Characterization and RNA interference-mediated silencing of tryptophan 2,3-dioxygenase gene in Carpophilus hemipterus (L.) (Coleoptera: Nitidulidae).

Dried fruit beetle, Carpophilus hemipterus (Linnaeus, 1758) (Coleoptera: Nitidulidae), is a serious pest of ripened fresh fruit in the orchard and dried fruit in postprocessing storage. Despite the economic impact and widespread distribution of C. hemipterus, there is a lack of functional genomics research seeking to elucidate features of molecular physiology for improved pest management. Here, we report the characterization of the gene named Vermilion in C. hemipterus (ChVer) that encodes for tryptophan 2,3-dioxygenase. The Vermilion is frequently used as a visual marker for genomics approaches as tryptophan 2,3-dioxygenase is involved in the biosynthesis of eye coloration pigments in insects. We identified 1628 bp long full-length transcript of ChVer from transcriptomic database of C. hemipterus. The expression analysis among adult body parts revealed peak ChVer expression in head compared to thorax and abdomen, which is consistent with its role. Among the C. hemipterus developmental stages, peak ChVer expression was observed in first instar larva, second instar larva, and adult male stages, whereas the lowest levels of expression were seen in third instar larva, prepupa, and pupa. The nanoinjection of ChVer double-stranded RNA in larval C. hemipterus resulted in a significant reduction in ChVer transcript levels as well as caused a loss of eye color, that is, the white-eyed phenotype in adults. Characterization of visually traceable marker gene and robust RNA interference response seen in this study will enable genomics research is this important pest.

Confirmatory evaluation of eFUME for control of brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae).

The governments of Australia and New Zealand require a phytosanitary treatment to control adult brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), as overwintering aggregations have been intercepted in the importation pathway of various nonhorticultural consignments, including vehicles. The commercialized fumigant, eFUME, a 16.7% by mass dilution of ethyl formate in carbon dioxide, resulted in complete control of ca. 1,000 field-collected, naturally diapausing adult H. halys in each of 3 independent trials at 10 ±â€…0.5 °C (x¯ ± 2s) when ethyl formate levels in enclosure headspace were maintained steady-state at ca. 14.5 mg/liter for 4 h to yield Ct exposures ranging from 57.9 to 63.1 mg/liter h. Consistent with previous findings where greenhouse reared H. halys were controlled using laboratory formulations of this ethyl formate-carbon dioxide mixture, these confirmatory methods and results further inform technical and operational features of commercial practice.

Ethyl formate dilution in carbon dioxide for fumigation control of the brown marmorated stink bug Halyomorpha halys, Stål (Hemiptera: Pentatomidae).

BACKGROUND: The brown marmorated stink bug (BMSB), Halyomorpha halys, has caused significant agricultural damage to numerous hosts, so agricultural producers seek to limit its spread. Where established, BMSB can also cause substantial urban and commercial disturbance, as overwintering adults may seek refuge inside dwellings, covered spaces, vehicles, and consignments. Phytosanitary authorities are most concerned with the importation of 'hitchhiking' adults in this refugia, with certain countries requiring a quarantine treatment to mitigate risk. This study explores fumigation with ethyl formate, applied as 16.7% by mass dilution in carbon dioxide, for control of adult BMSB. RESULTS: The induction of diapause, to simulate overwintering physiology, resulted in 2- and 3-fold increases in the tolerance of adults toward this ethyl formate fumigation at 10 ± 0.5 °C ( x ¯ ± 2 s ) lasting for 8 and 12 h, respectively. However, a decreased tolerance (0.7-fold) of diapausing specimens was observed for a 4-h duration. Diapausing and nondiapausing adult BMSB can be controlled at the probit 9 level if the headspace concentration of ethyl formate, [EF], in the carbon dioxide mixture is maintained ≥7.68 mg L-1 for 12 h at 10 ± 0.5 °C ( x ¯ ± 2 s ). If the duration is shortened to 4 h, [EF] must be maintained ≥14.73 mg L-1 over the course of fumigation. CONCLUSION: The toxicity of ethyl formate in this mixture can be distinct for different physiological states of the same life stage, as evidenced by a ca. 3-fold increase in the Haber's z parameter for adult BMSB when in diapause. Respective to the physiological state of adults, this study identifies how the applied dose and/or treatment duration can be modulated (i.e. tuned) to ensure adequate toxicological efficacy toward BMSB infesting hosts or refuge at temperatures ca. >10 °C. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

A sorghum genome-wide association study (GWAS) identifies a WRKY transcription factor as a candidate gene underlying sugarcane aphid (Melanaphis sacchari) resistance.

MAIN CONCLUSION: A WRKY transcription factor identified through forward genetics is associated with sorghum resistance to the sugarcane aphid and through heterologous expression reduces aphid populations in multiple plant species. Crop plant resistance to insect pests is based on genetically encoded traits which often display variability across diverse germplasm. In a comparatively recent event, a predominant sugarcane aphid (SCA: Melanaphis sacchari) biotype has become a significant agronomic pest of grain sorghum (Sorghum bicolor). To uncover candidate genes underlying SCA resistance, we used a forward genetics approach combining the genetic diversity present in the Sorghum Association Panel (SAP) and the Bioenergy Association Panel (BAP) for a genome-wide association study, employing an established SCA damage rating. One major association was found on Chromosome 9 within the WRKY transcription factor 86 (SbWRKY86). Transcripts encoding SbWRKY86 were previously identified as upregulated in SCA-resistant germplasm and the syntenic ortholog in maize accumulates following Rhopalosiphum maidis infestation. Analyses of SbWRKY86 transcripts displayed patterns of increased SCA-elicited accumulation in additional SCA-resistant sorghum lines. Heterologous expression of SbWRKY86 in both tobacco (Nicotiana benthamiana) and Arabidopsis resulted in reduced population growth of green peach aphid (Myzus persicae). Comparative RNA-Seq analyses of Arabidopsis lines expressing 35S:SbWRKY86-YFP identified changes in expression for a small network of genes associated with carbon-nitrogen metabolism and callose deposition, both contributing factors to defense against aphids. As a test of altered plant responses, 35S:SbWRKY86-YFP Arabidopsis lines were activated using the flagellin epitope elicitor, flg22, and displayed significant increases in callose deposition. Our findings indicate that both heterologous and increased native expression of the transcription factor SbWRKY86 contributes to reduced aphid levels in diverse plant models.

Greenhouse Rearing Methods for Brown Marmorated Stink Bug (Hemiptera: Pentatomidae) on Live Cowpea Plants.

Brown marmorated stink bug (BMSB), Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is a pest of concern that must be controlled for market access of host material and regulated articles to certain countries. This work outlines a rearing system for BMSB on live cowpea plants, Vigna unguiculata (L.) Walp. (Fabales: Fabaceae), including methods to induce adults to both enter and exit diapause. This scalable system affords continuous access to >600 specimens per week of each life stage and/or age group, which is particularly advantageous when developing treatment efficacy data.

Management of Xanthomonas fragariae with Pre- and Postharvest Treatments to Overcome Trade Barriers for California Strawberries.

Xanthomonas fragariae, the causal agent of angular leaf spot (ALS) of strawberry, is a quarantine pathogen in some export markets, causing trade restrictions and economic loss to the California fresh-market strawberry industry. Preharvest chemical management options are limited to copper, and there are no postharvest treatments available that reduce populations of the pathogen if ALS is detected at an export destination. Here, we report high preharvest efficacy for the experimental bactericide amino thiadiazole and the commercial product zinc thiadiazole, alone and in mixtures with low rates of copper or the antibiotic kasugamycin, with average disease incidence reduction of up to 92.8% compared with the control. Although effective against quarantine insect pests of strawberry, postharvest methyl bromide fumigation was ineffective against X. fragariae in diseased plant tissue at a standard commercial rate. Postharvest propylene oxide fumigation, used for decades by the California nut industries for insect and microbial disinfestation, significantly reduced X. fragariae populations in infected leaflet tissues by at least 2.5-log compared with controls at a dose of ≥142 µg/ml for 2 h at 15 to 20°C. Fumigated leaflets showed little to no phytotoxicity at effective rates, and fumigated fruit were not significantly affected in appearance or susceptibility to postharvest gray mold or Rhizopus rot following storage at 2°C for 3 days and at 15°C for an additional 5 days. Together, these new treatments offer potential strategies for establishing a systems approach with preharvest treatments significantly reducing the risk of ALS on plants and, in response to quarantine detections, a postharvest fumigation treatment that reduces viable pathogen populations in existing lesions.

Predicting Larval Stage Distribution of Lobesia botrana (Lepidoptera: Tortricidae) at Three Constant Temperatures.

European grapevine moth, Lobesia botrana (Denis and Schiffermüller), is an invasive pest species subject to regulatory control under the International Plant Protection Convention. An extensive infestation of this insect detected in California vineyards in October 2009 resulted in the initiation of postharvest research intended to ensure continuing trade of California table grapes by preventing the unintentional introduction of L. botrana to uninfested export destinations. Having the ability to accurately predict and confirm the particular stadia of larval development of insects obscured from direct observations, such as those being mass-reared in diet for use in phytosanitary control experiments, is necessary to be able to ensure that the most tolerant life stage is accurately targeted. Lobesia botrana were reared from egg to eclosion at three different temperatures: 16, 20, and 24°C (60% RH; 16:8 [L:D] h). Daily samples (≥15) were taken of larvae and pupae to determine the chronological distribution of life stages until daily samples comprised 100% pupae, after which only adults were counted. Head capsule measurements of all sampled larvae were taken (n = 2,820) and analyzed using nonlinear least squares (NLLS) fitting to Gaussian curves to describe stadia-specific head capsule demarcations and the probability of misclassifying instars due to overlapping life stage distributions.

Sublethal exposure, insecticide resistance, and community stress.

Insecticides are an invaluable pest management tool and anthropogenic stressors of widespread environmental occurrence that are subject to biased perceptions based on the targeted application, market value of use, and regulatory requirements. As a result, short-term and simplistic efforts focusing on lethal effects toward individual species and populations prevail. Holistic and comprehensive studies exploring rather common sublethal insecticide exposures are rare, particularly considering their potential role in structuring populations and communities in diverse environmental settings and potentially interfering in a range of ecological interactions. Studies on insecticide resistance, for example, do not go beyond population-based studies, disregarding temporal and spatial effects in the associated community, and rarely considering the whole of sublethal exposure. Some of these knowledge gaps are here recognized and explored.

Development of an Activated Carbon-Based Electrode for the Capture and Rapid Electrolytic Reductive Debromination of Methyl Bromide from Postharvest Fumigations.

Due to concerns surrounding its ozone depletion potential, there is a need for technologies to capture and destroy methyl bromide (CH3Br) emissions from postharvest fumigations applied to control agricultural pests. Previously, we described a system in which CH3Br fumes vented from fumigation chambers could be captured by granular activated carbon (GAC). The GAC was converted to a cathode by submergence in a high ionic strength solution and connection to the electrical grid, resulting in reductive debromination of the sorbed CH3Br. The GAC bed was drained and dried for reuse to capture and destroy CH3Br fumes from the next fumigation. However, the loose GAC particles and slow kinetics of this primitive electrode necessitated improvements. Here, we report the development of a cathode containing a thin layer of small GAC particles coating carbon cloth as a current distributor. Combining the high sorption potential of GAC for CH3Br with the conductivity of the carbon cloth current distributor, the cathode significantly lowered the total cell resistance and achieved 96% reductive debromination of CH3Br sorbed at 30% by weight to the GAC within 15 h at -1 V applied potential vs standard hydrogen electrode, a time scale and efficiency suitable for postharvest fumigations. The cathode exhibited stable performance over 50 CH3Br capture and destruction cycles. Initial cost estimates indicate that this technique could treat CH3Br fumes at ∼$5/kg, roughly one-third of the cost of current alternatives.

Quantifying Residues from Postharvest Propylene Oxide Fumigation of Almonds and Walnuts.

A novel analytical approach involving solvent extraction with methyl tert-butyl ether (MTBE) followed by GC was developed to quantify residues that result from the postharvest fumigation of almonds and walnuts with propylene oxide (PPO). Verification and quantification of PPO, propylene chlorohydrin (PCH) [1-chloropropan-2-ol (PCH-1) and 2-chloropropan-1-ol (PCH-2)], and propylene bromohydrin (PBH) [1-bromopropan-2-ol (PBH-1) and 2-bromopropan-1-ol (PBH-2)] was accomplished with a combination of electron impact ionization MS (EIMS), negative ion chemical ionization MS (NCIMS), and electron capture detection (ECD). Respective GC/EIMS LOQs for PPO, PCH-1, PCH-2, PBH-1, and PBH-2 in MTBE extracts were [ppm (µg/g nut)] 0.9, 2.1, 2.5, 30.3, and 50.0 for almonds and 0.8, 2.2, 2.02, 41.6, and 45.7 for walnuts. Relative to GC/EIMS, GC-ECD analyses resulted in no detection of PPO, similar detector responses for PCH isomers, and >100-fold more sensitive detection of PBH isomers. NCIMS did not enhance detection of PBH isomers relative to EIMS and was, respectively, approximately 20-, 5-, and 10-fold less sensitive to PPO, PCH-1, and PCH-2. MTBE extraction efficiencies were >90% for all analytes. The 10-fold concentration of MTBE extracts yielded recoveries of 85-105% for the PBH isomers and a concomitant decrease in LODs and LOQs across detector types. The recoveries of PCH isomers and PPO in the MTBE concentrate were relatively low (approximately 50 to 75%), which confound improvements in LODs and LOQs regardless of detector type.

Destruction of methyl bromide sorbed to activated carbon by thiosulfate or electrolysis.

Methyl bromide (CH3Br) is widely used as a fumigant for postharvest and quarantine applications for agricultural products at port facilities due to the short treatment period required, but it is vented from fumigation chambers to the atmosphere after its use. Due to the potential contributions of CH3Br to stratospheric ozone depletion, technologies for the capture and degradation of the CH3Br are needed to enable its continued use. Although granular activated carbon (GAC) has been used for CH3Br capture and thiosulfate has been used for destruction of CH3Br in aqueous solution, this research explored techniques for direct destruction of CH3Br sorbed to GAC. Submerging the GAC in an aqueous thiosulfate solution achieved debromination of CH3Br while sorbed to the GAC, but it required molar concentrations of thiosulfate because of the high CH3Br loading and produced substantial concentrations of methyl thiosulfate. Submergence of the GAC in water and use of the GAC as the cathode of an electrolysis unit also debrominated sorbed CH3Br. The reaction appeared to involve a one-electron transfer, producing methyl radicals that incorporated into the GAC. Destruction rates increased with decreasing applied voltage down to ∼-1.2 V vs the standard hydrogen electrode. Cycling experiments conducted at -0.77 V indicated that >80% debromination of CH3Br was achieved over ∼ 30 h with ∼ 100% Coulombic efficiency. Sorptive capacity and degradation efficiency were maintained over at least 3 cycles. Capture of CH3Br fumes from fumigation chambers onto GAC, and electrolytic destruction of the sorbed CH3Br could mitigate the negative impacts of CH3Br usage pending the development of suitable replacement fumigants.

Activated carbons from end-products of tree nut and tree fruit production as sorbents for removing methyl bromide in ventilation effluent following postharvest chamber fumigation.

End-products of tree nuts and tree fruits grown in California, USA were evaluated for the ability to remove methyl bromide (MB) from ventilation effluent following postharvest chamber fumigation. Activated carbon sorbents from walnut and almond shells as well as peach and prune pits were prepared using different methods of pyrolysis, activation, and quenching. Each source and preparation was evaluated for yield from starting material (%, m/m) and performance on tests where MB-containing airstreams were directed through a columnar bed of the activated carbon in an experimental apparatus, termed a parallel adsorbent column tester, which was constructed as a scaled-down model of a chamber ventilation system. We report the number of doses needed to first observe the breakthrough of MB downstream of the bed and the capacity of the activated carbon for MB (%, m/m) based on a fractional percentage of MB mass sorbed at breakthrough relative to mass of the bed prior to testing. Results were based on a novel application of solid-phase microextraction with time-weighted averaging sampling of MB concentration in airstreams, which was quantitative across the range of fumigation-relevant conditions and statistically unaffected by relative humidity. Activated carbons from prune pits, prepared either by steam activation or carbon dioxide activation coupled to water quenching, received the greatest number of doses prior to breakthrough and had the highest capacity, approximately 12-14%, outperforming a commercially marketed activated carbon derived from coconut shells. Experimental evidence is presented that links discrepancy in performance to the relative potential for activated carbons to preferentially sorb water vapor relative to MB.

Effects of phosphine fumigation on survivorship of Epiphyas postvittana (Lepidoptera: Tortricidae) eggs.

Light brown apple moth, Epiphyas postvittana (Walker), eggs were subjected to phosphine fumigations under normal atmospheric and elevated oxygen levels in laboratory-scale chamber experiments to compare their susceptibilities to the two different fumigation methods. In fumigations conducted under atmospheric oxygen at 5 and 10 degrees C, egg survivorship decreased with increase in phosphine concentration but then increased at a concentration of 3,000 ppm; this increase was significant at 10 degrees C. Based on egg survivorship data, phosphine fumigations conducted in a 60% oxygen atmosphere were significantly more effective than those conducted under atmospheric oxygen conditions. Oxygenated phosphine fumigations at 5 and 10 degrees C killed all 1,998 and 2,213 E. postvittana eggs treated, respectively, after 72 h of exposure. These results indicate the great potential of oxygenated phosphine fumigation for the control of E. postvittana eggs.

Postharvest treatment of fresh fruit from California with methyl bromide for control of light brown apple moth (Lepidoptera: Tortricidae).

Methyl bromide (MB) chamber fumigations were evaluated for postharvest control of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in fresh fruit destined for export from California. To simulate external feeding, larvae were contained in gas-permeable cages and distributed throughout loads of peaches, plums, nectarines (all Prunus spp.), apples (Malus spp.), raspberries (Rubus spp.), or grapes (Vitis spp.). Varying the applied MB dose and the differential sorption of MB by the loads resulted in a range of exposures, expressed as concentration x time cross products (CTs) that were verified by gas-chromatographic quantification of MB in chamber headspace over the course of each fumigation. CTs > or = 60 and > or = 72 mg liter(-1) h at 10.0 +/- 0.5 and 15.6 +/- 0.5 degrees C (x +/- s, average +/- SD), respectively, yielded complete mortality of approximately 6,200 larvae at each temperature. These confirmatory fumigations corroborate E. postvittana mortality data for the first time in relation to measured MB exposures and collectively comprise the largest number of larval specimens tested to date. In addition, akinetic model of MB sorption was developed for the quarantine fumigation of fresh fruit based on the measurement of exposures and how they varied across the fumigation trials. The model describes how to manipulate the applied MB dose, the load factor, and the load geometry for different types of packaged fresh fruit so that the resultant exposure is adequate for insect control.

Quantifying host potentials: indexing postharvest fresh fruits for spotted wing Drosophila, Drosophila suzukii.

Novel methodology is presented for indexing the relative potential of hosts to function as resources. A Host Potential Index (HPI) was developed as a practical framework to express relative host potential based on combining results from one or more independent studies, such as those examining host selection, utilization, and physiological development of the organism resourcing the host. Several aspects of the HPI are addressed including: 1) model derivation; 2) influence of experimental design on establishing host rankings for a study type (no choice, two-choice, and multiple-choice); and, 3) variable selection and weighting associated with combining multiple studies. To demonstrate application of the HPI, results from the interactions of spotted wing drosophila (SWD), Drosophila suzukii Matsumura (Diptera: Drosophilidae), with seven "reported" hosts (blackberries, blueberries, sweet cherries, table grapes, peaches, raspberries, and strawberries) in a postharvest scenario were analyzed. Four aspects of SWD-host interaction were examined: attraction to host volatiles; population-level oviposition performance; individual-level oviposition performance; and key developmental factors. Application of HPI methodology indicated that raspberries ( (mean)HPIvaried  = 301.9±8.39; rank 1 of 7) have the greatest potential to serve as a postharvest host for SWD relative to the other fruit hosts, with grapes ( (mean)HPIvaried  = 232.4±3.21; rank 7 of 7) having the least potential.

Quantification, localization, and speciation of selenium in seeds of canola and two mustard species compared to seed-meals produced by hydraulic press.

Brassica plants accumulate selenium (Se) especially in seeds when grown in soils laden with Se. We report a chemical analysis of Se in Brassica seeds (canola, Indian mustard, and white mustard) and in their hydraulically pressed seed meals, which are used as a Se supplement in livestock animal feeds. Complementary techniques were used to measure total Se concentrations, to map the localization of Se, and to quantify different Se forms. Seeds and hydraulically pressed seed meals contained an average of 1.8 and 2.0 µg Se g(-1) DW, respectively. Selenium was primarily located in cotyledons and roots of seed embryos. Microfocused Se K-edge XANES and bulk XANES showed that seeds contained 90% of Se as C-Se-C forms. Hydraulically pressing seeds for oil caused changes in the forms of Se as follows: 40-55% C-Se-C forms, 33-42% selenocystine, 5-12% selenocysteine, and 11-14% trimethylselenonium ion. Aqueous extracts of seed and seed meals were also analyzed by SAX-HPLC/ICPMS and found to contain mainly the C-Se-C form SeMet, but also another C-Se-C form MeSeCys, which is of dietary pharmacological interest for cancer inhibition. In addition, SAX-HPLC/ICPMS also detected selenocystine and selenocysteine, further confirming the results obtained by XANES analyses.

Remediation of fungicide residues on fresh produce by use of gaseous ozone.

Ozone fumigation was explored as a means for degrading organic fungicide residues on fresh produce. Fungicides sorbed onto model abiotic glass surfaces or onto grape berries were fumigated separately in a flow-through chamber. Gaseous ozone at a constant concentration of 150 ± 10 ppmv (µL·L(-1)) selectively oxidized fungicides sorbed to model surfaces. Over 140 min, boscalid and iprodione levels did not change significantly based on a single-factor analysis of variance (ANOVA) at the 95% level of confidence (p = 0.05); however, pseudo-first-order losses resulted in observable rate constants of ozonolysis, k(ozonolysis) (min(-1)), of 0.0233 ± 0.0029 (t(1/2) ≈ 29.7 min), 0.0168 ± 0.0028 (t(1/2) ≈ 41.3 min), and 0.0127 ± 0.0010 (t(1/2) ≈ 54.6 min) for fenhexamid, cyprodinil, and pyrimethanil, respectively. The relative degradation of fungicides on berries at gaseous ozone concentrations of 900 ± 12 ppmv (µL·L(-1)) over 2 h was similar to that on glass; decreases in residue concentration were observed for only fenhexamid (∼ 64%), cyprodinil (∼ 38%), and pyrimethanil (∼ 35%) with corresponding k(ozonolysis) (min(-1)) of 0.0085 ± 0.0021 (t(1/2) ≈ 81.5 min), 0.0039 ± 0.0008 (t(1/2) ≈ 177.7 min), and 0.0036 ± 0.0007 (t(1/2) ≈ 192.5 min). Heterogeneous rate constants of gaseous ozone reacting with a sorbed fungicide, k(O(3)) (M(-1)·min(-1)), were calculated for both surfaces and indicate losses proceed ∼ 15-fold slower on grapes. The kinetics and mechanism of fungicide removal, supported by gas chromatography- and liquid chromatography-mass spectrometry product analyses, is discussed in the context of facilitating compliance with maximum residue level (MRL) tolerances for fresh produce.

Selenium accumulation, distribution, and speciation in spineless prickly pear cactus: a drought- and salt-tolerant, selenium-enriched nutraceutical fruit crop for biofortified foods.

The organ-specific accumulation, spatial distribution, and chemical speciation of selenium (Se) were previously unknown for any species of cactus. We investigated Se in Opuntia ficus-indica using inductively coupled plasma mass spectrometry, microfocused x-ray fluorescence elemental and chemical mapping (µXRF), Se K-edge x-ray absorption near-edge structure (XANES) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS). µXRF showed Se concentrated inside small conic, vestigial leaves (cladode tips), the cladode vasculature, and the seed embryos. Se K-edge XANES demonstrated that approximately 96% of total Se in cladode, fruit juice, fruit pulp, and seed is carbon-Se-carbon (C-Se-C). Micro and bulk XANES analysis showed that cladode tips contained both selenate and C-Se-C forms. Inductively coupled plasma mass spectrometry quantification of Se in high-performance liquid chromatography fractions followed by LC-MS structural identification showed selenocystathionine-to-selenomethionine (SeMet) ratios of 75:25, 71:29, and 32:68, respectively in cladode, fruit, and seed. Enzymatic digestions and subsequent analysis confirmed that Se was mainly present in a "free" nonproteinaceous form inside cladode and fruit, while in the seed, Se was incorporated into proteins associated with lipids. µXRF chemical mapping illuminated the specific location of Se reduction and assimilation from selenate accumulated in the cladode tips into the two LC-MS-identified C-Se-C forms before they were transported into the cladode mesophyll. We conclude that Opuntia is a secondary Se-accumulating plant whose fruit and cladode contain mostly free selenocystathionine and SeMet, while seeds contain mainly SeMet in protein. When eaten, the organic Se forms in Opuntia fruit, cladode, and seed may improve health, increase Se mineral nutrition, and help prevent multiple human cancers.

Quaternary amines as nitrosamine precursors: a role for consumer products?

Nitrosamine formation has been associated with wastewater-impacted waters, but specific precursors within wastewater effluents have not been identified. Experiments indicated that nitrosamines form in low yields from quaternary amines, and that the nitrosamines form from the quaternary amines themselves, not just lower order amine impurities. Polymeric and benzylated quaternary amines were more potent precursors than monomeric quaternary alkylamines. Pretreatment of quaternary amines with ozone or free chlorine, which deactivate lower order amine impurities, did not significantly reduce nitrosamine formation. The nitrosamine formation pathway is unclear but experiments indicated that transformation of quaternary amines to lower order amine precursors via Hofmann elimination was not involved. Experiments suggest that the pathway may involve quaternary amine degradation by amidogen or chloramino radicals formed from chloramines. Quaternary amines are significant constituents of consumer products, including shampoos, detergents, and fabric softeners. Although quaternary amines may be removed by sedimentation during wastewater treatment, their importance should be evaluated on a case-by-case basis. The high loadings from consumer products may enable the portion not removed to serve as precursors.

Cell stimulation with optically manipulated microsources.

Molecular gradients are important for various biological processes including the polarization of tissues and cells during embryogenesis and chemotaxis. Investigations of these phenomena require control over the chemical microenvironment of cells. We present a technique to set up molecular concentration patterns that are chemically, spatially and temporally flexible. Our strategy uses optically manipulated microsources, which steadily release molecules. Our technique enables the control of molecular concentrations over length scales down to about 1 microm and timescales from fractions of a second to an hour. We demonstrate this technique by manipulating the motility of single human neutrophils. We induced directed cell polarization and migration with microsources loaded with the chemoattractant formyl-methionine-leucine-phenylalanine. Furthermore, we triggered highly localized retraction of lamellipodia and redirection of polarization and migration with microsources releasing cytochalasin D, an inhibitor of actin polymerization.