Echium plantagineum L.) is an alternative oilseed crop in summer-wet temperate regions that provides floral resources to pollinators. Its seed oil is rich in omega-3 fatty acids, such as stearidonic acid, which is desired highly by the cosmetic industry. Seeds were sown in field plots over three years in western Minnesota in spring (early-sown) or early summer (late-sown), and flower abundance, pollinator visitation, and seed yields were studied. Initial flowering commenced 41 to 55 d after sowing, and anthesis duration (first flowering to harvest) was 34 to 70 d. Late sowing dates delayed anthesis, but increased the intensity of visitation by pollinators. Cumulative flower densities ranged from 1 to 4.5 billion ha−1. Flowers attracted numerous honey bees (Apis mellifera L.), as many as 35 per minute of observation, which represented about 50% of all insect visitors. Early-sown echium produced seed yields up to 750 kg ha−1, which were 2–29 times higher than those of late-sown echium. Early sowing of echium in Minnesota provides abundant floral resources for pollinators for up to two months and simultaneously produces seed yields whose profits rival those of corn (Zea mays L.).Echium ( Echium plantagineum L., Boraginaceae) is a winter annual weed in Mediterranean climates Echium (Echium seeds contain about 27% oil that is enriched with high levels of stearidonic acid (SDA) and gamma-linolenic acid (GLA) In addition to its seed oil value, echium is highly attractive to pollinating insects, especially honey bees. Echium is a common melliferous (honey producing) plant native to the Mediterranean Basin and naturalized throughout southern Australia, where it is used widely for honey production −1 for autumn-sown echium in Chillan Province, Chile. Echium also has been grown successfully as a spring-sown crop in the United Kingdom, with yields of 200 to 300 kg ha−1−1 and averaged 251±29.5 kg ha−1 across 12 site-years Reports on growing echium as a crop are rare. Berti et al. via high-value oilseed crops in the Upper Midwest and Northern Great Plains of the USA. This region supplies a large proportion of the nation's transient honey bee colonies, and such colonies suffer high yearly losses. Summer-time nutrition is thought to play an important role for subsequent over-winter survival and vigor during the colonies' fruit and nut pollination activities in California and other southern locations Our interest in echium arose from a desire to extend floral resources for pollinators Experiments were performed at the USDA-ARS Swan Lake Research Farm, Stevens County, MN  on a Barnes loam soil  during 2011, 2012, and 2013. Treatments (planting dates) were arranged in a randomized complete block design with three replications in 2011 and 2012. Plots were 3.1 by 12.2 m. In 2013, data were collected from four replications of each of two separate but proximal experiments involving nine oilseed crops, including echium. In the first experiment (May-sowing) the plots were 6.1 by 18.3 m, and in the other (June and July sowings) the plots were 2.4 by 3.1 m. The variability in plot sizes is unlikely to have influenced flowering dynamics, but potentially could have affected pollinator visitation and seed yields.Glycine max L. [Merr]) in 2010 and wheat (Triticum aestivum L.) otherwise. Fertilizer was applied at a rate of 77-33-33 kg ha−1 of N-P-K and incorporated with the field cultivator or a no-till drill at sowing. Fertilizer applications were based on those recommended in the NNFCC crop fact sheet N,N-dipropyl-4-(trifluoromethyl)aniline) was applied preplanting at 0.75 kg ai ha−1 through a 3.1-m tractor-mounted boom that delivered 187 L ha−1 at 207 kPa pressure. Weeds that escaped herbicide control were removed by hand.Previous crops were soybean (−1 depending upon viability (TCI growing guide for echium recommends a seeding rate of 11 kg viable seed ha−1 based on germination percent of seed stocks).Echium seeds were obtained originally from Technology Crops International (TCI) in 2008. Harvested seeds were cleaned and resown each year thereafter. Seeds were sown 1.5 to 2.5 cm deep in rows spaced by 20 cm and at a rate of 11 to 17 kg seed haSowing dates were May 26 and July 7, 2011; April 25 and June 15, 2012; and May 14, June 13, and July 8, 2013 . Early shttp://www.ars.usda.gov/Services/docs.htm?docid=3512). Cumulative soil thermal times  between January 1 and the sowing dates each year were 474 and 1270 (2011), 393 and 1268 (2012), and 201, 659, 1238 (2013).Weather data were collected throughout the growing season at the Swan Lake Research Farm Weather Station (Percent ground cover of echium flowers (flower area) was estimated visually in each plot from the time open flowers were first visible through the end of anthesis for all three years. Visual estimates were made by two observers by viewing the crop from above and approximating the percent of the total area that was covered by flowers. Cumulative flower area was estimated by calculating the areas under the curves throughout anthesis. These integrals represent the coverage time (%t) of flower area. Anthesis duration was measured from the start of flowering to the time of swathing; because echium is an indeterminate crop, anthesis persisted until the crop was harvested.−1) were performed using Transect Point Density software −1) was calculated by integrating the area under the curve.In 2013, number of open flowers were measured once per week during anthesis using the Batcheler corrected point distance estimation 2 (PAR = 479), with a mean RH of 71%, and no measurable rainfall 24 h before nectar was removed. On the latter date the mean temperature was 18.2°C, mean solar radiation was 0.30 kwh/m2 (PAR = 525), with a mean RH of 74%, and 0.64 cm of rainfall 24 h before nectar was removed. Nectar was extracted from 10 open flowers using a 1, 5, or 10 µL microcapillary. The length of the nectar column in the microcapillary was measured immediately after extraction. Degree Brix  was recorded for each flower using an Eclipse low volume handheld refractometer . “Standing crops” of nectar were removed at three time points: 900, 1200, and 1500 h, from 10 different flowers at each time, and the flowers were tagged and isolated with insect exclusion bags. After 2 h of exclusion, the flowers were resampled for nectar volume and °Bx. Nectar volume was calculated by dividing the length of the nectar column by the total length of the microcapillary and multiplying by the microcapillary volume. Sugar content was calculated from the Corbet et al. Nectar analysis was performed on early-sown echium flowers on July 16 and August 7, 2013, following protocols described by Corbet et al. giving the total sucrose produced in 2 h by a single echium flower. The µg of sucrose produced was averaged across the two sampling dates for the 10 flowers sampled during each time interval. The daily sucrose production per echium flower was approximated by summing the average µg of sucrose produced during each of the three time intervals. The sum of flowers produced per hectare throughout anthesis was multiplied by the daily sucrose production of a single echium flower to calculate the potential sucrose production of a hectare of echium throughout anthesis.−1, when the temperature >5°C and the sky was ≥50% clear or when the temperature was >10°C with any sky cover. All insect counts were divided by the number of observers and the minutes of each observation period giving number of insects observed by one observer in 1 min. Integrating under the curves of pollinator-time relationships allowed calculation of pollinator visitation time (pvt) throughout anthesis. A ratio of insect visitation to flower area was derived by dividing pvt by %t for each planting date. A larger value indicates more insects per flower area. The 52-ha research farm harbored one commercial-grade honey bee colony in 2011 and 2012, and four colonies in 2013. In addition, a commercial apiary with 34 hives was located within two miles of the research farm, which supported consistent bee visitation each year. Otherwise, the research farm was surrounded by narrow strips of semi-natural vegetation , which likely provided nesting habitat for native pollinators.Pollinator visitations to the plots were recorded from the time open flowers were first visible through the end of anthesis. Visitation was documented by investigators walking along the length of each plot for 1–2 minutes and listing the number of individuals observed in each of the following insect categories: honey bee, bumble bee, other bee, fly, butterfly, and other insects. Insect counts were conducted between 1100 and 1400 h on rainless days, with wind speeds <7 m sEchium seeds were harvested by swathing sections in each plot on September 15 and October 25, 2011; August 12 and September 12, 2012; for the early and late planted plots, respectively and August 23, 2013 for the early planted plot. The 2013 mid planted plot was harvested on September 11 by straight combining without swathing. The 2013 late planted plot was harvested on October 10 by hand harvesting two rows, allowing the plant material to dry, and hand threshing the seed. Variation in harvesting techniques in 2013 was unavoidable due to unforeseen problems with machinery and labor availability. Swaths typically were 1.5 m wide and the length of the plot. Swathing and direct harvesting occurred when the bottom third of former flowers along branches of the cyme-type infructescence had black seeds (dried and fully mature), the middle third had grayish seeds , and the upper third had green seeds or still-maturing flowers P-value <0.15 were included in the model. Standardized regression coefficients (SRC) are computed in SAS 9.3 by dividing a parameter estimate by the ratio of the sample standard deviation of the dependent variable to the sample standard deviation of the regressor (SAS 9.3). The SRC indicates how many standard deviations a given variable will change per standard deviation increase in the predictor variable independent of scale.Estimation of open flower density was done using Transect Point Density software Time from sowing to first flower averaged 45 d and varied little across sowing dates. Flowering ended at swathing, typically in late August but extended into early October for late sowings. The average of 55 d for anthesis duration in the relatively wet climate of Minnesota was similar to flowering of cultivated and weedy populations in Mediterranean-like environments. The flowering period for echium is about 30 to 40 d in its native Mediterranean Basin Flowering peaked at or near 40% cover within 2 to 3 weeks of the start of flowering, and remained above 10% for most of anthesis . In 2011Coverage times of flower area (%t) for early- and late-sowing dates were 1577 and 765%t in 2011, 769 and 535%t in 2012, and 1113 and 972%t in 2013 . Thus, l−1 for the early, mid, and late sown echium in 2013 . Echium flowers produced nectar from 900 to 1700 h, providing a food resource to foraging insects from dawn to dusk. Sucrose production for each time point was not significantly different between the two sampling dates, despite varying weather conditions ectively .−1 of sugar A healthy honey bee colony requires approximately 100–200 kg yr−1 yr−1, which is about half of what we predicted for our early sown crop in 2013. They also reported that nectar secretion in echium is affected by density of flowers, with higher densities of flowers having lower secretion rates. We extracted nectar before peak flower density was reached in the 2013 early sown crop, thus nectar production later in the season may have decreased as the flower density increased. These assumptions likely led to overestimations of sucrose production. More detailed investigations into nectar production throughout anthesis, as well as pollen production, and foraging by honey bees, are needed to give a more accurate recommendation for hive densities that can be supported near echium fields. Regardless of inaccuracies, however, the results indicate that echium not only can support high densities of honey bee colonies, but can supply most of their annual energy needs during a single one- to two-month flowering/foraging season.Corbet and Delfosse −1 of nectar, and the sugar concentration of soybean nectar extracted directly from bee's stomachs was 36.2±1.94%, giving approximately 5 µg sugar per flower. Thus, at a flower density of 100–800 flowers per plant −1 we calculated approximately 0.5 to 4 L ha−1 of nectar and 0.25 to 2 kg ha−1 of sugar would be available to nectar-gathering insects. Similarly, Severson and Erickson −1 for a range of cultivars. These values convert to 2 to 54 kg ha−1 of sugar at the same flower density mentioned above. In brief, even the highest value of nectar sugar production for soybean is an order of magnitude lower than that for echium.Comparison of sugar production values by echium to those of soybean reveals the potential value of having echium on the landscape. Erickson −1 were observed. The numbers of pollinators visiting echium were much higher than those reported for other oilseed crops, such as Thlaspi arvense L. (1.1 pollinators min−1) and Camelina sativa L. Crantz. (3.1 pollinators min1) Brassica napus L. (20 pollinators min−1) B. rapa L. (10 to 50 pollinators min−1) Insect visitors were present immediately upon initial observations of open flowers . At time−1) increased in the later sown echium relative to the early sown echium each year (Pollinator visitation times (pvt) were 271 and 356 pvt in 2011, 376 and 897 pvt in 2012, and 1787, 1774, and 2662 pvt in 2013  for earlach year . ResultsHoney bees comprised the majority of pollinators often representing more than half of the observed insects  g–i. ExcAs a comparison to commodity crops, the pvt for corn and soybean in 2013 were 177 and 378, which were four and two times less than the lowest pvt for echium across all years, which shows that presence of echium provides valuable insect forage in a landscape dominated by corn and soybean. Honey bee visits to corn and soybean only accounted for 5 and 4%, respectively, of all insects observed, which was over 8 times lower than the average proportion of honey bee visits of 42% in echium (data not shown).A caveat regarding echium as a forage resource for honey bees is that its pollen contains pyrrolizidine alkaloids, which is potentially harmful to mammals −1. Early-sown echium had 285, 386, and 167% higher yield than late-sown echium in 2011, 2012, and 2013, respectively . Since rainfall during anthesis was the most significant term in determining yield, the manipulation of sowing time can be used to time echium growth with rainfall during anthesis, thereby increasing yield. Consequently, sowing echium in May, and assuming 45 days to first flower, synchronizes anthesis with higher summer rainfall and would be expected to achieve highest combined flower production and seed yield.−1 and were as high as 425 kg ha−1−2 and had a 100-seed weight of 0.38 g −1. Plants in nearby grazed and mowed pastures produced about half of this amount (500 and 650 kg ha−1). These Australian seed production values included seeds retained on harvested plants as well as those in the litter and soil (2.5 cm depth). The average overall seed yield for the current experiments of 330 kg ha−1 is much lower than that for southern Australia, but higher than that for North Dakota, and it was above the minimum production goal of 250 kg ha−1 (see below).Echium yields for several experiments across North Dakota averaged 251 kg ha−1, whereas net returns were $894 and $526 ha−1 according to the Farm Financial Database for Minnesota and the surrounding region (http://www.finbin.umn.edu). Net returns represented 32% of gross returns for both crops. To garner attention by growers, echium likely would have to match the net returns of corn or soybean. Assuming that the gross return to net return ratio is the same for echium as for corn and soybean, and that the contract price for echium seed is $5.30 kg−1−1 to match the net return of soybean and 527 kg ha−1 to match corn in 2012. Corn and soybean prices in 2012 were uncommonly high, thus an echium seed yield of 250 to 300 kg ha−1 probably represents a reasonable minimum production goal for profitability and grower acceptance.Corn and soybean are the two main crops in the Upper Midwest and Northern Great Plains. Median gross returns (N = 2553 and 2209 farms) for these crops in 2012 were $2776 and $1628 ha−1, early sown echium crops in 2011, 2012, and 2013 would have generated gross returns of $3710, $1733, and $2279 ha−1, respectively. In 2010, 2011 and 2012 the average gross returns for corn were $2020, $2260 and $2776 ha−1 (http://finbin.umn.edu), which overall makes echium a seemingly viable alternative crop from an economic perspective. In addition to its economic value, oilseed echium provides valuable ecosystem services  that cannot be matched by crops such as corn, soybean, and wheat. Early sown echium had high insect visitation and did not suffer the yield losses of late sown crops. From an agro-ecosystem perspective, early sown echium offers a high yielding crop to farmers and provides a valuable forage resource for pollinating insects.When sown in April or May in the Upper Midwest and Northern Great Plains, oilseed echium produces high seed yields whose value can rival those of standard commodity crops. With echium seed valued at $5.30 kg


ALOXE3 (NM_001165960.1) in the proband and discovered that his parents are both heterozygous carriers of the variant. The clinical manifestations of the proband’s skin were consistent with NBCIE, and detailed histopathological assessment revealed epidermal bulla formation and Majocchi’s granuloma. Infection with Trichophyton rubrum was confirmed by culture. The patient responded to oral terbinafine antifungal treatment. Decreased skin barrier function, such as that caused by hereditary disorders of keratinization, can increase the risk of severe cutaneous fungal infections and the formation of Majocchi’s granuloma and associated alopecia. Patients with NBCIE should be alerted to the possible predisposition for developing dermatophytoses and warrant close clinical follow-up.Non-bullous congenital ichthyosiform erythroderma (NBCIE) is a hereditary disorder of keratinization caused by pathogenic variants in genes encoding enzymes important to lipid processing and terminal keratinocyte differentiation. Impaired function of these enzymes can cause pathologic epidermal scaling, significantly reduced skin barrier function. In this study, we have performed a focused, genetic analysis of a probrand affected by NBCIE and extended this to his consanguineous parents. Targeted capture and next-generation sequencing was performed on NBCIE associated genes in the proband and his unaffected consanguineous parents. We identified a homozygous nonsense variant c.814C>T (p.Arg272*) in  Autosomal recessive congenital ichthyosis (ARCI) represents a group of hereditary disorders of keratinization with clinical manifestations of diffuse, widespread thickened epidermal scaling and varies degree of erythroderma . ARCI isABCA12, ALOX12B, ALOXE3, CERS3, CYP4F22, NIPAL4, PNPLA1, and TGM1 [ALOXE3 gene.Pathogenic variants in genes encoding enzymes involved in keratinocyte lipid processing, including and TGM1 , have beAn 11-year-old boy of Chinese extraction was brought to our clinic by his consanguineous parents (first cousins) for the evaluation of multiple dermatologic issues. The patient lives in an isolated, rural, mountainous region of southern China with limited access to medical care. The patient’s parents recall that the patient’s total body surface appeared “pink and smooth” at birth. Further details regarding the first five years of life were difficult to obtain, given that the child’s parents, his father a coal miner, had little access to the child during this time. However, over the past six years, the boy suffered from severe, diffuse epidermal scaling and, over the past year, had also developed multifocal hair loss and erythema. Initially, the patient had developed red, scaly lesions on the bilateral ankles that eventually progressed to involve the entire body. The patient also suffered from severe xerosis and pruritus, particularly during the winter season that was particularly worse on the ankles, popliteal fossa, and groin. In addition, the patient was also noted to produce limited sweat, even during warmer seasons, and would occasionally be noted to be febrile, despite lacking any sign of underlying infection. The patient had not received any form of systemic or dermatologic treatment prior to his presentation.Upon physical examination, there were multiple, widespread round, erythematous, polycyclic macules with the overlying scale involving 95% of the total body surface area. There was marked, thick, cornification diffusely involving the bilateral palmoplantar surfaces. Erythematous macules and papules with overlying scale were also present on the left scalp, right cheek, and neck, and were noted to have been present for one year . In addiBased on the constellation of clinical and physical exam findings, a preliminary diagnosis of congenital ichthyosis was made. In addition, based on the presence of significant erythema and scaling, there was also concern for an overlying, superficial fungal infection. Thus, a potassium hydroxide preparation, punch biopsies , and tissue cultures were obtained. Moreover, the patient and his parents were consented to enroll in a research protocol approved by the Beijing Genomics Institute at Shenzhen (BGI) to identify potentially clinically-relevant DNA sequence variants in specific targeted genes .Microscopic examination of the buttock skin biopsy of H and E stained sections revealed marked hyperkeratosis and focal parakeratosis with mild hypergranulosis within a diffusely acanthotic epidermis A. AcanthTrichophyton rubrum. The findings were later confirmed by tissue culture  within this consanguineous family  in 1 out of 4406 African American chromosomes by the National Hart, Lung, and Blood Institute Exome Sequencing Project  and 1 out of 10,404 African chromosomes by the Exome Aggregation Consortium   variant in s family . The pros family . Based oute.org) . This loThe patient received a two-week course of oral terbinafine, which led to dramatic resolution of the erythema as well as papules and pustules . A topicALOXE3, which was complicated by a superimposed Trichophyton rubrum infection and associated alopecia. ALOXE3 encodes the arachidonate lipoxygenase 3 enzyme, which functions as a hydroperoxide isomerase that oxidizes the 9R-hydroperoxide derivative of esterified ω-hydroxyacyl-sphingosine (EOS) to synthesize a unique epoxy alcohol important for activating the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). This pathway is critical for proper epidermal differentiation. The ALOXE3 gene is located on chromosome 17p13.1 and contains 15 exons [We describe the case of a pediatric patient of Chinese extraction, born to consanguineous parents, who was diagnosed with autosomal recessive congenital ichthyosis of the NBCIE subtype. Our sequencing results revealed that the genodermatosis was caused by a homozygous nonsense variant c.814C>T (p.(Arg272*)) in 15 exons ,17,18. T15 exons . We specALOXE3 have been reported since the gene was first identified as an ARCI gene in 2002 [et al. described two point mutations and one deletion found in ALOXE3 in a family of consanguineous lineage from the Mediterranean basin [et al. described the molecular and clinical findings in 17 families with ARCI originating from Central Europe, Turkey, and the Indian subcontinent, and identified four different inactivating variants in ALOXE3 [et et al. also found 11 previously unreported variants in ALOX12B and ALOXE3 in 21 ARCI patients from 19 unrelated families and demonstrated that variants in these two genes were the second most common cause for ARCI in their cohort of patients [ALOX12B and two mutational hotspots in ALOXE3 in 2009 [et al. also identified three patients with germline variants in ALOXE3: one Swedish patient was homozygous for a T>C transition at cDNA position 1676 in exon 10 (p.(Leu559Pro)) while the others were compound heterozygotes [ALOXE3 and ALOX12B genes usually present with a NBCIE phenotype [ALOX12B gene show more limited, whitish desquamation compared with those with ALOXE3 variants. In our case, the patient exhibited scaling of a grey to dark hue.Thirteen variants in  in 2002 ,11,12. T in 2002 ,19. Jobapatients . Examina in 2009 . VahlquiALOXE3 gene. The variant found in our patient has not been reported in the Human Gene Mutation Database and represents the results of serendipitous contributions from consanguineous parents, who were both carriers. The patient presented to us with severe ichthyosiform skin lesions and was ultimately diagnosed with NBCIE. However, unlike most existing reported cases of ACRI, we identified multiple microscopic, inner epidermal bullae, which were negative on direct and indirect immunofluorescence studies. We hypothesize that the robust inflammatory reaction, secondary to heavy dermatophyte infestation led to the formation of intraepidermal bullae in our ARCI patient. In addition, the patient’s alopecia resolved after anti-fungal therapy, implying that this manifestation was secondary to the infection and not the underlying ichthyosis. Furthermore, we speculate that the homozygous nonsense variant in ALOXE3 led to severe impairment in skin barrier function, which predisposed our patient to this severe cutaneous fungal infection.Taken together, our patient’s phenotype, including the presence of generalized erythema and the thick dark scaling over the joints in our patient, was reflective of his germline homozygous nonsense c.814C>T (p.(Arg272*)) variant in the The study was conducted in accordance with the guiding principles of the Declaration of Helsinki. Collection of samples was approved by the ethical committees of Peking Union Medical College Hospital, and informed written consent was obtained from the parents. The parents completely understood that their peripheral blood samples would be used in this research and requested genetic counseling. We followed all regulations for the enrollment of patients, sample collection and informed consent for the purpose of research.Routine histology was performed by microscopic examination of 5 µm Hematoxylin and Eosin (H&E) stained sections of the skin biopsy obtained from the buttock region. Direct immunofluorescence was performed to evaluate for IgG and C3 deposition. Fungal elements were detected by potassium hydroxide (KOH) preparation as well as Periodic acid-Schiff (PAS) special stains.Serum antibodies for desmoglein 1 and 3 detection were performed with standard laboratory testing .Escherichia coli DNA polymerase to fill 5′ overhangs and remove 3′ overhangs. Terminal A residues were added following a brief incubation with dATP and the Klenow 3′–5′ exo-enzyme following standard Illumina protocols. Subsequently, adapters were ligated to both ends of the resulting fragments. The adapter-ligated templates were purified by the Agencourt AMPure SPRI beads.Genomic DNA was extracted from peripheral blood samples of the patient’s family members using QIAamp Blood DNA mini Kit  according to the manufacturer’s instructions. After quality control via gel electrophoresis, 1 µg of high molecular weight genomic DNA sample was randomly fragmented to approximately 250 bp using a Covaris focused-ultrasonicator. Next, Gel-purified DNA was end-repaired with T4 DNA polymerase, T4 phosphonucleotide kinase and the Klenow fragment of A 2.1M Human Array targeting seven NBCIE disease-causing genes encoding exons and 10 bp flanking intronic sequences was designed with Roche NimbleGen . The adapter-ligated DNA templates were amplified by ligation-mediated polymerase chain reaction (LM-PCR), purified, pooled, and hybridized to the array for enrichment. Captured ligation mediated-PCR products were subjected to Agilent 2100 Bioanalyzer and real-time quantitative PCR on ABI StepOne Real-time PCR system  to estimate the magnitude of enrichment. Qualified library was sequenced by Illumina HiSeq2000 Analyzers (following the manufacturer’s protocols) for 90 cycles per read to generate paired-end reads. Image analysis, error estimation, and base calling were performed using Illumina Pipeline software (version 1.3.4) to generate raw data. The “clean reads” derived from targeted sequencing and filtering were then aligned to the human reference genome (hg19/GRCh37) using the BWA (Burrows Wheeler Aligner) Multi-Vision software package. We used SOAPsnp software and Samtools to detect single nucleotide variants and indels [www.premierbiosoft.com) and synthesized by BGI-Beijing, Beijing, China. All potential variants identified by NGS of the affected individual were confirmed by Sanger sequencing. Sequencing reactions were performed by mixing 5 μL of purified PCR products , 0.75 µM of primers and 1 μL of BigDye Terminator v1.1 Cycle Sequencing kit  and were run in an ABI 3730 Genetic Analyzer (Applied Biosystems).Sanger sequencing was used to determine whether any of the variants co-segregated with the disease phenotype in the patient’s family. Primers were designed using Primer 6.0 ) in


Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and 15N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2–3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production.Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow ( However, the use of nitrogen (N) fertilizer during cultivation requires energy (during manufacture) and can contribute to GHG emissions through nitrous oxide emissions . Nitroge US corn  and 80–1gar cane ; and 188eed rape . In conteed rape .Salix spp.), grown as short rotation coppice (SRC), are among the important commercially grown biomass trees in temperate regions. Typical fertilizer inputs for SRC willow are low: 30–80 kg N ha−1 applied after cutback  and after every harvest (every 2–3 years), giving average inputs of only 10–30 kg N ha−1 year−1 , placed SRC willow second after sugarcane, ahead of Miscanthus, sugar beet, oil palm, birch, poplar, switchgrass, corn, sweet sorghum and algae. Trials in willow that have compared different fertilization regimes  genotypic variation occurs in primary N allocation with regard to canopy or root system establishment; (ii) shifts occur in the patterns of N allocation during the growing season in relation to biomass accumulation; and (iii) genotypic differences occur in allocation and remobilization of N between organs.The main aim of the current study was to investigate how successfully different SRC willow genotypes assimilate and allocate N during establishment and the growing season as well as remobilize and consequently recycle N during leaf senescence before the onset of winter dormancy. To address this, allocation and remobilization of N to and between different organs were assessed in the parents and 12 progeny of a genetic mapping population using elemental analysis and Salix viminalis ×  hybrids of grandparents, S. viminalis ‘Astrid’ and ) , and 12 progeny (referred to as genotypes 1–12) of a mapping population (K8) in which the parents S3 and R13 are two full-sib diploid erinii))  (Table 1erinii))  and usederinii))  and rusterinii)) .Table 115N) and three harvest dates replicated three times, giving a total of 252 individual pots of willow used in the experiment. These were arranged in a trial design comprising randomized blocks. The three harvests were taken in June, August and October after 2, 4 and 6 months of growth, respectively. Average hours of sunshine and max/min air temperature over the 2 months preceding each harvest were: 194 h 16.4/7.6 °C, 158 h 20.5/12.7 °C and 134 h 15.4/7.6 °C, respectively. The trees were fertilized with ammonium nitrate, either 14NH414NO3 or 15NH415NO3 (containing 10% 15N and 90% 14N). The natural abundance of 15N is 0.3676% and the fate of the 10% 15N applied was followed using isotope ratio mass spectrometry. Of the 252 potted cuttings, 45 failed to grow, of which genotypes 3 (8719) and 7 (8861) had the lowest number of survivors.In April 2008, the cuttings were planted in 12-l pots filled with a 1 : 1 ratio of sand and perlite and placed outside in a roofed and caged area at RRes. Before planting, the 20-cm cuttings were first soaked in water overnight. There were 28 treatments (14 genotypes × 2 N treatments: with or without 15N so that the remobilization of (the previously labelled) N during early senescence could be detected. Although lower total N applications per tree have been shown to be sufficient for the initial growth season of some willow varieties . The trees were given 200 ml of water every 2 days for the first 2 months. This was increased to 400 ml for the remaining 4 months.Each fertilizer application was made in 50 ml of water that also included a constant nutrient mix: , root, total and growth of biomass yields (g DM) were recorded for each individual after 2, 4 and 6 months (June/August/October). As the cutting represented a significant proportion of each tree's total weight, especially at the first harvest, the total growth in DM excluding the cutting is also presented here as ‘growth’ DM.® A10 Analytical Grinder . Prior to grinding, all samples were air-dried at room temperature for 2 days. A sample of 150 mg of ground biomass was then oven-dried over-night at 105 °C in order to assess the moisture content allowing the calculation of organ and total DM per tree. Then 100 mg of oven-dried sample from each organ was sent to Iso-Analytical Ltd Crewe, UK, for 15N stable isotope ratio mass spectrometry as well as total N assessment.At each of the three harvest time points, each of the four organs  from each tree was ground independently using a water cooled IKAP < 0.05, ANOVA F-test) were revealed for the DM of each organ, as well as for total and growth DM at each harvest point , when all the trees were considered together as one group, 56% of growth DM (excluding the cutting) was leaf, only 38% was stem and 6% root. By August, the proportions had changed, with only 34% of the growth DM as leaf, 54% stem and 12% root. By the final harvest, in October, the tree biomass was 27% leaf, 52% stem and 21% root  from the pot trial, at both August and October harvests, had strong and significant positive correlations with the recorded field above-ground biomass yields from three field sites: RRes (harvested in 2005), LARS (harvested in 2003) and Woburn Table h.Table 15N ratio, which enabled quantification of assimilated N. The 15N ratio revealed <2% of total tree N had been assimilated from the growth medium (containing the labelled fertilizer) by June for all the trees. This cutting N (non-assimilated) was used as a genotype-specific baseline for tree UpE, which was subtracted from later harvests to establish assimilated N levels.As little root growth (average 6% growth DM) had occurred by June Figure e, the maThe UpE was found to strongly correlate to root DM at both the August and October harvests . A strong genotype effect was observed . UpE was highest during August and the amount of available N was close to limiting for highest yielding genotypes, the parents, in August (between 70 and 80% assimilation of available N); however, this dropped sharply in October as fertilizer application was increased.Once the genotypes were further categorized into biomass yield groups there was a clear and significant segregation for UpE Figure c ; low yielders had a larger N remobilization away from leaves (9%) and into stems (7.5%), whereas medium yielders had a reduced difference with only a small remobilization of N away from leaves (3%) and into stems (1.5%). The two parental genotypes, here the highest biomass yielding, showed the reverse, with a remobilization of N into the leaves (4%) and away from the stems (3.5%)  Figure b.15N was used a label to investigate allocation and remobilization of N in different SRC willow genotypes from cutting establishment up to leaf senescence, before the onset of winter dormancy. It was hypothesized that genotypic variation in primary N allocation would occur in relation to canopy or root system establishment, that shifts in the patterns of N allocation relating to biomass accumulation would take place during the growing season, and that genotypic differences would be present in allocation and remobilization of N between organs. Genotype-specific variation was observed for all traits measured and all three hypotheses were supported by the results found.In the present study, The pot trial was grown outside in a covered cage (and not in glasshouse conditions) using a defined supply of fertilizer that was intended to approximate field conditions as much as possible. In pot conditions, relative biomass yields by genotype were similar to those obtained in the field. Overall, correlations were comparable to (or even stronger than) those previously reported in willow Table  Lindega. MoreoveAs expected growth DM steadily increased over the three harvest points; however, the allocation of mass to each organ showed a more complex pattern. By the first harvest in June the majority of the trees' resources had been committed to producing leaves and there was very little or no observable root development. When considered in conjunction with the small amount of growth medium-derived (assimilated) N in June, there is strong evidence that the cutting acted as the major source of N for that entire period of growth. By June the majority of the trees' resources had been committed to producing leaves Figure . Weih anSome work has been performed showing that both increased diametre and/or length of a planted cutting can have a positive impact on subsequent growth performance in a genotype-specific manner . HoweverBy August, a shift from a high growth rate of leaves to a high growth rate of stems was seen as gradually the stems became the largest organ of the tree Figure a. Priori−1 available) resulted from reduced root development in June, initially marginalized as a consequence of the principal investment into canopy establishment. This relationship is further supported by the strong correlation, observed between root development and UpE in both August and October  and increased biomass production has previously been demonstrated in willow by Seasonal storage and mobilization is well characterized in SRC willow for starch, the plant's primary non-permanent carbon store, with the roots being the major storage organ over winter dormancy . As evidA small but clear trend became apparent when data from all the trees were pooled Figure a, of N m15N data is that remobilization of resources seemed to be primarily a shift from leaves to stems, with only a small amount being remobilized from above-ground biomass to below-ground biomass  to below-ground (non-harvested) biomass over a pre-harvest winter if subsequent harvest yields are to be maintained while minimizing agricultural land inputs. The potential physiological benefits for substantial N remobilization from stems to roots, which is well understood in grasses such as Miscanthus where the rhizome is the perennial organ . Bark protein and bark N concentrations, which also varied significantly between clones within families, were positively correlated to BSP amounts within several of the families  in 14 genotypes of a genetic family. Initial canopy establishment was almost entirely fuelled by resources remobilized from the cutting, showing that the cutting acted as a primary resource hub for ∼8 weeks after planting. Root biomass accumulation and N assimilation (UpE) were strongly associated and varied between genotypes but an earlier cessation of the dependency on the cutting for N was linked to higher biomass yields.The combined use of elemental analysis and duration over the season is a major factor in biomass accumulation.Through observation of both organ biomass accumulation and N allocation, a clear sequence of growth priorities was identified. In the first 2 months, canopy establishment took precedence over root development yet after this time, long before leaf senescence, resource investment shifted towards the stems and roots. Earlier canopy development, in conjunction with reduced (or delayed) N remobilization from the leaves to the stems and roots by October, resulted in increased biomass yields. This provides supporting evidence that increased canopy N retention, with the hope of reducing agricultural land inputs and opening up more low-nutrient land for cultivation, research should be directed towards further increasing the roots or stools as N sinks before harvest without deleteriously effecting inter-harvest bud burst in spring.Little variation in root N allocation was observed although root biomass production was highly varied. Evidence was also found that the stem is the major N reserve during winter dormancy of SRC willow. To increase None declared.http://www.porteralliance.org.uk/) for a studentship awarded to N.J.B.B. F.E.P. was supported through a FQRNT postdoctoral research fellowship from the Government of Quebec, Canada.The authors gratefully acknowledge the funding support from the Rothamsted Cropping Carbon Institute Strategic Programme Grant and from the Porter Alliance (


Objective. To report the rate of progression of keratectasia after primary crosslinking (CXL) and evaluate the safety and efficiency of CXL redo. Materials and Methods. We conducted a retrospective analysis of the patients who underwent CXL between 2010 and 2013 at the Beirut Eye Specialist Hospital, Lebanon. Progression of keratectasia was based on the presence of an increase in maximum keratometry of 1.00 D, a change in the map difference between two consecutive topographies of 1.00 D, a deterioration of visual acuity, or any change in the refraction. Primary and redo CXL were done using the same protocol. Results. Among the 221 eyes of 130 patients who underwent CXL, 7 eyes (3.17%) of five patients met the criteria of progression. All patients reported a history of allergic conjunctivitis and eye rubbing and progressed within 9 to 48 months. No complications were noted and all patients were stable 1 year after CXL redo. Conclusion. Allergic conjunctivitis and eye rubbing were the only risk factors associated with keratoconus progression after CXL. A close followup is thus mandatory, even years after the procedure. CXL redo seems to be a safe and efficient technique to halt the progression after a primary CXL. Keratoconus (KC) is a noninflammatory corneal disease characterized by corneal deformation and thinning caused by structural changes in the corneal collagen, inducing irregular astigmatism, myopia, and protrusion, which leads to mild to marked impairment in vision quality . CornealK value of more than 58.0 D as a risk factor for progression [Corneal collagen crosslinking (CXL) was introduced in 2003 by Wollensak et al. to halt the progression of keratectasia –8. Duringression . In addiTo our knowledge, no previous study evaluated the safety and efficiency of CXL redo. We hence report the rate of progression and the risk factors after CXL in our private practice and evaluate the technique, safety, and efficiency of CXL redo after primary CXL.We conducted a retrospective analysis of the patients with progressive keratectasia who underwent CXL between March 2010 and March 2013 at the Beirut Eye Specialist Hospital Beirut, Lebanon. Diagnosis of keratoconus was based on a combination of computed slit-scanning videokeratography of the anterior and posterior corneal surfaces, keratometric readings, and corneal pachymetry. Keratoconus was classified into four stages based on corneal power, astigmatism, corneal transparency, and corneal thickness, according to the classification of Amsler-Krumeich .All patients included in this study had a history of progressive keratectasia either from keratoconus or from corneal ectasia after LASIK in one or both eyes and underwent a primary CXL in order to stabilize the disease.μm. Central corneal thickness (CCT) and thinnest corneal location were measured using Pentacam topography with epithelium on prior to the procedure. Because of the potential deswelling effect in the corneal stroma of the dextran in riboflavin solution [μm was reached.All patients included in this study had a central corneal thickness >400 solution , the CCTExclusion criteria for primary and redo CXL were preoperative corneal opacities, ocular pathology other than keratectasia, especially the cornea guttata or other endothelial irregularities, age younger than 18 years, actual or intended pregnancy, not available for follow-up examinations for 1 year, and connective tissue disease.K readings in several consecutive recordings in the last 6 months with or without progressive corneal thinning as well as deterioration in visual acuity and manifest refraction. We evaluated the progression of KC after CXL based on the presence of 2 or more of the following criteria: increase (≥1 D) in K value , a change in the map difference between two consecutive topographies of ≥1 D, a deterioration of VA defined as a drop of one or more lines, or any change in the refraction as a change of 0.5 D.Progressive keratectasia was suspected by an increase in maximum k-reading over time with no further steepening happening 6 months after CXL treatment in stable keratoconus. Any steepening in k-reading that happens 6 months after primary CXL is considered as sign of keratoconus progression, mainly when it was associated with one of the aforementioned criteria of keratoconus progression. Therefore, the effect of CXL is considered only 6 months after primary CXL and corneal stability is judged after that.Based on our observation, the cornea may endure major topographic changes in the first 6 months after CXL with significant changes in manifest refraction and visual acuity. We set a baseline corneal topography 6 month after CXL treatment and we noticed no further clinically significant changes are happening then after apart from a nonsignificant minor flattening of All patients undergoing CXL were followed up closely in the postoperative period at day one, one week, one month, 3 months, sixth months, and one year and then half yearly after. Corneal topography was repeated at each visit starting 3 months after CXL and then 6 months after CXL corneal topography is considered as the baseline topography after CXL based on which the progression of KC is considered. Thus, all patients are evaluated at 6 months after CXL by a complete ophthalmic workup including assessment of uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest and cycloplegic refractions, and anterior and posterior segment evaluation with dilated fundus examination, as well as an anterior/posterior topography. Corneal topography  was conducted with undilated pupils under scotopic conditions by a single experienced technician. Baseline strategy for the treatment of recurrent allergic episodes was based on topical mild steroids (Fluorometholone 0.1%) to be used four times a day for 10 to 14 days as needed. In case of severe exacerbations, topical antihistamine and topical cyclosporine 0.1% were added to the regimen.2. The required irradiance of 3.0 mW/cm2 was calibrated prior to each treatment using a UVA meter . During UVA administration, riboflavin drops were applied to the cornea every 2 minutes. Thinnest and central pachymetry were continuously monitored to ensure that none of the two parameters dropped below 400 μm. After treatment, the eye surface was washed with balanced salt solution and two drops of gatifloxacin 0.3% were instilled, followed by the placement of a bandage soft contact lens. Postoperatively, patients received acetaminophen 500 mg twice daily for 3 days, one drop of gatifloxacin 0.3% six times daily for 7 days along with one drop of tobramycin-dexamethasone 0.1% four times daily for 10 days, followed by one drop of Loteprednol 0.5% 5 times daily, slowly tapered over 5 weeks. The bandage soft contact lens was removed on postoperative day 4, and the eye examined by slit-lamp microscopy to confirm complete corneal epithelialization.Primary and redo corneal collagen crosslinking (CXL) were done using the same protocol. All surgeries were performed by the same surgeon (EJ). The eye to be treated was anesthetized by applying proparacaine hydrochloride 0.5% drops on three occasions spaced by five minutes. After positioning the patient under the operating microscope, a lid speculum was inserted and the central 9 mm corneal epithelium was removed with a blunt spatula. A mixed riboflavin 0.1% dextran solution  was instilled every 2 minutes until the riboflavin penetrated the cornea, that is, after approximately 30 minutes. The ultraviolet (UV) lamp  was then focused on the apex of the cornea at a distance of 5 cm for a total of 30 minutes, providing a radiant energy of 3.0 ± 0.3 mW/cmComplications after CXL redo such as significant stromal haze, sterile corneal infiltrates, recurrent erosion syndrome, corneal edema, Desçemets membrane folds, corneal melting and perforation were noted if present. Stability after CXL redo was also assessed.Two hundred twenty-one eyes of 130 patients underwent a corneal collagen CXL for progressive keratoconus or post-LASIK ectasia in our department between March 2010 and March 2013. The demographic and topographic data of the initial 221 eyes are mentioned in K value was > 58.0 D in 3 eyes and <58 D in 4 eyes (mean of 58.6 D). Two eyes (of one patient) had CXL alone, 4 eyes had CXL subsequent to ICRS implantation, and one eye had simultaneous CXL with PRK. Four eyes had a stage 2 keratoconus, 2 eyes had a stage 4 keratoconus, and one eye had a post-LASIK ectasia.Although the majority of the eyes remained stable after primary CXL (according to the aforementioned criteria), seven eyes (3.17%) of five patients met the criteria of progression and necessitated a CXL redo. Patients characteristics are summarized in Progression of KC was noticed more than one year (14 to 48 months) after the original CXL in 6 eyes of four patients  and one eye  was diagnosed with KC progression 9 months after the original CXL. The mean time of KC progression after original CXL was 29.14 months. Progression was noted by all the patients after a decrease in CDVA and was evidenced by progression in corneal topography . ProgresNo major complications after CXL do and redo such as significant stromal haze, sterile corneal infiltrates, recurrent erosion syndrome, corneal edema, Desçemets membrane folds, corneal melting and perforation were noted in any patient. At one year after CXL redo, all patients remained stable by either UDVA, CDVA, or topographic readings. The characteristics of the 5 patients are summarized in Tables k readings of less than 1.00 D (0.9 in eye 1 and 0.68 in eye 2). Thus, a change in the map difference between two consecutive topographies of 1.00 D (or maybe less), a deterioration of visual acuity (excluding other possible non-cornea-related reasons for deterioration), or any change in the refraction must be taken into account when evaluating the stability after CXL. Any of these indicators are considered as progression and necessitate a redo of CXL. The failure of CXL as a continued progression of keratoconus during the first year postoperatively has been reported in several studies [Despite the proven effect of CXL in halting the progression of KC and corneal ectasia with stabilization in the majority of cases , 19, 20, studies , 23, 24. studies . Hersh e studies . In our  studies –28. Such studies . This ch studies , 30. AccP = 0.048), and preoperative maximum K reading of less than 58.00 D was found to reduce the failure rate to 3% [K max (K max >58 D in 2 eyes and <58 D in 3 eyes). Finally, one out of the five patients in our study with CXL failure in our study had a post-LASIK ectasia. Post-LASIK ectasia might have a higher rate of failure. Hersh et al. reported a reduced effect of CXL in cases of post-LASIK ectasia compared with keratoconus [Risk factors associated with progression after primary CXL remain unclear. In our practice, a history of allergic conjunctivitis with eye rubbing was found to be a common risk factor to all patients in the progression group. However, because of the small number of patients with keratoconus progression, we could not conduct a multifactorial analysis to determine other risk factors. Further prospective studies with multifactorial analysis are thus necessary to determine other risk factors associated with progression of keratoconus after a primary CXL. Similarly, Raiskup-Wolf et al. reported progression in 2 patients with neurodermatitis, a condition in which constant skin and ocular rubbing is present . In factte to 3% . In our atoconus . It was atoconus . Finallyatoconus , 35, 36.The majority of the studies report the failure of CXL during the first year postoperatively , 22, 24.In the literature, the safety, efficiency, and the technique of CXL redo were not previously evaluated. To our knowledge, this is the first report to assess long-term safety and efficacy of CXL redo. We performed the primary and redo corneal collagen CXL using the same classical protocol, and the 7 eyes we treated with CXL redo were stable 1 year after the second CXL. No complications after CXL redo such as significant stromal haze, sterile corneal infiltrates, recurrent erosion syndrome, corneal edema, Desçemets membrane folds, corneal melting and perforation were noted in any of the patients we treated with CXL redo. However, we did not perform an endothelial cell count preoperatively and after CXL and the effect of CXL redo on the endothelial health was not evaluated which constitutes a limitation to our study.Recently, Kanellopoulos and Asimellis introduced a novel, noninvasive, quantitative technique utilizing anterior segment OCT images to quantitatively assess the depth and cross-sectional area of CXL in the corneal stroma. Despite the usefulness of the aforementioned method, OCT was not performed systematically in all patients who underwent CXL treatment in our study; therefore, the value of OCT in determining the depth and effectiveness of CXL treatment was not studied in our group, and this is considered as a limitation factor of our study .In conclusion, according to our understanding of keratocytes turnover in the cornea, the effect of CXL may be transient and progression of KC after primary CXL may happen. Thus, a close followup is mandatory in patients after CXL, even after a stability of years after the procedure and CXL redo procedures for those cases who progressed seems to be a safe and efficient technique to halt the progression of keratoconus or post-LASIK corneal ectasia after a failed primary CXL.


Stegodyphus dumicola, colonies' boldest individuals wield a disproportionately large influence over colony behaviour. We experimentally manipulated keystones' tenure in laboratory-housed colonies and tracked their legacy effects on collective prey capture following their removal. We found that bolder keystones caused more aggressive collective foraging behaviour and catalysed greater inter-individual variation in boldness within their colonies. The longer keystones remained in a colony, the longer both of these effects lingered after their departure. Our data demonstrate that, long after their disappearance, keystones have large and lasting effects on social dynamics at both the individual and colony levels.The collective behaviour of social groups is often strongly influenced by one or few individuals, termed here ‘keystone individuals’. We examined whether the influence of keystone individuals on collective behaviour lingers after their departure and whether these lingering effects scale with their tenure in the group. In the social spider,  The coordinated gliding of fish schools when evading predators or the emergent nest structures of social insects represent collective adaptations that afford groups advantages that are not achievable for solitary individuals . Such cokeystone individuals (or just ‘keystones’). Though one may reason that keystone individuals might be a relatively rare phenomenon, a recent literature review on the topic identified more than 80 case studies where just one or a few highly influential group members shape group behaviour and success [We define individuals that exhibit a disproportionate large influence over collective behaviour as  success . Movemen success –19, know success –22, hype success , catalyt success ,25 and d success  represenThe reliance of a group on one influential individual may reduce its robustness to perturbation. For instance, groups' ideal collective phenotype could be compromised if their keystone individuals leave or die . Such grStegodyphus are a superb model for the study of keystone individuals. Social spiders live in multi-female societies that are inbred and exhibit female-biased sex ratios [Stegodyphus dumicola , colonies differ substantially in their collective aggressiveness during prey capture [S. dumicola, colonies’ collective aggressiveness and success are both predicted by the behavioural phenotype of the single boldest individual within the colony. Colonies with just one very bold individual exhibit heightened aggressiveness during prey capture, increased within-colony behavioural variation and gain mass more rapidly in laboratory conditions relative to all-shy colonies [Social spiders of the genus x ratios ,34 and ix ratios ,36. In S capture ,38. Some capture , group-l capture , and eve capture ,42, thou capture ,44. For  capture . Additio capture . In S. dcolonies . When thcolonies . Recent Here we explore what controls the tenure of the keystone individual's long-lasting impacts on group collective behaviour. We term the lingering behavioural changes imposed by the keystone individual ‘legacy effects’. We reason that the longer keystone individuals remain within groups, the greater their influence will be on the behaviour of their fellow group members. Specifically, we test the following hypotheses: (H1) the influence of keystone individuals will linger following their departure but these legacy effects will dissipate with time; (H2) the duration of keystones' legacy effects will scale positively to the amount of time that they spent within the group prior to removal and (H3) keystones with longer tenure will create a larger shift in the behaviour of their fellow group members.2.(a)N = 35) of S. dumicola were collected in February 2014 along the southern Kalahari Desert near Upington, Northern Cape, South Africa (S 28°27′11.8″ E 21°22′51.8″). Colonies ranged in size from 232 to 689 individuals. Spiders were transported to the laboratory at the University of Pittsburgh where our experiments were conducted. Only mature females were used in the studies reported here. Colonies were provided a maintenance diet of ad libitum domestic crickets twice weekly. All of our experimental colonies were established within three weeks of returning to laboratory as detailed below.Whole colonies ((b)Stegodyphus respond to this stimulus by drawing in their legs in the form of a death feign. We then recorded spiders' latency to resume a normal posture and move one complete body length. Trials were terminated after 10 min (600 s). Some individuals resume normal activity rapidly, which we refer to as bold behaviour, and others fail to resume normal activity even after 10 min, which we refer to as shy behaviour. In S. dumicola, individuals' latency to resume movement is highly repeatable over several weeks [We assessed individuals' boldness using an established aversive stimulus assay ,48. Triaal weeks ,51,52 an(c)Experimental colonies were housed in 490 ml deli containers each containing a tangled ball of poultry wiring to facilitate web construction. Colonies were established 20 days prior to the start of our removal experiment. ‘Keystone removal’ colonies were created with 19 very shy individuals that each exhibited a boldness index score of zero. To this group of 19 spiders, one haphazardly selected, putative keystone individual of variable boldness was added to the group. In some groups, this individual was only slightly bolder than its fellow colony members (e.g. boldness score 5–150) whereas in others it was extraordinarily bold (e.g. boldness scores 400–600). In natural colonies, the vast majority of individuals exhibit a shy behavioural type, thus, the phenotypic ratios used in our study represent a reasonable approximation of those seen in nature . All indN = 30 colonies per treatment group): (1) a keystone individual was added on the day of colony establishment, i.e. 20 days before its subsequent removal (added on day-20); (2) a keystone was added 10 days after colony establishment (added on day-10) or (3) a keystone was added 15 days after colony establishment (added on day-5). On the same day (day 0), we removed the keystone from all colonies. Thus, colonies harboured their keystone for 20, 10 or 5 days. This procedure held the date of removal and the duration of group tenure constant among treatments. To control for changes to group size, we created paired control colonies containing 18 shy individuals and one putative keystone 20 days prior to the start of the experiment. We then added one very shy individual 20, 10 or 5 days before the start of the experiment (N = 30 colonies per control treatment). Then, on the same day that we removed the keystones in the treatment groups, we removed the added shy control individuals from their colonies .To determine the effect of keystones' tenure on their legacy effects, we established three treatment groups that varied in the duration of exposure to the keystone individual  individual, we assayed the collective aggressiveness during staged prey capture events for all colonies 1, 3, 6, 12 and 24 days after removal. Colony prey capture was assessed by placing a 1.5 × 1.5 cm piece of white paper within the capture web and vibrating it using a handheld vibratory device (GoVibe), producing a controlled and consistent stimulus across all trials. Colonies were not exposed to this vibratory stimulus until day 1 of our experiment . Thus, the vibrating paper represented a novel prey stimulus for all of our colonies. We recorded the number of attackers that emerged in response to the stimulus over the next 10 min, and noted if the keystone individual was among the attackers. We then provided colonies a four-week-old domestic cricket as a reward for attacking the stimulus. In nature, the number of individuals that respond to prey is positively related to the probability that prey will be successfully subdued –57. ThisTo determine whether the boldness of the keystone individual influenced the boldness of other colony members, and whether these effects depended on the duration of time that keystones remained within their colony, we conducted further individual boldness assays at the end of our study. After all collective prey capture assays were complete, we dismantled all of the colonies and haphazardly selected three to four shy individuals per colony. Each of these individuals was subjected to three boldness assays as described above, once per day for 3 consecutive days.(d)To examine whether the effects of the keystone individual dissipate overtime (H1) and whether the duration of the legacy effect varied according to the tenure of the keystone individual (H2), we tested if the number of attackers in the collective prey capture assays changed over time and among treatments using a repeated measures ANOVA. Day of collective prey capture trial, treatment  and the interaction between day and treatment were effects in the model. Because keystone boldness varied among colonies, and colony aggression scales with the boldness of the keystone individual , we furtFinally, we provide a list of statistics describing the boldness scores of non-keystone group members at the beginning versus the end of our experiments. These values were obtained by calculating the average and standard deviation in boldness scores of all non-keystone individuals at the start of our experiments and comparing these values to the average and standard deviation in boldness scores using three to four representative individuals per colony at the end of our experiments. Statistical analysis was conducted in ‘R’ v. 3.1.2 .3.a). There was a significant change in the number of attackers over time, and overall, the number of attackers was significantly lower in the colonies that contained a keystone for only 5 days : F2,88 = 4.904, p = 0.0095; test day (time): F4,352 = 22.336, p < 0.0001; time × treatment: F8,352 = 1.664, p = 0.106). No such patterns were noted in control colonies : F2,87 = 0.535, p = 0.588; test day (time): F4,351 = 1.314, p = 0.264; time × treatment: F8,351 = 1.342, p = 0.222). The data meet the sphericity assumptions according to Mauchly's test, p > 0.05, for both control and treatment colonies.Regardless of how long the keystone had been in the colony, the number of attackers that responded to prey tended to decrease over time following the removal of the keystone a. There b; electronic supplementary material, figure S1, Pearson's correlation: all r > 0.63, all ps < 0.0001). However, for keystone removal colonies, the correlation between the removed keystone's boldness and the aggressiveness of the colony decayed over time. This rate of decay was associated with the duration of time that the keystone had remained in the colony (figure 2a). Twelve days after the keystone was removed, the association between the keystone's boldness index and the number of attackers had dissipated in colonies that contained keystones for only 5 or 10 days. However, for colonies that contained a keystone for 20 days prior to its removal, the association between the keystone's boldness and the number of attackers lingered for the entirety of the study, 24 days after the keystone had been removed.As found in previous studies, the boldness of colonies' keystones was tightly associated with the number of spiders that attacked during staged prey capture trials. This association emerged in control colonies regardless of the day that we measured the colony . No difference in inter-individual variation was observed among control treatments . The colony average boldness index of formerly shy individuals did not differ among keystone removal treatments  or control removal treatments  at the end of experiments. Regardless of treatment, however, the average boldness of formerly shy individuals increased during our experiments. At the beginning of all treatments the mean ± s.d. boldness of the shy individuals was 0 ± 0. At the end of the experiments the average boldness for the various treatments was: keystone removal 5 = 249.63 ± 63.02; keystone removal 10 = 230.41 ± 57.47; keystone removal 20 = 265.87 ± 62.73; control removal 5 = 289.83 ± 31.5; control removal 10 = 253.54 ± 49.01 and control removal 20 = 247.19 ± 59.91.At the end of our study, inter-individual variation in boldness among formerly shy individuals was highest in colonies that had contained their keystones for longer durations (figure 3b). Thus, colony-wide tendency to attack did not decrease, just the tendency for keystones to participate. Notably, although keystones' participation in prey capture diminished over time, the association between the boldness index of the keystone and colonies' collective foraging aggressiveness did not decay . Thus, keystone individuals' influence over collective foraging behaviour is not contingent on their consistent participation.As seen in previous experiments, keystones' participation in foraging events tapered off rapidly over time in all of our control colonies . This is despite the fact that control colonies continued to attack prey with an equally large number of attackers b. Thus, 4.S. dumicola, bold keystones exhibit a strong influence over the collective foraging behaviour of their society: colonies containing bolder keystones attack prey with many more attackers than other colonies . Importantly, this effect does not immediately vanish when the keystone disappears. Instead, the impacts of keystone individuals echo for a variable amount of time following their departure . Second, we found that the association between keystone individuals' boldness and colony foraging behaviour persisted longer in colonies that contained their keystones for more time (a). These findings suggest that keystones shift the behaviour of their fellow colony members, causing semi-stable changes in collective foraging that persist after the keystone individual's departure. More broadly, these data suggest that keystone individuals  that persist in their roles for longer are more likely to have long-lasting effects on societal behaviour/culture, as seen in human societies and potentially some non-human primates [The duration of a keystone individual's legacy effects scaled positively to the amount of time that it had spent within its group. First, we found that colonies' collective aggressiveness during prey capture decreased over time following the departure of their keystone, and this decrease was more pronounced in colonies that contained their keystone for less time  and somprimates ) in whicb and 2b; electronic supplementary material, figure S1). Thus, keystone effects may be better explained by their catalysis of behavioural variation within their colony . Consistent with this hypothesis, preliminary data suggest that both network structure and within-colony variation in boldness change simultaneously with the presence of a keystone individual. However, the relative contribution of either factor to the phenomena observed here is yet unknown. Similar catalytic mechanisms have been proposed in other kinds of cooperative hunters, like chimpanzees [Keystone individuals may affect collective behaviour by either performing the task themselves and organizing others to perform it, or by catalysing long-lasting behavioural changes in colony members . Althoug1r colony . Such individual . Notablydividual ,48. Heredividual , changesmpanzees ,25.S. dumicola is important for the functioning and success of their societies. Indeed, the presence of just one bold colony member increases the collective mass gain of fellow colony members by 200–300% and reduces mortality rates by 40% relative to colonies of all-shy individuals [The ecological implications of keystone individuals' ability to enhance colony aggressiveness are substantial. Like wild dogs, social spiders are cooperative hunters that as a group can capture larger and more profitable prey than solitary individuals –57. Subdividuals . Fortunaa). In contrast, the within-group variation of colonies that housed a keystone individual for 20 days before its removal was nearly identical to the behavioural variation observed in the control colonies  at the end of the study. However, colonies that contained keystones for longer exhibited a more behaviourally diverse group composition at the end of our study  and thesfigure 3dumicola  and othegodyphus  have shoOur study offers several conceptual and empirical advancements for our understanding of how keystone individuals influence collective behaviour. While most behavioural ecologists are familiar with examples of how innovative behaviours emerge and spread culturally within a population –71, our 5.S. dumicola, the fact that keystones are able to generate long-lasting shifts in the behavioural tendencies (boldness) of their fellow colony members, even after their departure, highlights the extraordinary influence of keystone individuals on collective outcomes. Few scientists would have presupposed that the behavioural dynamics of social spiders would resemble the hysteresis of physical systems and the collective memory of human societies. The generality of our findings may thus change the kinds of questions asked regarding the interplay between individual variation, social dynamics and collective behaviour.We demonstrate that the legacy effects of keystone individuals have the potential to buffer their societies from radical shifts in collective behaviour associated with their sudden disappearance. However, these effects scale to the former keystone's tenure within the society: groups in which keystones persist for longer periods appear buffered against shifts in collective behaviour, perhaps by producing alternate keystone replacements, whereas societies with short-lived keystones are more likely to exhibit fluctuations in collective behaviour. Although a causal understanding of how keystone individuals exert their influence is still missing for


The aim of this study was to identify the factors which can affect the efficacy of corticosteroid (CORT) therapy in the management of hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome.Research articles reporting the efficacy of CORT therapy to HELLP syndrome patients were searched in several electronic databases including EMBASE, Google Scholar, Ovid SP, PubMed, and Web of Science. Study selection was based on predefined eligibility criteria. Efficacy was defined by the changes from baseline in HELLP syndrome indicators after CORT therapy. Meta-analyses were carried out with Stata software.Data of 778 CORT-treated HELLP syndrome patients recruited in 22 studies were used in the analyses. Corticosteroid treatment to HELLP syndrome patients was associated with significant changes from baseline in platelet count; serum levels of aspartate aminotransaminase, alanine transaminase, and lactic dehydrogenase (LDH); mean blood pressure; and urinary output. Lower baseline platelet count predicted higher change in platelet count after CORT therapy. Lower baseline platelet count and lower baseline urinary output predicted greater changes in LDH levels after CORT therapy. There was also an inverse relationship between the change from baseline in LDH levels and intensive care duration. Higher CORT doses were associated with greater declines in the aspartate aminotransaminase, alanine transaminase, and LDH levels. Incidence of cesarean delivery was inversely associated with the gestation age. The percentage of nulliparous women had a positive association with the intensive care stay duration.High-dose CORT therapy to HELLP syndrome patients provides benefits in improving disease markers and reducing intensive care duration, especially in cases such as mothers with much lower baseline platelet count and LDH levels. Pathological dynamics of the syndrome, that is, microangiopathic hemolytic anemia, liver dysfunction, and thrombocytopenia are not easier to understand at the earliest and therefore diagnosis is often late.5Three main options for the management of HELLP syndrome are as follows: immediate delivery at 34 weeks’ gestation or later; delivery within 48 hours after evaluation and stabilization of the maternal clinical condition and corticosteroid (CORT) treatment during 27 to 34 weeks of gestation; and conservative management for more than 48 to 72 hours in pregnant women before 27 weeks’ gestation.7 It is important to know which factors can affect the efficacy of CORT in improving HELLP syndrome markers to identify subgroups of patients that can be benefitted with CORT treatment. We therefore carried out a systematic review and metaregression analysis to identify the factors affecting the efficacy of CORT therapy by using data generated in the studies, which evaluated the efficacy of CORT therapy in HELLP patients.Use of CORT in the management of HELLP syndrome is frequent. Data generated in the past 2 decades suggest that the CORT treatment to HELLP patients is associated with improvements in biological markers including platelet count, serum aspartate aminotransaminase (AST), alanine transaminase (ALT), and lactic dehydrogenase (LDH) levels, without affecting overall morbidity and mortality.8 Eligibility criteria for the selection of studies are presented in Table Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines are followed for carrying out the present study.Several electronic databases including EMBASE, Google Scholar, Ovid SP, PubMed/Medline, and ASI Web of Science were searched for original research articles published before July 2015. The Medical Subject Headings and keywords specific to this research, including hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, corticosteroid therapy, dexamethasone, betamethasone, prednisolone, peripartum, antepartum, postpartum, lactic dehydrogenase (LDH), aspartate aminotransaminase (AST), and alanine transaminase (ALT), were used in logical combinations and phrases. Search strategy is described in the supplementary information file. Additional methods of search included the scrolling of cross-references of important research articles and exploration of the software corroborations.9Information regarding the endpoints and outcomes, dosage and mode of CORT administration, and clinical, obstetric, perinatal, and demographic characteristics were obtained from identified research articles and organized in Microsoft Excel datasheets. For estimation of the efficacy of CORT therapy, changes from baseline in disease indicators were extracted from respective research reports or calculated from raw data, if not provided. For studies which did not provide standard deviation values of the changes from baseline, these were imputed by using formulae that yield calculate the nearest approximate estimates.2 and the percentage of between-study heterogeneity was assessed with I2 index. For the identification of the predictors and prognostic factors of the efficacy of CORT therapy, the analyses were carried out systematically by testing each of the dependent variables  against several independent variables including the number of patients in a study, CORT total dose, mother's age, nulliparity, gestation week at the time of CORT treatment, baseline platelet count, baseline mean blood pressure, baseline urinary output, baseline AST levels, baseline ALT levels, and baseline LDH levels. A P value of less than 0.1 was considered to show a significant relationship.Metaregression analyses were carried out with Stata software . Restricted maximum likelihood method was used for the metaregression analyses. Between-study variance was tested with tauFor the assessment of publication bias, Begg funnel plot and Egger precision plot were studied, and trim and filled method was applied to estimate missing number of studies.31 which overall recruited 778 CORT-treated patients, were used for the analyses  was 26.12 ± 2.64 (23.2 ± 6 to 33.5 ± 4) years. Gestation age at the time of randomization or CORT treatment was 31.6 ± 2.22 (range 27 ± 1–35.1 ± 2.9) weeks. Of these patients, 41.6 ± 31.4% women were nulliparous. Total dose administered to these patients ranged from 30 to 150 mg, with an average of 60.8 ± 45.5 mg (12 studies data). Incidence of cesarean deliveries in CORT-treated HELLP syndrome patients was 68.9 ± 19.4% (range 33.3%–100%).Important findings of the meta-analysis are presented in Tables Change in platelet count from baseline had a significant inverse relationship with baseline platelet count Figure , Table 3Lower baseline platelet count and lower baseline urinary output also predicted higher changes in LDH levels after CORT therapy as a significant inverse relationship was observed between the changes from baseline in LDH levels and the baseline platelet count or urinary output Table . There wTotal CORT dose had significantly positive association with the changes from baseline in AST, ALT, and LDH levels. Thus, higher CORT doses were associated with more declines in these enzyme levels Table .Incidence of cesarean delivery was inversely associated with the gestation age Figure . The intThis pooled analysis, followed by the metaregression analyses, has found that the CORT treatment to HELLP syndrome patients significantly improves platelet count; AST, ALT, and LDH levels; and urinary output. Changes in platelet count and LDH levels after CORT therapy were more robust in patients with lower baseline platelet count, and these outcomes were also associated with lesser intensive care duration. However, intensive care duration was also positively associated with nulliparity. Cesarean delivery rate was negatively associated with gestation age.32 A statistically significant negative association between the change from baseline in platelet count and the baseline platelet count, and also baseline LDH levels observed in the present study suggests that CORT treatment can be more efficacious in patients with lower platelet count and thus CORT therapy to class 1 patients can be more beneficial. In a RCT also, a faster platelet recovery was found in the class 1 subgroup of HELLP patients after CORT therapy.11 After analyzing HELLP class 1 patients’ data retrospectively, Vigil-De Gracia29 also suggested high-dose CORT therapy for class 1 HELLP syndrome patients.Class 1 HELLP syndrome is associated with relatively higher maternal mortality and poor prognosis in delayed diagnosis cases.33 after analyzing 500 women with severe preeclampsia, have observed that whereas uncontrolled hypertension was a risk factor for cesarean section, there was no association between cesarean section and HELLP syndrome. Haddad et al,34 in a retrospective analysis of 183 HELLP syndrome cases, have found that except for the need for blood products transfusion, onset of HELLP syndrome at or less than 28 weeks’ gestation was not associated with an increased risk of adverse maternal or neonatal outcomes when they compared these cases with severe preeclampsia patients without the HELLP syndrome at a similar gestational age.In the present analysis, we have found that the incidence of cesarean deliveries was inversely associated with the gestation week, and intensive care duration was positively associated with the percentage of nulliparous women in the CORT-treated HELLP syndrome patients. Neither any of the baseline biological marker nor change in the biological markers after CORT therapy was significantly associated with cesarean delivery rate. Recently, Katz et al,35 in the same 183 HELLP syndrome cases, found that women with nadir platelet counts <50,000 cells/μL were at increased risk for adverse maternal outcomes in comparison with those with nadir platelet counts between 50,000 and 100,000 cells/μL. However, their logistic regression analyses could not identify significant associations between the nadir values of platelet count of <50,000/μL, serum AST level of >150 U/L, or serum LDH level of >1400 IU/L and maternal adverse outcome. There was also no significant association between laboratory markers and the adverse outcomes in a study of 44 HELLP syndrome patients.36 The present study also could not find any significant relationship between baseline platelet count, AST, ALT, or LDH, and the incidence of comorbidities in CORT-treated HELLP syndrome patients. Intensive care duration was negatively associated with gestation week and positively associated with nulliparity and percent patients having comorbidities in HELLP patients, but not with the severity of biological markers per se.Haddad et al,Some of the limitations of the present study may have compromised the overall outcomes to some extent. Firstly, studies with varying designs are included because no particular design could make available sufficient data. Secondly, clinical and methodological heterogeneity of the sample population in the form of factors such as the severity of HELLP syndrome, time of CORT administration, and dosage and duration of CORT administration in recruited patients may have impact on overall outcomes. Another factor to compromise the precision of the outcomes may be the handling of missing data. Statistical procedures used to impute measures of dispersal may also have slight impact as some study reports lacked such data.Corticosteroid administration to HELLP syndrome patients provides benefits in improving disease markers. Changes in platelet count and LDH levels after CORT therapy are found to be negatively associated with baseline platelet count, and also with intensive care duration in CORT-treated HELLP syndrome patients. Intensive care duration was, however, positively associated with nulliparity and percentage of patients with comorbidities. Incidence of cesarean delivery was negatively associated with gestation age, but not with any of the biological markers or their changes after CORT therapy. These results favor the use of high-dose CORT therapy to HELLP syndrome patients with greater reductions in platelet count.


Zea mays) seedlings. As the seedlings behavior in growth associate with yield under drought, it could partly reflect the potential of drought adaptability. Growth and physiological responses to progressive drought stress and recovery were observed in seedlings of 10 maize lines. The results showed that drought adaptability is closely related to drought recovery (r = 0.714**), but not to drought resistance (r = 0.332). Drought induced decreases in leaf water content, water potential, osmotic potential, gas exchange parameters, chlorophyll content, Fv/Fm and nitrogen content, and increased H2O2 accumulation and lipid peroxidation. After recovery, most of these physiological parameters rapidly returned to normal levels. The physiological responses varied between lines. Further correlation analysis indicated that the physiological bases of drought resistance and drought recovery are definitely different, and that maintaining higher chlorophyll content (r = 0.874***) and Fv/Fm (r = 0.626*) under drought stress contributes to drought recovery. Our results suggest that both drought resistance and recovery are key determinants of plant drought adaptation, and that drought recovery may play a more important role than previously thought. In addition, leaf water potential, chlorophyll content and Fv/Fm could be used as efficient reference indicators in the selection of drought-adaptive genotypes.Non-irrigated crops in temperate climates and irrigated crops in arid climates are subjected to continuous cycles of water stress and re-watering. Thus, fast and efficient recovery from water stress may be among the key determinants of plant drought adaptation. The present study was designed to comparatively analyze the roles of drought resistance and drought recovery in drought adaptation and to investigate the physiological basis of genotypic variation in drought adaptation in maize ( Drought stress imposes huge reductions in crop yield and is one of the greatest limitations to crop production outside present-day agriculture areas  lines were subjected to a progressive soil drought and subsequent re-watering treatment. Growth was measured during drought stress and re-watering cycle. Drought resistance, drought recovery and drought adaptability were estimated based on the relative growth. Furthermore, several physiological traits, including the relative water content, water potential, osmotic potential, gas exchange parameters, chlorophyll content, chlorophyll fluorescence, H2O2 content, lipid peroxidation, carbohydrate content, nitrogen content and C/N ratio were assessed to investigate the physiological basis of genotypic variation in drought adaptation and to characterize the physiological traits that may indicate plant capacity for drought recovery and drought adaptability.Yield is determined by growth and developmental processes and plant growth was considered as a measure of drought adaptive capacity Blum, . As the Nine maize inbred lines L1~6, L8~10) and the hybrid line Zhengdan 958 (L7), one of the most popular cultivar of maize in China in terms of total area planted, were used in this study. The experiment was conducted in a greenhouse at the Institute of Soil and Water Conservation, Northwest A&F University  with a natural photoperiod (approximately 14 h light) from April to June 2014. The daily mean air temperature during the experiment is shown in Supplementary Figure ~6, L8~10The youngest fully expanded leaves were removed and weighed immediately for measurement of fresh weight (FW). Turgid weight (TW) was determined after leaf segments were immersed in distilled water for 6 h, and dry weight (DW) was measured after leaf segments were dried at 70°C in an oven for 24 h. Each treatment included five replications. The relative water content (RWC) was calculated as: RWC The predawn water potential of the youngest fully expanded leaves was measured using a pressure chamber  between 5:30 and 6:30 a.m., based on the protocol of Chen et al. . Each trThe youngest fully expanded leaves were used for osmotic potential measurement. Frozen leaf samples were placed into 0.5 mL tubes and allowed to thaw at room temperature. After thawing, these 0.5 mL tubes were drilled at the bottom, placed inside 1.5 mL tubes, and then centrifuged at 4000 rpm for 5 min to gather the cell sap. The osmolarity of the collected sap was determined using a dew point microvolt meter , and the osmotic potential was calculated.−2 s−1; the flow rate through the chamber was 500 μmol s−1 and the leaf temperature was 28°C. The ambient CO2 concentration was approximately 380 μmol CO2 mol−1 air, and the vapor pressure deficit was approximately 2.0 kPa. Each treatment included five replications.The photosynthetic rates, stomatal conductance, and transpiration rates of individual leaves were measured between 9:00 and 11:00 a.m. using a portable photosynthesis system . The youngest fully expanded leaf was placed in the chamber at a photon flux density of 1000 μmol mChlorophyll fluorescence of individual leaves was analyzed using a pulse amplitude modulated chlorophyll fluorescence system  according to the method of Xu et al. . The you−1 DW) were then calculated.The pigments were extracted from frozen youngest fully expanded leaf samples (~0.2 g) using 80% acetone on a shaker at room temperature until the tissue was completely bleached. The extract was centrifuged at 5000 g for 5 min, and the supernatant was gathered for absorbance measurement at 646, 470, and 663 nm using a spectrophotometer . The concentration of each pigment was calculated according to the method of Lichtenthaler . The chl2O2 was quantified based on the standard curve.Hydrogen peroxide levels were determined according to the method of Yin et al. . Leaf tiLipid peroxidation was determined through measurement of the malondialdehyde (MDA) content. Leaf material (0.5 g) was homogenized in 5 ml 0.1% (w/v) TCA solution. The homogenate was centrifuged at 12,000 g for 20 min and 0.5 ml of the supernatant was added to 1 ml 0.5% (w/v) TBA in 20% TCA. The mixture was incubated in boiling water for 30 min; the reaction was stopped by placing the reaction tubes in an ice bath. The samples were then centrifuged at 10,000 g for 5 min and the supernatant was used for an MDA assay. The MDA content was measured according to the method of Yin et al. .Dried leaf samples were ground to a fine powder for carbohydrate analysis. Leaf powder (~0.2 g) was extracted with 6 ml of 80% (v/v) ethanol for 30 min in a water bath at 80°C under agitation and centrifuged at 5000 g for 10 min. The supernatant was then collected. The process was repeated three times. The supernatant was combined and diluted with water to 25 ml; 2 ml of this solution was taken and evaporated in a boiling water bath. The samples were dissolved in 10 ml distilled water, and the soluble sugars were measured using the anthrone reagent according to Yemm and Willis .The ethanol-insoluble residue was extracted for starch and measured using the anthrone reagent according to Clegg . The pel2SO4-H2O2. Nitrogen content was determined by the standard macro-Kjeldahl procedure using a Kjeltec 2300 analyzer unit . The C/N ratio was calculated as the ratio of TNC to nitrogen content according to Chen et al. (Finely ground leaf samples (~0.1 g each) were digested in Hn et al. .At each time point, the shoots of 10 seedlings per treatment in each line were harvested and dried in an oven at 70°C for 72 h to determine the total dry weight (DW).Drought resistance was estimated based on relative growth during drought stress.Drought recovery was estimated based on relative growth during drought recovery.Drought adaptability was estimated based on relative growth during the entire drought stress and recovery cycle.post-hoc test, and a P < 0.05 was considered significant. Principle component analysis (PCA) was performed using the relative values of all physiological traits to comprehensively evaluate the differences in plant physiological responses among the lines, and the final total score was calculated to represent physiological responses according to the method of An et al. . Data were analyzed by analysis of variance (ANOVA) using the least significant differences (LSD) n et al. . PearsonTo mimic natural drought stress in the field, a cycle of progressive drought stress and subsequent recovery was imposed by withholding and then reintroducing watering. Soil water content in all the pots in each treatment was adjusted to the same level based on daily measurements of pot weight for all maize lines Figure . In ordeWe next addressed the impact of progressive drought and subsequent re-watering on gas exchange parameters Figure . PhotosyFollowing our analysis of the gas exchange parameters, we investigated the changes in chlorophyll fluorescence and contents of photosynthetic pigment, chlorophyll and carotenoid under progressive drought and subsequent re-watering. As shown in Figure 2O2) and lipid peroxidation  were extracted, and together these dimensions explained over 90 and 94% of the total variability under drought stress and recovery, respectively Figure . The datAs shown in Figures The principal components reflected different aspects of physiological responses. To display the genotypic variation of the physiological responses more intuitively, scores representing total responses to drought and recovery were calculated for each line Table . Under dThe tissues above ground were harvested at the start of treatment, at the end of drought stress, and again at the end of recovery. As shown in Figure To enable a comparative analysis of the roles of drought resistance and drought recovery in drought adaptation, each line's drought resistance, recovery and adaptability were estimated based on relative growth during drought stress, recovery and the entire cycle, respectively. As shown in Figure r2 = 0.11) between resistance and recovery. This result indicates that drought recovery was independent of drought resistance.Next, in order to analyze the relationship between drought resistance and recovery, drought recovery was regressed against drought resistance Figure . There wr2 = 0.11) with resistance but was highly correlated with recovery (r2 = 0.51**). This result indicates that drought recovery played a more important role than resistance in drought adaptation among the maize lines we examined.Strong drought adaptability can result from either strong resistance or strong recovery. To determine which factor contributes more to drought adaptability, drought adaptability was regressed against drought resistance Figure  and agai2O2 content and osmotic potential increased in all lines, while the Fv/Fm, chlorophyll content, effective PSII quantum yield, leaf RWC, transpiration rate, stomatal conductance and photosynthetic rate dropped in all lines; other responses varied among the lines . Then, to characterize the physiological traits possibly related to drought-adaptive capabilities in greater detail, the correlations between relative values of physiological changes and drought-adaptive capability were analyzed , National Key Technology Support Program of China (2015BAD22B01) and National Basic Research Program of China (2015CB150402).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.