Functional characterization of four antenna-biased chemosensory proteins in Dioryctria abietella reveals a broadly tuned olfactory DabiCSP1 and its key residues in ligand-binding.

The orientation of the oligophagous cone-feeding moth Dioryctria abietella (Lepidoptera: Pyralidae) to host plants primarily relies on olfactory-related proteins, particularly those candidates highly expressed in antennae. Here, through a combination of expression profile, ligand-binding assay, molecular docking and site-directed mutagenesis strategies, we characterized the chemosensory protein (CSP) gene family in D. abietella. Quantitative real-time PCR (qPCR) analyses revealed the detectable expression of all 22 DabiCSPs in the antennae, of which seven genes were significantly enriched in this tissue. In addition, the majority of the genes (19/22 relatives) had the expression in at least one reproductive tissue. In the interactions of four antenna-dominant DabiCSPs and different chemical classes, DabiCSP1 was broadly tuned to 27 plant-derived odors, three man-made insecticides and one herbicide with high affinities (Ki < 6.60 µM). By contrast, three other DabiCSPs (DabiCSP4, CSP6 and CSP17) exhibited a narrow odor binding spectrum, in response to six compounds for each protein. Our mutation analyses combined with molecular docking simulations and binding assays further identified four key residues (Tyr25, Thr26, Ile65 and Val69) in the interactions of DabiCSP1 and ligands, of which binding abilities of this protein to 12, 15, 16 and three compounds were significantly decreased compared to the wildtype protein, respectively. Our study reveals different odor binding spectra of four DabiCSPs enriched in antennae and identifies key residues responsible for the binding of DabiCSP1 and potentially active compounds for the control of this pest.

Expression profile and functional characterization of odorant binding proteins in a forest pest, Dioryctria abietella (Lepidoptera: Pyralidae).

In the forest ecosystem dominated by the Pinaceae plants, this boring pest Dioryctria abietella is subject to a variety of odorants derived from host and nonhost plants, in which olfactory-related proteins enriched in antennae are key behavioral modulators for the orientation of feeding and ovipositing hosts. Here, we addressed the odorant binding protein (OBP) gene family in D. abietella. Expression profiles revealed that the majority of OBPs were abundantly expressed in the antennae at a female-biased level. A male-antenna-biased DabiPBP1 was a strong candidate for detecting type I and type II pheromones of D. abitella female moths. Using a prokaryotic expression system combined with affinity chromatography, we harvested two antenna-dominant DabiOBPs. In the ligand-binding assays, the two DabiOBPs exhibited different odorant response spectra, as DabiOBP17 was tuned to most odorants with higher affinities compared to DabiOBP4. Of these, DabiOBP4 could strongly bind syringaldehyde and citral (dissociation constants (Ki) < 14 µM). A floral volatile, benzyl benzoate (Ki = 4.72 ± 0.20 µM), was the best ligand for DabiOBP17. Remarkably, several green leaf volatiles were found to strongly interact with DabiOBP17 (Ki < 8.5 µM), including Z3-hexenyl acetate, E2-hexenol, Z2-hexenal and E2-hexenal that may mediate a repellent response to D. abietella. Structural analyses of ligands revealed that the binding of the two DabiOBPs to odorants was associated with carbon-chain lengths and functional groups. Molecular simulations identified several key residues involved in the interactions of DabiOBPs and ligands, suggesting specific binding mechanisms. This study highlights olfactory roles of two antennal DabiOBPs in D. abietella, helping the identification of potentially behavioral compounds for the population control of this pest.

Identification and expression profiling of chemosensory membrane protein genes in Achelura yunnanensis (Lepidoptera: Zygaenidae).

During the past decade, antennal transcriptome sequencing has been applied to at least 50 species from 16 families of the Lepidoptera order of insects, emphasizing the identification and characterization of chemosensory-related genes. However, little is known about the chemosensory genes in the Zygaenidae family of Lepidoptera. Herein, we report the transmembrane protein gene repertoires involved in chemoreception from Achelura yunnanensis (Lepidoptera: Zygaenidae) through transcriptome sequencing, bioinformatics, phylogenetics and polymerase chain reaction (PCR) approaches. Transcriptome analysis led to the generation of 555.47 million clean reads and accumulation of 83.30 gigabases of data. From this transcriptome, 132 transcripts encoding 69 odorant receptors (ORs), 33 gustatory receptors (GRs), 26 ionotropic receptors (IRs), and four sensory neuron membrane proteins (SNMPs) were identified, 69 of which were full-length sequences. Notably, the number of SNMPs in A. yunnanensis was the largest set in Lepidoptera to date. Phylogenetic analysis combined with sequence homology highlighted several conserved groups of chemoreceptors, including pheromone receptors (a so-called pheromone receptor (PR) clade: AyunOR50 and novel PR members: AyunOR39 and OR40), a phenylacetaldehyde-sensing OR (AyunOR28), carbon dioxide receptors (AyunGR1-3), and antennal IRs (13 A-IRs). In addition, a Zygaenidae-specific OR expansion was observed, including 15 A. yunnanensis members. Expression profiles revealed 99 detectable chemosensory genes in the antennae and 20 in the reproductive tissues, some of which displayed a sex-biased expression. This study identifies potential olfactory molecular candidates for sensing sex pheromones, phenylacetaldehyde or other odorants, and provides preliminary evidence for the putative reproductive function of chemosensory membrane protein genes in A. yunnanensis.

Combined transcriptomic, proteomic and genomic analysis identifies reproductive-related proteins and potential modulators of female behaviors in Spodoptera litura.

The common cutworm, Spodoptera litura, is a polyandrous moth with high reproductive ability. Sexual reproduction is a unique strategy for survival and reproduction of population in this species. However, to date available information about its reproductive genes is rare. Here, we combined transcriptomics, genomics and proteomics approaches to characterize reproductive-related proteins in S. litura. Illumina sequencing in parallel with the reference genome led to the yields of 12,161 reproductive genes, representing 47.83% of genes annotated in the genome. Further, 524 genes of 19 specific gene families annotated in the genome were detected in reproductive tissues of both sexes, some of which exhibited sex-biased and/or tissue-enriched expression. Of these, manual efforts together with the transcriptome analyses re-annotated 54 odorant binding proteins (OBPs) and 23 chemosensory proteins (CSPs) with an increase of 18 OBPs and one CSP compared to those previously annotated in the genome. Interestingly, at least 35 OBPs and 22 CSPs were transcribed in at least one reproductive tissue, suggestive of their involvement in reproduction. Further proteomic analysis revealed 2381 common proteins between virgin and mated female reproductive systems, 79 of which were differentially expressed. More importantly, 74 proteins exclusive to mated females were identified as transferred relatives, coupled with their specific or high expression in male reproductive systems. Of the transferred proteins, several conserved protein classes across insects were observed including OBPs, serpins, trypsins and juvenile hormone-binding proteins. Our current study has extensively surveyed reproductive genes in S. litura with an emphasis on the roles of OBPs and CSPs in reproduction, and identifies potentially transferred proteins serving as modulators of female post-mating behaviors.

Identification and characterization of detoxification genes in two cerambycid beetles, Rhaphuma horsfieldi and Xylotrechus quadripes (Coleoptera: Cerambycidae: Clytini).

The longhorned beetles, Rhaphuma horsfieldi and Xylotrechus quadripes, are two polyphagous insects with larvae feeding on different host plants. In this study, we identified and characterized three gene superfamilies of cytochrome P450s (CYPs), carboxylesterases (COEs) and glutathione-S-transferases (GSTs) involved in the detoxification of endobiotics (e.g., hormones and steroids) and xenobiotics (e.g., insecticides, sex pheromones and plant allelochemicals) through a combination approach of bioinformatics, phylogenetics, expression profiles and genomics. Transcriptome analyses led to the identification of 281 transcripts encoding 135 P450s, 108 COEs and 38 GSTs from the two beetles, coupled with comparative studies of detoxification genes among coleopteran species, suggesting a correlation between host range and the sizes of P450 or COE gene repertoires. The P450s of two beetles were phylogenetically classified into four clades, representing the majority of genes in the CYP3 clan. The COEs from R. horsfieldi and X. quadripes were separately grouped into 11 and 10 clades, and the GST superfamily was assigned into six clades. Expression profiles revealed that the detoxification genes were broadly expressed in various tissues as an implication of functional diversities. Ultimately and more importantly, five alternative splicing events in the Epsilon GSTs, including RhorGSTe7.1/GSTe7.2 and XquaGSTe3.1/GST3.2, were acquired in Coleoptera, in which these genes and their orthologs shared highly conserved gene structure. Our current study has complemented the resources for the detoxification genes in the family Cerambycidae, and allows for functional experiments to identify candidate molecular targets involved in pest resistance to insecticides like organophosphates, organochlorines and pyrethroids.

Melatonin rescues impaired penetration ability of human spermatozoa induced by mitochondrial dysfunction.

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also 'rescued' the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.

Regional whole-course trauma care - Experiences from a county-level hospital.

Fifty percent of the deaths caused by severe trauma occur within 1 h after injury. With the concepts of "golden 1 h" and "platinum 10 min", the professionals in the field of emergency trauma treatment have agreed on the necessity of establishing a rapid and efficient trauma rescue system. However, due to the size of the hospital, the population in the neighborhood, the local economic conditions and geographical features, how to establish an optimal trauma rescue system remains an issue. In this paper, we introduced our experiences in a county-level hospital located in middle-and high-income areas.

[Effects of defoliation on the allocation of non-structural carbohydrates in roots of Fraxinus mandshurica seedlings.] / åŽ»å¶å¯¹æ°´æ›²æŸ³è‹—æœ¨æ ¹ç³»éžç»“æž„æ€§ç¢³æ°´åŒ–åˆç‰©åˆ†é çš„å½±å“.

Global climate changes would lead to outbreaks of leaf-feeding insects. Leaf loss could reduce photosynthate production, with consequences on non-structural carbohydrates (NSC) storage and allocation in trees. In this study, the responses of NSC and its compartment concentrations in tap-, coarse- and the first to fifth order fine roots of 2-year-old seedlings of Fraxinus mandshurica to defoliation (40% loss of leaf area) were measured from June to October. The results showed that NSC and its compartment concentrations in roots exhibited distinct seasonal dynamics in both control and defoliation treatments. Following defoliation, NSC concentration decreased in tap- and coarse roots by 3.8% and 30.7%, respectively, while increased in the first five order roots by 1.2%-23.5%, to which starch contributed majorly for each root compartment. Soluble sugar concentration was enhanced by defoliation in tap- and coarse roots by 7.1% and 62.3%, respectively, but decreased in the first to fifth order roots by 2.7%-42.8%. Defoliation had different influences on starch and soluble sugar, with positive effects on the ratio of soluble sugar to starch concentrations in tap- and coarse roots but negative effects on the first to fifth order roots. Overall, defoliation decreased photosynthate production in leaves, leading to the remobilization of starch in tap- and coarse roots and the transportation as soluble sugar to fine roots, as well as the following storage in these roots, which would facilitate the resistance of fine roots to the low temperature in winter.

[Effects of elevated N availability on anatomy, morphology and mycorrhizal colonization of fine roots: A review.] / æ°®æœ‰æ•ˆæ€§å¢žåŠ å¯¹ç»†æ ¹è§£å‰–ã€å½¢æ€ç‰¹å¾å’ŒèŒæ ¹ä¾µæŸ“çš„å½±å“.

Increase of nitrogen (N) availability can greatly affect the structure and function of forest ecosystems. Fine root is critical to belowground ecological processes, thus its response to elevated N availability has become a focus of ecological researches. This article reviewed the trend and under-lying mechanism of fine root response to elevated N availability, including root anatomy, morphology and mycorrhizal colonization. We found that cross-sectional areas of root stele and xylem and the diameter, number and wall thickness of xylem vessel (tracheid) all increased, however, cortex thickness, the number of cortical cell layer, diameter of cortical cell and number of passage cell in exodermis decreased with higher N availability. The response of root anatomy was closely related to plant hormones. In addition, mycorrhiza colonization rate decreased after N fertilization, while specific root length (root length per unit root mass) generally decreased in arbuscular mycorrhizal species, while increased in ectomycorrhizal species. By contrast, the root diameter and tissue density varied widely among species under higher N status. These findings based on individual roots and species provided deeper understanding of carbon and nutrient cycles in terrestrial ecosystems. In addition, we discussed some knowledge gaps and proposed several research outlooks for guiding future researches.

Identification of peptides from soybean protein, glycinin, possessing suppression of intracellular Ca2+ concentration in vascular smooth muscle cells.

In this study, we challenged to identify vasoactive peptides in soybean 11S glycinin hydrolysate by thermolysin to regulate intracellular Ca(2+) concentration ([Ca(2+)]i) that can induce constrictive vascular tension. As a function of the inhibition of elevated [Ca(2+)]i by 10 µM angiotensin (Ang) II in vascular smooth muscle cells (VSMCs), eleven peptides were successfully identified from the hydrolysate, among which His-Gly-Lys exhibited the most potent inhibition against [Ca(2+)]i elevation in Ang II-stimulated VSMCs (inhibition at 300 µM: 46.5±8.0% vs. control). The biological capacity of His-Gly-Lys analogues as an [Ca(2+)]i inhibitor was also proven when His-Lys and His-Gly-Arg elicited a significant reduction in [Ca(2+)]i. In contrast, less reduction of [Ca(2+)]i by His-Gly-Ile and His-(3-methyl)-Gly-Lys indicated the importance of the imino proton in His, along with basic amino acids positioned at C-terminal for the effect.

[Seasonal dynamics of carbon and nitrogen concentrations in Phellodendron amurense fine roots].

Taking a 23 years old Phellodendron amurense plantation as test object, the first five order roots of P. amurense were sampled to study the seasonal dynamics of their total carbon (TC), total nitrogen (TN), total non-structural carbohydrates (TNC), and soluble N concentrations, with the correlations among these parameters analyzed. In the first five order roots, the TNC occupied 49% of TC, and the soluble N accounted for 26% of TN. Within the growth season, the rate of TNC to TC increased from 42% in the first order roots to 52% in the fifth order roots, and the rate of soluble N to TN decreased from 28% to 21% correspondingly. All the first five order roots had the lowest concentration of TC but the highest concentration of TN in spring, and the lowest concentrations of TNC and soluble N in summer. The increase of the TC concentration in the roots decreased the concentrations of TNC and soluble N, whereas the increase of the TN concentration decreased the TNC concentration significantly and increased the concentration of soluble N. From the first to the fifth order roots, the TC and TN had an increasing correlation with TNC but a decreasing correlation with soluble N, suggesting the close correlations of TNC and soluble N with TC and TN in P. amurense fine roots.

[Seasonal variations of fine root production and mortality in Larix gmelinii plantation in 2004-2008].

Minirhizotron approach was employed to investigate the seasonal variations of fine root production and mortality in Larix gmelinii plantation in 2004-2008. At the same time, air temperature, precipitation, and soil temperature and moisture at 10 cm depth were recorded. The overall aim of this study was to determine the seasonal patterns of fine root production and mortality in the plantation, and their relationships with the four environmental factors. On an annual basis, the fine root length production ranged from 0.20 to 0.78 mm x cm(-2), while the mortality varied from 0.26 to 0.72 mm x cm(-2). The mean fine root production and mortality in 2004-2006 were 0.67 mm x cm(-2) and 0.59 mm x cm(-2), respectively, being greater than the corresponding values (0.37 mm x cm(-2) and 0.39 mm x cm(-2)) in 2007-2008. During growth season (from May to October), the fine root production in late spring and early summer (June and July) occupied 51% -68% of total, while that in late autumn (October) only occupied 1% -4%. The root mortality in late summer (August) and autumn (September and October) ranged from 59% to 70%, but that in early spring (May) only ranged from 1% to 5%. Correlation analysis indicated that 66% of the variation in fine root production could be explained by air temperature, and only 24% and 27% could be explained by the soil temperature at 10 cm depth and precipitation, respectively. Fine root mortality only showed an exponential positive correlation with the soil temperature at 10 cm depth.

Residue dynamics of procymidone in leeks and soil in greenhouses by smoke generator application.

A modified QuEChERS-GC-MS method for analysis of procymidone residue in leeks and soil was developed and validated. Procymidone residue dynamics and residues in supervised field trials at GAP conditions in leeks and soil in greenhouses were studied. Leek samples were treated under microwave radiation for 1min before homogenization, followed by extracting with acetonitrile and clean-up with reverse solid phase dispersion by primary and secondary amine (PSA). Procymidone residue was determined by GC-MS in selected ion monitoring (SIM) mode. At fortification levels of 0.02, 0.2 and 2mg/kg in leeks and soil, it was shown that recoveries ranged from 74.9% to 100.8% with relative standard deviations of 1.3-8.5%. At four geographical experimental plots, procymidone residue in leeks and soil showed a relatively fast dissipation rate, with half-lives of 4.52-8.76 days for leeks and 3.76-5.65 days for soil. At pre-harvest intervals of 21-30 days, procymidone residue ranged from 0.033 to 0.17mg/kg in leeks, and 0.020-1.75mg/kg in soil. Residues persistence varied in leeks and soil in four field trials, suggesting that it might be affected by some physical and chemical factors, growth dilution factor, soil characteristics and microorganisms.

[Effects of nitrogen fertilization on seasonal dynamics of soil microbial biomass carbon and nitrogen in Larix gmelinii and Fraxinus mandshurica plantations].

This paper studied the seasonal dynamics of soil microbial biomass C (Cmic) and N (Nmic), and of bacteria (cfu(b)), fungi (cfu(f)), and actinomyces (cfu(a)) in Larix gmelinii and Fraxinus mandshurica plantations in 2007-2008 under N fertilization. The two-year averaged soil Cmic and Nmic in L. gmelinii plantation were 13.8% and 18.3% lower than those in F. mandshurica plantation, respectively, but the soil Cmic and Nmic in the two plantations had similar seasonal patterns, being the lowest in May and the highest in September. The Cmic and Nmic, and the cfu(b), cfu(r), and cfu(a), were all greater in topsoil (0-10 cm) than in subsoil (10-20 cm), but the seasonal patterns of cfu(b), cfu(f), and cfu(a), were different from those of Cmic and Nmic. N fertilization decreased the Cmic and Nmic, and the cfu(b), cfu(f), and cfu(a), significantly, with the decrements of Cmic and Nmic being 24% and 63% in L. gmelinii plantation, and 51% and 68% in F. mandshurica, respectively, which suggested that N fertilization limited soil microbial biomass and altered soil microbial communities in the two plantations.

[Effects of nitrogen fertilization on fine root lifespan of Fraxinus mandshurica and Larix gmelinii].

Root observation tubes (minirhizotrons) were used to study the effects of nitrogen addition on the fine root growth of Fraxinus mandshurica and Larix gmelinii, with the correlations between the fine root lifespan and nitrogen availability analyzed. After the nitrogen addition, the fine root number of F. mandshurica and L. gmelinii had a decreasing trend, but the fine root diameter became larger and the branching ratio decreased. The survival rate of F. mandshurica fine roots increased, and the median root lifespan prolonged 105 days, compared with the control. No significant responses to the nitrogen addition were observed in the survival rate of L. gmelinii fine roots. The first-order fine roots with smaller diameter, the roots in surface soil (0-15 cm), and the fine roots newly born in spring and summer were vulnerable to extend their lifespan by nitrogen addition, suggesting that the fine roots with higher physiological activity were easily to be affected by nitrogen fertilization.

[Interactive effects of light intensity and nitrogen supply on fraxinus mandshurica seedlings growth, biomass, and nitrogen allocation].

With sand culture in greenhouse, the responses of Fraxinus mandshurica seedlings growth, biomass, and N allocation to 2 levels of light intensity and 4 levels of N supply were studied. The results showed that under low light intensity, the seedlings shoot/root ratio (S/R) and net N uptake rate (NNUR) increased significantly (P < 0.01), but their relative growth rate (RGR) and net assimilation rate (NAR) had a significant decrease (P < 0.01). The biomass of root, stem, leaf, and total plant under low light was decreased by 36.8% (P < 0.01), 1.7%, 12.7% (P < 0.05) , and 24.3% (P < 0.01), respectively, and the N allocation to leaf increased but that to root was in adverse. At the two light levels, N supply had an obvious promotion effect on the seedlings growth, and the S/R and the N allocation to leaf were increased obviously with increasing N supply. Significant interactive effects of light and N supply were observed on the seedlings diameter, S/R, RGR, and biomass allocation.

[Effects of nitrogen fertilization on ectomycorrhizal infection of first order roots and root morphology of Larix gmelinii plantation].

In this paper, the first order roots of Larix gmelinii plantation under N fertilization were sampled from different soil depths in different seasons to study their morphology under effects of ectomycorrhizal fungi. The results showed that the infection rate of ectomycorrhizal fungi on the first order roots was significantly affected by soil N availability, soil depth, and season. N fertilization induced a decrease of the infection rate, and the decrement varied with soil depth and season. In comparing with the control, the infected first order roots had an obvious variation of their morphology, e. g., averagely, root diameter increased by 18.7%, root length decreased by 23.7%, and specific root length decreased by 16.3%, which differed significantly with N application rate, soil depth, and season. The infection of ectomycorrhizal fungi changed the first order root morphology of L. gmelinii, which might substantially affect the physiological and ecological processes of host plant fine roots.

[Physiological processes and major regulating factors of nitrogen uptake by plant roots].

Soil nitrogen (N) is one of the mineral elements absorbed in large amount by plant roots, while global change could affect its availability, and furthermore, affect the carbon (C) allocation in terrestrial ecosystem. Therefore, the study of plant root N uptake and regulation becomes an important issue in predicting the structure and function of ecosystem. In the biosphere, plants are exposed to different N forms, and long-term biological evolution and environmental adaptation resulted in a significant distinction of plant root N uptake regions and metabolic processes, as well as the regulation of the N uptake. However, plant has formed different mechanisms and strategies for N uptake, because of their living in the soil with dominant sole N form for generations. In this paper, the research advances on how plant root absorbs N and which factors control the N absorption processes were reviewed, with the biological availability of different soil N forms (nitrate, ammonium and organic N), N uptake regions in root, N loading and transport in xylem, and uptake mechanisms of different N forms emphasized. The signal regulation of N uptake and the effects of environmental factors were also considered. Several issues about the present researches on plant root N uptake were discussed.

[Effects of fertilization on fine root biomass of Larix kaempferi plantation].

With 16 years old Larix kaempferi plantation in eastern mountain area of Liaoning Province as test object, this paper studied its total fine root biomass, fine root biomass at different soil depths, and biomass of different root orders under effects of fertilization. The results showed that compared with no fertilization, applying N decreased the total fine root biomass significantly (P < 0.05), while the difference between applying P and N + P was not significant. The fine root biomass in top soil (0-10 cm) was significantly (P < 0.01) higher than that in subsoil (10-20 cm), and in all treatments, it accounted for 64%-73% of the total. The effects of fertilization on fine root biomass varied with soil depth and root orders. In top soil, N fertilization decreased five orders (except the second order) fine root biomass significantly (P < 0.05), while P and P + N fertilization had no significant effects (P > 0.05), except the fifth order root in treatment P, and the second order root in treatment P + N (P < 0.05). In subsoil, treatments N and P had no effects on five orders fine root biomass (P > 0.05), while treatment N + P increased the first order fine root biomass significantly (P < 0.05).