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Argentine ants, Linepithema humile (Mayr) (Hymenoptera: Formicidae), form mutualisms with hemipteran pests in crop systems. In vineyards, they feed on honeydew produced by mealybugs and soft scales, which they tend and protect from natural enemies. Few options for controlling Argentine ants are available; one of the more effective approaches is to use liquid baits containing a low dose of an insecticide. Knowledge of ant foraging patterns is required to estimate how many bait stations to deploy per unit area. To measure how far ants move liquid bait in vineyards, we placed bait stations containing sugar water and a protein marker in plots for 6 d, and then collected ants along transects extending away from bait stations. The ants moved an average of 16.08 m and 12.21 m from bait stations in the first and second years of the study, respectively. Marked ants were found up to 63 m from bait stations; however, proportions of marked ants decreased exponentially as distance from the bait station increased. Results indicate that Argentine ants generally forage at distances <36 m in California vineyards, thus suggesting that insecticide bait stations must be deployed at intervals of 36 m or less to control ants. We found no effect of insecticide on distances that ants moved the liquid bait, but this may have been because bait station densities were too low to affect the high numbers of Argentine ants that were present at the study sites.

To mitigate the effects of heat and drought stress, a better understanding of the genetic control of physiological responses to these environmental conditions is needed. To this end, we evaluated an upland cotton ( Gossypium hirsutum L.) mapping population under water-limited and well-watered conditions in a hot, arid environment. The elemental concentrations (ionome) of seed samples from the population were profiled in addition to those of soil samples taken from throughout the field site to better model environmental variation. The elements profiled in seeds exhibited moderate to high heritabilities, as well as strong phenotypic and genotypic correlations between elements that were not altered by the imposed irrigation regimes. Quantitative trait loci (QTL) mapping results from a Bayesian classification method identified multiple genomic regions where QTL for individual elements colocalized, suggesting that genetic control of the ionome is highly interrelated. To more fully explore this genetic architecture, multivariate QTL mapping was implemented among groups of biochemically related elements. This analysis revealed both additional and pleiotropic QTL responsible for coordinated control of phenotypic variation for elemental accumulation. Machine learning algorithms that utilized only ionomic data predicted the irrigation regime under which genotypes were evaluated with very high accuracy. Taken together, these results demonstrate the extent to which the seed ionome is genetically interrelated and predictive of plant physiological responses to adverse environmental conditions., (Copyright © 2018 Pauli et al.)

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. In some previously studied strains of three major lepidopteran pests, resistance to Bt toxin Cry1Ac is associated with mutations disrupting the extracellular or cytoplasmic domains of cadherin proteins that bind Cry1Ac in the midgut of susceptible larvae. Here we report the first case of a cadherin transmembrane mutation associated with insect resistance to Bt. We discovered this mutation in a strain of the devastating global cotton pest, the pink bollworm (Pectinophora gossypiella), derived from a field population in the Yangtze River Valley of China. The mutant allele analyzed here has a 207 base pair deletion and encodes a cadherin protein lacking its transmembrane domain. Relative to a susceptible strain, a strain homozygous for this allele had 220-fold resistance to Cry1Ac and 2.1-fold cross-resistance to Cry2Ab. On transgenic cotton plants producing Cry1Ac, no susceptible larvae survived, but the resistant strain completed its life cycle. Inheritance of resistance to Cry1Ac was autosomal, recessive and tightly linked with the cadherin gene. Transportation of cadherin protein to the cell membrane and susceptibility to Cry1Ac occurred in transfected insect cells expressing the wild type cadherin allele, but not in transfected insect cells expressing the mutant cadherin allele. The results imply that the mutant allele analyzed here confers resistance to Cry1Ac by disrupting cellular trafficking of cadherin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A study was conducted that compared the effectiveness of a sweepnet versus a vacuum suction device for collecting arthropods in cotton. The study differs from previous research in that body-mounted action cameras (B-MACs) were used to record the activity of the person conducting the arthropod collections. The videos produced by the B-MACs were then analyzed with behavioral event recording software to quantify various aspects of the sampling process. The sampler's speed and the number of sampling sweeps or vacuum suctions taken over a fixed distance (12.2 m) of cotton were two of the more significant sampling characteristics quantified for each method. The arthropod counts obtained, combined with the analyses of the videos, enabled us to estimate arthropod sampling efficiency for each technique based on fixed distance, time, and sample unit measurements. Data revealed that the vacuuming was the most precise method for collecting arthropods in the relatively small cotton research plots. However, data also indicates that the sweepnet method would be more efficient for collecting most of the cotton-dwelling arthropod taxa, especially if the sampler could continuously sweep for at least 1 min or ≥80 m (e.g., in larger research plots). The B-MACs are inexpensive and non-cumbersome, the video images generated are outstanding, and they can be archived to provide permanent documentation of a research project. The methods described here could be useful for other types of field-based research to enhance data collection efficiency.

In most moth species, including Mamestra brassicae, pheromone biosynthesis activating neuropeptide (PBAN) regulates pheromone production. Generally, PBAN acts directly on the pheromone gland (PG) cells via its specific G protein-coupled receptor (i.e. PBANR) with Ca 2+ as a second messenger. In this study, we identified cDNAs encoding three variants (A, B and C) of the M. brassicae PBANR (Mambr-PBANR). The full-length coding sequences were transiently expressed in cultured Trichoplusia ni cells and Sf9 cells for functional characterization. All three isoforms dose-dependently mobilized extracellular Ca 2+ in response to PBAN analogs with Mambr-PBANR-C exhibiting the greatest sensitivity. Fluorescent confocal microscopy imaging studies demonstrated binding of a rhodamine red-labeled ligand (RR10CPBAN) to all three Mambr-PBANR isoforms. RR10CPBAN binding did not trigger ligand-induced internalization in cells expressing PBANR-A, but did in cells expressing the PBANR-B and -C isoforms. Furthermore, activation of the PBANR-B and -C isoforms with the 18 amino acid Mambr-pheromonotropin resulted in co-localization with a Drosophila melanogaster arrestin homolog (Kurtz), whereas stimulation with an unrelated peptide had no effect. PCR-based profiling of the three transcripts revealed a basal level of expression throughout development with a dramatic increase in PG transcripts from the day of adult emergence with PBANR-C being the most abundant., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Drought is the No. 1 factor that limits agricultural production in the world, thus, making crops more drought tolerant is a major goal in agriculture. Many genes with functions in abiotic stress tolerance were identified, and overexpression of these genes confers increased drought tolerance in transgenic plants. The isopentenyltransferase gene (IPT) that encodes a rate limiting enzyme in cytokinin biosynthesis is one of them. Interestingly, when IPT-transgenic cotton was field-tested at two different sites, Texas and Arizona, different results were obtained. To explain this phenomenon, reduced irrigation experiments with different timing in applying water deficit stress were conducted. It was found that the timing of water deficit stress is critical for IPT-transgenic cotton to display its yield advantage over control plants (i.e. wild-type and segregated non-transgenic plants). If water deficit stress occurs before flowering (vegetative phase), IPT-transgenic cotton would outperform control plants; however, if water deficit stress occurs at or after flowering (reproductive phase), there would not be a yield difference between IPT-transgenic and control cotton plants. This result suggests that an early induction of IPT expression (before first flowering) is needed in order to realize the benefits of IPT-expression in transgenic plants that face water-deficit stress later in development.

Cabbage looper, Trichoplusia ni (Hübner) is an important lepidopteran pest on many vegetable and greenhouse crops, and some field crops. Although there are no commercial transgenic Bt vegetable or greenhouse crops, T. ni is a target of Bollgard II cotton, which produces Cry1Ac and Cry2Ab. We expand on previous work that examined the effect of Bt crops on parasitoids using Bt-resistant lepidopteran populations as hosts. Cry1Ac/Cry2Ab-resistant T. ni larvae were used to eliminate host quality effects and to evaluate the direct effects of Bt cotton on the parasitoids Copidosoma floridanum (Ashmead) and Cotesia marginiventris (Cresson). These tri-trophic studies confirm that Bt cotton had no significant impact on development, success of parasitism, survival and adult longevity of C. marginiventris when using Bt-resistant T. ni fed on Bt cotton. Similarly, this Bt cotton had no significant impact on the development, mummy weight and the number of progeny produced by C. floridanum. Our studies verified that lyophilized Bt crop tissue maintained its insecticidal bioactivity when incorporated into an artificial diet, demonstrating that hosts and parasitoids were exposed to active Cry proteins. The egg-larval parasitoid C. floridanum, or similar species that consume their entire host, should be considered useful surrogates in risk assessment of Bt crops to non-target arthropods.

Recent studies have shown that it is possible to engineer substantial increases in triacylglycerol (TAG) content in plant vegetative biomass, which offers a novel approach for increasing the energy density of food, feed, and bioenergy crops or for creating a sink for the accumulation of unusual, high-value fatty acids. However, whether or not these changes in lipid metabolism affect plant responses to biotic and/or abiotic stresses is an open question. Here we show that transgenic Arabidopsis thaliana plant lines engineered for elevated leaf oil content, as well as lines engineered for accumulation of unusual conjugated fatty acids in leaf oil, had similar short-term responses to heat stress (e.g., 3 days at 37°C) as wild-type plants, including a reduction in polyunsaturated fatty acid (PUFA)-containing polar lipids and an increase in PUFA-containing neutral lipids. At extended time periods (e.g., 14 days at 37°C), however, plant lines containing accumulated conjugated fatty acids displayed earlier senescence and plant death. Further, no-choice feeding studies demonstrated that plants with the highest leaf oil content generated cabbage looper (Trichoplusia ni) insects with significantly heavier body weights. Taken together, these results suggest that biotic and abiotic responses will be important considerations when developing and deploying high-oil-biomass crops in the field.

Generalist insect predators play an essential role in regulating the populations of Bemisia tabaci and other pests in agricultural systems, but may be affected negatively by insecticides applied for pest management. Evaluation of insecticide compatibility with specific predator species can provide a basis for making treatment decisions with the aim of conserving natural enemies. Eleven insecticides representing six modes of action groups were evaluated for toxicity against four predator species and at different developmental stages. Full-concentration series bioassays were conducted on laboratory-reared or insectary-supplied predators using Petri dish and systemic uptake bioassay techniques. Highest toxicities were observed with imidacloprid and clothianidin against first and second instar nymphs of Geocoris punctipes (Say) (Hemiptera: Geocoridae). Later instar nymphs were less susceptible to neonicotinoid treatments based on higher LC50s observed with imidacloprid, thiamethoxam, and dinotefuran against third or fourth instar nymphs. The pyrethroid insecticide bifenthrin was highly toxic against adults of G. punctipes and Orius insidiosus (Say) (Hemiptera: Anthocoridae). Standard concentration/mortality evaluation of nonacute toxicity insecticides, including buprofezin, pyriproxyfen, spirotetramat, and spiromesifen, was inconclusive in terms of generating probit statistics. However, low mortality levels of insects exposed for up to 120 h suggested minimal lethality with the exception of pyriproxyfen that was mildly toxic to Chrysoperla rufilabris (Burmeister) (Neuroptera: Chrysopidae)., (Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady-state maintenance and turnover of plant LDs, particularly in non-seed tissues, are relatively unknown. Previously, we showed that the LD-associated proteins (LDAPs) are a family of plant-specific, LD surface-associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two-hybrid library using the Arabidopsis LDAP3 isoform as 'bait' in an effort to identify other novel LD protein constituents. One of the candidate LDAP3-interacting proteins was Arabidopsis At5g16550, which is a plant-specific protein of unknown function that we termed LDIP (LDAP-interacting protein). Using a combination of biochemical and cellular approaches, we show that LDIP targets specifically to the LD surface, contains a discrete amphipathic α-helical targeting sequence, and participates in both homotypic and heterotypic associations with itself and LDAP3, respectively. Analysis of LDIP T-DNA knockdown and knockout mutants showed a decrease in LD abundance and an increase in variability of LD size in leaves, with concomitant increases in total neutral lipid content. Similar phenotypes were observed in plant seeds, which showed enlarged LDs and increases in total amounts of seed oil. Collectively, these data identify LDIP as a new player in LD biology that modulates both LD size and cellular neutral lipid homeostasis in both leaves and seeds., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. We found that Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome-wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis Relative quantification of the changes in the lysine acetylation levels was determined on a proteome-wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1-like histone deacetylases in Arabidopsis , of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar-localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss-of-function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low-light conditions., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Septic systems may contribute micropollutants to shallow groundwater and surface water. We constructed two in situ conventional drainfields (drip dispersal and gravel trench) and an advanced drainfield of septic systems to investigate the fate and transport of micropollutants to shallow groundwater. Unsaturated soil-water and groundwater samples were collected, over 32 sampling events (January 2013 to June 2014), from the drainfields (0.31-1.07 m deep) and piezometers (3.1-3.4 m deep). In addition to soil-water and groundwater, effluent samples collected from the septic tank were also analyzed for 20 selected micropollutants, including wastewater markers, hormones, pharmaceuticals and personal care products (PPCPs), a plasticizer, and their transformation products. The removal efficiencies of micropollutants from septic tank effluent to groundwater were similar among three septic systems and were 51-89% for sucralose and 53->99% for other micropollutants. Even with high removal rates within the drainfields, six PPCPs and sucralose with concentrations ranging from <0.3 to 154 ng/L and 121 to 32,000 ng/L reached shallow groundwater, respectively. The human health risk assessment showed that the risk to human health due to consumption of groundwater is negligible for the micropollutants monitored in the study. A better understanding of ecotoxicological effects of micropollutant mixtures from septic systems to ecosystem and human health is warranted for the long-term sustainability of septic systems., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Insect sex pheromones (SPs) are central to mate-finding behaviour, and play an essential role in the survival and reproduction of organisms. Understanding the roles, biosynthetic pathways and evolution of insect chemical communication systems has been an exciting challenge for biologists. Compared with Lepidoptera, little is known about the mechanisms underlying pheromone biosynthesis in Hemiptera. In this study, we isolated and characterized two new desaturase-like genes, termed Asutdes1 and Asutdes2, from Adelphocoris suturalis, an important agricultural pest in China. Although the two genes encode an identical protein, Southern blot analysis revealed that they are duplicated genes. The Asutdes2 transcript is more abundant than Asutdes1 in the tissues tested, in particular the metathoracic scent gland and fat body. Silencing Asutdes expression in females by injecting double-stranded RNA (dsAsutdes) against a portion of the coding sequence shared by the two genes enhanced the production of (E)-4-oxo-2-hexenal, a component of the A. suturalis SP blend, and dramatically suppressed the sexual attractiveness of A. suturalis females. We conclude that dsAsutdes is associated with the SP biosynthetic pathway in A. suturalis., (© 2017 The Royal Entomological Society.)

Nature Plants 3, 17102 (2017); published online 17 July 2017; corrected online 27 September 2017.

Cucurbit yellow stunting disorder virus (CYSDV) is a whitefly-transmitted Crinivirus (Closteroviridae) that impacts melon production in many parts of the world including the USA. It has been responsible for melon crop loss in the southwestern U.S. since 2006 when it was first identified. Control strategies have revolved mainly around chemical control, but research to identify suitable products and approaches to implementing them have lagged. The current study investigated the performance of four systemic insecticides in the field while concurrently tracking CYSDV disease progression after controlled and natural whitefly inoculation of young melon plants. Assessments of virus incidence were made using two different visual observation methods in concert with ELISA analyses of leaf disks samples collected biweekly. Infection rates were consistently lowest in plots treated with the butenolide insecticide flupyradifurone while dinotefuran was second in efficacy measures. Flupyradifurone also held whitefly densities to their lowest numbers relative to the other treatments. Two other insecticides, imidacloprid and cyantraniliprole, exacerbated virus incidence in multiple trials. Further investigation into the anomalous finding of increased virus incidence due to insecticide application is ongoing., (Published by Elsevier B.V.)

The seeds of many nondomesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered seeds remains low and is often hampered by their inefficient exclusion from phospholipids. Recent studies have established the feasibility of increasing triacylglycerol content in plant leaves, which provides a novel approach for increasing energy density of biomass crops. Here, we determined whether the fatty acid composition of leaf oil could be engineered to accumulate unusual fatty acids. Eleostearic acid (ESA) is a conjugated fatty acid produced in seeds of the tung tree (Vernicia fordii) and has both industrial and nutritional end-uses. Arabidopsis thaliana lines with elevated leaf oil were first generated by transforming wild-type, cgi-58 or pxa1 mutants (the latter two of which contain mutations disrupting fatty acid breakdown) with the diacylglycerol acyltransferases (DGAT1 or DGAT2) and/or oleosin genes from tung. High-leaf-oil plant lines were then transformed with tung FADX, which encodes the fatty acid desaturase/conjugase responsible for ESA synthesis. Analysis of lipids in leaves revealed that ESA was efficiently excluded from phospholipids, and co-expression of tung FADX and DGAT2 promoted a synergistic increase in leaf oil content and ESA accumulation. Taken together, these results provide a new approach for increasing leaf oil content that is coupled with accumulation of unusual fatty acids. Implications for production of biofuels, bioproducts, and plant-pest interactions are discussed., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Increasing the accuracy of crop productivity estimates is a key element in planning adaptation strategies to ensure global food security under climate change. Process-based crop models are effective means to project climate impact on crop yield, but have large uncertainty in yield simulations. Here, we show that variations in the mathematical functions currently used to simulate temperature responses of physiological processes in 29 wheat models account for >50% of uncertainty in simulated grain yields for mean growing season temperatures from 14 °C to 33 °C. We derived a set of new temperature response functions that when substituted in four wheat models reduced the error in grain yield simulations across seven global sites with different temperature regimes by 19% to 50% (42% average). We anticipate the improved temperature responses to be a key step to improve modelling of crops under rising temperature and climate change, leading to higher skill of crop yield projections.

Passive mechanisms of mate guarding are used by males to promote sperm precedence with little cost, but these tactics can be disadvantageous for their mates and other males. Mated females of the plant bug Lygus hesperus are rendered temporarily unattractive by seminal fluids containing myristyl acetate and geranylgeranyl acetate. These antiaphrodisiac pheromones are gradually released from the female's gonopore, declining until they no longer suppress male courtship. Because starting quantities of these compounds can vary widely, the repellant signal becomes less reliable over time. Evidence was found of a complimentary mechanism that more accurately conveys female mating status. Once inside the female, geranylgeranyl acetate is progressively converted to geranylgeraniol then externalized. Geranylgeraniol counteracts the antiaphrodisiac effect despite having no inherent attractant properties of its own. This is the first evidence for such an anti-antiaphrodisiac pheromone, adding a new element to the communication mechanisms regulating reproductive behaviors.

Diverse midgut cadherin mutations confer resistance to Cry1A toxins in at least three lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Most of these cadherin mutations are inherited as recessive alleles and result in changes within the cadherin repeat (CR) regions of the extracellular protein domain. However, the H. armigera r 15 cadherin mutation results in a deletion of 55 amino acid residues within the cytoplasmic domain, and Cry1A resistance is inherited as a non-recessive trait. Here, eight recombinant H. armigera cadherin (HaCad) proteins, including seven variants containing different combinations of CRs and the cytoplasmic domain, were expressed in cultured insect cells using a baculovirus expression system and were analyzed for Cry1Ac binding and toxicity. Cells expressing either the wild-type HaCad or a mutant lacking only the region corresponding to the first nine CRs bound Cry1Ac and were equally susceptible to Cry1Ac. Cells expressing mutant HaCad proteins without the Cry1A toxin binding region (TBR) located in the CR nearest the plasma membrane did not bind Cry1Ac and were not killed by the toxin. Among the mutant proteins, loss of toxicity was observed in all cells producing HaCad variants lacking the amino acids 1422-1440, indicating that this TBR motif is important for both toxin binding and to confer susceptibility to Cry1Ac. Cells expressing the HaCad variant lacking the entire cytoplasmic domain retained Cry1Ac binding, but were significantly less susceptible to Cry1Ac than the cells producing either wild-type HaCad or HaCad lacking the first nine CRs. These results suggest that both the extracellular and the cytoplasmic domains of HaCad participate in Cry1Ac intoxication., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lipid droplets (LDs) in plants have long been viewed as storage depots for neutral lipids that serve as sources of carbon, energy, and lipids for membrane biosynthesis. While much of our knowledge of LD function in plants comes from studies of oilseeds, a recent surge in research on LDs in non-seed cell types has led to an array of new discoveries. It is now clear that both evolutionarily conserved and kingdom-specific mechanisms underlie the biogenesis of LDs in eukaryotes, and proteomics and homology-based approaches have identified new protein players. This review highlights some of these recent discoveries and other new areas of plant LD research, including their role in stress responses and as targets of metabolic engineering strategies aimed at increasing oil content in bioenergy crops., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Biogenic amines regulate the proximate mechanisms underlying most behavior, including those that contribute to the overall success of complex societies. For honey bees, one crucial set of behaviors contributing to the welfare of a colony is involved with nest thermoregulation. Worker honeybees cool the colony by performing a fanning behavior, the expression of which is largely influenced by response thresholds modulated by the social environment. Here, we examined how changes in biogenic amines affect this group-performed thermoregulatory fanning behavior in honeybees. Concentrations of two biogenic amines, octopamine and tyramine, are significantly lower in active fanners than in non-fanners, but there is no difference in dopamine and serotonin concentrations. Direct feeding of octopamine and tyramine induced a decrease in fanning responses, but only when both amines were included in the treatment. This is the first evidence that fanning behavior is influenced by these two biogenic amines, and this result is consistent with the typical role of these neurotransmitters in regulating locomotor activity in other insects. Individual variation in amine expression also provides a mechanistic link that helps to explain how this group behavior might be coordinated within a colony., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

In insect societies, both queens and workers produce chemicals that reliably signal caste membership and reproductive status. The mechanisms that help to maintain the honesty of such queen and fertility signals, however, remain poorly studied. Here we test if queen signal honesty could be based on the shared endocrine control of queen fertility and the production of specific signals. In support of this "hormonal pleiotropy" hypothesis, we find that in the common wasp, application of methoprene (a juveline hormone analogue) caused workers to acquire a queen-like cuticular hydrocarbon profile, resulting in the overproduction of known queen pheromones as well as some compounds typically linked to worker fertility. By contrast, administration of precocene-I (a JH inhibitor) had a tendency to have the opposite effect. Furthermore, a clear gonadotropic effect of JH in queens was suggested by the fact that circulating levels of JH were ca. 2 orders of magnitude higher in queens than those in workers and virgin, non-egg-laying queens, even if methoprene or precocene treatment did not affect the ovary development of workers. Overall, these results suggest that queen signal honesty in this system is maintained by queen fertility and queen signal production being under shared endocrine control.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Many strategies for controlling insect pests require an understanding of their hormonal signaling agents, peptides being the largest and most diverse single class of these molecules. Lygus hesperus is a pest species of particular concern, as it is responsible for significant damage to a wide variety of commercially important plant crops. At present, little is known about the peptide hormones of L. hesperus. Here, transcriptomic data were used to predict a peptidome for L. hesperus. Fifty-three L. hesperus transcripts encoding peptide precursors were identified, with a subset amplified by PCR for sequence verification. The proteins deduced from these transcripts allowed for the prediction of a 119-sequence peptidome for L. hesperus. The predicted peptides include isoforms of allatostatin A, allatostatin B (AST-B), allatostatin C, allatotropin, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, GSEFLamide, insulin-like peptide, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pyrokinin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide. Of note were several isoforms of AST-B that possess -WX 7 Wamide carboxyl-termini rather than the stereotypical -WX 6 Wamide (e.g., KWQDMQNPGWamide), an allatotropin ending in -SARGFamide rather than -TARGFamide (GLKNGPLNSARGFamide), a GSEFLamide ending in -GTEFLamide (TVGTEFLamide), several orcokinins with PMDEIDR- rather than NFDEIDR- amino-termini (e.g., PMDEIDRAGFTHFV), and an eight rather than 12 amino acid long isoform of SIFamide (PPFNGSIFamide). Collectively, the L. hesperus peptidome predicted here provides a resource for initiating physiological investigations of peptidergic signaling in this species, including studies directed at the biological control of this agricultural pest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors of the phytopathogenic bacterium Xylella fastidiosa (Wells), a causal agent of Pierce's disease of grapevines and other crop diseases. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. To develop a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support survival on a nutritionally austere dietary source, transcriptome assemblies for three sharpshooter species and one spittlebug species were produced., Findings: Trinity-based de novo transcriptome assemblies were generated for all four xylem-sap feeders using raw sequencing data originating from whole-insect preps. Total transcripts for each species ranged from 91 384 for Cuerna arida to 106 998 for Homalodisca liturata with transcript totals for Graphocephala atropunctata and the spittlebug Clastoptera arizonana falling in between. The percentage of transcripts comprising complete open reading frames ranged from 60% for H. liturata to 82% for C. arizonana. Bench-marking universal single-copy orthologs analyses for each dataset indicated quality assemblies and a high degree of completeness for all four species., Conclusions: These four transcriptomes represent a significant expansion of data for insect herbivores that feed exclusively on xylem sap, a nutritionally deficient dietary source relative to other plant tissues and fluids. Comparison of transcriptome data with insect herbivores that utilize other dietary sources may illuminate fundamental differences in the biochemistry of dietary specialization., (Published by Oxford University Press on behalf of GIGSCI 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

In its defense against herbivores, cotton ( Gossypium sp.) relies in part on the production of a set of inducible, non-volatile terpenoids. Under uniform damage levels, in planta allocation of induced cotton terpenoids has been found to be highest in youngest leaves, supporting assumptions of the optimal defense theory (ODT) which predicts that plants allocate defense compounds to tissues depending on their value and the likelihood of herbivore attack. However, our knowledge is limited on how varying, and thus more realistic, damage levels might affect cotton defense organization. We hypothesized that the allocation of terpenoids and densities of terpenoid-storing glands in leaves aligns with assumptions of the ODT, even when plants are subjected to temporally, spatially and quantitatively varying caterpillar ( Heliothis virescens ) damage. As expected, cotton plants allocated most of their defenses to their youngest leaves regardless of damage location. However, defense induction in older leaves varied with damage location. For at least 14 days after damage treatments ended, plants reallocated defense resources from previously young leaves to newly developed leaves. Furthermore, we observed a positive hyperbolic relationship between leaf damage area and both terpenoid concentrations and gland densities, indicating that cotton plants can fine-tune defense allocation. Although it appears that factors like vascular constraints and chemical properties of individual defense compounds can affect defense levels, our results overall demonstrate that induced defense organization of cotton subjected to varying damage treatments is in alignment with key assumptions of the ODT.

We report n-6 monounsaturated primary alcohols (C 26:1 , C 28:1 , and C 30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Key Message: Molecular markers linked to QTLs controlling post-harvest fruit water loss in pepper may be utilized to accelerate breeding for improved shelf life and inhibit over-ripening before harvest. Bell pepper (Capsicum annuum L.) is an important vegetable crop world-wide. However, marketing is limited by the relatively short shelf life of the fruit due to water loss and decay that occur during prolonged storage. Towards breeding pepper with reduced fruit post-harvest water loss (PWL), we studied the genetic, physiological and biochemical basis for natural variation of PWL. We performed quantitative trait locus (QTL) mapping of fruit PWL in multiple generations of an interspecific cross of pepper, which resulted in the identification of two linked QTLs on chromosome 10 that control the trait. We further developed near-isogenic lines (NILs) for characterization of the QTL effects. Transcriptome analysis of the NILs allowed the identification of candidate genes associated with fruit PWL-associated traits such as cuticle biosynthesis, cell wall metabolism and fruit ripening. Significant differences in PWL between the NILs in the immature fruit stage, differentially expressed cuticle-associated genes and differences in the content of specific chemical constituents of the fruit cuticle, indicated a likely influence of cuticle composition on the trait. Reduced PWL in the NILs was associated with delayed over-ripening before harvest, low total soluble solids before storage, and reduced fruit softening after storage. Our study enabled a better understanding of the genetic and biological processes controlling natural variation in fruit PWL in pepper. Furthermore, the genetic materials and molecular markers developed in this study may be utilized to breed peppers with improved shelf life and inhibited over-ripening before harvest.

The boll weevil (Anthonomus grandis grandis Boheman) has been eradicated from much of the United States, but remains an important pest of cotton (Gossypium spp.) in other parts of the Americas. Where the weevil occurs, the pheromone trap is a key tool for population monitoring or detection. Traditional monitoring programs have placed traps in or near the outermost cotton rows where damage by farm equipment can cause loss of trapping data. Recently, some programs have adopted a trap placement adjacent to but outside monitored fields. The effects of these changes have not been previously reported. Captures of early-season boll weevils by traps near (≤1 m) or far (7-10 m) from the outermost cotton row were evaluated. In 2005, during renewed efforts to eradicate the boll weevil from the Lower Rio Grande Valley of Texas, far traps consistently captured more weevils than traps near cotton. Traps at both placements indicated similar patterns of early-season weevil captures, which were consistent with those previously reported. In 2006, no distinction between trap placements was detected. Early-season patterns of captures in 2006 were again similar for both trap placements, but captures were much lower and less regular compared with those observed in 2005. These results suggest magnitude and likelihood of weevil capture in traps placed away from cotton are at least as high as for traps adjacent to cotton. Therefore, relocation of traps away from the outer rows of cotton should not negatively impact ability to monitor or detect the boll weevil., (Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by a US Government employee and is in the public domain in the US.)

Social insects frequently engage in oral fluid exchange - trophallaxis - between adults, and between adults and larvae. Although trophallaxis is widely considered a food-sharing mechanism, we hypothesized that endogenous components of this fluid might underlie a novel means of chemical communication between colony members. Through protein and small-molecule mass spectrometry and RNA sequencing, we found that trophallactic fluid in the ant Camponotus floridanus contains a set of specific digestion- and non-digestion related proteins, as well as hydrocarbons, microRNAs, and a key developmental regulator, juvenile hormone. When C. floridanus workers' food was supplemented with this hormone, the larvae they reared via trophallaxis were twice as likely to complete metamorphosis and became larger workers. Comparison of trophallactic fluid proteins across social insect species revealed that many are regulators of growth, development and behavioral maturation. These results suggest that trophallaxis plays previously unsuspected roles in communication and enables communal control of colony phenotypes., Competing Interests: The authors declare that no competing interests exist.

The ability to mark individuals is a critical feature of many entomological investigations, including dispersal studies. Insect dispersal is generally investigated using mark-release-recapture techniques, whereby marked individuals are released at a known location and then captured at a measured distance. Ectoparasite dispersal has historically been challenging to study, in part because of the ethical concerns associated with releasing marked individuals. Here, we introduce the protein self-marking technique, whereby ectoparasites mark themselves in the field by feeding on the blood of an introduced host. We demonstrate the potential of this technique using laboratory-reared bed bugs (Cimex lectularius L.) that marked themselves by feeding on either rabbit or chicken blood. We then used enzyme-linked immunosorbent assays to detect host-specific blood serum proteins in bed bugs. We assessed these protein markers' ability to 1) distinctively identify marked individuals, 2) persist following multiple feedings on an alternate diet, 3) persist over time across a range of temperatures, and 4) transfer from marked to unmarked individuals. Protein markers were detectable in bed bugs before and after molting, remained detectible after multiple feedings on an alternate diet, persisted regardless of whether an individual was starved or fed on an alternate diet following original mark acquisition, and did not transfer between individuals. The duration of detectability depended on temperature. Our results suggest that protein self-marking is an effective technique for marking bed bugs and holds promise for use in dispersal studies of ectoparasitic insects., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The whitefly (Bemisia tabaci) causes tremendous damage to cotton production worldwide. However, very limited information is available about how plants perceive and defend themselves from this destructive pest. In this study, the transcriptomic differences between two cotton cultivars that exhibit either strong resistance (HR) or sensitivity (ZS) to whitefly were compared at different time points (0, 12, 24 and 48 h after infection) using RNA-Seq. Approximately one billion paired-end reads were obtained by Illumina sequencing technology. Gene ontology and KEGG pathway analysis indicated that the cotton transcriptional response to whitefly infestation involves genes encoding protein kinases, transcription factors, metabolite synthesis, and phytohormone signalling. Furthermore, a weighted gene co-expression network constructed from RNA-Seq datasets showed that WRKY40 and copper transport protein are hub genes that may regulate cotton defenses to whitefly infestation. Silencing GhMPK3 by virus-induced gene silencing (VIGS) resulted in suppression of the MPK-WRKY-JA and ET pathways and lead to enhanced whitefly susceptibility, suggesting that the candidate insect resistant genes identified in this RNA-Seq analysis are credible and offer significant utility. Taken together, this study provides comprehensive insights into the cotton defense system to whitefly infestation and has identified several candidate genes for control of phloem-feeding pests., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Septic systems, a common type of onsite wastewater treatment systems, can be an important source of micropollutants in the environment. We investigated the fate and mass balance of 17 micropollutants, including wastewater markers, hormones, pharmaceuticals and personal care products (PPCPs) in the drainfield of a septic system. Drainfields were replicated in lysimeters (1.5m length, 0.9m width, 0.9m height) and managed similar to the field practice. In each lysimeter, a drip line dispersed 9L of septic tank effluent (STE) per day (equivalent to 32.29L/m(2) per day). Fourteen micropollutants in the STE and 12 in the leachate from drainfields were detected over eight months. Concentrations of most micropollutants in the leachate were low (<200ng/L) when compared to STE because >85% of the added micropollutants except for sucralose were attenuated in the drainfield. We discovered that sorption was the key mechanism for retention of carbamazepine and partially for sulfamethoxazole, whereas microbial degradation likely attenuated acetaminophen in the drainfield. This data suggests that sorption and microbial degradation limited transport of micropollutants from the drainfields. However, the leaching of small amounts of micropollutants indicate that septic systems are hot-spots of micropollutants in the environment and a better understanding of micropollutants in septic systems is needed to protect groundwater quality., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Agricultural crops have a long history of being irrigated with recycled wastewater (RW). However, its use on vegetable crops has been of concern due to the potential prevalence of microcontaminants, such as pharmaceuticals and personal care products (PPCPs) in the latter, which represents a possible health hazard to consumers. We investigated the uptake of three PPCPs (atenolol, diclofenac, and ofloxacin), at three different concentrations in irrigation water (0.5, 5, and 25 μg L -1 ) in relation to three varying volumetric soil moisture depletion levels of 14 % (-4.26 kPa), 10 % (-8.66 kPa), and 7 % (-18.37 kPa) by various vegetable crop species. Experiments were conducted in a split-split block completely randomized design. PPCPs were extracted using a developed method of accelerated solvent extraction and solid phase extraction and analyzed via liquid chromatography mass spectrometry (LCMS). Results indicate that all treated crops were capable of PPCP uptake at nanogram per gram concentrations independent of the applied soil moisture depletion levels and PPCP concentrations. Ofloxacin was the chemical with the highest uptake amounts, followed by atenolol and then diclofenac. Although the results were not statistically significant, higher concentrations of PPCPs were detected in plants maintained under higher soil moisture levels of 14 % (-4.26 kPa).

The Middle East-Asia Minor 1 (MEAM1) whitefly, Bemisia tabaci (Gennadius) is an economically important pest of food, fiber, and ornamental crops. This pest has evolved a number of adaptations to overcome physiological challenges, including 1) the ability to regulate osmotic stress between gut lumen and hemolymph after imbibing large quantities of a low nitrogen, sugar-rich liquid diet; 2) the ability to avoid or prevent dehydration and desiccation, particularly during egg hatching and molting; and 3) to be adapted for survival at elevated temperatures. One superfamily of proteins involved in the maintenance of fluid homeostasis in many organisms includes the aquaporins, which are integral membrane channel proteins that aid in the rapid flux of water and other small solutes across biological membranes. Here, we show that B. tabaci has eight aquaporins (BtAqps), of which seven belong to the classical aquaporin 4-related grade of channels, including Bib, Drip, Prip, and Eglps and one that belongs to the unorthodox grade of aquaporin 12-like channels. B. tabaci has further expanded its repertoire of water channels through the expression of three BtDrip2 amino-terminal splice variants, while other hemipteran species express amino- or carboxyl-terminal isoforms of Drip, Prip, and Eglps. Each BtAqp has unique transcript expression profiles, cellular localization, and/or substrate preference. Our phylogenetic and functional data reveal that hemipteran insects lost the classical glp genes, but have compensated for this by duplicating the eglp genes early in their evolution to comprise at least three separate clades of glycerol transporters., (Published by Elsevier Ltd.)

Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1-16h in the presence of 0-2.0% (w/v) n-dodecyl-β-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The mechanistic basis of tolerance to heat stress was investigated in Oryza sativa and two wild rice species, Oryza meridionalis and Oryza australiensis. The wild relatives are endemic to the hot, arid Australian savannah. Leaf elongation rates and gas exchange were measured during short periods of supra-optimal heat, revealing species differences. The Rubisco activase (RCA) gene from each species was sequenced. Using expressed recombinant RCA and leaf-extracted RCA, the kinetic properties of the two isoforms were studied under high temperatures. Leaf elongation was undiminished at 45°C in O. australiensis. The net photosynthetic rate was almost 50% slower in O. sativa at 45°C than at 28°C, while in O. australiensis it was unaffected. Oryza meridionalis exhibited intermediate heat tolerance. Based on previous reports that RCA is heat-labile, the Rubisco activation state was measured. It correlated positively with leaf elongation rates across all three species and four periods of exposure to 45°C. Sequence analysis revealed numerous polymorphisms in the RCA amino acid sequence from O. australiensis. The O. australiensis RCA enzyme was thermally stable up to 42°C, contrasting with RCA from O. sativa, which was inhibited at 36°C. We attribute heat tolerance in the wild species to thermal stability of RCA, enabling Rubisco to remain active., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (GPAT) genes encode enzymes involved in glycerolipid biosynthesis in plants. Ten GPAT homologues have been identified in Arabidopsis. GPATs 4-8 have been shown to be involved in the production of extracellular lipid barrier polyesters. Recently, GPAT9 was reported to be essential for triacylglycerol (TAG) biosynthesis in developing Arabidopsis seeds. The enzymatic properties and possible functions of GPAT9 in surface lipid, polar lipid and TAG biosynthesis in non-seed organs, however, have not been investigated. Here we show that Arabidopsis GPAT9 exhibits sn-1 acyltransferase activity with high specificity for acyl-coenzyme A, thus providing further evidence that this GPAT is involved in storage lipid biosynthesis. We also confirm a role for GPAT9 in seed oil biosynthesis and further demonstrate that GPAT9 contributes to the biosynthesis of both polar lipids and TAG in developing leaves, as well as lipid droplet production in developing pollen grains. Conversely, alteration of constitutive GPAT9 expression had no obvious effects on surface lipid biosynthesis. Taken together, these studies expand our understanding of GPAT9 function to include modulation of several different intracellular glycerolipid pools in plant cells., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Juvenile hormone (JH) is a key insect growth regulator frequently involved in modulating phenotypically plastic traits such as caste determination in eusocial species, wing polymorphisms in aphids, and mandible size in stag beetles. The jaw morphology of stag beetles is sexually-dimorphic and condition-dependent; males have larger jaws than females and those developing under optimum conditions are larger in overall body size and have disproportionately larger jaws than males raised under poor conditions. We have previously shown that large males have higher JH titers than small males during development, and ectopic application of fenoxycarb (JH analog) to small males can induce mandibular growth similar to that of larger males. What remains unknown is whether JH regulates condition-dependent trait growth in other insects with extreme sexually selected structures. In this study, we tested the hypothesis that JH mediates the condition-dependent expression of the elaborate horns of the Asian rhinoceros beetle, Trypoxylus dichotomus. The sexually dimorphic head horn of this beetle is sensitive to nutritional state during larval development. Like stag beetles, male rhinoceros beetles receiving copious food produce disproportionately large horns for their body size compared with males under restricted diets. We show that JH titers are correlated with body size during the late feeding and early prepupal periods, but this correlation disappears by the late prepupal period, the period of maximum horn growth. While ectopic application of fenoxycarb during the third larval instar significantly delayed pupation, it had no effect on adult horn size relative to body size. Fenoxycarb application to late prepupae also had at most a marginal effect on relative horn size. We discuss our results in context of other endocrine signals of condition-dependent trait exaggeration and suggest that different beetle lineages may have co-opted different physiological signaling mechanisms to achieve heightened nutrient-sensitive weapon growth., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Background: The pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) is one of the world's most important pests of cotton. Insecticide sprays and transgenic cotton producing toxins of the bacterium Bacillus thuringiensis (Bt) are currently used to manage this pest. Bt toxins kill susceptible insects by specifically binding to and destroying midgut cells, but they are not toxic to most other organisms. Pink bollworm is useful as a model for understanding insect responses to Bt toxins, yet advances in understanding at the molecular level have been limited because basic genomic information is lacking for this cosmopolitan pest. Here, we have sequenced, de novo assembled and annotated a comprehensive larval midgut transcriptome from a susceptible strain of pink bollworm., Findings: A de novo transcriptome assembly for the midgut of P. gossypiella was generated containing 46,458 transcripts (average length of 770 bp) derived from 39,874 unigenes. The size of the transcriptome is similar to published midgut transcriptomes of other Lepidoptera and includes up to 91 % annotated contigs. The dataset is publicly available in NCBI and GigaDB as a resource for researchers., Conclusions: Foundational knowledge of protein-coding genes from the pink bollworm midgut is critical for understanding how this important insect pest functions. The transcriptome data presented here represent the first large-scale molecular resource for this species, and may be used for deciphering relevant midgut proteins critical for xenobiotic detoxification, nutrient digestion and allocation, as well as for the discovery of protein receptors important for Bt intoxication.

The allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia, and America. We detected strong population structure broadly concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits.

The application of high-throughput plant phenotyping (HTPP) to continuously study plant populations under relevant growing conditions creates the possibility to more efficiently dissect the genetic basis of dynamic adaptive traits. Toward this end, we employed a field-based HTPP system that deployed sets of sensors to simultaneously measure canopy temperature, reflectance, and height on a cotton (Gossypium hirsutum L.) recombinant inbred line mapping population. The evaluation trials were conducted under well-watered and water-limited conditions in a replicated field experiment at a hot, arid location in central Arizona, with trait measurements taken at different times on multiple days across 2010-2012. Canopy temperature, normalized difference vegetation index (NDVI), height, and leaf area index (LAI) displayed moderate-to-high broad-sense heritabilities, as well as varied interactions among genotypes with water regime and time of day. Distinct temporal patterns of quantitative trait loci (QTL) expression were mostly observed for canopy temperature and NDVI, and varied across plant developmental stages. In addition, the strength of correlation between HTPP canopy traits and agronomic traits, such as lint yield, displayed a time-dependent relationship. We also found that the genomic position of some QTL controlling HTPP canopy traits were shared with those of QTL identified for agronomic and physiological traits. This work demonstrates the novel use of a field-based HTPP system to study the genetic basis of stress-adaptive traits in cotton, and these results have the potential to facilitate the development of stress-resilient cotton cultivars., (Copyright © 2016 Pauli et al.)

Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Most organisms are constantly faced with environmental changes and stressors. In diverse organisms, there is an anticipatory mechanism during development that can program adult phenotypes. The adult phenotype would be adapted to the predicted environment that occurred during organism maturation. However, whether this anticipatory mechanism is present in eusocial species is questionable because eusocial organisms are largely shielded from exogenous conditions by their stable nest environment. In this study, we tested whether food deprivation during development of the honey bee (Apis mellifera), a eusocial insect model, can shift adult phenotypes to better cope with nutritional stress. After subjecting fifth instar worker larvae to short-term starvation, we measured nutrition-related morphology, starvation resistance, physiology, endocrinology and behavior in the adults. We found that the larval starvation caused adult honey bees to become more resilient toward starvation. Moreover, the adult bees were characterized by reduced ovary size, elevated glycogen stores and juvenile hormone (JH) titers, and decreased sugar sensitivity. These changes, in general, can help adult insects survive and reproduce in food-poor environments. Overall, we found for the first time support for an anticipatory mechanism in a eusocial species, the honey bee. Our results suggest that this mechanism may play a role in honey bee queen-worker differentiation and worker division of labor, both of which are related to the responses to nutritional stress., (© 2016. Published by The Company of Biologists Ltd.)

The primary strategy for delaying the evolution of pest resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) entails refuges of plants that do not produce Bt toxins and thus allow survival of susceptible pests. Recent advances include using refuges together with Bt crop 'pyramids' that make two or more Bt toxins effective against the same pest, and planting seed mixtures yielding random distributions of pyramided Bt and non-Bt corn plants within fields. We conclude that conditions often deviate from those favoring the success of pyramids and seed mixtures, particularly against pests with low inherent susceptibility to Bt toxins. For these problematic pests, promising approaches include using larger refuges and integrating Bt crops with other pest management tactics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.