Deciphering cellular heterogeneity in Spodoptera frugiperda midgut cell line through single cell RNA sequencing.

Using the 10x Genomics Chromium single-cell RNA sequencing (scRNA-seq) platform, we discovered unexpected heterogeneity in an established cell line developed from the midgut of the Fall armyworm, Spodoptera frugiperda, a major global pest. We analyzed the sequences of 18,794 cells and identified ten unique cellular clusters, including stem cells, enteroblasts, enterocytes and enteroendocrine cells, characterized by the expression of specific marker genes. Additionally, these studies addressed an important knowledge gap by investigating the expression of genes coding for respiratory and midgut membrane insecticide targets classified by the Insecticide Resistance Action Committee. Dual-fluorescence tagging method, fluorescence microscopy and fluorescence-activated cell sorting confirmed the expression of midgut cell type-specific genes. Stem cells were isolated from the heterogeneous population of SfMG-0617 cells. Our results, validated by KEGG and Gene Ontology analyses and supported by Monocle 3.0, advance the fields of midgut cellular biology and establish standards for scRNA-seq studies in non-model organisms.

Comparative transcriptional analysis of metabolic pathways and mechanisms regulating essential oil biosynthesis in four elite Cymbopogon spp.

Cymbopogon is an important aromatic and medicinal grass with several species of ethnopharmaceutical importance. The genus is extremely rich in secondary metabolites, monoterpenes like geraniol and citral being principal constituents, also used as biomarker for classification and identification of Cymbopogon chemotypes. In the light of this, present study involved RNA sequencing and comparison of expression profiles of four contrasting Cymbopogon species namely C. flexuosus var. Chirharit (citral rich and frost resistant), C. martinii var. PRC-1 (geraniol rich), C. pendulus var. Praman (the most stable and citral-rich genotype), and Jamrosa (a hybrid of C. nardus var. confertiflorus × C. jwarancusa (rich in geraniol and geranyl acetate). The transcriptome profiles revealed marked differences in gene expression patterns of 28 differentially expressed genes (DEGs) of terpenoid metabolic pathways between the four Cymbopogon sp. The major DEGs were Carotenoid Cleavage Dioxygenases (CCD), Aspartate aminotransferase (ASP amino), Mevalonate E-4 hydroxy, AKR, GGPS, FDPS, and AAT. In addition, few TFs related to different regulatory pathways were also identified. The gene expression profiles of DEGs were correlated to the EO yield and their monoterpene compositions. Overall, the PRC-1 (C. martinii) shows distinguished gene expression profiles from all other genotypes. Thus, the transcriptome sequence database expanded our understanding of terpenoid metabolism and its molecular regulation in Cymbopogon species. Additionally, this data also serves as an important source of knowledge for enhancing oil yield and quality in Cymbopogon and closely related taxa. KEY MESSAGE: Unfolding the new secretes surrounding EO biosynthesis and regulation in four contrasting Cymbopogon species.

A database of crop pest cell lines.

We have developed an online database describing the known cell lines from Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera that were developed from agricultural pests. Cell line information has been primarily obtained from previous compilations of insect cell lines. We conducted in-depth Internet literature searches and drew on Internet sources such as the Cellosaurus database (https://web.expasy.org/cellosaurus/), and inventories from cell line depositories. Here, we report on a new database of insect cell lines, which covers 719 cell lines from 86 species. We have not included cell lines developed from Drosophila because they are already known from published databases, such as https://dgrc.bio.indiana.edu/cells/Catalog. We provide the designation, tissue and species of origin, cell line developer, unique characteristics, its use in various applications, publications, and patents, and, when known, insect virus susceptibility. This information has been assembled and organized into a searchable database available at the link https://entomology.ca.uky.edu/aginsectcellsdatabase which will be updated on an ongoing basis.

RNAi-based gene silencing in Phenacoccus solenopsis and its validation by in planta expression of a double-stranded RNA.

BACKGROUND: Cotton is a cash crop majorly affected by many hemipteran pests, among them the cotton mealybug, Phenacoccus solenopsis. Cotton mealybug attack has a devastating effect on cotton production and causes huge yield losses. RESULTS: In this study, 25 potential RNA interference (RNAi) target genes were selected from the iBeetle database and a transcriptome data set for P. solenopsis. To assess the effectiveness of the selected target genes, three methods were utilized to deliver double-stranded (ds)RNA (ingestion, artificial diet bioassay and transient gene silencing). dsRNA molecules at different concentrations were fed to insects and insect mortality was recorded for each target gene. Based on the mortality data, three genes, Krüppel homologue-1, ADP-ATP/Translocase and IDGF-1, were selected for further gene expression studies using a reduced concentration of dsRNA (5 µg/ml). Of the three genes, Krüppel homologue-1 showed significantly downregulated expression (by 70.81% and 84.33%) at two different time points (8 and 14 days). An RNAi silencing construct was designed for Krüppel homologue-1 under control of the double enhancer CamV35S promoter in the plant binary vector. Significant downregulation of gene expression, by 66.69% and 81.80%, was found for Krüppel homologue-1 using transient gene silencing at the same time intervals. CONCLUSION: This work provides the first evidence for targeting the Krüppel homologue-1 gene in a hemipteran pest, P. solenopsis, using RNAi technology through oral delivery and in planta-based transient gene silencing methods. © 2020 Society of Chemical Industry.

De novo characterization of Phenacoccus solenopsis transcriptome and analysis of gene expression profiling during development and hormone biosynthesis.

The cotton mealybug Phenacoccus solenopsis is a devastating pest of cotton causing tremendous loss in the yield of crops each year. Widespread physiological and biological studies on P. solenopsis have been carried out, but the lack of genetic information has constrained our understanding of the molecular mechanisms behind its growth and development. To understand and characterize the different developmental stages, RNA-Seq platform was used to execute de-novo transcriptome assembly and differential gene expression profiling for the eggs, first, second, third instar and adult female stages. About 182.67 million reads were assembled into 93,781 unigenes with an average length of 871.4 bp and an N50 length of 1899 bp. These unigenes sequences were annotated and classified by performing NCBI non-redundant (Nr) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG), Gene ontology (GO), the Swiss-Prot protein database (Swiss-Prot), and nearest related organism Acyrthosiphon pisum (pea aphid) database. To get more information regarding the process of metamorphosis, we performed a pairwise comparison of four developmental stages and obtained 29,415 differentially expressed genes. Some of the differentially expressed genes were associated with functional protein synthesis, anti-microbial protection, development and hormone biosynthesis. Functional pathway enrichment analysis of differentially expressed genes showed the positive correlation with specific physiological activities of each stage, and these results were confirmed by qRT-PCR experiments. This study gives a valuable genomics resource of P. solenopsis covering all its developmental stages and will promote future studies on biological processes at the molecular level.

Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but for normalization of data requires the use of stable reference genes. However, suitable reference genes are still not known in the case of Phenacoccus solenopsis under variable experimental treatments. The present study focused on the identification of stable housekeeping genes as a reference for analysis under different abiotic and biotic factors in P. solenopsis. We analyzed the relative expression of six commonly used candidate reference genes in different developmental stages, host-feeding assay, temperature treatments and field distribution conditions. Expression stabilities were analyzed by geNorm, NormFinder, and RefFinder. Under developmental and field distribution conditions, ß-Tubulin was found to be most stable reference genes followed by rpl32 and α-Tubulin. In the case host feeding treatment conditions, ß-Tubulin and α-tubulin identified to be the most stable reference genes, while in temperature stress, a combination of α-Tubulin and rpl32 found to be suitable for normalizing the RT-qPCR data. Further, the above-identified genes were validated using RT-qPCR based gene expression analysis of four objective genes namely, Myoinhibitory peptides (MIPs), Zinc_metalloprotease (Zn_Mp), fatty acid synthase (fas) and alpha-glucosidase. Identified reference genes will facilitate gene expression studies in future under different stress treatments in P. solenopsis.