The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two‐step PCR assay to detect Wolbachia in wild‐collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop‐mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia‐positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.
BackgroundLow temperature is a major abiotic stress affecting the production of rapeseed in China by impeding plant growth and development. A comprehensive knowledge of small-RNA expression pattern in Brassica rapa under cold stress could improve our knowledge of microRNA-mediated stress responses.ResultsA total of 353 cold-responsive miRNAs, 84 putative novel and 269 conserved miRNAs, were identified from the leaves and roots of two winter turnip rape varieties ‘Longyou 7’ (cold-tolerant) and ‘Tianyou 4’ (cold-sensitive), which were stressed under − 4 °C for 8 h. Eight conserved (miR166h-3p-1, miR398b-3p, miR398b-3p-1, miR408d, miR156a-5p, miR396h, miR845a-1, miR166u) and two novel miRNAs (Bra-novel-miR3153-5p and Bra-novel-miR3172-5p) were differentially expressed in leaves of ‘Longyou 7’ under cold stress. Bra-novel-miR3936-5p was up-regulated in roots of ‘Longyou 7’ under cold stress. Four and five conserved miRNAs were differentially expressed in leaves and roots of ‘Tianyou 4’ after cold stress. Besides, we found two conserved miRNAs (miR319e and miR166m-2) were down-regulated in non-stressed roots of ‘Longyou 7’ compared with ‘Tianyou 4’. After cold stress, we found two and eight miRNAs were differentially expressed in leaves and roots of ‘Longyou 7’ compared with ‘Tianyou 4’. The differentially expressed miRNAs between two cultivars under cold stress include novel miRNAs and the members of the miR166 and miR319 families. A total of 211 target genes for 15 known miRNAs and two novel miRNAs were predicted by bioinformatic analysis, mainly involved in metabolic processes and stress responses. Five differentially expressed miRNAs and predicted target genes were confirmed by quantitative reverse transcription PCR, and the expressional changes of target genes were negatively correlated to differentially expressed miRNAs. Our data indicated that some candidate miRNAs (e.g., miR166e, miR319, and Bra-novel-miR3936-5p) may play important roles in plant response to cold stress.ConclusionsOur work indicates that miRNA and putative target genes mediated metabolic processes and stress responses are significant to cold tolerance in B. rapa.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1242-4) contains supplementary material, which is available to authorized users.
A comprehensive description of flavonoids and hydroxycinnamic acid derivatives in Brassica napus L. var. napus seeds is important to improve rapeseed quality. HPLC-PDA-ESI(-)-MS(n)/HRMS has been broadly applied to study phenolic compounds in plants. In the present study, crude phenolic compounds extracted from rapeseed were subjected to column chromatography, alkaline hydrolysis, and HPLC-PDA-ESI(-)-MS(n)/HRMS analysis. A total of 91 flavonoids and hydroxycinnamic acid derivatives were detected, including 39 kaempferol derivatives, 11 isorhamnetin derivatives, 5 quercetin derivatives, 6 flavanols and their oligomers, and 30 hydroxycinnamic acid derivatives. A total of 78 of these compounds were tentatively identified; of these, 55 were reported for the first time in B. napus L. var. napus and 24 were detected for the first time in the genus Brassica. This research enriches our knowledge of the phenolic composition of rapeseed and provides a reliable guide for the selection of rapeseed with valuable breeding potential.
Breeders have focused on yellow-seeded Brassica napus (rapeseed) for its better quality compared with the black-seeded variety. Moreover, flavonoids have been associated with this kind of rapeseed. In this study, we applied lipid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS(n)) to compare flavonoids in developing seeds of natural black-seeded B. napus and yellow-seeded introgression lines selected from progenies of B. napus-Sinapis alba somatic hybrids. Aside from the most abundant phenolic compounds (sinapine and sinapic acid) and 1, 2-disinapoylglucose, 16 different flavonoids were identified and quantified, including (-)-epicatechin, five monocharged oligomers of (-)-epicatechin ([DP 2](-), [DP 3](-), [DP 4] [DP 2](-) B2 and [DP 2](-) B5), quercetin, kaempferol, isorhamnetin-dihexoside, kaempferol-sinapoyl-trihexoside, isorhamnetin-sinapoyl-trihexoside, isorhamnetin-hexoside-sulfate, and isorhamnetin-3-O-glucoside. Most of the flavonoids accumulated with seed development, whereas some rapidly decreased during maturation. The content of these flavonoids were lower in the yellow-seeded materials than in the black seeds. In addition, variations of insoluble procyanidin oligomers and soluble phenolic acids were observed among both rapeseed varieties. Transcriptome changes of genes participating in the flavonoid pathway were discovered by quantitative reverse transcription polymerase chain reaction analysis. Consistent with flavonoid changes identified by high performance liquid chromatography analysis, the expression of most genes in the flavonoid biosynthetic pathway was also downregulated.
Brassica napus L. is rich in phenolic components and it has natural antioxidant characteristics which are important to human health. In the present study, the total phenolic and flavonoid contents of developing seeds of yellow- and black-seeded B. napus were compared. Both phenolic and flavonoid contents were significantly higher at 5 weeks after flowering (WAF) in black seeds (6.44 ± 0.97 mg EE/g phenolics and 3.78 ± 0.05 mg EE/g flavonoids) than yellow seeds (2.80 ± 0.13 mg/g phenolics and 0.83 ± 0.01 mg/g flavonoids). HPLC–DAD–ESI/MS analysis revealed different content of 56 phenolic components between yellow and black-seeded B. napus, including kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, quercetin-3-O-sophoroside, procyanidin B2 ([DP 2]), which were significantly reduced in yellow seeds compared with black seeds. Applying the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical assay, we found maximum clearance of DPPH and ABTS in the late developmental stages of yellow and black seeds. Additionally, the ferric reducing antioxidant power (FRAP) value maximized at 5 WAF in black seeds (432.52 ± 69.98 μmol Fe (II)/g DW) and 6 WAF in yellow seeds (274.08 ± 2.40 μmol Fe (II)/g DW). Generally, antioxidant ability was significantly reduced in yellow-seeded B. napus compared to black rapeseed, and positive correlations between antioxidation and flavonoid content were found in both yellow- and black-seeded B. napus.
BackgroundPolyploidy is an important evolutionary mechanism in flowering plants that often induces immediate extensive changes in gene expression through genomic merging and doubling. Brassica napus L. is one of the most economically important polyploid oil crops and has been broadly studied as an example of polyploid crop. RNA-seq is a recently developed technique for transcriptome study, which could be in choice for profiling gene expression pattern in polyploids.ResultsWe examined the global gene expression patterns of the first four generations of resynthesized B. napus (F1–F4), its diploid progenitors B. rapa and B. oleracea, and natural B. napus using digital gene expression analysis. Almost 42 million clean tags were generated using Illumina technology to produce the expression data for 25959 genes, which account for 63% of the annotated B. rapa genome. More than 56% of the genes were transcribed from both strands, which indicate the importance of RNA-mediated gene regulation in polyploidization. Tag mapping of the B. rapa genome generated 19023, 18547, 24383, 20659, 18881, 20692, and 19955 annotated genes for the B. rapa, B. oleracea, F1–F4 of synthesized B. napus, and natural B. napus libraries, respectively. The unambiguous tag-mapped genes in the libraries were functionally categorized via gene ontological analysis. Thousands of differentially expressed genes (DEGs) were identified and revealed the substantial changes in F1–F4. Among the 20 most DEGs are DNA binding/transcription factor, cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs suggested approximately 120 biological pathways.ConclusionsThe systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of early generations of synthesized B. napus. This information broadens our understanding of the mechanisms of B. napus polyploidization and contributes to molecular and genetic research by enriching the Brassica database.
Genetic factors contribute significantly to the etiology of febrile seizures (FS), the most common type of seizures in childhood. However, in most patients with FS, the causative gene is unknown. The purpose of this study was to explore the relationship between human brain-specific gene SEZ-6 and FS. Through amplification of genomic DNA by PCR and sequencing of the resulting products, we screened 75 subjects for mutations in the coding region (17 exons) of the SEZ-6 gene. Fifteen subjects were healthy individuals and 60 subjects had FS. Patients with FS could be divided into sub-groups based on seizure type (42 simple and 18 complex) and family history (41 had a positive family history). All patients have been followed to date to evaluate seizure recurrence and the development of epilepsy. No mutations were found in healthy controls, but 21 of the patients with FS had mutations in SEZ-6, and the most common type of mutation was a heterozygous, cytosine insertion (frame shift mutation) at position 1435 of the cDNA. The mutation incidence was significantly higher in patients with complex FS (vs. simple FS) and in patients with a positive family history. Sixteen of 42 patients with simple FS experienced seizure recurrence during the 1-5-year follow-up period. Fifteen of 18 patients with complex FS also experienced a recurrence during this period. Among these patients with recurrences, five patients with simple FS and six patients with complex FS have developed epilepsy. The mutation incidence among these epileptic patients is 72.7%. The human SEZ-6 gene is related to the occurrence and development of FS and may be a novel candidate gene for epilepsy. Screening for mutations in SEZ-6 may be valuable in predicting FS recurrence or the development of epilepsy.
Sand fly Phlebotomus chinensis is a primary vector of transmission of visceral leishmaniasis in China. The sand flies have adapted to various ecological niches in distinct ecosystems. Characterization of the microbial structure and function will greatly facilitate the understanding of the sand fly ecology, which would provide critical information for developing intervention strategy for sand fly control. In this study we compared the bacterial composition between two populations of Ph. chinensis from Henan and Sichuan, China. The phylotypes were taxonomically assigned to 29 genera of 19 families in 9 classes of 5 phyla. The core bacteria include Pseudomonas and enterobacteria, both are shared in the sand flies in the two regions. Interestingly, the endosymbionts Wolbachia and Rickettsia were detected only in Henan, while the Rickettsiella and Diplorickettsia only in Sichuan. The intracellular bacteria Rickettsia, Rickettsiella and Diplorickettsia were reported for the first time in sand flies. The influence of sex and feeding status on the microbial structure was also detected in the two populations. The findings suggest that the ecological diversity of sand fly in Sichuan and Henan may contribute to shaping the structure of associated microbiota. The structural classification paves the way to function characterization of the sand fly associated microbiome.
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