Horticulture

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Non-agricultural land use types include residential, industrial, natural habitat, and other. For the analysis, I join artichokes with vegetable row crops, blueberries and vine crops with caneberries, and cover crops and unknown agricultural land with fallow ground, given the limited number of parcels in each of these categories. These detailed land survey data are coupled with tax assessor ownership and parcel boundary data from the County Assessor offices to form appropriate decision units. These data delineate property boundaries and enable assignment of land use and groundwater quality to each farm at the land parcel level, designated by the Assessor’s Parcel Number in the tax assessor data. The average size of a parcel is 33.94 acres. I also use ownership data from the County Tax Assessor to aggregate parcels to the ownership level . This helps both with crop decision-making , and with water attribution. Since not every parcel has a well or recycled water turnout, but almost all parcels use groundwater or recycled water, I want to assign that parcel water from the most likely source,danish trolley which would be a well or turnout on another parcel owned by the same person. The definition of “agricultural land” is therefore a parcel with documented ownership information that has been designated by PVWMA at some point to have produced a crop, and has known access to water.

To be classified as having known access to water, the parcel must contain a well or a recycled water turnout, or the owner of the parcel has a different parcel with well/turnout access. In order to provide a comparison point on how restrictive this definition is, we look at the tax assessor land use classifications. After filtering out land uses that involve residences, businesses, and industry, across Santa Cruz and Monterey counties there are 1653 parcels that could plausibly be in agriculture. After restricting the dataset to parcels that also have a clearly linked source of water, I’m left with 1048 parcels. Therefore, this is a relatively restrictive set of qualifications. While this could lead to an overestimation of water applied per acre for the parcels classified as being in agriculture, as described below, there is no evidence that this is biased in a particular direction for growers receiving delivered water. Moreover, the difference-in-differences analysis and event studies do not use quantities of recycled water delivered in their analysis. There are 978 documented wells across Pajaro Valley and 102 recycled water turnouts in the DWZ, all with quarterly measurements of how much water was pumped or delivered. For all of the wells owned by the same owner, I pool the total water pumped and area-weight the water across all parcels planted in a crop during that year. I follow the same procedure for all turnouts owned by the same owner.

This assumes that water needs across all crops are similar, which is largely the case for crops grown in the Pajaro Valley, which require between 2-3 acre-feet a year. I classify a parcel as using water if they are listed for an agricultural purpose that is not fallow. This includes “unknown agriculture”. This does not include parcels that have no information on their agricultural status, making the assumption that PVWMA is capturing all agricultural fields. I gather outside data to estimate crop prices and weather patterns. Data on temperature and precipitation are from PRISM Climate Data, which incorporates coastal weather patterns and land elevation into its projections. The data projections have approximately an 800 m resolution. I use the gridded data cell that lines up with the centroid of each of the parcels. I use averages of monthly mean temperatures and total precipitation in the spring of each year, at the parcel level. Since several of the land use types include multiple crops, I take area-weighted averages of crop prices and revenue for each land use category, and average these across Santa Cruz and Monterey counties.The following section evaluates the impact that recycled water has on growers that receive water deliveries using a discrete crop choice model. Growers receiving recycled water may see benefits of two forms: their yields may improve with higher quality water, and they may be able to grow more salt-sensitive crops.

To capture these benefits in tandem, I use a revealed preference, panel mixed logit model that evaluates the impacts of salinity and recycled water on crop choice, and calculates the willingness to pay for improvements in salinity. This framework also allows for a simulation to find the counterfactual crop choices without recycled water deliveries. The modeling framework, empirical strategy, and results of the model are detailed below. Spatially and time-varying data on groundwater quality, recycled water deliveries, and land use allow for the estimation of a panel mixed logit model to understand the impacts of salinity and recycled water on crop choices. The research design relies on observable changes in groundwater quality and recycled water deliveries, along with controls for observable and unobservable factors that may be correlated with both salinity, recycled water, and crop choice. I take a similar approach to the working paper by Sears, Bruno, and Hanemann 2022 that estimates damages associated with groundwater salinity. Building on these methods, this approach differs in a few key ways. Most importantly, I consider only parcels that can be clearly linked to a source of water, since the analysis specifically accounts for recycled water deliveries. Additionally, part of the water assignment structure involves adding in tax assessor data to the model, and standard error clustering at the ownership level. This helps account for decision-making on crop rotations, allocation of water across parcels, and crop diversification that may occur. Finally, I allow for an unbalanced panel of parcels in agriculture: a parcel is able to leave agriculture and is still counted in the analysis up until the point of departure. where the parameters can be estimated by maximum likelihood. The panel mixed logit structure model avoids invoking the Independence of Irrelevant Alternatives assumption that troubles multinomial and conditional logit models , which is particularly important in a setting with perennial crops and repeated observations over time. The mixed logit model provides the framework for the direct welfare estimates of the delivered program. Under this structure, a simulation is run that estimates growers’ crop choices if they did not have access to recycled water, and estimates welfare with and without the water deliveries. In addition, the estimates from the mixed logit model provide willingness-to-pay estimates for improved water quality by crop, by dividing the marginal utility of reducing salinity by the absolute value of the parameter estimate on crop price α. Identification of the WTP hinges on the assumption that, conditional on the suite of relevant spatial and time-varying parcel-level observables and an annual time trend,vertical aeroponic tower garden unobservable factors are not correlated with both salinity levels and crop prices.One potential concern with this analysis is that groundwater salinity may be endogenous to crop choice, if salinity is controllable by an individual farmer.

While soil salinity problems can often be managed by an individual grower, provided that they have enough freshwater to leach salts out of the root zone, groundwater salinity is harder to influence. Groundwater salinity is largely based on unobserved hydrological and transmissivity properties of the underlying aquifer, along with recharge rates from precipitation and runoff, distance to the coast, and aggregate basin-wide groundwater pumping. In the crop choice model, I explicitly control for groundwater pumping, depth to groundwater, temperature, and precipitation. It is also possible that basin-wide groundwater pumping may be influenced by other unobserved economic factors that also impact crop choice. I control for the distance to the coastline, since this is an important determining factor of soil texture and the spatial distribution of salinity in Pajaro Valley, and because coastal micro-climates determine how well crops grow in certain areas. Features of the parcel, such as its size and crop history, are also likely to influence planting decisions and could be correlated with salinity. The inclusion of a linear time trend accounts for basin-wide unobservables that may trend linearly with both salinity and crop choice over time. Finally, the inclusion of county fixed effects and time and parcel random effects can combat regional effects that may be correlated with salinity. To identify the impacts of recycled water on crop choice, there may also be concerns about how recycled water is allocated. Conditional on being in the delivered water zone, assignment of recycled water is somewhat random: parcels that lie on the currently constructed portion of the Coastal Delivery System are the only parcels able to receive water deliveries. Growers apply for their recycled water turnout to be turned on. Application acceptance is conditional on whether there is excess recycled water available to serve the needs of all current users, and on underlying groundwater salinity. Recycled water availability depends on facility capacity, which has slowly increased over time. Finally, while underlying groundwater salinity does impact application acceptance, a parcel’s groundwater salinity is not highly correlated with its impact on the aquifer salinity: i.e. the positive or negative externality from recycled water deliveries or groundwater pumping to neighboring parcels is not well-linked to a parcel’s own salinity levels. This is especially important for the identification of the DiD and event studies, since salinity is explicitly controlled for in the crop choice model.

Finally, it’s important to note that the outside option in the choice model is fallow land, rather than land that leaves agriculture. I drop parcels from the sample after they leave agriculture permanently. This is standard in the crop choice modeling literature, largely because it is difficult to think about comparing annual profits to a lump sum payment received when exiting agriculture. However, it does limit the model to thinking about relatively short-run effects of salinity damages. It is plausible that a grower experiencing dramatic increases in groundwater salinity may not believe that water quality will improve or that they will receive recycled water on a fast-enough timeline to remain in business. In the appendix, I use linear probability models to test this hypothesis, finding that increases to salinity are not significantly linked to parcels leaving agriculture.Results from the crop choice model are presented in Table 3.3. Column 1 shows the basic conditional logit estimates and Column 2 presents the mixed logit results, which is the preferred specification and has a lower AIC. The table reports crop-specific estimates of the effect of groundwater salinity, where salinity is measured as the total dissolved solids from March-May of the growing season. In the mixed logit, the coefficients on the crop indicator variables are treated as random variables and are allowed to vary across parcels. Their coefficients and estimated standard deviations are also reported in the table, while coefficients on additional independent variables are suppressed. Standard errors are clustered by owner. I control for parcel-specific factors that directly affect salinity, including the depth to the groundwater table, groundwater pumping, the cumulative precipitation from March-May of the growing year, and the average mean daily temperature from March May of the growing year. Also included are factors related to the parcel that may affect crop choice, including the parcel size, recycled water deliveries, distance to the coastline, and additional aggregate controls for agency’s water pumping fee and a linear annual trend. All reported coefficients are relative to fallow ground, which increases in response to an increase in salinity. Results demonstrate that, compared to fallow ground, an increase in groundwater salinity decreases the probability that a farmer will grow a high-value, salt sensitive crop. We see the largest effects among vegetables, strawberries, and caneberries, the most profitable and some of the most salt-sensitive crops grown in the region. Since not all crops in our choice set are grown in the delivered water zone, the clearest way to evaluate the impact that recycled water has on crop choice is to look at the marginal effects of changes in salinity on crop choice for parcels that do and do not receive recycled water deliveries. These results are presented in Figure 3.8. Results are presented for each of the major crops that are grown both inside and outside of the delivered water zone, for three levels of TDS: 500, 1000, and 1500 mg/L. These are all relatively moderate levels of salinity: enough to impact yields, but not enough to completely destroy a crop.

In 1980, the California Department of Water Resources defined 447 groundwater basins, sub-basins, and other water storage types, and highlighted the Pajaro Valley water basin to be one of 11 basins facing severe overdraft . Due to this classification, the Pajaro Valley Water Management Agency was established in 1984 to combat overdraft and salinity problems by bringing several different groups of stakeholders together. There is a seven member board of directors for the PVWMA. Four are elected every four years by the public , and the other three are appointed by Santa Cruz County, Monterey Bay County, and the City of Watsonville, respectively. The appointed individuals must receive a majority of their income through agriculture, and serve two year terms. There are no term limits. The PVWMA can manage groundwater resources, but it is not authorized to deliver potable water. The PVWMA completed its first basin management plan in 1994. In the early 1990s, the Pajaro Valley Water Management Agency started a water supply system that combines surface water, groundwater,blueberry grow pot and recycled water to provide water to farms as well as to recharge the aquifer to prevent seawater intrusion. Today, under the Sustainable Groundwater Management Act, PVWMA is tasked with bringing the water basin into “balance” by 2040, where the water entering the aquifer matches the annual quantity removed.

Total water use in the Pajaro Valley is approximately 55,000 acre-feet a year , with 52,000 AFY sourced from groundwater2 . Estimates from the PVHM suggest that overdraft in the groundwater basin is approximately 12,000 AFY . Through acombination of water conservation and alternative water resources , PVWMA is tasked to ensure that overdraft is reduced to zero. The Pajaro Valley Water Management Agency has embarked upon several strategies to fight the depletion of groundwater in the Pajaro Valley. Their primary objective is to reduce seawater intrusion in the valley, and this is partially accomplished by providing alternate water sources for farmers along the coast. There are three primary programs that have been carried out by the PVWMA, including the Watsonville Recycled Water Facility, the Harkins Slough Recharge and Recovery Facility, and the building of the Coastal Distribution System. The facility began operation in 2009, and has increased the volume of water deliveries from 2700 AFY in 2010 to 5500 AFY in 2020 . The recycled water is tertiary treated to meet California’s regulations required for the water to be applied to agricultural land. This involves the removal of all solids greater than 10 microns, and the removal of pathogens using UV filters. After the tertiary treatment process, the main differences between recycled water and potable water are the salinity, nitrate, and phosphate levels3 .

Nitrates and phosphates are useful for irrigated agriculture, as they are the main nutrients in fertilizers, and are therefore not removed during the recycling process. Salinity levels are carefully monitored to not exceed 590 mg/L before being delivered to growers. If salinity levels are too high, the recycled water is blended with water from inland wells, in order for it to fall below the threshold. The water storage at the Watsonville Recycled Water Facility has expanded over time, and additional storage tanks have been approved to be built . The Harkins Slough Recharge and Recovery Facility is another project by the PVWMA, where water is recharged and stored until needed for agricultural use. It has been in existence since 2002, and was the first groundwater recharge project constructed by the Agency. It stores winter surface water flow as well as irrigation water runoff in a 14 acre percolation basin to use for irrigation during the summer . While the PVWMA has a permit to pump 2000 AFY from the Harkins Slough, the reality has been closer to 1000 AFY , due to a lack of flow through the Slough and the limited capacity in the recharge pond . There are plans in place to improve the Harkins Slough Project, by installing new shallow extraction wells, upgrading the pump system and filters, and establishing a new recharge basin. The Coastal Distribution System is the delivery mechanism for the recycled water and recovered Harkins Slough water to the section of the Pajaro Valley that has the highest levels of salinity from groundwater intrusion, called the “Delivered Water Zone”. The CDSis a pipeline system that primarily serves farms in coastal southern Santa Cruz and northern Monterey counties.

The total length of the pipeline was approximately 20 miles long in 2020, and provided water to 5100 acres. There are plans to extend the CDS further, but all expansion plans are within the confines of the Delivered Water Zone . The location of the DWZ is depicted in Figure 3.2. There is a policy commitment to not deliver alternative water outside of the zone until groundwater pumping is no longer necessary within the DWZ. This is unlikely to happen in the near term: 2019 was the first year that growers in the DWZ used more alternative water than they pumped. Additional projects are on the docket for the PVWMA, mostly pertaining to expanding recycled water storage and increasing recharge. There are plans for additional recharge basins on the San Andreas Terrace and near Murphy Crossing . The College Lake Project is another plan, designed to add additional surface water from an existing lake to the CDS . In 2016, the Pajaro Valley Board of Directors approved ReNeM, a five year pilot project that develops multiple Managed Aquifer recharge projects using storm water . In addition, PVWMA does have rights to 19,900 AFY of Central Valley Project water, a pipeline was never built to the Pajaro Valley, and they currently lease the water rights to other sources. The length of pipeline required is financially unfeasible. In total, the PVWMA’s management strategy, combining the CDS, Watsonville Recycled Water Facility, and Harkins Slough, provided 5200 AF in 2020, which is more than ten percent of the annual groundwater used in the region . Targeting this water as a substitute for groundwater in coastal areas, as well as recharging the groundwater can lead to a mitigation of seawater intrusion. The Pajaro Valley Water Management Agency has the ability to regulate and limit pumping directly, but has opted for pricing policies and the supplemental water projects described above as an alternative to a command and control program.

The price of water varies in the Pajaro Valley depending on where the water is sourced , as well as if the well is metered or unmetered4 , and if the well is located within the delivered water zone . New rate structures have to be approved by residential, commercial,square plastic pot and agricultural water users. In addition, revenues for water pricing must not exceed the proportionate costs of the property-related service attributable to the parcel that is charged. Pajaro Valley Water Management Agency has found themselves in a few legal battles over the water pricing structure. PVWMA v. Amrhein was a lawsuit in 2007 that declared that all water prices were property-related, and therefore fell under the rules of Proposition 218. This means that prices charged for water need to be presented at a public hearing, and if a majority of affected owners file written protests at the hearing, the fees are not assessed. In response, the water pricing structure shifted into the tiered pricing structure that is still in use today. Griffith v. PVWMAfound that the revenue from water prices could pay for activities associated with implementing the groundwater program, including water conservation. Basically, the court found that the revised water augmentation charges did meet the requirements of Proposition 218, and that the charges benefited the entire basin. This led to upholding the revised augmentation charges in 2010. Dependence on groundwater as a freshwater source has increased significantly over time . It now accounts for over a third of irrigation water withdrawals worldwide and about half of the domestic needs of the world’s population . Meanwhile, climate change is bringing warmer temperatures, sea-level rise, and more frequent and extreme weather events . The increased variability of precipitation and the reduction in surface water supplies due to warming temperatures puts additional pressure on groundwater resources. Additionally, sea-level rise will flood agricultural lands, as well as degrade water quality through saltwater intrusion into coastal aquifers . Since both the quality and reliability of water supplies are crucial for agricultural productivity, this poses a threat to global food production. To date, the focus of the economics literature on groundwater resources has been on issues related to groundwater supply. Groundwater is a classic common-pool resource whereby, in the absence of well-defined property rights, individual pumpers’ actions cause direct, external effects to their neighbors. Recent work is advancing our understanding of the magnitude and nature of the stock and pumping cost externalities associated with extraction , and evaluating the potential for and impacts of different mechanisms to overcome this market failure . In addition, over pumping groundwater can also degrade water quality, particularly in coastal agricultural regions, because it moves seawater into freshwater zones as cones of depression are formed in areas around tube wells .

Largely absent from this recent and growing body of groundwater work are questions about water quality. This paper empirically estimates the marginal damages associated with increasing groundwater salinity. Our approach is based on the idea that the willingness of farmers to switch to more salt-tolerant crops reveals how much they value a change in groundwater salinity. Crop yields decline as salinity increases and switching to a more salt-tolerant crop is the primary mechanism to mitigate these costs . Thus, our understanding of the marginal damages of sea-level rise and the willingness to pay to avoid saltwater intrusion hinges on an unbiased estimate of the effect of changing water quality on crop choice. In this paper, we use a unique panel dataset on micro-level land use and groundwater quality from California’s central coast to provide a credible estimate of the likelihood that farmers switch crops in response to changes in groundwater salinity. Controlling for basin wide trends and relevant parcel-level characteristics, we estimate the impact of changes in salinity on crop choice with a panel mixed logit model. Then, we use our estimates to derive measures of the marginal WTP and simulate crop changes under salinity conditions likely to occur from continued groundwater overdraft and sea-level rise under climate change. Our empirical setting – the Pajaro Valley, California – provides a rare opportunity to estimate the WTP for changes in groundwater salinity using a revealed preference approach. Best known for its berries and vegetables, this productive agricultural area has experienced decades of seawater intrusion due to its dependence on groundwater for irrigation and its proximity to the coast. As a result, the water agency that services this region has been monitoring groundwater quality with an extensive network of monitoring wells for decades. The density of monitoring wells allows us to spatially interpolate water quality with minimal error, which is important to a research design that relies on these observable changes in quality over time and across space. Second, we are able to use high-quality geospatial land use data collected by the agency to pair water quality measurements with acres planted in a variety of crops that vary in their salt sensitivity.We take advantage of these observations over both space and time to apply panel discrete choice methods to this agricultural context. Our results show that an increase in groundwater salinity decreases the likelihood that a farmer will grow cash crops, relative to leaving the land idle, and that the impacts are greater among the more salt-sensitive crops. Our model indicates that strawberry and vegetable growers are willing to pay an average of $1,613 and $3,084 per acre, respectively, for a 10 mg/L reduction in total dissolved solids. Blackberry and raspberry producers are willing to pay $16,369 per acre on average. As a result, a simulation of changes in regional crop production due to a doubling in groundwater salinity projects that blackberry and raspberry production will experience the greatest decline in the region as sea-level rise exacerbates existing saltwater intrusion problems. We estimate that a change in salinity of this magnitude would cost $140 million, roughly 10% of annual agricultural revenues in the region.

Interestingly, a global reduction by approximately 20% in gray matter CBF was not only observed two hours after the intake of a single dose of 184 mg caffeine , but also following the consumption of 2820 mg black tea solids containing 184 mg caffeine that is equivalent to about six cups of tea.This suggests that flavonoids in black tea did not affect the acute decrease in CBF following the intake of caffeine in healthy male subjects with a mean age of 24 years. Even though a robust blood flow decrease in response to acute caffeine consumption was observed, neural activity was enhanced, as adenosine’s effects are antagonized. Cognitive performance, however, was not changed. This might partly be explained by results from calibrated blood oxygenation level-dependent functional MRI experiments. It was shown that after caffeine intake, the cerebral metabolic rate of oxygen consumption remained stable in some, but not all studies,dutch buckets because the decrease in CBF was compensated for by an increase in oxygen extraction. More importantly, long-term effects are unclear. Vascular adenosine receptors may be upregulated during long-term caffeine use to preserve the CBF at a level that would have existed in a caffeine-naïve state. Another main issue are the effects of caffeine withdrawal.

CBF may be abnormally high due to overnight withdrawal from caffeine or abnormally low due to recent caffeine ingestion. Due to its widespread use, caffeine is therefore a potential confounder in many cerebral perfusion studies, complicating the interpretation of the study results. Interestingly, the observed effects of caffeine on CBF may explain its efficacy as a contrast booster in functional MRI studies. In fact, by acting as a cerebral vasoconstrictor, caffeine causes an increase in the concentration of deoxyhemoglobin, and thus decreases the BOLD baseline resting signal. During activation, the human vasculature responds from below-normal baseline levels with a normal increase in blood flow, resulting in an overall increase in the BOLD contrast. The benefit of an increased BOLD signal contrast can be used to improve, for example, image resolution, the acquisition scheme, or the task design of functional MRI experiments. Neuroimaging studies clearly indicate that alcohol increases cerebral perfusion. In fact, alcohol may affect CBF directly via its vasodilatory effects on arterial vessels and may induce changes in brain metabolism that trigger indirect vasodilatory mechanisms. As reviewed by Bjork and Gilman, earlier studies using the dynamic SPECT technique with inhalation of Xenon-133 have indicated significant increases in mean gray matter CBF at low, moderate, and high alcohol doses.

Similar effects were found on global cortical CBF measured with [99mTc]-HMPAO SPECT following alcohol intoxication. Volkow et al. performed blood flow measurements using 15O-labeleld water and PET. They demonstrated increased perfusion in the temporal and prefrontal cortex, while consumption of 1.0 g/kg of alcohol reduced blood flow in the cerebellum in a group of thirteen normal drinkers consuming 1 to 2 mixed drinks or 2 to 4 beers per week. More recent intervention trials using MRI methods based on ASL perfusion contrast have further established that moderate alcohol doses of about 0.6 g/kg breath alcohol concentration increase CBF, although the between-subject and regional variabilities are large. In fact, Rickenbacher and colleagues indicated an increased perfusion in bilateral frontal regions after acute alcohol administration in men, but not in women, while Khalili-Mahani reported a global perfusion increased in response to intravenously administered alcohol in only half of the subjects. In a trial involving a total of 88 healthy young adult social drinkers, cerebral perfusion increased after alcohol as compared to placebo in only frontal brain regions, whereas other RCTs also reported significant treatment differences in other brain regions. The purpose of the study by Marxen et al. was to reduce potential sources of inter-subject variability. In 48 young adults aged either 18 or 19 years, CBF was monitored continuously during the rise of BrAC up to 0.6 g/kg and a steady BrAC of 0.6 g/kg for two hours. Interestingly, global perfusion increased after alcohol consumption by about 7% as Blood Alcohol Concentrations approached 60 mg %, and CBF increases and changes in BrAC were tightly coupled in time.

Except for the occipital lobe, significant increases in CBF were observed in most regions of the brain. These findings are in agreement with a more recent study that showed widespread and dose-dependent increases in cortical perfusion following intravenous alcohol administration. In addition, measurements were performed at a higher BAC concentration of 80 mg %, which represents a clinically relevant threshold for heavy episodic alcohol exposure, as defined by the National Institute on alcohol abuse and alcoholism . It was reported that increases in cerebral perfusion were significantly more pronounced at 80 mg % versus 40 mg % for specific frontal brain regions, suggesting that these brain regions are particularly sensitive to the vasoactive effects of acute alcohol intake. Evidence for the chronic effects of alcohol on CBF is limited, but observational studies found that long-term alcohol consumption was associated with reduced CBF in the sober state. Decreased perfusion levels were observed both globally and regionally in the frontal, temporal, parietal, and occipital cortices, as well as in the thalamus. As reviewed by Ogoh and Ainslie, cerebral perfusion increased during mild to moderate exercise intensities up to about 60% of maximal oxygen uptake. This is due not only to increases in cardiac output, but also to changes in neuronal activity and metabolism in regions associated with central command and skeletal muscle afferents. However, CBF during exercise is largely dependent on exercise intensity. In contrast to mild to moderate exercise, higher exercise intensities first increase cerebral perfusion, after which CBF reduces towards resting values, possibly due to hyperventilation-induced cerebral vasoconstriction. The effects of long-term exercise on CBF have hardly been studied. In 307 healthy men aged 18 to 79 years, regular aerobic-endurance training was significantly associated with higher mean blood flow velocity in the MCA, measured with transcranial Doppler ultrasonography. In fact, a difference of approximately 17% in blood flow velocity was observed at rest between exercise-trained and sedentary men, independently of possible confounding variables. The effects of a six-month aerobic exercise-based cardiac rehabilitation program on regional CBF were investigated in coronary artery disease patients. In this trial using ASL, CBF increased in the bilateral anterior cingulate region, which is involved in cognitive processing. In addition, Chapman et al. found increased CBF in the anterior cingulate region, and related increase of memory performance in 37 cognitively healthy sedentary adults after three months of supervised aerobic-based exercise training.

This indicates that improvements in cerebrovascular function may be a mechanism underlying the beneficial effects of exercise on cognitive function. In summary, CBF is a sensitive physiological marker of cerebrovascular function that can be quantified, for example, by the non-invasive MRI method ASL. ASL provides a highly repeatable quantitative measure of human brain perfusion. Cerebral perfusion is positively associated with cognitive performance. Furthermore, lifestyle factors,grow bucket including dietary composition and physical exercise, can significantly increase CBF, thereby improving cognitive performance. A limited number of studies have observed the beneficial effects of acute intakes of dietary nitrate and polyphenols on CBF. However, evidence for a significant relationship between these effects and improvements in cognitive functioning is limited, possibly because acute perfusion changes are not related to improvements in cognitive function in healthy young adults, who were involved in most of these intervention studies. Furthermore, long-term trans-resveratrol supplementation enhanced CBF in populations at increased risk of accelerated cognitive decline. A long-term supplementation of n-3 LC-PUFAs may also increase CBF, but the related effects on cognitive performance have not yet been found in these supplementation studies. Consistent evidence exists for the acute effects of caffeine and alcohol. Decreases in CBF are induced by commonly consumed amounts of caffeine, while alcohol significantly increases cerebral perfusion in a dose-dependent way. Long-term effects of caffeine and alcohol, however, are not clear. Finally, long-term exercise training may be a promising future approach to improve CBF, as it is thought that increases in perfusion contribute to the beneficial effects on cognitive functioning following increased physical activity levels.Future well-designed RCTs are still warranted to further investigate the potential of diet and exercise on CBF. Of particular use would be longer-term intervention studies focusing on middle-aged and elderly adults at increased vascular risk who are also known to be at increased risk of cognitive impairment and dementia. Furthermore, the specific effects of lifestyle changes on other physiological measures of cerebrovascular function are largely unknown. These effects are also of major interest, since other aspects of an impaired brain vascular function may also consist of key pathological events that precede the development of impaired cognitive function. Understanding the potential of lifestyle factors to affect these outcome measurements may further help to determine effective strategies to mitigate the adverse effects of human aging on cognitive performance. Future research should thus broaden its focus, considering emerging MRI techniques for the non-invasive assessment of additional brain vascular measures. For example, the dietary and physical exercise effects on the compliance of the major cerebral arteries measured non-invasively with short inversion time ASL and the cerebrovascular reactivity to hypercapnia are of great interest.

An example arterial compliance map is shown in Figure 3, with compliance in units of percentage change in arterial blood volume per millimeter of mercury . From these maps, measures of average arterial compliance can be obtained in major cerebral arteries, including the left middle, right middle, left posterior, right posterior, and anterior cerebral arteries, in a region just superior to the circle of Willis that supplies blood to the brain.Drosophila suzukii Matsumura is an economic pest of small and stone fruit in major production areas including North America, Asia and Europe. Female D. suzukii oviposit into suitable ripening fruits using a serrated ovipositor. This is unique compared to other drosophilids, including the common fruit fly, D. melanogaster, which oviposit into overripe or previously damaged fruit. Developing fruit fly larvae render infested fruit unmarketable for fresh consumption and may reduce processed fruit quality and cause downgrading or rejection at processing facilities. In Western US production areas, D. suzukii damage may cause up to $500 million in annual losses assuming 30% damage levels , and $207 million in Eastern US production regions. Worldwide, the potential economic impacts of this pest are staggering. Pesticide applications have been the primary control tactic against D. suzukii both in North America and in Europe. The most effective materials are those that target gravid females, including pyrethoids, carbamates, and spinosyns. These applications are timed to prevent oviposition in susceptible ripening host crops. In the Pacific Northwest, many growers have adopted scheduled spray intervals of 4–7 days. This prophylactic use of insecticide is unsustainable as growers have a limited selection of products and modes of action. This could ultimately lead to D. suzukii becoming resistant and may cause secondary pest problems because of negative effects on beneficial organisms. Furthermore, production costs have increased substantially in crops where D. suzukii must be managed. Effective sampling methodology for D. suzukii is lacking despite extensive efforts to improve trap technology or determine effective fruit infestation sampling protocols. Theoretically, traps to capture adult flies should aid growers in the timing of spray applications so that insecticides could be used more judiciously. Traps baited with apple cider vinegar or a combination of sugar-water and yeast are currently used to monitor adult D. suzukii flight patterns. However, without standard methods for trapping or management thresholds based on trap count data, it is questionable how much is gained by establishing and monitoring traps in crops. Establishing, monitoring, and maintaining traps is very labor intensive and the costs do not justify the benefits for many growers. Historically, trap data has not provided a reliable warning against D. suzukii attack, especially for susceptible crops in high-density population areas where considerable oviposition can occur in short time periods. Currently, no significant differences are found in any traps used for monitoring D. suzukii given differences between crops and environments where traps have been tested. Monitoring fruit infestation levels to guide management may also be impractical. Furthermore, by the time larvae are detected in the fruit, it is too late for management action and damage has already occurred. No detailed studies could be found using monitoring for fruit infestation for this pest, and precision of sampling methodology is currently unavailable.

Various antimicrobial compounds are correlated with AFR resistance, including 1-acetoxy-2-hydroxy- 4-oxoheneicosa-12,15-diene and avocadynone acetate in avocado, and dopamine and dopamine-derivatives in banana. In blueberry, resistance was previously hypothesized to come from the interaction of several compounds, including phenolics and organic acids . Supportive of this hypothesis, two non-anthocyanin f lavonoids from blueberry fruit, quercetin 3-Orhamnoside and putative syringetin-rhamnoside, were found to have antifungal activity against the causative agent of AFR. Alternatively, resistant fruits may prompt active resistance mechanisms such as inducible defense-related proteins, including chitinases and β-1-3-glucanases that degrade the cell wall. Expression of defense-related proteins in response to anthracnose fungi has been previously reported in pepper , apple, raspberry, tomato, and blueberry . Putative defense genes in blueberry include those that encode cell wall degrading protein, pathogenesis-related protein 10 , metallothionein-like protein, and monodehydroascorbate reductase. Moreover,hydroponic nft channel resistant fruits may actively combat anthracnose infection through the formation of reactive oxygen species .

Production of ROS in response to anthracnose has been documented in tomato, strawberry, and blueberry. In all three crops, ROS occurs at or near the time of attempted penetration of the pathogen. Concurrently, infected fruits upregulate expression of oxidative stress response genes to minimize potential harmful effects of the ROS on the host’s own tissues. In this study, we sought to identify genomic loci associated with resistance to AFR by examining a genetic mapping population with tremendous variation in susceptibility. In order to capture allele and sub-genome-specific variation that might be associated with resistance, we mapped to a fully haplotype resolved genome assembly with no explicit association made between the locations of the equivalent bases in the different subgenomes/haplotypes. Cultivated high bush blueberry has a rich history of introgression from multiple other wild blueberry species from breeding efforts, plus natural gene f low that has previously shown to occur among sympatric blueberry species. We have an unpublished analysis, as part of another study, that suggests that the genome of cultivar ‘Draper’, one of the parents analyzed here, has minimally 23% introgressed from at least three other Vaccinium species. This may, in part, be the reason why we identified significant candidate genomic regions in only one of the homoeologous chromosomes. Homoeologous chromosomesets are shown in Supplemental Figure S5.

The different homoeologous chromosomes of ‘Draper’ exhibit high sequence divergence. In effect, the tetraploid was treated in alignment terms as a single fully expanded haploid. QTL mapping tools are available that function for polyploids, but they frequently depend on other tools such as polymap that require dosage information that we are not exploring in the current work or require rather specific types of population that were not generated as a part of this study. Meanwhile, the GWAS approaches are necessarily somewhat robust to variable levels of population relatedness, explicitly incorporating this in approach-specific ways, while more recent GWAS approaches adopt elements of QTL mapping to improve their sensitivity. Both the FARMCPU approach and BLINK approach for example exploit the generation of pseudo-markers and marker aggregation, FARMCPU using block-interval marker aggregation with block-size determined by MLE and BLINK marker aggregation in discovered bayesian linkage groups to recover essentially much of the power lost in approaches such as GWAS GLM/MLM where markers are considered essentially individually. Nonetheless, the different GWAS approaches selected very similar sites with only the association strength being notably different. BLINK and FARMCPU in this respect achieved higher levels of significance at these consistent sites likely due to their mapping-like marker aggregation. We have added to the supplementals a QTL mapping study results that includes standard interval mapping, composite interval mapping, and multiple QTL mapping to test whether the QTL and GWAS approaches are essentially in agreement.

The QTL approaches largely support the GWAS sites albeit with the limitations on marker selection discussed above not always leading to greatly enhanced levels of significance relative to FARMCPU/BLINK . In addition, we reran GWAS and QTL analysis to examine results with a binary categorization of susceptibility . There was no evident increase in association power when classifying the genotypes in a binary form . We identified three loci located on pseudomolecules 17, 23, and 28 in the V. corymbosum ‘Draper’ genome that are significantly associated with the resistance phenotype . Having several candidate causal SNPs is consistent with a polygenic resistance trait that may partly contribute to the observed continuous fruitrot susceptibility distribution. Variants in several linked biosynthesis pathways, including cytokines, anthocyanins, and other f lavonoids such as quercetin, may have become established in the population due to a protective role gained over time against multiple fungal pathogens. The identification of these loci will be useful for developing a molecular marker-assisted selection protocol for blueberry, which will greatly facilitate future screening for AFR resistance early in the breeding and selection process. Several candidate genes within these QTLs were previously associated with resistance against pathogens and/or f lavonoid biosynthesis, including anthocyanins. In comparing gene expression of blueberry fruits infected with C. fioriniae to uninfected fruits, numerous biological processes showed differential expression across the infection time course. Within 1 hour of infection, infected samples began upregulating genes related to metabolic pathways, including starch and sucrose metabolism and biosynthesis of secondary metabolites. In the following days, genes involved in metabolic pathways and biosynthesis of secondary metabolites continued to be upregulated. Additionally, the biosynthesis and metabolism of other primary and specialized metabolites and plant hormones were upregulated. Notably, many of the differential KEGG pathways show associations with the metabolite marker quercetin 3-Orhamnoside. Most clearly, phenylpropanoid and f lavonoid biosynthesis can be tied directly to quercetin 3-O-rhamnoside content as this metabolite falls within the phenylpropanoid and f lavonoid families of specialized metabolites. Furthermore, the biosynthesis of amino acids may also be connected to quercetin 3-Orhamnoside, as L-phenylalanine, an amino acid, is a precursor of quercetin 3-O-rhamnoside.

Our transcriptome analyses identified an enrichment of significantly differentially expressed genes associated with certain specialized metabolic pathways and pathogen resistance. Plus, several candidate genes within these QTLs were previously associated with resistance againstpathogens and/or f lavonoid biosynthesis, including anthocyanins. However, it’s important to note that none of these genes were identified as significantly differentially expressed. Low expressed genes, including certain regulators ,may not be identified as significantly differently expressed but can cause a cascade of transcriptional changes,nft growing system including inducing certain genetic pathways, and impact certain phenotypic traits. Thus, it’s possible that our transcriptome analyses were unable to identify them based on the current data and significance thresholds.We hope that the community will engage in combining these results with their own to further explore the data and identify additional potential candidate genes. Lastly, we identified a f lavonol glycoside with accurate mass, fragmentation, and absorbance characteristics consistent with a quercetin rhamnoside, whose abundance is significantly greater in berries from resistant individuals. These findings confirm data previously reported by Miles et al. , which identified quercetin 3-O-rhamnoside as an antifungal component of blueberry fruit. Several efforts were taken to normalize any variation in metabolite content due to fruit location on the bush, collection time, and fruit ripeness. Fruits within each subset were ground together and homogenized, resulting in three separate homogeneous mixtures of powdered fruit from each individual. Variability of the quercetin rhamnoside abundances was observed within resistant and susceptible individuals . Furthermore, levels of quercetin were previously shown to vary greatly between different harvests – indicating that the biosynthesis of these compounds may be inf luenced by the environment. In general, f lavonol glycosides are known to have antioxidant activity , and blueberry fruit f lavonol extracts demonstrate antioxidative activity against peroxyl and superoxide anion radicals. Taken with the hypotheses of Cipollini and Stiles , these findings suggest that quercetin 3-O-rhamnoside, a phenolic compound, is likely a major component of resistance to AFR in blueberry, but not the only component. Perhaps this molecule acts with other resistance mechanisms to protect the fruits from AFR. Traditional breeding efforts in blueberry have contributed to major improvements of various target traits, but it is a lengthy and expensive process for perennial crops. The use of molecular markers to guide breeding efforts has long been shown to greatly accelerate the development of superior cultivars, including selecting disease-resistant individuals.

There has been a strong community effort to develop and implement cost-effective methods for blueberry breeding programs. Collectively, we hope that these findings reported in this study will allow breeders to develop new resistant cultivars to AFR more efficiently and rapidly by leveraging these new genetic regions to identify and select resistant individuals in their breeding programs. The phenotype predictive power from the combination of the three markers on chromosomes 17, 23 and 28 was assessed using a multiple regression approach. Individually the Pearson product moment between phenotype on the 0–5 scale and genotype ranged from a low of 0.25 to a high of 0.30 , jointly a predictive model generated from all 3 sites had an r of 0.49 . In summary, to the best of our knowledge, this is the first study to identify potential markers associated with resistance to AFR in blueberry. We anticipate that additional markers and candidate genes will likely be identified as part of future studies of this important target trait in blueberry. We see the research presented in this manuscript as a stepping stone toward uncovering the underlying mechanism that contributes to anthracnose resistance in fruit of northern high bush blueberry.Farmland covers more than 35% of Earth’s ice-free terrestrial area, and agriculture is expanding and intensifying in many regions to meet the growing demands of human populations . This trend threatens biodiversity and the ecosystem services on which agriculture depends, including crop pollination . Indeed, recent reviews have highlighted how multiple anthropogenic pressures lead to a decline in wild pollinators such as bees, flies, beetles, and butterflies . However, practices to enhance wild pollinators in agroecosystems are still in development , and considerable uncertainty remains regarding their effects on crop yield and farmers’ profits. Here we review recent research on the topic, including the impacts of certain practices on wild pollinators, crop pollination, yield, and profits . We focus on practices that enhance the carrying capacity of habitats for wild insect assemblages that may then provide crop pollination services; practices to conserve or manage a particular pollinator species are outside our scope although they have received attention elsewhere . We offer general science-based advice to land managers and policy makers and highlight knowledge gaps. Throughout, we emphasize the need to consider population-level processes, rather than just short-term behavioral responses of pollinators to floral resources.Plant–pollinator interactions are typically very general, with many pollinators being rewarded with pollen, nectar, or other resources from several plant species , and with most angiosperms being pollinated by multiple insect species . Humans benefit from this generalized nature of pollination systems, as exotic crops brought far from their ancestral ranges can find effective pollinators within native insect assemblages . Accordingly, a synthesis of 600 fields from 41 crop systems showed that only two of the 68 most frequent pollinators globally were specialist species: the weevil Elaeidobius kamerunicus pollinating oil palm and the squash-bee Peponapis pruinosa pollinating pumpkin .Because of differences in species functional traits, greater pollinator richness can lead to foraging complementarity or synergy, improving the quantity and quality of pollination and therefore increasing both the proportion of flowers setting fruits and product quality . Across crop species, insects with contrasting mouth part lengths may be needed for the pollination of flowers not only with easily accessible rewards but also with rewards hidden at the bottom of a tubular corolla . Within a crop species, social and solitary bees visited flowering radish plants at different times of day, suggesting temporal complementarity among these pollinator groups . Flower visiting behavior also differs among pollinators of different body sizes, and visits by a range of differently sized pollinator species increase pumpkin pollination . In addition to functional traits, inter specific differences in response traits to climate and land-use change can increase resilience of pollination services . The role of diverse assemblages of wild insects in crop pollination is also evident from recent global analyses. Worldwide, incomplete and variable animal pollen delivery decreases the growth and stability of yields for pollinator-dependent crops . This lower yield growth has been compensated for by greater land cultivation to sustain production growth .

Although Sitka spruce was the second most important species in sapling regeneration in the affected forests, this species played a less important role in the conifer community composition in subsequent stages of stand development. Spruce has been found to have lower regeneration than western hemlock on disturbed sites but higher survival rates; however, western hemlock regeneration that existed prior to disturbance formed a significant and dominant position in the future stand . In old mortality, the relatively high density of small, dead trees, low density of big, live trees, and high percentage of dead basal area suggest that although the Sitka spruce regenerates on plots less open, the species may be out competed by western hemlock over time. At the fine-scale, species traits can play important and even predictable roles in determining the establishment and growth of individuals under changing site conditions. Our study documented significant losses in the yellow-cedar population but not extirpation. These directional changes likely extend beyond the temporal period of our chronosequence due to the diminished regeneration.

The temporal dynamics of decreased yellow-cedar importance in each tree size class across the chronosequence suggest that yellow-cedar decline may be more likely to affect smaller trees first,blueberry grow pot whereas larger tree mortality occurs in a staggered process. Previous research has shown that surviving yellow-cedar trees in declining stands can produce larger growth rings but with greater inter annual variability after the onset of decline , and that climate thresholds for the survival and reproduction of individuals can vary across life-history stages . Reductions in yellow-cedar sapling occurrence and abundance in forests affected by decline indicate significant, long-term reductions in species abundance across larger size classes. Whether these reductions are caused by seed limitation, changes in seedling germination conditions, herbivory, or other mortality mechanisms is beyond the scope of our study. Although we did not count individuals in the 10–99 cm size class or dead saplings, our seedling and sapling findings indicate reductions in yellow-cedar over time and consistent rank-order of other conifer species competing in sapling stage in the affected forests. Ramage et al. similarly found tanoak unlikely to regenerate successfully in forests affected by sudden oak death, a disease disturbance. Yellow-cedar appear maladapted to forests affected by decline for the foreseeable future. We observed a process of stand development similar to forests affected by host-specific insect or disease disturbance, distinguished by an increase in regeneration while surviving trees release and saplings advance into the over story.

The results of our comparisons of total sapling density across the chronosequence indicate a reinitiation phase that occurs post-decline. We found no significant difference between canopy openness in live forests compared with old mortality, significant increases in canopy openness in recent and midrange mortality, and an overall steady increase in stand density across the chronosequence. These results document stand advancement toward canopy closure over time, indicating development of a relatively mature forest distinguished by changes in conifer community composition. We were unable to date the precise onset of widespread mortality at each plot; our estimates of temporal dynamics of stand re-initiation and advancement are informed by time-sincedeath estimates for snags observed at each plot. Snag class estimates suggest that widespread mortality began 81.4 6 22.0 years ago for the old mortality cedar decline status and indicate approximately 50–100 years for stand advancement toward canopy closure. Consistent with our hypotheses, we found an increase in the importance of western hemlock across all size classes as the importance of yellow-cedar was greatly reduced over time in the affected forests. It has been previously hypothesized that declining yellow-cedar forests on the northern extent of the yellow-cedar population distribution may convert to scrub forest or open bog, as western hemlock is not able to exploit some soil conditions favorable to yellow-cedars . In contrast but consistent with prolific hemlock regeneration found in old-growth hemlock-spruce forests affected by stand-replacing disturbances and minor wind throw disturbance , our results show that western hemlock regenerates vigorously on plots affected by decline, and changes in community composition, as described by IVs, show a turnover to western hemlock-dominated forests.

However, because our sampling frame was restricted to plots with a total live and dead basal area of 35 m2 /ha, our results are not generalizable to dynamics on poorly drained, acidic soils that may be more likely to convert to scrub forests. Chronosequences and associated space-for time substitutions serve as a useful tool for studying temporal dynamics of plant communities that occur across time . Despite critiques as to whether there are predictable links between sites and at what rates characteristics actually change over time , Walker et al. assert that chronosequences are well-suited for studying plant communities that have low biodiversity, rapid species turnover, and low frequencies and severity of disturbance. We applied this methodology to coastal temperate forests with relatively low vascular plant diversity and restricted our sites to those where widespread mortality was evident as a significant disturbance , avoiding wind throw. Based on current understanding of the pathway to decline, varying snow pack conditions were likely the cause of the spatial pattern of mortality observed across the study area ; old mortality occurred in the more southerly plots and live further north . We acknowledge that there may have been confounding factors, such as variability in deer populations, snow conditions, or relative abundance of trees species associated with the spatial distribution, that affected the patterns we observed across the chronosequence. Specifically, seedling and sapling abundance at sites located in GLBA may have been positively affected by geographic location at the northern limit of Sitka black-tailed deer . The observed increase in volume of forage species post-decline may also have a negative feedback on yellow-cedar regeneration, as deer browsing can be a barrier to seedling performance . As there were no affected forests identified in GLBA by aerial survey, plots measured increased our sample size of live forests and extended our chronosequence further north across a relatively limited spatial extent. Our biophysical controls used for site selection helped reduce the likelihood of other factors driving variation in forest structure and community composition among the cedar status categories.Our study provides evidence of a dynamic under story response that occurs as forests become affected by decline. Changes in community composition were elucidated by the increase in Shannon diversity for functional groups and the dissimilarities in functional group abundance across the chronosequence : live forests were primarily differentiated by bryophyte abundance, recent mortality by graminoids, and old mortality by shrubs. We found forests affected by decline can lead to increased forage production over time, potentially increasing deer carrying capacity. As variations in snow-depth are known to effect browse availability ,hydroponic bucket reduced snow pack that triggers tree mortality may also expand winter habitat for deer. An extensive literature on widespread mortality of a single dominant tree species from other types of disturbances indicates that over story loss typically alters under story plant communities . Recent studies on the effects of a species die back associated with climate change on under story vegetation showed: increases in species richness and abundance seven years after a major drought-induced mortality event of Juniperus monosperma ; shifts in community composition with decreases in herbaceous species, cover, and volume; and increases in shrub abundance, cover, and volume over a two-year time period in stands affected by sudden aspen decline .

Typically characterized by frequency, duration, severity, size and spatial pattern, disturbance regimes provide critical information to understand stand formation and subsequent development . Timing and intensity of yellow-cedar mortality plays a critical role in determining plant community responses. Although we observed an increase in the vascular plant taxa identified across the chronosequence, our ability to make inferences to changes in species richness and diversity as forest become affected by decline was limited by taxonomic resolution . We did not identify sedges , grasses , clubmosses , horsetails , and bryophytes at the species level, but the abundance of the associated functional groups differed substantially across the chronosequence. As such, taxonomic richness and diversity for these vascular plant taxa and the bryophytes group would play an important role in overall dynamics of species diversity.Our ability to identify and quantify the trajectories of ecological communities is important for monitoring and maintaining biodiversity, as well as other ecosystem services. Because of the remoteness and inaccessibility of the Archipelago in southeast Alaska, monitoring and active forest management, such as harvesting or planting, may occur only on a small portion of forest experiencing yellow-cedar decline . We documented far-reaching effects of climate change on protected wilderness lands, affirming that areas once set-aside for conservation may be insufficient for species preservation in a changing climate . Given expected climate change in the current century, many vegetation types and individual species may lose representation in protected areas . Effective conservation strategies require understanding the changing plant community dynamics and assessing habitat where species are more likely to survive on both managed and protected lands. Managers and conservation planners operating in other ecosystems may need to consider impacts of climate change on plant communities in protected areas to evaluate broad-scale implications of activities on actively managed lands. Whether this species decline will expand northward into the live forests in Glacier Bay National Park and Preserve is currently unknown; this study’s plots offer opportunities for directly monitoring future changes in affected and unaffected forests to date. The changes observed across the chronosequence can have a range of cascading effects on ecosystem services. Conversion to western-hemlock dominated forests represents a long-term reduction in culturally valued trees and a loss of the cultural services these trees provide. Forests affected by decline may sequester less carbon in the long-term, given western hemlock’s relatively shorter lifespan and wood deterioration rates . Researchers are just beginning to understand the influence of dead cedars on watershed nutrient export . By replacing yellow-cedar trees with the most abundant tree species in the region , yellow cedar decline can lead to a loss of conifer diversity at the landscape level, yet may provide increased forage availability for deer hunted throughout the region. Although our research occurred on protected lands at the northern reaches of yellow-cedar decline, dynamics observed can provide forest managers on the Tongass National Forest with a better understanding of the processes of stand development and conifer species most likely to dominate impacted forests over time. In consideration of salvage activity or thinning on managed lands affected by decline, our results suggest that managers should recognize that western hemlock is more likely to out compete other species, and that favoring spruce or mountain hemlock individuals may help maintain conifer diversity. Further research could evaluate whether forests affected by decline support a greater deer population or if shifting hunting pressures to these areas could have a positive impact on yellow-cedar regeneration. Our study also underscores the importance of maintaining yellow-cedar populations at higher elevations, given the decreased likelihood for yellow-cedar regeneration once low-elevation, coastal forests become affected by the die back.The relationship between diversity and the stability of communities and ecosystems is a fundamental question in theoretical and experimental ecology . Beginning in the 1990s, and sparked by clear experimental results showing that species richness decreased the temporal variance of ecosystem function , biodiversity research contributed to a broader discussion about how worldwide declines in biodiversity would affect ecosystem services on which humans rely . A major goal of biodiversity research has involved separating the relative importance of richness , composition , and abundance as drivers of temporal variance in ES. However, both the study designs and the analytical approaches used vary between experimental and observational studies. At smaller scales, the field of biodiversity-ecosystem function research has used controlled experiments and a well-developed body of mathematical theory to explore how species richness and composition affect temporal variance. In contrast, at larger scales, the field of biodiversity-ecosystem services research has been built mostly on correlative studies conducted in real-world systems —where ‘real-world’ means communities that are not directly manipulated—in which it is difficult to rigorously separate the causal roles of richness, composition and abundance . Because species loss continues to occur at high rates worldwide , it is critical to gain a better understanding of how species richness affects temporal variance of ES. This requires the development of novel analytical approaches that can separate the effects of richness, composition, and abundance without experimental manipulations, which are difficult if not impossible to conduct at landscape scales.

Methylamine and ethylamine, the degradation products of glycine and alanine, are detected in increasingly high abundances with age in ancient sulfate samples up to ~40Ma. The comparison of these concentrations to their parent amino acids allow for the calculation of glycine and alanine decarboxylation rates and are assumed to be indicative of these rates in sulfate matrices. Because sulfate minerals have been detected in high abundance on Mars, these rates are extrapolated to Mars’ average surface temperatures to offer an estimate of the rates that might be expected on Mars. Chapter 4 analyzes modern sulfate samples from Southern Australia for amino acids and extrapolates these concentrations to equivalent bacterial concentrations . The low abundances of methylamine and ethylamine in these modern samples show consistency with previous findings . Chapter 5 focuses on a new analog to the Martian hematite blueberries that have been detected on Mars in the Meridiani Planum region. These ironstones are ubiquitous in San Diego county, and by analyzing samples from various deposits,nft hydroponic system they are dated using amino acid racemization based on the measured D/L-ratios and a sample calibration method. Similar concretions on Mars would show good preservation in such environments despite the fact that they’re iron rich.

The ironstone formation seems to be mediated by enhanced precipitation as well as the possibility of bacterially induced mineralization within these deposits. Chapter 6 investigate an extreme environment deep within a South African subterranean gold mine. These filtrates show extremely low bio-densities and is of interest because it might represent the extremely low levels of biomass and extremely slow turnover times that push the limits of analytical sensitivity. A simple steady-state model corroborates the long turnover times that have been reported for these samples. Two well known Mars analog sites comprise the next two chapters, rock samples from the Antarctic dry valley Deserts and surface soils from the Atacama Desert, Chile , which have been suggested to be the best terrestrial Mars soil analog . The fact that life can persist in these extreme climates is impressive enough, however the extremely cold samples from Antarctica show amazing preservation while the opposite is observed in the Atacama Desert surface and near-surface samples. The inferred cell counts based on total amino acid abundances within cryptoendolithic microbial life agree well with previous bio-density enumerations in similar environments. The Atacama Desert shows extremely degraded organic material at the surface and shows drastic variations in amino acid distributions and diagenetic state as a function of depth and surface micro-environment habitability. One of the premier instruments for advanced in situ Mars life detection experiments is the Urey – Mars Organic and Oxidant detector. Chapter 9 focuses on research and development of the instrument over the past 3 years, specifically the optimization of the extraction system for Mars exploration. Both laboratory and field experiment data is discussed in detail and exhibits efficient extraction of target bio-molecules, specifically amino acids and total organic carbon.

The use of sublimation as a second-stage extraction method for extract purification and concentration is evaluated for recoveries using various mineral matrices.The astrobiological studies have broad implications for the search for life on Mars over the next decade. Two missions will be launched within the next 6 years: NASA’s Mars Science Laboratory and the ESA’s ExoMars . The studies herein validate the importance of targeting amino acids in life detection studies, emphasize their importance as an indicator of bio-density, and demonstrate the potential for sequestration within Mars mineral deposits. Not to be dismissed is the fact that the detection limits of the Urey Mars Organic Detector are many orders of magnitude greater than necessary for the detection of amino acid biomarkers in some of the most uninhabitable places in the world such as the Atacama Desert. Sulfate minerals are highly abundant on Mars and our studies suggest that they can offer enhanced preservation on the Martian surface based on the estimated rates observed for in situ diagenetic reactions.Bacterial bio-density can be quantified by a number of traditional cell staining methods. Many of these methods target intact DNA, such as 4′,6-diamidino-2-phenylindole , acridine orange , or ethidium bromide. These methods target only intact nucleoid containing cells . Other staining methods, such as trypan blue, target cell membranes and will react with both living and dead cells. Cell staining can lead to erroneous bio-density calculation due to human error and interfering medium . Analyses of individual cellular molecular components can be used to accurately determine cell concentrations in natural samples. Two of these methods are cell enumerations based on total adenosine triphosphate and phospholipid fatty acid analyses, and these have been shown to be accurate at determining cell concentrations in natural samples . ATP is a nucleotide and a ubiquitous component of bacterial life while the majority of phospholipids and fatty acids are present as components within microbial cell walls. One unique aspect of PLFA analyses is that they can distinguish between different types of bacteria depending on the distribution of the target molecules. Similarly, any biomarker can be used to quantify bacteria such as amino acids or nucleobases.Quantifying total hydrolyzable amino acids yields accurate determination of the total protein content in bacterial colonies.

Traditional wet chemistry extraction and analytical methods for amino acid quantification involve acid hydrolysis followed by desalting, pre-column derivatization using a fluorescent chiral adduct, separation by reverse-phase high performance liquid chromatography , and quantification by fluorescence detection against standards of known concentration . Most amino acids are stable during traditional acid-hydrolysis methods as only the peptide bonds within proteins are cleaved during this treatment. The exception is tryptophan which is completely destroyed during acid hydrolysis while arginine, tyrosine, threonine, serine, methionine, and cysteine are degraded to a small degree during longer hydrolysis times .These degradation mechanisms involve the conversion of the carboxamide side groups to carboxyl groups through the incorporation of water and liberation of ammonia.Ortho-phthaldialdehyde/N-acetyl-L-cysteine was first used to derivatize amino acids by Aswad and later applied to amino acid quantification in geological samples . The fluorescent derivatizing chiral adduct is made by combiningOPA and NAC into an alkaline borate buffered solution to form a cyclic fluorescent derivative .This fluorescent chiral adduct reacts with primary amines to form fluorescent derivatives . The fluorogen reacts with all primary amines, so it targets all 20 protein amino acids except for proline. Highly specific fluorescence detection is accomplished at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. This highly specific derivatization allows for low interference during quantification.In order to determine the amino acid composition and distribution of a typical bacterial culture,hydroponic nft system and to test the THAA method for cell enumeration, samples of cultured E. coli cells were run through traditional wet chemistry extraction and analytical protocols. This allowed for determination of the most important targets for the search for amino acid bio-signatures derived from bacterial proteins in the study of geological samples and for the purposes of life detection.Cultured E. coli cells were obtained and added to a sterilized crushed serpentine medium. Cell bio-densities were measured by traditional methods on the inoculated sample and a procedural blank growth medium that did not contain E. coli cells. The OD460 of the E. coli growth medium was measured to be 0.65 which corresponded to 6.5 x 109 E. coli cells in 10 ml of LB growth medium with a 5% measurement error. Because physiological variation may changes in cell size, capsule formation, or aggregation, small differences in the conversion between OD and total cell counts may be observed. For this reason, the total number of E. coli cells as determined from the OD reading was independently confirmed by measuring the mass of a solid E. coli pellet generated by overnight growth and centrifugation of a volume of LB medium identical to that used to inoculate the serpentine. If it is assumed that the E. coli cells were homogenously mixed into the 0.5-g crushed serpentine sample, a concentration of 1.3 x 1010 cells/g for the serpentine inoculated with E. coli was inferred. ~200 mg of the inoculated serpentine cell media and a serpentine growth medium blank were hydrolyzed and desalted according to the procedures of Zhao and Bada . The sample was vapor-phase hydrolyzed under 6M doubly-distilled HCl for 24 hours in a flame-sealed test tube after flushing with nitrogen. The hydrolyzed residue was loaded onto an equilibrated desalting column of ~2.5 mL of BioRad AG50W-X8 resin in a pasteur pipette. The sample was rinsed with ~6 column volumes of doubly-distilled water before eluting the amino acid fraction with 3mL of ~3M doubly-distilled ammonium hydroxide .

These fractions were concentrated on a vacuum centrifuge under 60°C heat into 1.5mL mini-eppendorf vials. These residues were resuspended into 100µL of ddH2O for derivatization and analysis by RP-HPLC. The OPA/NAC fluorescent derivative was prepared with chemicals purchased from Sigma. 4 mg of OPA was first dissolved into 300µL of methanol, and 250µL of borate buffer was added to the solution, followed by the addition of 435µL of double-distilled water. The last step is the addition of 15µL of 1M NAC solution . This derivatizing solution has a final concentration of ~0.03M OPA and ~0.015M NAC before they react. The final concentration of the cyclical derivative in the OPA/NAC solution is 0.015M OPA/NAC. The reaction between OPA/NAC and primary amines has been demonstrated to be linear over large concentration ranges for reaction with amino acids and biogenic amines . 10µL aliquots of diluted fractions of the desalted hydrolyzed E. coli extracts and procedural blanks were first dried down on a vacuum centrifuge at room temperature with 10uL of borate buffer to remove any residual ammonia from the NH4OH carried through from the desalting stage. These residues were brought up in 20µL of ddH2O and derivatized for 1 minute with 5µL of the 0.015M OPA/NAC solution. After this pre-column derivatization, the samples were separated by RP-HPLC and quantified with a fluorescence detector. The RP-HPLC setup utilizes an Hitachi L6200 Intelligent HPLC pumps, rheodyne sample injectors, coupled with a Phenomenex Luna-C18 RP-HPLC column and a Shimadzu fluorescence detector . Data sampling and analysis, including automatic and manual Gaussian peak integrations, were performed using Thermo Scientific Grams/AI software. Sample peak intensities were quantified against 100-1000x diluted commercial standards of known concentration , and the trace amounts of D-enantiomers ratio-normalized against racemic laboratory standards of similar concentration. The HPLC conditions included a stationary-phase buffer, 50mM sodium acetate solution with 8% methanol, with methanol as the mobile phase. Two gradient protocols were necessary to resolve the 16 amino acids extracted and purified by these methods . The traditional amino acid separation protocol developed over the last 20 years has been used in a variety of studies including those on natural samples , meteorites , and hydrolyzed bacteria . This RP-HPLC protocol was primarily developed to well separate the primary amino acids present in geological samples as well as their enantiomers. A slower methanol elution gradient was developed that was necessary to resolve the coeluting peaks of glycine and arginine, and to better resolve threonine as a shoulder of glycine and tyrosine from alanine. The trace amounts of D-enantiomers did not interfere with the peak separation as the D/L-enantiomer ratios were too small to be significant. This is expected of any extant bacterial community and the hydrolysis not harsh enough to cause a significant degree of racemization.The OPA/NAC fluorescent derivatizing reagent tags only primary amines, so proline does not react, tryptophan is completely destroyed during hydrolysis in 6N HCl for 24 hours, and asparagine and glutamine degrade to aspartic acid and glutamic acid during acid-catalyzed hydrolyis, respectively. This results in the derivatization of 16 of 20 total protein amino acids.Aspects of this study have been investigated before. Similar procedures were utilized by Glavin et al. to determine the amino acid composition in similarly treated fraction of hydrolyzed E. coli cultures, however they analyzed a more limited set of amino acids during their experiments. More recently, the recoveries of adenine from sublimed E. coli colonies were used to enumerate bacterial bio-density , so this study provides verification of procedures for THAA determination and accurate enumeration of cell bio-density.

Surveys conducted during the first two years of the study throughout the entirety of the six research blocks showed that the prevalence of PD in control vines actually declined slightly from the first to the second year , but not due to an increase in replanting efforts or vine death , Rather, this decline in prevalence likely reflects overwinter recovery of mild cases of the disease . Thus, the observed return of symptoms in most severely pruned vines does not appear to be explained by reinfection with X. fastidiosa after clearing of infection during the severe-pruning process. Our results indicate that the apparent effectiveness of severe pruning depended on the initial disease severity, and the effectiveness weakened over time. This suggests at least two constraints exist regarding the general utility of pruning as a PD management tool. First, severe pruning does not appear to be useful for mild cases of PD, as many of those same vines would recover from the infection over the winter . Second, there appears to be little value in pruning severely diseased vines; the high frequency of symptom return within a few years indicates that even severe pruning does not clear most vines of X. fastidiosa infection.

That leaves a statistically significant window with respect to intermediate severity cases,blueberry grow bag size which may benefit from severe pruning. The apparent benefit for this category of diseased vines would stem from infections that are not so localized that they are highly susceptible to natural recovery over the winter, but also not fully systemic such that the infection has developed below the pruning point . Reliable identification of this narrow class of diseased vines may require substantial experience with PD scouting, detailed record keeping, and an appreciation for variability in symptoms or infection dynamics based on grapevine cultivar and environmental conditions . Research in other bacterial plant pathosystems has evaluated the potential benefit of pruning and whether pruning extent is related to its effectiveness at clearing hosts of infection . A study of the citrus disease huanglongbing, associated with infection by Candidatus Liberibacter spp., evaluated two levels of pruning severity, neither of which showed promise as a disease management tool . In this pathosystem, it is plausible that a very protracted incubation period may undermine the effectiveness of pruning, because by the time the first symptoms are visible, the infection may have already moved throughout much of the tree. Collectively, our results are more similar to a study of citrus variegated chlorosis .

In this study, the presence of X. fastidiosa in plant tissues at different distances from symptomatic leaves was determined for varying levels of disease severity. X. fastidiosa was more widely distributed in trees with severe disease symptoms compared to those with early stage foliar symptoms. Although ColettaFilho et al. did not test whether pruning at various distances proximal to symptomatic leaves would eliminate X. fastidiosa infections, the current recommendation is to prune citrus material if early symptoms are present, and to not prune plants with severe disease symptoms . Citrus plant age is also an important consideration; Coletta-Filho and de Souza recommend that symptomatic citrus trees up to three-years-old be removed rather than pruned, whereas trees four-years-old or older should be pruned. We did not examine vine age as a factor in this study, but the biology of citrus and grape differ in terms of the overwinter recovery that can occur in grape and the apparently slower movement of X. fastidiosa in citrus compared to grape. Anecdotally, the two most mature plots in our study showed the most rapid return of disease, and the youngest plot showed the slowest return. More studies of the effect of vine age are needed before concluding that interactive effects of plant age and pruning differ between the PD and citrus variegated chlorosis pathosystems.The length of lifespan of a particular normal cell of any organism is predetermined. Similarly, the length of lifespan of all organisms is pre-determined by their genetic makeup and their external and internal environments and diet-related factors specific to an organism.

Therefore, the length of lifespan can be increased or decreased by manipulating the environment, diet and genetic factors only by small extent. The length of lifespan of the organisms can also be impacted by differential rates of senescence of cells and organs that ultimately lead to the death of the organisms. The differential rates of cellular senescence are influenced by several confounding factors, such as external and internal environments, diet and genetic factors. Because of these confounding factors that can impact rates of progression of degenerative changes in the organs, it is almost impossible to study aging in the absence of organ pathology. Based on numerous studies on aging in vertebrates and invertebrates, a recent informative review has suggested that oxidative stress theory of aging can only be applied to conditions in which age associated pathologies are included. Furthermore, it was suggested that in environment with minimal stress, oxidative damage plays little role in aging. This suggestion can be argued on the fact that little oxidative damage may take longer time to deregulate protective transcription factors, adaptive responses to stressors, and repair mechanisms, and thereby extending the lifespan of the organisms more than that produced by higher oxidative damage which can deregulate above biological functions in shorter time. Using vertebrate and invertebrate models, some major biochemical and genetic factors that are associated with aging processes have been identified. They include increased oxidative stress and chronic inflammation, decreased adaptive response to stressors, post-translational protein modifications, mitochondrial dysfunction, decreased of proteasome and lysosomal-mediated proteolytic activity, shortening of telomeres and transcriptional deregulation.

Among these, the theory of oxidative stress is most extensively investigated in various experimental models, using pharmacological agents, antioxidants, anti-inflammatory agents and deletion of one or more antioxidant enzymes as well as of mitochondrial complexes. Depending upon the experimental models, experimental designs, and substrate used to assay oxidative stress and criteria of oxidative stress, the role of oxidative stress in aging has been substantiated or questioned. We hypothesized that increased oxidative stress may be one of the primary early events that causes chronic inflammation, transcriptional deregulation, post-translational protein modifications, mitochondrial dysfunction, decreased of proteasome and lysosomal-mediated proteolytic activity and shortening of telomeres. Invertebrate models, such as Caenorhabditis eleganshas been extensively used to evaluate the role of oxidative stress in aging primarily due to shorter lifespan of about 3 days and ease of genetic manipulation. This review analyzes recent published studies on C. elegans on the role of oxidative stress in determining the length of lifespan by generating mutants that show suppression of mitochondrial function or lack of superoxide dismutase . Caenorhabditis elegans has been extensively used to investigate the role of oxidative stress in aging by measuring the length of lifespan. Mitochondria are considered the major sites for the production Reactive oxygen species ,blueberry box although ROS are also produced outside the mitochondria. In order to demonstrate the impact of oxidative stress, several mutants of C. elegans were generated. They include mutations in four clock genes , mutation in the iron sulfur protein of mitochondrial complex III, mutation in the gene NUO-6 and mutation in the gene daf-2. The effects of mutations on oxidative stress and lifespan are summarized in Table 1. The clk-1 gene encodes an enzyme that is necessary for the biosynthesis of ubiquinone that is required by the mitochondria to generate energy. Mutation in the clk-1 gene increases the life span by slowing down mitochondrial activity due to reduced availability of ubiquinone. This slowing of the electron transport chain would reduce oxidative stress. The role of reduced oxidative stress in extending the lifespan is further supported by the fact that overexpression of clk-1 gene in wild-type C. elegans increased mitochondrial activity and shortened the lifespan. A mutation in the iron sulfur protein of mitochondrial complex III causes low oxygen consumption, reduced oxidative stress and increased lifespan. Mutation in the daf-2 gene which codes for a member of insulin receptor family increased lifespan and enhanced resistance to oxidative stress. In this daf-2 mutant, expression of the SOD-3 gene, which encodes mitochondrial Mn-superoxide dismutase, was much higher than in the wild type. This implies that the increased levels of SOD-3 in the daf-2 mutant reduced oxidative stress and thereby increased lifespan. Mutation in the gene NUO-6 which encodes complex I of mitochondria increases life span of C. elegans by decreasing the mitochondrial function. Mutation in the age-1 increased lifespan by two folds. This mutant wormhad increased catalase and Cu/Zn SOD activities which may account for the increased resistance to the paraquat, a superoxide generating chemical.

The mutants C. elegans support the view that the levels of oxidative stress is one of the important determinant factors in determining the length of lifespan.Superoxide anions are produced enzymatically outside the mitochondria by different oxidases and nonenzymatically inside the mitochondria. SOD detoxifies superoxide to hydrogen peroxide , which is converted to water and oxygen by catalase. There are five superoxide dismutase isoforms SOD-1, SOD-2, SOD-3, SOD-4 and SOD-5 in C. elegans. However, in most organisms there are only 3 SODs. SOD-1 is present in the cytoplasm and represents the majority of SOD activity, whereas SOD-2 and SOD-3 are present in mitochondria. Increased levels of superoxide were observed in SOD deleted wild type worms or in ISP-1 and NOU-6 mutants. The effect of deletion of SOD on C. elegans lifespan is shown in Table 2. The impact of SOD deletion on the lifespan appears to be contradictory, depending upon the C. elegans model used. For example, SOD2 deletion markedly increased the lifespan of mutant clk-1 worms, but it decreased the lifespan of mutant isp-1worms. In addition, deletion of individual SOD genes from wild type C. elegans did not decrease the lifespan of these worms. This is in sharp contrast to other model, such as yeast, flies, and mice in which deletion of cytoplasmic or mitochondrial SOD caused decreased in the lifespan. In another study, it was demonstrated that the levels of superoxide were elevated in nou-6 mutant and isp-1 mutant and they lived longer than the wild type, however, the oxidative stress was low and overall levels of ROS did not change. From these results, it was concluded that elevation of superoxide is sufficient to increase the lifespan of these mutant worms. Based on these results, the role of oxidative stress in aging was questioned. It should be pointed out that if superoxide is precursor of ROS, the levels of ROS and oxidative stress should have been increased. This was not observed in the above study, suggesting that reduced oxidative stress possibly due to adaptive response by other antioxidant enzymes, such as catalase and glutathione peroxidase and improved repair mechanisms was responsible for the extension of lifespan of these mutant worms. In the same article, it was observed that addition of N-acetylcysteine and vitamin C individually abolished the effect of superoxide on life extension and other associated changes in nou-6 and isp-1 mutants. The antioxidant effect of NAC is mediated via glutathione, which in the presence of the high superoxide environment of mutant worms can be oxidized and then act as a pro-oxidant. Therefore, observed abolition of the effect of superoxide on life extension could be related to pro-oxidant effects of NAC and vitamin C. In order to assess the role of SOD on life extension further, a model of C. elegans was developed in which all five SODs were deleted, was established. The results showed that SOD 12345 worms were viable and exhibited a normal lifespan similar to that of wild-type despite increased sensitivity to multiple stressors. However, these SOD lacking worms showed reduced fertility, slow development, slower defecation cycle and decreased movement . From these results, it was concluded that SOD is dispensable for normal lifespan of C. elegans. This is in sharp contrast to mammals in which SOD is considered indispensable for survival. Thus, the results obtained on some genetic models of C. elegans cannot readily be extrapolated to the genetic models of mammals. If SOD is dispensable for the survival and lifespan of C. elegans as reported recently, overexpression of SOD should have no impact on the lifespan of these worms. On the contrary, it was reported that over expression of the major cytosolic Cu/Zn-SOD increased lifespan of wild type worms which was not related to reduced lipid oxidation or glycation.