ABSTRACT
Understanding the selenium tolerance of different sweet potato [Dioscorea esculenta (Lour.) Burkill] is essential for simultaneously for breeding of new selenium-tolerant varieties and improving the selenium content in sweet potato. Therefore, a greenhouse experiment was conducted from February to April 2022 to evaluate the effect of sweet potato cultivars and selenium (Na2SeO3) concentrations (0-40 mg/L) on plant growth, physiological activities and plant selenium content distribution. The results showed that when the selenium concentration was more than 3 mg/L, the plant growth was significantly affected and the plant height and root length were significantly different compared to the control. While the selenium concentration was 20 and 40 mg/L had the greatest effect on plant growth when the number of internodes and leaves of the plant decreased, the root system stopped growing and the number of internodes of the plant, the number of leaves and the dry-to-fresh weight ratio of the plant a very significant level compared to reached control. The relative amount of chlorophyll in leaves under treatment with a selenium concentration of 1 mg/L was increased, and the relative amount of chlorophyll in 3 mg/L leaves gradually increased with the increase in the selenium concentration. The values of the maximum photochemical efficiency PSII (fv/fm) and the potential activity of PSII (fv/fo) compared to the control under treatment with 40 mg/L selenium concentration and photosynthesis of plants was inhibited. The selenium content in root, stem and leaf increased with the increase in selenium concentration, and the distribution of selenium content in the plant was leaf
ABSTRACT
KEY MESSAGE: Background-independent (BI) and stably expressed (SE) quantitative trait loci (QTLs) were identified using two sets of introgression lines across multiple environments. Genetic background more greatly affected fiber quality traits than environmental factors. Sixty-one SE-QTLs, including two BI-QTLs, were novel and 48 SE-QTLs, including seven BI-QTLs, were previously reported. Cotton fiber quality traits are controlled by QTLs and are susceptible to environmental influence. Fiber quality improvement is an essential goal in cotton breeding but is hindered by limited knowledge of the genetic basis of fiber quality traits. In this study, two sets of introgression lines of Gossypium hirsutum × G. barbadense were used to dissect the QTL stability of three fiber quality traits (fiber length, strength and micronaire) across environments using 551 simple sequence repeat markers selected from our high-density genetic map. A total of 76 and 120 QTLs were detected in the CCRI36 and CCRI45 backgrounds, respectively. Nine BI-QTLs were found, and 78 (41.71%) of the detected QTLs were reported previously. Thirty-nine and 79 QTLs were SE-QTLs in at least two environments in the CCRI36 and CCRI45 backgrounds, respectively. Forty-eight SE-QTLs, including seven BI-QTLs, were confirmed in previous reports, and 61 SE-QTLs, including two BI-QTLs, were considered novel. These results indicate that genetic background more strongly impacts on fiber quality traits than environmental factors. Twenty-three clusters with BI- and/or SE-QTLs were identified, 19 of which harbored favorable alleles from G. barbadense for two or three fiber quality traits. This study is the first report using two sets of introgression lines to identify fiber quality QTLs across environments in cotton, providing insights into the effect of genetic backgrounds and environments on the QTL expression of fiber quality and important information for the genetic basis underlying fiber quality traits toward QTL cloning and molecular breeding.
Subject(s)
Chromosome Mapping , Cotton Fiber , Genetic Linkage , Gossypium/genetics , Quantitative Trait Loci , Alleles , Crosses, Genetic , Genome, Plant , Microsatellite Repeats , Phenotype , Plant BreedingABSTRACT
The plants in the genus Gardenia (Rubiaceae) have long been used as traditional medicines in China. In this study, two new 3,4-seco-cycloartane triterpenes, sootepin J (1) and sootepin K (2), and a novel nor-3,4-seco-cycloartane triterpenes, sootepin L (3), together with two known compounds (4-5), were isolated from the methanolic extract of the leaves and twigs of Gardenia sootepensis. The structures of the new compounds were elucidated by combinations of 1D, 2D NMR experiments and HR-MS data, while the known compounds were identified by comparison of the NMR data with previously published data.
Subject(s)
Gardenia/chemistry , Triterpenes/isolation & purification , China , Molecular Structure , Plant Leaves/chemistry , Spectrum Analysis , Triterpenes/chemistryABSTRACT
MBI9915 is an introgression cotton line with excellent fiber quality. It was obtained by advanced backcrossing and continuous inbreeding from an interspecific cross between the upland cotton (Gossypium hirsutum) cultivar CCRI36 as the recurrent parent and the sea island cotton (G. barbadense) cultivar Hai1, as the donor parent. To study the genetic effects of the introgressed chromosome segments in G. hirsutum, an F2 secondary segregating population of 1537 individuals was created by crossing MBI9915 and CCRI36, and an F2:3 population was created by randomly selecting 347 individuals from the F2 generation. Quantitative trait locus (QTL) mapping and interaction for fiber length and strength were identified using IciMapping software. The genotype analysis showed that the recovery rate for MBI9915 was 97.9%, with a total 6 heterozygous segments and 13 homozygous segments. A total of 18 QTLs for fiber quality and 6 QTLs for yield related traits were detected using the two segregating generations. These QTLs were distributed across 7 chromosomes and collectively explained 0.81%-9.51% of the observed phenotypic variations. Six QTLs were consistently detected in two generations and 6 QTLs were identified in previous studies. A total of 13 pairs of interaction for fiber length and 13 pairs of interaction for fiber strength were identified in two generations. Among them, 3 pairs of interaction for fiber length and 3 pairs of interaction for fiber strength could be identified in all generations; 4 pairs of interactions affected fiber length and fiber strength simultaneously. The results clearly showed that 5 chromosome segments (Seg-5-1, Seg-7-1, Seg-8-1, Seg-20-2 and Seg-20-3) have important effects on fiber yield and quality. This study provides the useful information for gene cloning and marker-assisted breeding for excellent fiber related quality.
Subject(s)
Chromosomes, Plant , Cotton Fiber , Gossypium/genetics , Phenotype , Quantitative Trait Loci , Chromosome Mapping , Crosses, Genetic , Genetic Markers , Genome, Plant , Gossypium/classification , Gossypium/growth & developmentABSTRACT
BACKGROUND: How to develop new cotton varieties possessing high yield traits of Upland cotton and superior fiber quality traits of Sea Island cotton remains a key task for cotton breeders and researchers. While multiple attempts bring in little significant progresses, the development of Chromosome Segment Substitution Lines (CSSLs) from Gossypium barbadense in G. hirsutum background provided ideal materials for aforementioned breeding purposes in upland cotton improvement. Based on the excellent fiber performance and relatively clear chromosome substitution segments information identified by Simple Sequence Repeat (SSR) markers, two CSSLs, MBI9915 and MBI9749, together with the recurrent parent CCRI36 were chosen to conduct transcriptome sequencing during the development stages of fiber elongation and Secondary Cell Wall (SCW) synthesis (from 10DPA and 28DPA), aiming at revealing the mechanism of fiber development and the potential contribution of chromosome substitution segments from Sea Island cotton to fiber development of Upland cotton. RESULTS: In total, 15 RNA-seq libraries were constructed and sequenced separately, generating 705.433 million clean reads with mean GC content of 45.13% and average Q30 of 90.26%. Through multiple comparisons between libraries, 1801 differentially expressed genes (DEGs) were identified, of which the 902 up-regulated DEGs were mainly involved in cell wall organization and response to oxidative stress and auxin, while the 898 down-regulated ones participated in translation, regulation of transcription, DNA-templated and cytoplasmic translation based on GO annotation and KEGG enrichment analysis. Subsequently, STEM software was performed to explicate the temporal expression pattern of DEGs. Two peroxidases and four flavonoid pathway-related genes were identified in the "oxidation-reduction process", which could play a role in fiber development and quality formation. Finally, the reliability of RNA-seq data was validated by quantitative real-time PCR of randomly selected 20 genes. CONCLUSIONS: The present report focuses on the similarities and differences of transcriptome profiles between the two CSSLs and the recurrent parent CCRI36 and provides novel insights into the molecular mechanism of fiber development, and into further exploration of the feasible contribution of G. barbadense substitution segments to fiber quality formation, which will lay solid foundation for simultaneously improving fiber yield and quality of upland cotton through CSSLs.
Subject(s)
Chromosomes, Plant/genetics , Cotton Fiber , Gene Expression Profiling , Gossypium/growth & development , Gossypium/genetics , Hybridization, Genetic , Cell Wall/metabolism , Gossypium/cytology , Phenotype , Reproducibility of ResultsABSTRACT
As high-strength cotton fibers are critical components of high quality cotton, developing cotton cultivars with high-strength fibers as well as high yield is a top priority for cotton development. Recently, chromosome segment substitution lines (CSSLs) have been developed from high-yield Upland cotton (Gossypium hirsutum) crossed with high-quality Sea Island cotton (G. barbadense). Here, we constructed a CSSL population by crossing CCRI45, a high-yield Upland cotton cultivar, with Hai1, a Sea Island cotton cultivar with superior fiber quality. We then selected two CSSLs with significantly higher fiber strength than CCRI45 (MBI7747 and MBI7561), and one CSSL with lower fiber strength than CCRI45 (MBI7285), for further analysis. We sequenced all four transcriptomes at four different time points postanthesis, and clustered the 44,678 identified genes by function. We identified 2200 common differentially-expressed genes (DEGs): those that were found in both high quality CSSLs (MBI7747 and MBI7561), but not in the low quality CSSL (MBI7285). Many of these genes were associated with various metabolic pathways that affect fiber strength. Upregulated DEGs were associated with polysaccharide metabolic regulation, single-organism localization, cell wall organization, and biogenesis, while the downregulated DEGs were associated with microtubule regulation, the cellular response to stress, and the cell cycle. Further analyses indicated that three genes, XLOC_036333 [mannosyl-oligosaccharide-α-mannosidase (MNS1)], XLOC_029945 (FLA8), and XLOC_075372 (snakin-1), were potentially important for the regulation of cotton fiber strength. Our results suggest that these genes may be good candidates for future investigation of the molecular mechanisms of fiber strength formation and for the improvement of cotton fiber quality through molecular breeding.
Subject(s)
Gene Expression Regulation, Plant , Gossypium/genetics , Chromosomes, Plant , Cotton Fiber , Crosses, Genetic , Gene Expression Profiling , Genotype , PhenotypeABSTRACT
Two new cyclic nonapeptides, named clausenlanins A (1) and B (2), were isolated from the roots and rhizomes of Clausena lansium. Their structures were elucidated as cyclo-(Gly1-L-Leu2-L-Ile3-L-Leu4-L-Leu5-L-Leu6-L-Leu7-L-Leu8-L-Leu9) (1) and cyclo-(Gly1-L-Leu2-L-Val3-L-Leu4-L-Leu5-L-Leu6-L-Leu7-L-Leu8-L-Leu9) (2) respectively on the basis of extensive spectroscopic analysis, particularly 2D NMR spectra taken at the temperature of 338 or 303 K and MS.
ABSTRACT
BACKGROUND: Pectin methylesterase (PME, EC 3.1.1.11) is a hydrolytic enzyme that utilizes pectin as substrates, and plays a significant role in regulating pectin reconstruction thereby regulating plant growth. Pectin is one of the important components of the plant cell wall, which forms the main structural material of cotton fiber. In this research, cotton genome information was used to identify PMEs. RESULTS: We identified 80 (GaPME01-GaPME80) PME genes from diploid G. arboreum (A genome), 78 (GrPME01-GrPME78) PME genes from G. raimondii (D genome), and 135 (GhPME001-GhPME135) PME genes from tetraploid cotton G. hirsutum (AD genome). We further analyzed their gene structure, conserved domain, gene expression, and systematic evolution to lay the foundation for deeper research on the function of PMEs. Phylogenetic data indicated that members from the same species demonstrated relatively high sequence identities and genetic similarities. Analysis of gene structures showed that most of the PMEs genes had 2-3 exons, with a few having a variable number of exons from 4 to 6. There are nearly no differences in the gene structure of PMEs among the three (two diploid and one tetraploid) cotton species. Selective pressure analysis showed that the Ka/Ks value for each of the three cotton species PME families was less than one. CONCLUSION: Conserved domain analysis showed that PMEs members had a relatively conserved C-terminal pectinesterase domain (PME) while the N-terminus was less conserved. Moreover, some of the family members contained a pectin methylesterase inhibitor (PMEI) domain. The Ka/Ks ratios suggested that the duplicated PMEs underwent purifying selection after the duplication events. This study provided an important basis for further research on the functions of cotton PMEs. Results from qRT-PCR indicated that the expression level of different PMEs at various fiber developmental stages was different. Moreover, some of the PMEs showed fiber predominant expression in secondary wall thickening indicating tissue-specific expression patterns.
Subject(s)
Carboxylic Ester Hydrolases/genetics , Cotton Fiber , Gene Expression Regulation, Plant , Genome, Plant , Genome-Wide Association Study , Gossypium/classification , Gossypium/genetics , Phylogeny , Carboxylic Ester Hydrolases/metabolism , Cluster Analysis , Enzyme Activation , Gene Expression Profiling , Multigene Family , Reproducibility of Results , TranscriptomeABSTRACT
Chromosome segment substitution lines MBI9804, MBI9855, MBI9752, and MBI9134, which were obtained by advanced backcrossing and continuously inbreeding from an interspecific cross between CCRI36, a cultivar of upland cotton (Gossypium hirsutum) as the recurrent parent, and Hai1, a cultivar of sea island cotton (G. barbadense) as the donor parent, were used to construct a multiple parent population of (MBI9804×MBI9855)×(MBI9752×MBI9134). The segregating generations of double-crossed F1 and F2 and F2:3 were used to map the quantitative trait locus (QTL) for fiber quality and yield-related traits. The recovery rate of the recurrent parent CCRI36 in the four parental lines was from 94.3%-96.9%. Each of the parental lines harbored 12-20 introgressed segments from Hai1across 21 chromosomes. The number of introgressed segments ranged from 1 to 27 for the individuals in the three generations, mostly from 9 to 18, which represented a genetic length of between 126 cM and 246 cM. A total of 24 QTLs controlling fiber quality and 11 QTLs controlling yield traits were detected using the three segregating generations. These QTLs were distributed across 11 chromosomes and could collectively explain 1.78%-20.27% of the observed phenotypic variations. Sixteen QTLs were consistently detected in two or more generations, four of them were for fiber yield traits and 12 were for fiber quality traits. One introgressed segment could significantly reduce both lint percentage and fiber micronaire. This study provides useful information for gene cloning and marker-assisted breeding for excellent fiber quality.
Subject(s)
Breeding , Cotton Fiber , Gossypium/genetics , Quantitative Trait Loci/genetics , Chromosome Mapping , Chromosomes, Plant/genetics , Genetic Linkage , Genome, Plant , Gossypium/growth & development , Microsatellite Repeats/genetics , Phenotype , TextilesABSTRACT
BACKGROUND: Upland Cotton (Gossypium hirsutum) is one of the most important worldwide crops it provides natural high-quality fiber for the industrial production and everyday use. Next-generation sequencing is a powerful method to identify single nucleotide polymorphism markers on a large scale for the construction of a high-density genetic map for quantitative trait loci mapping. RESULTS: In this research, a recombinant inbred lines population developed from two upland cotton cultivars 0-153 and sGK9708 was used to construct a high-density genetic map through the specific locus amplified fragment sequencing method. The high-density genetic map harbored 5521 single nucleotide polymorphism markers which covered a total distance of 3259.37 cM with an average marker interval of 0.78 cM without gaps larger than 10 cM. In total 18 quantitative trait loci of boll weight were identified as stable quantitative trait loci and were detected in at least three out of 11 environments and explained 4.15-16.70 % of the observed phenotypic variation. In total, 344 candidate genes were identified within the confidence intervals of these stable quantitative trait loci based on the cotton genome sequence. These genes were categorized based on their function through gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and eukaryotic orthologous groups analysis. CONCLUSIONS: This research reported the first high-density genetic map for Upland Cotton (Gossypium hirsutum) with a recombinant inbred line population using single nucleotide polymorphism markers developed by specific locus amplified fragment sequencing. We also identified quantitative trait loci of boll weight across 11 environments and identified candidate genes within the quantitative trait loci confidence intervals. The results of this research would provide useful information for the next-step work including fine mapping, gene functional analysis, pyramiding breeding of functional genes as well as marker-assisted selection.
Subject(s)
Chromosome Mapping/methods , Chromosomes, Plant/genetics , Gossypium/genetics , High-Throughput Nucleotide Sequencing/methods , Quantitative Trait Loci/genetics , Analysis of Variance , DNA, Plant/chemistry , DNA, Plant/genetics , Genes, Plant/genetics , Genetic Linkage , Genetic Markers , Genome, Plant/genetics , Genotype , Gossypium/classification , Phenotype , Polymorphism, Single Nucleotide , Species SpecificityABSTRACT
Two new 3,4-seco-cycloartane triterpenes, named sootepins H (1) and I (2), were isolated from the ethyl acetate extract of the leaves and twigs of Gardenia sootepensis. Their structures were elucidated on the basis of 1D- and 2D-nuclear magnetic resonance (NMR) analysis, as well as high-resolution mass spectrometry (HR-MS), infrared (IR), and ultra violet (UV).
Subject(s)
Drugs, Chinese Herbal/isolation & purification , Drugs, Chinese Herbal/pharmacology , Gardenia/chemistry , Triterpenes/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Drug Screening Assays, Antitumor , Drugs, Chinese Herbal/chemistry , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Plant Leaves/chemistry , Plant Stems/chemistry , Structure-Activity Relationship , Triterpenes/chemistryABSTRACT
Three new C20 quassinoids nigakilactone P (1), picraqualide F (2), nigakilactone Q (3), along with eight known quassinoids (4-11), were isolated from the 95% EtOH extract of the stems of Picrasma quassioides. The structures of the new compounds were elucidated by means of HRESIMS and different NMR techniques. Assignments of relative and absolute configurations for these compounds were achieved on the basis of ROESY spectra and quantum chemical ECD calculation. In vitro activity assays, none of the compounds showed cytotoxic (IC50>50 µM) and NO production-inhibitory activities (IC50>30 µM), and the structure-activity relationships of quassinoids were summarized. In addition, the chemotaxonomic significance of the isolated compounds was also discussed.
Subject(s)
Picrasma/chemistry , Plant Extracts/chemistry , Quassins/chemistry , Hep G2 Cells , Humans , Inhibitory Concentration 50 , MCF-7 Cells , Molecular Structure , Nitric Oxide/chemistry , Plant Stems/chemistry , Quassins/isolation & purification , Structure-Activity RelationshipABSTRACT
Two novel naphthohydroquinone dimers with unprecedented skeletons, rubialatins A (1) and B (2), were isolated from the herbal plant Rubia alata together with their precursor, mollugin (3). The structures were elucidated on the basis of NMR spectra and crystal X-ray diffraction. Compound 1, a racemate, was separated by chiral column chromatography, and the absolute configurations of the enantiomers were determined by the computational methods. Cytotoxicity of 1-3 was evaluated as well as the effect on the NF-κB pathway. Compound (+)-1 showed cytotoxicity and could inhibit NF-κB pathway. Meanwhile, 2 showed cytotoxicity and a synergistic effect with TNF-α on NF-κB activation.
Subject(s)
Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Hydroquinones/chemistry , Hydroquinones/pharmacology , Hydroquinones/toxicity , NF-kappa B/chemistry , NF-kappa B/drug effects , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plant Roots/chemistry , Pyrans/chemistry , Rubia/chemistry , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/drug effects , Cell Line, Tumor , Humans , Hydroquinones/isolation & purification , Magnetic Resonance Spectroscopy , Molecular Structure , Pyrans/isolation & purification , Stereoisomerism , Tumor Necrosis Factor-alpha/metabolism , X-Ray DiffractionABSTRACT
To investigate monoterpenes and sesquiterpenes of the stems and leaves of Clausena excavata, an AcOEt fraction of the methanol extract was subjected on column chromatographies including silica gel and RP-18, as well as preparative HPLC. The structures of compounds isolated were identified on the basis of spectroscopic data as excamonoterpene (1), (6R, 9S)-9, 10-dihydroxy-4-megastigmen-3-one (2), (3R, 6R, 7E) -3-hydroxy-4, 7-megastigmadien-9-one (3), (3S) -3-hydroxy-7, 8-dihydro-beta-ionone (4), (3S, 5R, 6S) -3-hydroxy-5,6-epoxy-beta-ionone (5), (6R, 9R) -9-hydroxy-4-megastigmen-3-one (6), (3S, SR) -dihydroxy-6, 7-megstigmadien-9-one(7), (-)-loliolide(8), caryolane-1, 9alpha-diol(9) and 2, 6-dihydroxyhumula-3 (12), 7 (13), 9(E)-triene (10), were isolated from the stems and leaves of C. excavata. Compound 1 is a new monoterpene, named as excamonoterpene. Compounds 2-10 were isolated from this plant for the first time.
Subject(s)
Chromatography, High Pressure Liquid/methods , Clausena/chemistry , Monoterpenes/analysis , Sesquiterpenes/analysis , Magnetic Resonance Spectroscopy , Methanol/chemistry , Molecular Structure , Monoterpenes/chemistry , Plant Leaves/chemistry , Plant Stems/chemistry , Sesquiterpenes/chemistry , Spectrometry, Mass, Electrospray IonizationABSTRACT
A new phenethanol, (2'R)-4-(2', 3'-dihydroxy-3'-methyl-butanoxy)-phenethanol (1), along with other eleven known benzene derivatives (2-12) were isolated from the roots, stems and leaves of Clausena excavata (Rutaceae). Compounds 3 and 4 are new natural products, and compounds 5-8, 10-12 were isolated from C. excavata for the first time. Their structures were elucidated on the basis of MS, 1D and 2D NMR spectroscopic analyses including HSQC, COSY and HMBC experiments. 1 was tested for its cytotoxicities against A549, HeLa and BGC-823 cancer cell lines, and antimicrobial activities against Candida albicans and Staphylococcus aureus. The results showed that 1 did not exhibit cytotoxic and antimicrobial activities.
Subject(s)
Benzene Derivatives/chemistry , Clausena/chemistry , Candida albicans/drug effects , Cell Line, Tumor , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , Plant Leaves/chemistry , Plant Roots/chemistry , Plant Stems/chemistry , Staphylococcus aureus/drug effectsABSTRACT
One new carbazole alkaloid, excavatine A (1), and two additional new alkaloids, excavatine B (2) and excavatine C (3), were isolated from the stems and leaves of Clausena excavata Burm.f. (Rutaceae). Their structures were determined on the basis of detailed spectroscopic analyses, especially 2D-NMR and HR-EI-MS data. Compounds 1-3 were tested for their cytotoxic activities against A549, HeLa, and BGC-823 cancer cell lines, and for their antimicrobial activities against Candida albicans and Staphylococcus aureus. Only 1 exhibited cytotoxicity against A549 and HeLa cell lines with the IC50 values of 5.25 and 1.91â µg/ml, respectively.
Subject(s)
Alkaloids/chemistry , Anti-Infective Agents/chemistry , Anti-Infective Agents/toxicity , Carbazoles/chemistry , Clausena/chemistry , Alkaloids/isolation & purification , Alkaloids/toxicity , Anti-Infective Agents/isolation & purification , Candida albicans/drug effects , Carbazoles/isolation & purification , Carbazoles/toxicity , Cell Line, Tumor , Cell Survival/drug effects , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Molecular Conformation , Plant Leaves/chemistry , Plant Stems/chemistry , Staphylococcus aureus/drug effectsABSTRACT
The wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), is one of the most serious pests of wheat worldwide. It overwinters as cocooned larvae in diapause and non-diapause forms. The cocoon is made of silk from the salivary glands. The silk glands, therefore, play an important role in the S. mosellana diapause. In the present study, the ultra-structures of the silk glands between diapause and non-diapause larvae were examined by electro and light-microscopically. The silk glands consist of 156 cells organized like moniliform particles. Although silk gland cells of both diapause and non-diapause larvae contain developed organelles, including the endoplasmic reticulum, dictyosome, mitochondria, and lipid droplet, the organelles in non-diapause larvae are more developed than those in diapause larvae. These morphological characteristics of the silk glands in the diapause and non-diapause larvae can be used to distinguish the diapause status of the larvae.