A bHLH transcription factor,CsSPT,regulates high-temperature resistance in cucumber

High-temperature stress threatens the growth and yield of crops. Basic helix-loop-helix(bHLH) transcription factors(TFs) have been shown to play important roles in regulating high-temperature resistance in plants. However, the bHLH TFs responsible for high-temperature tolerance in cucumbers have not been identified. We used transcriptome profiling to screen the high temperature-responsive candidate bHLH TFs in cucumber. Here, we found that the expression of 75 CsbHLH genes was altered under high-temperature stress. The expression of the CsSPT gene was induced by high temperatures in TT(Thermotolerant) cucumber plants. However, the Csspt mutant plants obtained by the CRISPR-Cas9 system showed severe thermosensitive symptoms, including wilted leaves with brown margins and reduced root density and cell activity.The Csspt mutant plants also exhibited elevated H_(2)O_(2) levels and down-regulated photosystem-related genes under normal conditions.Furthermore, there were high relative electrolytic leakage(REC), malondialdehyde(MDA), glutathione(GSH), and superoxide radical(O_(2)^(·-)) levels in the Csspt mutant plants, with decreased Proline content after the high-temperature treatment. Transcriptome analysis showed that the photosystem and chloroplast activities in Csspt mutant plants were extremely disrupted by the high-temperature stress compared with wildtype(WT) plants. Moreover, the plant hormone signal transduction, as well as MAPK and calcium signaling pathways were activated in Csspt mutant plants under high-temperature stress. The HSF and HSP family genes shared the same upregulated expression patterns in Csspt and WT plants under high-temperature conditions. However, most bHLH, NAC, and bZIP family genes were significantly down-regulated by heat in Csspt mutant plants. Thus, these results demonstrated that CsSPT regulated the high-temperature response by recruiting photosynthesis components, signaling pathway molecules, and transcription factors. Our results provide important insights into the heat response mechanism of CsSPT in cucumber and its potential as a target for breeding heat-resistant crops.

Genetics,resistance mechanism,and breeding of powdery mildew resistance in cucumbers(Cucumis sativus L.)

Cucumber is an important vegetable worldwide,and powdery mildew(PM)is a common and serious disease of cucumbers.Breeding disease-resistant cucumber varieties is the most advantageous strategy to control this disease.In recent years,exploration and identification of cucumber PM resistance genes have achieved great advancement,and many genes have been cloned and verified using different methods.However,the resistance mechanism of cucumber PM is still unclear,and many ambiguities need to be elucidated urgently.In this review,we summarized the research advances in PM resistance in cucumbers,including genetic analysis,quantitative trait locus mapping,map-based cloning,transcriptomics,mlo-mediated PM resistance,and mining of noncoding RNAs involved in resistance.Finally,the research directions and the problems that need to be solved in the future were discussed.

Transcriptomic analysis on cucumber tendril reveals GLRs play important roles in thigmotropism and thigmomorphogenesis

Thigmotropism and thigmomorphogenesis are two related and pervasive processes that play crucial roles in plant adaptation to the environment.However,there have been few investigations into the molecular regulatory mechanisms of these phenomena.Cucumber(Cucumis sativus L.)tendrils are ideal material for studying thigmotropism and thigmomorphogenesis because they display a combination of the two processes.Here,we generated the transcriptome profiles of cucumber tendrils at the young,stretch,and coiling stages.Genes related to receptor proteins,transmembrane transport,and ion transport were significantly enriched among those differentially expressed between stages.Pharmacological assays illustrated that three GLUTAMATE RECEPTOR(GLR)genes might play a vital function in perceiving or transducing touch stimulation signals.Comparing the transcriptomes of tendrils and roots after touch stimulation,we found that genes related to extracellular stimulus and xyloglucan metabolism might have conserved functions in the regulation of thigmomorphogenesis.The transcriptome atlas of thigmotropism and thigmomorphogenesis of cucumber tendrils constructed in this study will help further elucidate the molecular mechanisms behind these processes.

The intrinsic developmental age signal defines an age-dependent climbing behavior in cucumber

The tendril is a climbing organ in cucurbits and functions in physical support and to avoid shading by neighboring vegetation.However,how cucurbits produce tendrils to obtain climbing ability is largely unknown.In this study,tendril phenotypes were investigated during different developmental stages.Our results revealed that tendril growth exhibited an age-dependent pattern in cucurbits.Tendril growth was inhibited,and the tendril was formed as a short tendril[nonfunctional tendril(nonF-tendril),approximately 0.1 cm]during the seedling stage.In contrast,enhanced cell proliferation and cell expansion led to rapid elongation of the tendril during the climbing stage,and the tendril formed as a functional tendril(F-tendril,approximately 30 cm)to obtain climbing ability.RT-qPCR detection showed that age-dependent tendril growth correlated negatively with the abundance of the conserved age regulator CsmiR156.Defoliation induced CsmiR156 to inhibit CsSPLs,and F-tendril formation and climbing ability were delayed in defoliated cucumbers,which confirmed the role of CsmiR156 in regulating tendril growth in vivo.Additionally,exogenous gibberellin(GA)treatment showed that GA positively regulated tendril growth,and RT-qPCR detection showed that the GA bio-synthetic genes and metabolic genes were affected by age pathway,suggesting that the age pathway depended on GA bio-synthetic and metabolic pathway to regulate cell expansion to determine tendril growth.In summary,our work reveals that change in tendril type is an important marker of phase transition in cucumber,and tendril growth is regulated by an intrinsic developmental age signal,ensuring that the cucumber obtains climbing ability at a suitable age.

The bHLH transcription factor CsPIF4 positively regulates high temperature-induced hypocotyl elongation in cucumber

High temperature-induced hypocotyl elongation is a typical thermomorphogenesis trait that may significantly affect early seedling growth and subsequent crop yield.The ambient temperature and endogenous auxin are two critical factors that regulate hypocotyl growth.However,the mechanism of temperature and auxin integration in horticultural plants remains poorly understood.In this study,the roles of the basic helix-loop-helix transcription factor CsPIF4 in regulating auxin biosynthesis genes and the auxin content in the hypocotyl of cucumber(Cucumis sativus L.)seedlings under high temperature were investigated.qRT-PCR and in situ hybridization analysis revealed that expression of CsPIF4 was enhanced in the epidermis and vascular bundles in the hypocotyl of cucumber seedlings in response to high temperature.qRT-PCR and HPLC analysis showed that CsPIF4 positively regulated transcription of the auxin biosynthesis gene CsYUC8 and the auxin content in the hypocotyl under high temperature(35℃).The CRISPR/Cas9-mediated knockout of CsPIF4 resulted in a shorter hypocotyl compared with that of the wild type,together with decreased expression of CsYUC8 and lower auxin content in response to high temperature.Furthermore,biochemical assays showed that CsPIF4 could bind directly to the G-box motif of the CsYUC8 promoter and thereby activate CsYUC8 expression.These findings provide insight into the molecular mechanism of high temperature-mediated hypocotyl elongation in cucumber.

A mutation in the promoter of the yellow stripe-like transporter gene in cucumber results in a yellow cotyledon phenotype

Leaf color mutants in higher plants are considered to be ideal materials for studying the chlorophyll biosynthesis,photosynthesis mechanism and chloroplast development.Herein,we identified a spontaneous mutant,yc412,in cultivated cucumber that exhibited yellow cotyledons.The yellow-lethal mutant was diagnosed with an abnormal chloroplast ultrastructure,and reduced photosynthetic capacity and pigment content.Through bulked segregant analysis-based whole-genome sequencing and fine genetic mapping,we narrowed the yellow cotyledons (yc) locus to a 96.8 kb interval on chromosome 3.By resequencing and molecular cloning,we showed that Csyc is a potential candidate gene,which encodes a yellow stripe-like (YSL) transporter.The T to C mutation in the promoter region of Csyc caused the yellow cotyledon phenotype in yc412.Compared to YZU027A (WT),the expression of Csyc was significantly downregulated in the cotyledons of yc412.Silencing of Csyc in cucumber via virus-induced gene silencing resulted in chlorotic leaves,mainly by suppressing the chlorophyll content.Furthermore,a comparative transcriptome analysis revealed that chloroplast-related genes and chlorophyll biosynthesis genes were significantly downregulated in yc412 cotyledons.Our results provide new insights into the molecular function of the YSL transporter in plant chloroplast development and chlorophyll synthesis.

Sea Cucumber Fishery in Aurora, Philippines

This study was conducted to provide a preliminary assessment of the sea cucumber fishery in Aurora, Philippines. This was conducted from February to June 2013. All sea cucumber gatherers and traders, obtained through snowball sampling, in each identified sea cucumber fishery area in six coastal municipalities (Baler, Dipaculao, Dinalungan, Casiguran, Dilasag and Dingalan) were interviewed using a structured interview schedule and focused group discussions (FGD). Results revealed that gathering sea cucumbers were accomplished by handpicking, skin diving or compressor diving. A total of 15 commercial species were regularly gathered. Out of this, three species are of very high economic value (≥Php 4000/kg) (Holothuria nobilis, Holothuria scabra, and Actinopyga lecanora). The average number of kilograms harvested per hour per trip ranges from 0.21 (Holothuria fuscopunctata) to 2.71 (Holothuria scabra), while the average size of commonly gathered sea cucumber varies among species with Holothuria lecanora showed an average length (29.26 cm) greater than recorded 24 cm. Processing mainly involves washing, slicing, boiling, drying and packing. Processed sea cucumbers or trepan were sold to middlemen or direct buyers who usually offered higher prices (36.36% up to 69.20%) than the former. Problems encountered by sea cucumber gatherers and traders include a decline in total catch, a shift in area of collection from intertidal zones to deeper waters, an increase in the number of hours spent in finding and gathering sea cucumbers, the use of compressors in gathering, the decline of larger species and gathering of smaller sizes in huge amount, the absence of conservation program for sea cucumber, and the lenient implementation of government policies with regards to coastal resources management.

Building of lightweight Nb_(2)CT_(x) MXene@Co nitrogen-doped carbon nanosheet arrays@carbon fiber aerogels for high-efficiency electromagnetic wave absorption in X and Ku bands inspired by sea cucumber

The problems of electromagnetic wave(EMW)pollution in X and Ku bands(8–18 GHz)are becoming more and more serious.Therefore,it is urgent to design EMW absorbing materials with high-efficiency such as thin thickness,lightweight,wide bandwidth and strong EMW absorption.Inspired by the biomorph of sea cucumber,Nb_(2)CT_(x) MXene@Co nitrogen-doped carbon nanosheet arrays@carbon fiber aerogels(Nb_(2)CT_(x)@Co-NC@CFA,Nb_(2)CT_(x)=niobium carbide)were constructed by self-assembly,in-situ chemical deposition and subsequent pyrolysis.The carbon fiber aerogel,as the basic skeleton of sea cucumber,forms lightweight three-dimensional interconnected conductive network,enhances the dielectric loss and extends the multiple reflection and absorption paths of EMW.As the tentacles of sea cucumber surface,Nb_(2)CT_(x) MXene and Co nitrogen-doped carbon nanosheet arrays exist rich heterogeneous interfaces,which play an important role in improving EMW polarization loss and optimizing impedance matching.The minimum reflection loss(RLmin)of Nb_(2)CT_(x)@Co-NC@CFA reaches−54.7 dB at 9.84 GHz(2.36 mm)with a low filling ratio of 10 wt.%and the effective absorption bandwidth(EAB)of Nb_(2)CT_(x)@Co-NC@CFA reaches 2.96 GHz(8.48–11.44 GHz)with 2.36 mm and 5.2 GHz(12.8–18 GHz)with 1.6 mm,covering most of X and Ku bands by adjusting thickness.The radar cross section(RCS)value of Nb_(2)CT_(x)@Co-NC@CFA is 26.64 dB·m^(2),which is lower than that of the perfect electrical conductor(PEC),indicating that Nb_(2)CT_(x)@Co-NC@CFA can effectively decrease the probability of the target being detected by the radar detector.This work provides ideas for design and development of EMW absorbing materials with high-efficiency EMW absorption in X and Ku bands.

Effects of Exogenous Glycine Betaine on Oxidation Metabolism in Cucumbers during Low-temperature Storage

[Objective] This study aimed to analyze the effects of different concentrations of glycine betaine（GB） on oxidation metabolism in cucumbers under low-temperature stress and to investigate the possible mechanism of low-temperature resistance in cucumber during low-temperature storage. [Method] Cucumber cultivar Zhongnong No.8 was treated with 0, 5, 10 and 15 mmol/L GB solutions for 15 min and stored at 4 ℃. Changes in oxidative metabolism-related parameters were observed. [Result] Increasing exogenous GB concentration could enhance GB content in cucumbers, decline lipoxygenase（LOX） activity, improve peroxidase（POD） and catalase（CAT） activities, remove effectively hydrogen peroxide（H2O2） and reduce the accumulation of malondialdehyde（MDA）. [Conclusion] Treating cucumbers with10 mmol/L GB exhibited the most remarkable effect.

Determination of Forchlorfenuron Dissipation and Residue in Cucumbers and Red Soil

To assess the risks of forchlorfenuron after application, a residue analysis method for ferchlorfenuron in cucumbers and the red soil of Southern China was established, and the dissipation behavior and residue characteristics of forchlorfenuron were studied under field conditions. The field trials, including dissipation and residue experiments, were conducted in Hunan, Yunnan and Hainan Provinces. Forchlorfenuron was applied at 32 a.i.g ha^-1 rate for the dissipation study and both 21 and 32 a.i.g ha^-1 for the residue study. Representative cucumbers and soil samples were collected at predetermined intervals for HPLC for UV- detector analysis. The average recoveries of the method were 80.8%-92.0% with coefficients variation （CV） between 0.4% and 11.8%. The detection limit of forchlorfenuron in cucumbers and soil samples was 0.003 mg·kg^-1 and the minimum detectable amount was 3.0× 10^-10 g. The half life of forchlorfenuron in cucumbers in the three provinces changed as： Yunnan （5.50 days）, Hunan （5.88 days）, and Hainan （6.53 days） and in the soil was observed： Hainan （6.54 days）, Hunan （7.64 days）, and Yunnan （8.39 days）. The maximum terminal residue in cucumbers at 32 a.i.g ha^-1 rate after 5 days was 0.009 mg·kg^-1 （Yunnan） followed by 0.0085 mg·kg^-1 （Hainan） and 0.0082 mg·kg^-1（Hunan） which was below MRL value of 0.01 mg·kg^-1 established by the USA, EPA. So a waiting period of 5 days should be established after spaying on cucumbers to avoid hazard to human beings.

Bioactive compounds of sea cucumbers and their therapeutic effects

Abstract Sea cucumbers belong to the Class Holothuroidea of marine invertebrates. They are commercially valuable and prized as a food and folk medicine in Asia. Nutritionally, sea cucumbers have an impressive profile of valuable nutrients such as vitamins, minerals and amino acids. A number of unique biological and pharmacological activities/properties, including anticancer, anticoagulant/antithrombotic, antimicrobial, antioxidant, antihyperlipidemic, antihyperglycemic, anti-inflammatory, antihypertension and radioprotective, have been ascribed to various compounds isolated from sea cucumbers. The therapeutic properties and medicinal benefits of sea cucumbers can be linked to the presence of a wide array ofbioactives, especially triterpene glycosides, acid mucopolysaccharide, sphingoid bases, glycolipids, fucosylated chondroitin sulfate, polysaccharides, phospholipids, cerebrosides, phosphatidylcholines, and other extracts and hydrolysates. This review highlights the valuable bioactive components as well as the multiple therapeutic properties of sea cucumbers with a view to exploring their potential uses as functional foods and a natural source of new multifunctional drugs.

Effects of Neodymium Application Mode on Resistance to Fusarium Wilts in Cucumbers

The effects of neodymium application mode on the disease index, PAL, POD and PPO activity, content of lignin and generation rate of ·O2^- in cucumbers were investigated by pot experiment. The results suggest that the disease index decreases after spraying Nd^3＋. Relative protection effects of at-the-same-time-group, induced-group and therapy-group are 7.51%, 27.14% and 11.51%. The PAL activity of Nd ＋ Fo treatment in at-the-same-time-group, induced-group and therapy-group increases by 9.86%, 48.57% and 4.97% respectively compared with that of Fo treatment. The POD activity decreases by 27.52%, 35.87% and 7.67%. The PPO activity increases by 5.75%, 36.58% and 52.19%. The ·O2^- generation rate decreases by 9.49%, 28.08% and 25. 21%.

Effects of fresh-cut and storage on glucosinolates profile using broccoli as a case study

Glucosinolates(GLS) contribute to the unique flavour, nutrition, and plant defence of the Cruciferous vegetables. Understanding the GLS changes through postharvest processing is essential for defined preservation. In this study, four different fresh-cut types, whole flower(W),floret(F), quarterly cut floret(QF) and shredded floret(FS) of broccoli, were stored for 0, 1, 2 and 3 day(s) to explore GLS responses to postharvest treatments. As a result, seven GLS were identified, mainly including glucoraphanin(RAA), neoglucobrassicin(NEO), and glucobrassicin(GBC)and accounting for 52.69%, 20.12% and 14.99% of the total GLS(21.92 ± 0.48) μmol · g ^(-1 )DW, respectively. FS had the sharpest decrease in GLS after three days of storage(6.55 ± 0.37) μmol · g-1DW, while QF had the least(10.16 ± 0.33) μmol · g ^(-1 )DW. All GLS components decreased over storage, except for 4-methoxyglucobrassicin(4 ME) in FS and QF, suggesting its key role in serious wound defence. The results suggested certain postharvest approaches influenced the flavour and nutrition of broccoli.

Insights into flavonoid biosynthesis during cucumber fruit peel coloration based on metabolite profiling and transcriptome analyses

The fruit peel color is a crucial trait of cucumber.To better understand the molecular mechanisms underlying cucumber peel coloration,we compared the UPLC-ESI-MS/MS-based flavonoid metabolomic and RNA sequencing-based transcriptomic profiling of the brown peeled cucumber line‘PW’at six developmental stages.A total of 210 flavonoid metabolites were identified.Of which,117 flavonoid metabolites were differentially accumulated.In this study,weighted gene co-expression network analysis combined with Kyoto Encyclopedia of Gene and Genomes enrichment analysis revealed key genes coding for seven enzymes and eight transcription factors(TFs)associated with flavonoid biosynthesis.Among them,the R2R3MYB CsaV3_4G001130 is the best candidate gene that is responsible for controlling mature fruit colors in cucumber.Sanger sequencing revealed one nonsynonymous SNP in the exon of CsaV3_4G001130 among the selected 11 cucumber lines,which introduced a premature stop codon,generating a truncated protein in pale yellow or creamy peeled fruits.Yeast two-hybrid assays showed a direct interaction of CsaV3_4G001130 with the bHLH TF CsaV3_1G002260 and the WD40 protein CsaV3_5G001800.However,the interactions were influenced by the nonsynonymous SNP we identified.Our finding revealed that the integrated transcriptome and metabolome analysis further demonstrated that the abundance of some pigmented flavonoids(especially anthocyanins and chalcones)contributed to the coloration of‘PW’fruits.These findings pave the way for elucidation of flavonoid biosynthesis and improvement of cucumber peel color in the future.

A novel mutation in ACS11 leads to androecy in cucumber

Sex determination in plants gives rise to unisexual flowers. A better understanding of the regulatory mechanism underlying the production of unisexual flowers will help to clarify the process of sex determination in plants and allow researchers and farmers to harness heterosis. Androecious cucumber(Cucumis sativus L.) plants can be used as the male parent when planted alongside a gynoecious line to produce heterozygous seeds, thus reducing the cost of seed production. The isolation and characterization of additional androecious genotypes in varied backgrounds will increase the pool of available germplasm for breeding. Here, we discovered an androecious mutant in a previously generated ethyl methanesulfonate(EMS)-mutagenized library of the cucumber inbred line ‘406’. Genetic analysis, whole-genome resequencing, and molecular marker-assisted verification demonstrated that a nonsynonymous mutation in the ethylene biosynthetic gene 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 11(ACS11) conferred androecy. The mutation caused an amino acid change from serine(Ser) to phenylalanine(Phe) at position 301(S301F). In vitro enzyme activity assays revealed that this S301F mutation leads to a complete loss of enzymatic activity. This study provides a new germplasm for use in cucumber breeding as the androecious male parent, and it offers new insights into the catalytic mechanism of ACS enzymes.

Comparison of Physiological Properties Between Dwarf and Vinetype Cucumbers(Cucumis sativus Linn.)

Dwarfism is one of the most important traits in crop breeding. In this study, a dwarf cucumber D0462 and a vine NA129 were taken as experimental materials. The metabolism of hydrogen peroxide （H2O2） and superoxide anion radical （O2-·, activities of antioxidant enzymes, and the levels of endogenous hormones of the two cultivars were compared. In the whole growth period, D0462 maintained higher H2O2 and O2-· levels than NA129. The activities of SOD （superoxide dismutase）, CAT （catalase）, APX （ascorbate peroxidase） and IAAO （indoleacetic acid oxidase） in D0462 were found higher than those in NA129. The contents of gibberellic acid （GA3）, indole-3-acetic acid （IAA）, zeatin riboside （ZR） and jasmonic acid （JA） in intemode were determined by enzyme-linked immunosorbent assay （ELISA）. The intemode ofD0462 remained lower levels of GA3, IAA, and ZR. In contrast, JA level in internode of D0462 was higher than that of NA129. Compared with NA129, the cell mumbers in intemode of D0462 did not change, but cell size evidently decreased. The chloroplasts in mature mesophyll D0462 exhibited swollen filled with starch grains instead of regular oval ones, and thylakoids of chloroplast swelled and became ambiguous.

Species Distribution, Diversity, and Abundance of Sea Cucumbers in Tropical Intertidal Zones of Aurora, Philippines

The distribution, diversity, and abundance of sea cucumbers in intertidal zones of Aurora were studied from February to June 2013 to obtain baseline data. A one-hour timed-search survey covering an approximate one kilometer over a ten-meter wide area was conducted in six coastal municipalities to obtain a comprehensive list of sea cucumber species found in the zones. Only four sampling stations (Baler, Dipaculao, Dilasag, and Dingalan) were selected for belt transect survey, 50 × 50 m, based on the availability of coralline, sandy, muddy sand, and rocky habitats which were laid perpendicular and parallel to each intertidal zone. Timed-search survey revealed a total of 15 species of sea cucumbers distributed among two Orders (Order Aspidochirotida and Order Apodida) and four Families (Family Holothuriidae, Family Stichopodidae, Family Synaptidae, and Family Chirotidae) were recorded. Synapta maculata is most common and has widespread distribution. Belt-tranect surveys showed Holothuria leucospilota is among the most frequently occurring species both during day time (58%) and night time (75%). The Dilasag sampling station had the most diverse species (0.71) while the Dipaculao sampling station had the densest species of Holothuria leucospilota (10,014 in∙ha−1) and abundant (95.91%) species.

Genome-wide association study reveals candidate genes for gummy stem blight resistance in cucumber

Gummy stem blight(GSB),caused by Didymella bryoniae,is a serious fungal disease that leads to decline in cucumber yield and quality.The molecular mechanism of GSB resistance in cucumber remains unclear.Here,we investigated the GSB resistance of cucumber core germplasms from four geographic groups at the seedling and adult stages.A total of 9 SNPs related to GSB resistance at the seedling stage and 26 SNPs at the adult stage were identified,of which some are co-localized to previously mapped Quantitative trait loci(QTLs)for GSB resistance(gsb3.2/gsb3.3,gsb5.1,and gsb-s6.2).Based on haplotype analysis and expression levels after inoculation,four candidate genes were identified within the region identified by both Genome-wide association study(GWAS)and previous identified QTL mapping,including Csa3G129470 for gsb3.2/gsb3.3,Csa5G606820 and Csa5G606850 for gsb5.1,and Csa6G079730 for gsb-s6.2.The novel GSB resistant accessions,significant SNPs,and candidate genes facilitate the breeding of GSB resistant cucumber cultivars and provide a novel idea for understanding GSB resistance mechanism in cucumber.

Bacterial community composition in gut content and ambient sediment of two tropical wild sea cucumbers(Holothuria atra and H.leucospilota)

Sea cucumbers play an important role in sediment bioturbation in coral reef and rocky intertidal ecosystems,and bacteria were a commonly-reported component of holothuroid diets.Bacterial community composition in the foregut and hindgut of two common tropical sea cucumbers(Holothuria atra and H.leucospilota)and the ambient surface sediment were investigated using high throughput sequencing based on 16S rRNA gene analysis.A total of 5584 operational taxonomic units(OTUs)were obtained from 25 samples based on a 97%threshold,and more than half of the OTUs(n=3694,66.2%)were shared by the gut contents of two species of sea cucumbers and surrounding sediments.Bacterial richness and diversity in sediment samples were signifi cantly higher than those in the gut content samples(P<0.05).Proteobacteria was the predominant phylum in most samples showing 45.69%±8.61%,70.09%±4.03%,45.88%±5.38%,and 55.19%±0.79%reads in the foregut of H.leucospilota,hindgut of H.leucospilota,hindgut of H.atra,and sediment libraries,respectively,but Bacteroidetes was the predominant phylum with the relative content of 34.98%±5.52%in the foregut of H.atra.Among the dominant genera,reads related to the genera Anderseniella,Ilumatobacter,and Ruegeria were detected in all the gut contents and sediment libraries.A comparison of gut bacteria community between the two species of sea cucumbers suggested that H.atra had stronger feeding preference than H.leucospilota,and the same types of microbes escaped digestion of the two sea cucumber species.Obvious diff erent bacterial community composition in the foreguts of the two species of sea cucumbers and the surrounding sediments might result from the animal’s selective feeding for sediment patches.Bacterial community structure in hindgut contents of H.atra and H.leucospilota both diff ered clearly from adjacent sediments,which indicated feeding activity of deposit-feeding sea cucumbers could change the sedimental bacterial composition.In conclusion,from the perspective of bacteria,sea cucumber H.atra and H.leucospilota had diff erent feeding preferences,yet they could both aff ect bacterial composition in sediments by feeding activity.The motivation for selective feeding and sea cucumber-sediment interaction might be explored in the future.

An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.