served restaurant-style, pre-determined portions . This provided participants with approximately 12 meal options at any time of the day. Participants were not allowed to eat from the cafeteria-style line at work, nor were they allowed to eat from the graband-go venue, with the exception of the yogurt, milk, and cereal that was served at that venue. Participants were also not allowed to eat from the frozen yogurt stand or from the specialty coffee stand. Restricting participants from these foods had the benefit of allowing me to more accurately estimate their nutritional intake and to provide the experience of seeing food yet being unable to eat it. This experience, of seeing food and being unable to eat it, will not occur in outer space. Therefore, providing this experience in both the experimental and control conditions provided feelings of deprivation in both conditions, thereby effectively eliminating this deprivation from being a cause of different responses to the two conditions. Prior to starting the study, participants completed a Work-Specific Food Survey . This measure was used to screen participants and to provide descriptive information on the sample. This measure was not used as a moderator in any analyses. On Friday, participants also completed the Ten-Item Personality Inventory . This measure was also used to provide descriptive information on the sample and was not used as a moderator in any analyses.

In a laboratory setting, chlorantraniliprole was harmless to Orius laevigatusfor residues aged 1-14 d, in contrast to abamectin, which resulted in nearly 100% mortality up to 14 d . In a field setting, the half-life of chlorantraniliprole on rice straw was 3.5 d , and in sugarcane, was 8 d at both 1x and 2x the recommended field rate . Hence, the relatively short half-life of chlorantraniliprole compared to that of lambdacyhalothrin, further suggests that it had minimal effect on the acute mortality of H. convergens mortality in our study, which was still substantial by day 8 . The persistence of the toxicity effects of pyrethroid residues in the field appears to vary considerably among natural enemy species and active ingredients. For example, field-aged residues of esfenvalerate remained highly toxic to adult Trichogramma platneri Nagarkatti after 21 days, but were harmless after 7 days for Colpoclypeus florus. In addition, residues of permethrin and esfenvalerate on almond twigs were sufficient to cause 50% acute mortality of G. occidentalis even after 7 months of aging in the field . Nonetheless, pyrethroids are subject to photodegradation under field conditions. When exposed to UV light, lambda-cyhalothrin can degrade to 5% of the initial amount applied after 20 min . Similarly, when applied to a rice paddy, the half-lives of both components of the combination insecticide were determined to be 5 days . Thus rates of degradation of lambda-cyhalothrin under field conditions may vary with formulation, UV exposure within the canopy of the crop, and with residue substrate. Despite the potential for degradation of lambda-cyhalothrin under field conditions, the high level of sensitivity of the voltage-gated sodium channels of insects to disruption by pyrethroids resulted in the persistence of acute effects on H. convergens larvae in a walnut orchard after 38 days. From this study we have been able to show that acute mortality response of H. convergens to the insecticide combination of chlorantraniliprole and lambda-cyhalothrin was affected by residue age, setting, and life stage.

Adults and larvae both experienced high levels mortality when exposed to fresh residues of the combination insecticide. Chlorantraniliprole is frequently not toxic, whereas lambda-cyhalothrin is highly toxic,low round pots to a number of natural enemy species, and therefore the latter active ingredient was likely responsible for the high mortality of H. convergens. Larvae were more susceptible to the insecticide residues than adults, and aging had a greater effect for field-aged residues that were exposed to UV from natural daylight than for laboratory-aged residues that were exposed to fluorescent lighting. Thus, traditional laboratory bio-assays can yield greater estimates of the persistence of acute mortality responses among natural enemies than bio-assays based on field-aged residues. However, the acute mortality responses of H. convergens to fresh residues of the combination insecticide were similar for insects exposed to field-aged or laboratory-aged residues. This indicates that traditional laboratory bio-assays remain a useful tool for assessing acute toxicity for fresh residues, while bio-assays with field-aged residues are needed to accurately assess the persistence of toxic effects on natural enemy mortality. Integration of chemical signals at the peripheral sensory system remains one of the least understood mechanisms of insect olfaction, particularly in mosquitoes. Despite the great progress made in the last 2 decades in understanding how receptors form the basis of chemosensory perception in insects, how olfactory signals integrate at the periphery remains an enigma . ‘‘It is as if a new continent has been discovered but only the coastline has been mapped’’ . In the largest majority of reported cases , antennal neurons of Cx. quinquefasciatus displayed excitatory responses , but evidence for inhibitory responses , already known for Ae. aegypti , is now emerging for Cx. quinquefasciatus . It has been observed in moths , beetles , the fruit fly , and mosquitoes that activation of one neuron interferes with signaling of other olfactory receptor neurons . It has also been reported that a single compound caused reduction of nerve impulse followed by a transient post-stimulus excitation . Although Carlson and collaborators elegantly demonstrated that in the fruit fly lateral inhibition is most likely mediated by ephaptic coupling , the complete ensemble of the molecular mechanism of inhibition at the peripheral olfactory system of mosquitoes remains terra incognita.

A simple explanation of the ephaptic coupling is that, upon stimulation of an ORN, the potential declines. Consequently, per channel current generated by a cocompart mentalized neuron is reduced . This scenario argues that the firing of a neuron causes reduced spike frequency by a colocated neuron because of the close apposition of their neuronal processes. Although ephaptic coupling could explain lateral inhibition, other mechanisms of intraneuron inhibition may exist. While de-orphanizing odorant receptors expressed predominantly in Cx. quinquefasciatus female antennae, we serendipitously recorded currents from an OR that generate inhibition in response to certain odorants. Further studies unraveled a hitherto unknown mechanism of peripheral, intrareceptor inhibition in mosquito olfaction.To test whether the inhibitory responses were manifested in vivo at the periphery of the olfactory system, we generated transgenic flies, with CquiOR32 expression driven by DmelOrco promoter, and recorded electroantennogram responses by using a standard method . Not surprisingly, control flies gave strong response to -2-hexenal and weak response to methyl salicylate , whereas Orco-Gal4/UAS-CqOR32 flies gave robust response to methyl salicylate, with the strong response to -2-hexenal unchanged . w1118 flies gave very weak responses to eucalyptol at high doses, but interestingly Orco-Gal4/UAS-CqOR32 flies generated dose-dependent, inverse EAG responses . To further scrutinize this unusual reverse EAG responses, we used gas chromatography with electroantennographic detection . In GC-EAD analyses, injected mixtures are separated by GC and subjected to antennal preparations under the same condition thus ruling out any possibility of mechanical interference and minor sample contamination. Here, methyl salicylate responded with regular EAG responses, i.e., with the first phase , which is referred to as rise of the receptor potential, and the second phase starting at the end of the stimulus, commonly referred to as the decline of the receptor potential . This is analogous to the depolarization, repolarization, and hyperpolarization of a nervous impulse. As opposed to methyl salicylate, eucalyptol consistently gave inverse EAD responses thus corroborating what we observed in EAG analyses . Next, we recorded EAG responses when flies were challenged with odorants and an inhibitor. First, we compared the response of w1118 and Orco-Gal4/UAS-CqOR32 flies to -2-hexenal when it was delivered alone or in combination with eucalyptol. EAG responses from w1118 flies to 0.1% -2-hexenal alone or in combination with 10% eucalyptol did not differ significantly . By contrast, EAG responses from Orco-Gal4/UAS-CqOR32 flies to 0.1% -2-hexenal plus 10% eucalyptol were significantly lower than those elicited by 0.1% -2-hexenal alone . We then examined the dose-dependent effect of this inhibition by using Orco-Gal4/UAS-CqOR32 flies. Robust responses to 0.1% methyl salicylate were reduced in a dose-dependent manner with the addition of eucalyptol but remained unchanged at the end of the tests. Likewise, EAG responses to 0.01% -2-hexenal were reduced when coapplied with eucalyptol . Of note, -2-hexenal does not activate CquiOR32 .

Such inhibition presumably results from CquiOR32 indirectly inhibiting responses of the fly endogenous receptors to -2-hexenal. In these continuous experiments, a small difference between EAG responses before and after costimulus tests may be due to loss of this volatile semiochemical from the cartridge rather than adaptation. Taken together, these results further suggest that intrareceptor inhibition occurs in vivo as indicated by the inhibitory effect of eucalyptol on methyl salicylate responses. Additionally, the effect of eucalyptol on the response to -2-hexenal suggests that intraneuronal inhibition occurred. A few lines of evidence support this hypothesis. First and foremost,plastic pots 30 liters eucalyptol does not cause inhibition in control flies and -2-hexenal does not activate CquiOR32 . The simplest explanation is that, in Orco-Gal4/UAS-CqOR32 flies, all endogenous receptors are coexpressed with CquiOR32. Thus, CquiOR32 response to eucalyptol interferes with the response of DmelOR7a to -2-hexenal. In short, inhibitor and agonist are likely to be acting on different receptors in the same neurons, thus an intraneuron inhibition. To further test the notion of intraneuronal inhibition, we turned to single sensillum recordings .The best ligand for ab4A, the neuron in ab4 sensilla with a large spike amplitude, is -2-hexenal , although ab4A is also very sensitive to other ligands, including hexanal . Contrary to ab4B, ab4A houses only one OR, namely, DmelOr7a . Because expression of CquiOR32 was driven by DmelOrco, ab4A neurons in our transgenic flies house both DmelOr7a and CquiOR32. Coexpression was confirmed by a significantly stronger response to methyl salicylate recorded from Orco-Gal4/UAS-CquiOR32 than from WT flies , while retaining response to hexanal . It is known that methyl salicylate is the best ligand for DmelOr10a in ab1D but elicits only very low response in ab4A . The low response of WT flies to methyl salicylate did not differ significantly when the odorant was delivered alone or codelivered with eucalyptol . By contrast, responses recorded from Orco-Gal4/UAS-CquiOR32 flies were significantly lower when the two stimuli were delivered simultaneously from two different cartridges . Next, we tested whether CquiOR32 response to eucalyptol would affect DmelOR7a response to a cognate ligand, hexanal. Responses of WT flies to hexanal did not differ significantly when comparing hexanal alone with hexanal plus eucalyptol . Recordings from ab4 sensilla in the Orco-Gal4/UAS-CquiOR32 flies showed a slight, albeit not significant, increase in response to hexanal. This is unlikely to be due to hexanal activation of CquiOR32 . When hexanal and eucalyptol were delivered simultaneously firing of DmelOR7a was completely abolished . We also recorded from ab7 sensilla, which expresses DmelOR98a, in ab7A and for which butyl acetate is one of the best ligands . Eucalyptol elicited inhibitory response in ab7A neurons of Orco-Gal4/UASCquiOR32 flies . In the transgenic flies both methyl salicylate and butyl acetate generated excitatory responses , which were inhibited by eucalyptol . Because methyl salicylate and eucalyptol elicit inward and reverse currents in CquiOR32, this in vivo inhibition is not surprising. However, the consistent observation that eucalyptol inhibits the response of an endogenous receptor to a cognate ligand supports the notion that intraneuronal inhibition occurs when receptors are colocated in a neuron. Specifically, the inhibitory responses of CquiOR32 interferes with the activation of a collocated receptor by a cognate ligand.

For example, activation of DmelOR7a in ab4A neuron by hexanal and activation of DmelOR98a in ab7A neuron by butyl acetate were both inhibited by eucalyptol upon interaction with CquiOR32. Contrary to the fruit fly, which expresses only one receptor per neuron , mosquitoes can coexpress multiple ORs in the same neuron .Tissue fibrosis and cancer are two major causes of high human morbidity and mortality worldwide. Although there are multiple therapies for cancer, including chemotherapy, oncologic surgery, and radiation therapy, an effective therapeutic strategy is needed.1 Among these therapeutic strategies, chemotherapy is the main tool for curing various cancers. The therapeutic resistance of anticancer drugs, such as 5‐fluorouracil, gemcitabine, gefitinib, and trastuzumab, has been widely observed in the clinic.2 However, due to the lack of effective therapeutic drugs, tissue fibrosis still threatens human health. Although some drugs exhibit anti-fibrotic effects, including angiotensin‐ converting enzyme inhibitors, aldosterone inhibitors, statins, angiotensin II type 1 receptor blockers, endothelin receptors, β‐blockers, acetylsalicylic acid, and matrix metalloproteinase inhibitors, none of them are specifically designed to target fibrosis, and the related side effects limit their clinical use for treating fibrosis.Thus, anti-fibrotic and anticancer treatments are extremely urgent, and new therapeutic drugs should be designed based on specific targets that contribute to the progression of fibrosis and tumors. Epithelial‐mesenchymal transition is a reversible terminal differentiation process in which epithelial cells shed their properties and acquire a more mesenchymal phenotype.EMT is a fundamental process widely involved in the development and the progression of various diseases, and there are mainly three types of EMT. Type I EMT is involved in embryonic development and organ formation. Type II EMT is critical for wound healing and fibrosis. Type III EMT contributes to the progression and metastasis of tumors.Extensive studies revealed that EMT profoundly contributed to the production of myofibroblasts, which are the major cells producing massive amounts of collagen that leads to the deposition of collagen in the development of fibrosisIn addition, EMT confers increased motility and invasiveness in epithelial‐derived tumor cells and promotes tumor metastasis. Therefore, fibrosis and tumors share the common process of EMT, and drugs that specifically target EMT may exhibit both anti-fibrosis and antitumor effects, which will provide an effective strategy against fibrosis and tumors.

Copper hydroxide is the active ingredient in the pesticide marketed under the trade name Kocide 3000, and mancozeb is the active ingredient in the pesticide Manzate Pro-Stick. They are used to control both bacterial and fungal pathogens in numerous crops, including citrus, conifers, vegetables, tree crops, cereals, and small fruits. In high-value tree crops, they are used in combination to control pathogens such as apple scab, pear scab, fire blight, and powdery mildew . A mixture of copper hydroxide and mancozeb caused significantly high mortality in the predatory mite G. occidentalis, but at a concentration that was twice the maximum label rate. The same study found that, even at a diluted concentration, , exposing females to the pesticide mixture did not affect hatch rates, but, once hatched, the larvae had a higher mortality . On the other hand, a different predatory mite, Amblyseius cucumeris , appears to be compatible with copper hydroxide, in that exposure only causes high mortality at levels 10 times the recommended field rate. Copper hydroxide also had no significant effect on mortality or fecundity on the predatory mite Euseius victoriensis , whereas mancozeb significantly lowered mortality as well as fecundity, with no females laying eggs after treatment . With respect to parasitoids, copper hydroxide has shown little to no toxicity on the predator of citrus leafminer, Ageniaspis citricolaor Tamarixia radiata , a parasitoid of the Asian citrus psyllid . Mancozeb applications, suspected as the cause of low parasitism rates in Indonesian crops,hydroponic bucket demonstrated no toxicity to four different parasitoid species: Hemiptarsenus varicornis , Opius sp., Gronotoma micromorpha, and Diglyphus isaea.

These results suggest that the effects of copper hydroxide and mancozeb vary for different species, and their compatibility with natural enemies cannot be generalized. Lambda-cyhalothrin is a broad spectrum pyrethroid. One of its trade names is Warrior II, approved for use in a wide variety of crops, such as alfalfa, cereal crops, vegetables, tree nuts, tree fruits, cotton, and tobacco. In high-value tree crops, it is used to control many different pests: apple aphid, walnut aphid, codling moth, pear psylla, leafrollers, leafhoppers, and ants. This active ingredient is highly toxic to bees via direct application or residues on vegetation, and should not be applied when either crops or weeds are in bloom. Lambda-cyhalothrin is also highly toxic to aquatic organisms and wildlife, and areas subject to pesticide drift or runoff are considered hazardous . Lambda cyhalothrin has also demonstrated toxic effects on several natural enemies. The predatory mite, G. occidentalis, experienced 96% mortality at the full field rate. Even at 0.1x the full field rate, it was highly toxic to G. occidentalis, causing 76% mortality . In order to test for resistance, one study topically exposed 28 different populations of ladybird beetles to lambda cyhalothrin. However, the majority of populations tested displayed an LD50 below the maximum recommended field rate, including all populations of Coleomegilla maculata, Cycloneda sanguinea, Harmonia axyridis, and 6 of 8 H. convergens populations . Lamba-cyhalothrin is also toxic to both the larvae and adults of two green lacewing species, C. carnea and C. johnsoni . Additionally, it is highly toxic to the parasitoid, Diadegma insulare , which parasitizes diamondback moth, a major pest of brassica crops. Moreover, D. insulare can distinguish between host larvae fed on lambda-cyhalothrin treated plants versus non-treated plants, indicating a change in host preference .

Novaluron is an insect growth regulator, marketed under the trade name Rimon. It is approved for use in crops such as brassica, berries, potatoes, pome fruit, and stone fruit. In pear and apple orchards, it is used to control pear psylla, leafrollers, and codling moth. Its mode of action is to disrupt cuticle formation and deposition during molting. Although novaluron is not a conventional insecticide, it still may have non-target effects on beneficial insects, such as pollinators. For instance, exposure to novaluron may interfere with the development of honey bee larvae. Therefore, its application may not be compatible with beneficial insects when crops are in bloom. In addition, it has demonstrated toxicity to fish and aquatic organisms . Novaluron appears to have variable toxicity on several natural enemies. It demonstrated a low toxicity to O. insidiosus and Chrysoperla rufilabris , both of which are natural enemies in blueberry production . However, when exposed to novaluron residue aged 7 days, nearly 50% of H. convergens displayed acute effects, meaning they were either killed or knocked down. For G. occidentalis, an important predatory mite in apple orchards, novaluron is moderately toxic. Though it did not significantly reduce fertility, it caused reductions in fecundity as well as fertility . It has also demonstrated toxicity to green lacewing larvae, none of which survive to the adult life stage after treatment . Moreover, though it did not impact fecundity, it significantly reduced fertility and egg viability. Podisus maculiventris , a predator of the potato beetle, is susceptible through both direct contact and residual exposure . Additionally, when fed treated prey, 5th instar larvae were unable molt in to adults. Despite its impacts on survivor ship and reproduction, novaluron is still seen as a better alternative to pesticides with greater acute toxicity. For instance, when compared to brassica crops treated with broad spectrum pesticides , novaluron treated crops had higher yields and higher rates of parasitism in the pestiferous diamondback moth .

Spinetoram is chemical derived from the soil bacterium Saccharopolyspora spinosa. It is used as the active ingredient in an insecticide, marketed under the trade name Delegate, which is used to control foliage damaging pests such as leafminers, psyllids, and lepidopterous larvae. It is approved for use in a number of crops, including bananas and plantains, bushberries, hops, citrus, pome fruits, tree fruits, and tree nuts. In pome fruits, it is used to suppress populations of apple maggot, pear psylla, thrips, light brown apple moth and codling moth. In addition to targeting pests, spinetoram is toxic to bees exposed to treated crops, either though foraging on pollen-shedding or nectar producing plant parts. Delegate is recommended for use in integrated pest management programs and is advertised as being compatible with natural enemies, such as ladybird beetles, lacewings, assassin bugs, and spiders . Spinetoram has been demonstrated to be highly toxic to natural enemies and pollinators. For instance, T. triozae is a key parasitoid of the tomato psyllid, Bactericera cockerelli . T. triozae experiences 100% percent mortality when exposed to spinetoram via surface residue,stackable planters even when residue is aged as much as 15 days . Moreover, ingestion of spinetoram resulted in 100% mortality within a period of 12 hours. Additionally, spinetoram is highly toxic to eggs and larvae of the predatory mite G. occidentalis. It also caused 100% mortality in adult females after 72 h of exposure . Spinetoram is also toxic to pollinators via ingestion of treated sugar water, and it may cause mortality through exposure to either dry or wet residue. It is highly toxic to worker bees, and induces 100% mortality in bumble bees when applied at recommended field rates . Moreover, Neochrysocharis formosaand Ganaspidium nigrimanushave demonstrated susceptibility to spinetoram . These natural enemies are important parasitoids of Liriomyza trifolii , a major pest of vegetable and ornamental crops worldwide. Furthermore, spinetoram consistently lowers survival of both parasitoid species when exposed by direct application, residue, or ingestion. Sulfur is commonly used as both a fungicide and acaricide. Known as dry flowable sulfur or wettable sulfur, it is a dry powder that may be mixed with water and used as a foliar spray. It is the active ingredient in the pesticide marketed under the trade name Kumulus, which is used in an extensive variety of crops: ornamentals, turf, alfalfa, brassica, citrus, berries, cotton, hay, tree fruits, peanuts. In apple and pear orchards, sulfur is used to control plant pathogens, such as powdery mildew and scab, and pests, such as the two spotted mite and the red spider mite . It is also ranked as having variable impacts on natural enemies in tree fruits, ranging from low toxicity to aphid parasitoids to high toxicity on the western predatory mite, G. occidentalis . In apple orchards, G. occidentalis is a predator of tetranychid mites, such as the two spotted spider mite and the European red mite . Sulfur residue demonstrated a low toxicity to G. occidentalis adult females, and it had no effect on prey consumption. However, sulfur may have variable toxicity depending on the life stage of natural enemies, because the same study found that sulfur residue was highly toxic to larvae . It has also been shown to affect populations of another predatory mite, Iphiseiodes zuluagai , which preys on the coffee pest Oligonychus ilicis . The recommended field concentration caused approximately 60% mortality of I. zuluagai and decreased reproduction in adult females . Psyllobora vigintimaculatais a mycophagous coccinellid that may reduce the severity of the plant pathogen powdery mildew fungi. Additionally, sulfur was toxic when applied directly to P. vigintimaculata, resulting in 100% mortality within 24 h of exposure . Furthermore, when applied in commercial vineyards, sulfur reduced the density of P. vigintimaculata. Sulfur has also shown to affect multiple life stages of the predatory mired bug, D. brevis, causing acute toxicity in both nymphs and adults .

Exposure also decreased survival of nymphs to the adult life stage , fertility in adult females, and egg viability. Three main routes of exposure have been addressed in the literature: topical, residual, and oral. It is well known that natural enemy susceptibility to pesticide varies depending on exposure route . In a field setting, it is possible for natural enemies to be exposed to pesticide through all three aforementioned routes: topical exposure from direct spraying, residual exposure from a treated plant surface, and/or oral exposure from ingestion of a treated food source . Therefore, to more accurately assess the effects of pesticide exposure on natural enemies, it is necessary to examine multiple routes of exposure. There are several different methods for assessing the effects of pesticides on natural enemies . One route of exposure for assessing pesticide toxicity is to directly expose organisms via topical application. A common tool for topically applying pesticide is through the use of a Potter Spray Tower, which sprays a specific volume of the pesticide solution over a specified surface area . For instance, topical application via a Potter Spray Tower was used to assess the effects of buprofezin, an insect growth regulator that inhibits chitin synthesis, on several general predators, including H. axyridis, Stethorus punctum picipes , Orius tristicolor , Geocoris pallens, and Geocoris punctipes. In addition to direct spraying, there are other methods of topical application. For instance, to measure the effects of several conventional and biorational insecticides on the eggs of the generalist predator Chrysoperla externa , eggs were directly dipped into maximum field rate solutions for 25-s . While topical applications are a factor to consider when assessing pesticide risk, in a field setting, natural enemies are more likely to encounter pesticide via a combination of routes, including residual and oral exposure.Another approach is to measure pesticide toxicity via residual exposure, whereby a substrate is treated with a given pesticide, and the organism is then exposed to the treated substrate. For example, several pesticides frequently used in peach orchards were tested for their effects on the lacewing predator C. carnea using a Potter Spray Tower to apply residues to glass plates, and the predators were then exposed to the dried residue . Additionally, a Cornelis spray chamber was used to coat the sides of glass cages, to which the predatory stinkbug Picromerus bidenswas then added . Another study used a leaf dip bio-assay, in which pesticide residue was formed by dipping green bean leaves into pesticide solution for 5-s. In this manner, reduced-risk pesticides were then tested on multiple natural enemies: O. insidiosus, Amblyseius swirskii , and Eretmocerus eremicus. While most studies are based on fresh dried residues of pesticides, an equally important, but less commonly studied aspect of residues as a route of exposure for natural enemies is their persistence in the field . Several studies have begun to examine how the age of pesticide residue impacts natural enemies .

An accumulation of insect pest near windbreaks is more evident in windy weather as compared to calm weather conditions . Higher permeability of artificial windbreaks leads to higher wind speed in the leeward side, lower sheltering effects for insect pests, and lower insect pest density near the artificial windbreaks . Furthermore, higher windbreak permeability also shifts the positions of the highest insect pest density further away from the windbreak. Instead of maximal insect pest density at one wind break height for 0% permeable windbreak, the highest density was observed at distance equivalent to 2 to 3 windbreak height for thrips , aphids , fungus gnats , and gall midges . Besides artificial windbreaks, natural hedgerows also exert similar effects on insect pest distribution . In another experiment, using trace of rubidium element to mark egg parasitoids in the genus Aragus overwintering in French prune trees, Corbett and Rosenheim showed that the peak in Aragus density did not coincide with the original points of dispersal but some distance downwind, displaying certain degree of windbreak effect. Therefore, it can be inferred from these studies that the wake zone, which, in this case, can be considered as edge environment, traps insects along edges, resulting in their higher density along edges as compared to field interior.Several studies have examined effects of the combination of natural landscape fragmentation and microclimatic conditions and how such variables affect insect communities in non-agricultural habitats . The spatial distribution of a butterfly, Lopinga achine Scopoli ,hydroponic nft system showed distinct edge-biased distribution, which was explained by specific micro-climatic conditions leading to the ground cover of Carex montana L. having the highest abundance near forest edges .

In a study of bark beetle [Ips typographus L. ] infestations, Kautz et al. investigated the risk of infestation in three types of forest edges and compared the results of trees from the interior of the same forests. They concluded that the risk of bark beetle infestation was highest in forest patches cleared by sanitary logging measures, in particular along south-facing edges. Thus, the combination of a particular edge management practice and ambient temperature was highlighted as a key driver of bark beetle infestations. Although exclusively based on analyses of data derived from natural habitats, such studies strongly suggest that edge-biased distributions are often explained by micro-climatic differences between edge and interior locations. Using different types environmental sensors, it has been demonstrated how abiotic variables, including ground surface temperature and soil temperature, show considerable with-field variation and in particularly vary between edges and inwards into fields . Such micro-climatic variation can lead to spatial variation in crop growth and relative suitability of host plants and therefore lead to edge-biased distribution of herbivorous insects in agricultural systems.Although windbreaks and hedgerows are common features in many agricultural systems, sole reliance on the shelter effect and microclimate promoted by these features does not provide encompassing account for the occurrence of edge-biased distributions, especially for agricultural systems in which they are not present. Other factors that are more intrinsic to the agricultural environment, such as the nature of landscape matrix, have been shown to affect insect rate of dispersal and emigration and result in their aggregation along edges. In a marked release and re-captured experiment on movement of delphacid plant hopper, Prokelisia crocea Van Duzee , in patches of prairie cord grass, the rate of emigration from cord grass patches bordered with brome grass matrix was higher than that from cord grass patches bordered with mudflat .

The same study also noted the accumulation of plant hoppers along cord grass-mudflat edges, but no such aggregation of plant hoppers was observed along cord grass-brome edges. It was later confirmed that the lower permeability of cord grass-mudflat edges makes this type of edges a barrier to emigration since plant hoppers behaviorally avoid crossing highly contrast border between vegetated cord grass matrix to sparsely vegetated mudflat matrix . Moreover, the cord grass-mudflat border was clearly not a reflective edge, as plant hoppers did not show edge-avoidance behavior. Meanwhile, the distribution of plant hoppers within a homogenous patch of cord grass was found to be random . Therefore, although landscape matrix permeability may explain why insects can be contained in their original patches, it does not directly explain the aggregation of insects along edges, especially for the case of delphacid plant hoppers.In cases where agricultural systems are adjacent to natural forests, the edges can be described as an “ecotone” that marks the ecological change between two environments . The adjacency of vegetation of two different habitats results in an increase in floral diversity and vegetation heterogeneity . The increase in heterogeneity of vegetation along field edges has been shown to increase carabid species richness along field edges . For example, boreal forests with sparse ground vegetation were found to have lower numbers of herbivorous invertebrate prey for carabids . The interspersion between grasses, shrubs, and boreal trees as seen in the edges between farming lands and boreal forests can effectively increase the density of ground vegetation, resulting in greater abundance of herbivorous invertebrates which, in turn, leads to higher carabid abundance along edges . Similarly, the interspersion of vegetation types between the forest edges and urban habitat in north Ohio has been attributed to the increase in ant species richness .

Therefore, the ecotone environment observed along field edges could serve as a preferential habitat for some insect species.In the discussion of edge-biased distributions in relation to the vegetation heterogeneity, there seems to be an underlying assumption that vegetation homogeneity exists within a given habitat or agricultural system. Therefore, the heterogeneity of vegetation observed in the ecotone along edges is mainly caused by the interspersion between the vegetation types of two environments, while the within-system variation of vegetation is often ignored. However, despite cropping systems predominantly being monocultures and under a quasihomogeneous management regime, there is broad evidence of significant vegetational heterogeneity within agricultural fields. That means individual crop plants within an agricultural field may have differential growth rates, phenotypes, and quality. This issue was highlighted above as microclimatic conditions may vary within fields and show distinct gradients between edges and locations further inwards into fields . Edge-biased crop development has been observed and reported for almost 100 years in winter wheat fields,nft channel where wheat rows along the borders showed higher yields than those in the center . Over the years, rice , maize and climbing beans , wheat , millets, Sudan grass , soybean , cotton , rapeseed , carrots, cabbages, and onions have been shown to display edge-biased distribution of growth. In rice, the increase in yield of border rows compared to central portions of fields or plots ranged from 63 to 68% . The difference can be as large as 394% between edge and center crops . The major factors accounting for such edge-biased growth difference have been attributed to competition for nutrients and light among crop plants . The body of research documenting edge-biased distributions of crop development often regards the phenomenon as undesirable and seeks ways to curb the effect to avoid overestimation in yield calculations . However, from an entomological perspective, the research focus would be to study how within field vegetational heterogeneity contributes to the edge-biased distribution of insects in agricultural systems. We are unaware of any published study addressing this question directly, but the edge resource model by Ries and Sisk could partially explain this, as micro-climatic conditions near edges may affect host plant growth and quality . Nonetheless, given that edge crops can have significantly higher yield than center crops, it is reasonable to hypothesize that there are physiological, and therefore nutritional, differences between edge and center crops. If such physiological differences are linked to differences in suitability as hosts and insects are able to—via volatiles or visual cues—perceive such differences among crop plants, then vegetational heterogeneity may partially explain observed edge-biased distributions of insects. That is, vegetational heterogeneity may drive selection by favoring both behavioral attraction and oviposition behavior by insects to certain plants. In addition, difference in suitability of crop plants will likely lead to differential growth and survival rates of insects.

As an example, longevity and fecundity of Sitobion avenae and Rhopalosiphum padi L. have been shown to increase with the level of nitrogen fertilizer applied to wheat host plants . Meanwhile, poor-quality host plants can cause many female insects to resorb their own eggs to mobilize nutrients for their survival . In broader term, female insects have been shown to vary their oviposition and number of eggs laid in accordance to the suitability of host plants for the performance of future progeny . Therefore, the better-quality edge crops may be preferred by insects or provide better growing conditions for insects and their larval stages, resulting in a skewed distribution towards crop edges. However, using plant vigor to explain edge-biased distribution of insects may also involve important trade-offs. On one hand, more vigorous plants may provide insects and their larvae with more nutrients for their development. On the other hand, healthier plants can also produce more defensive chemicals such as alkaloids, glucosinolates, and phenolics to protect themselves from insect herbivores . Furthermore, the idea of plant quality as potential explanation for insect aggregation along edges is also contested by the data reported by Haynes and Cronin . Their study found that the nitrogen content of plants along mudflat-cord grass edges and bromecord grass edges was both higher than that of plants in the interior. Interestingly, delphacid plant hoppers aggregated along mudflat-cord grass edges but not along bromecord grass edges, implying that host plant quality may not be the main driving factor of edge effect. Thus, we argue that considerable research is needed to investigate the complex of factors affecting plant growth along edges compared to inwards into cropping systems and how differential plant growth may affect their role as suitable hosts for associated insect communities.Originally, skewed distributions of agricultural insects towards field edges were mostly regarded as a source of error when developing insect sampling plans. Therefore, the data collected along field edges were not considered representative of the actual insect population . However, edge-biased distributions of insects in agricultural systems can potentially provide opportunities to optimize sampling efforts. Currently, sampling units employed in sampling plans are often randomized and spread across the field. Such an approach can be highly labor intensive. Given the prevalence of edge effects in many insect species, stratified sampling plans with a greater focus along field edges than field interiors can reduce sampling efforts as well as increase sampling accuracy if a stable spatial pattern of insect distribution within an agricultural system is well studied and established. For example, increased performance of such a spatially targeted sampling approach was demonstrated for the wheat stem sawfly . In addition, Severtson et al. described a spatially optimized sequential sampling plan for cabbage aphids in canola fields. In their study, canola fields were divided into two types of sampling grids: inner grids and edge grids. Edge grids were defined as the areas within 20 m of the field edge, while inner grids were the rest of the sampling grids. Their sampling results showed that 9 out of 20 edge grids displayed infestation level above threshold while only 2 out of 20 inner grids showed infestation level above threshold. Taking the proposed edge effect distribution of aphids into account, Severtson et al. conducted stratified sampling analysis and managed to reduce spatial variability as well as to increase accuracy of infestation level. Nonetheless, we also acknowledge that edge effect distribution of insects in many agricultural systems can be highly seasonal and temporally dependent .Furthermore, applications of insecticides in agricultural fields have been proven to greatly change the edge-biased distribution of insects to random distribution across agricultural fields . Therefore, the mentioned limitations may jeopardize the robustness of edge-stratified sampling methods and highlight the importance of timing in proposed edge-stratified sampling methods. Broadly speaking, increased understanding of the mechanisms responsible for edge-biased distributions can be used to improve the practices of precision agriculture. As compared to conventional agriculture, precision agriculture focuses more on timely and targeted application of treatments to control insect pest infestations rather than field-wide and calendar-based spraying of chemicals . The knowledge of edge-biased distributions can help create targeted sampling plans to generate more reliable mapping of insect distributions and enable more targeted application of pesticides used in IPM programs .