Crinkled employs wingless pathway for wing development in Tribolium castaneum

Crinkled is associated with embryonic denticle formation and auditory organ development in Drosophila melanogaster. However, the functions of Crinkled have not been fully investigated. Additionally, the genes that participate in the Crinkled pathway are unknown. Phylogenetic analysis indicates that crinkled exhibits a one‐to‐one orthologous relationship in insects. In Tribolium castaneum, the crinkled gene is 6,498 bp in length and consists of six exons. Crinkled expression peaked during two phases in Tribolium: late embryonic and pupal stages. High levels of crinkled mRNA were detected in the fat body, head, epidermis, ovary, and accessory gland of late adults. Knockdown of crinkled using RNA interference (RNAi) severely affected wing morphogenesis in T. castaneum. We further showed that crinkled silencing reduced forked expression through wingless and shaven‐baby, and RNAi of forked phenocopied the effects of crinkled knockdown in T. castaneum. This study investigated the development role of crinkled in postembryonic stages and indicated that forked mediates the functions of crinkled during wing morphogenesis in T. castaneum.     

Evaluation of inhibitor of apoptosis genes as targets for RNAi-mediated control of insect pests

Apoptosis has been widely studied from mammals to insects. Inhibitor of apoptosis (IAP) protein is a negative regulator of apoptosis. Recent studies suggest that iap genes could be excellent targets for RNA interference (RNAi)-mediated control of insect pests. However, not much is known about iap genes in one of the well-known insect model species, Tribolium castaneum. The orthologues of five iap genes were identified in T. castaneum by searching its genome at NCBI ( https://www.ncbi.nlm.nih.gov/ ) and UniProt ( https://www.uniprot.org/ ) databases using Drosophila melanogaster and Aedes aegypti IAP protein sequences as queries. RNAi assays were performed in T. castaneum cell line (TcA) and larvae. The knockdown of iap1 gene induced a distinct apoptotic phenotype in TcA cells and induced 91% mortality in T. castaneum larvae. Whereas, knockdown of iap5 resulted in a decrease in cell proliferation in TcA cells and developmental defects in T. castaneum larvae which led to 100% mortality. Knockdown of the other three iap genes identified did not cause a significant effect on cells or insects. These data increase our understanding of iap genes in insects and provide opportunities for developing iap1 and iap5 as targets for RNAi-based insect pest control.     

Larval RNA Interference in the Red Flour Beetle,Tribolium castaneum

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.     

Effects of targeting eye color in Tenebrio molitor through RNA interference of tryptophan 2,3‐dioxygenase (vermilion): Implications for insect farming

The gene vermilion encodes tryptophan 2,3‐dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA‐Seq was used to analyze the impact of vermilion‐specific RNA interference on gene expression. There was a 425‐fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G‐protein‐coupled receptor signaling, cell‐cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white‐eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.     

Identification and functional characterization of methyl-CpG binding domain protein from Tribolium castaneum

Methyl-CpG binding domain proteins (MBD) can specifically bind to methylated CpG sites and play important roles in epigenetic gene regulation. Here, we identified and functionally characterized the MBD protein in Tribolium castaneum. T. castaneum genome encodes only one MBD protein: TcMBD2/3. RNA interference targeting this gene at different developmental stages caused lethal phenotypes including metamorphosis deficiency in larvae and pupae, gastrointestinal system problems and fecundity deficiency in adult. Moreover, Tcmbd2/3 knockdown adult showed progressive reduced locomoter activity, a typical neurodegeneration phenotype. This is a common feature of DNA methylation in mammals and has not been found in other insects. However, band shift assays demonstrated that TcMBD2/3 could not bind to methylated DNA, indicating the essential roles of TcMBD2/3 is independent of DNA methylation. Our study provides Tcmbd2/3 plays important roles in T. castaneum and gives new insights into the potential mechanism of action of MBD proteins in insect.     

CRISPR-dCas9-mediated knockdown of prtR,an essential gene in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a widely distributed non-fermentative Gram-negative opportunistic pathogen that is often responsible for nosocomial infections. Gene interference is a potentially valuable tool for investigating essential genes in P. aeruginosa. To establish a gene interference platform in P. aeruginosa, CRISPR system was used with an inactive Cas9 protein. The CRISPR-dCas9 system was cloned into pHERD20T, a shuttle vector with arabinose inducible promoter, and was further modified to target a regulatory gene prtR that is essential for the viability of P. aeruginosa. Cells expressing the prtR-targeting CRISPR interference (CRISPRi) showed growth defect in an arabinose dose-dependent manner. A high-throughput RNA sequencing analysis of bacterial cells with or without the CRISPRi-mediated prtR inhibition indicated that prtRis a global regulator affecting multiple biological processes. In conclusion, the CRISPR-dCas9-based gene knockdown system has been successfully implemented in P. aeruginosa and demonstrated to be an effective tool in the investigation of essential or difficult-to-inactivate genes in this species.     

A CysB regulator positively regulates cysteine synthesis,expression of type III secretion system genes,and pathogenicity in Ralstonia solanacearum

A syringe-like type III secretion system (T3SS) plays essential roles in the pathogenicity of Ralstonia solanacearum, which is a causal agent of bacterial wilt disease on many plant species worldwide. Here, we characterized functional roles of a CysB regulator (RSc2427) in R. solanacearum OE1-1 that was demonstrated to be responsible for cysteine synthesis, expression of the T3SS genes, and pathogenicity of R. solanacearum. The cysB mutants were cysteine auxotrophs that failed to grow in minimal medium but grew slightly in host plants. Supplementary cysteine substantially restored the impaired growth of cysB mutants both in minimal medium and inside host plants. Genes of cysU and cysI regulons have been annotated to function for R. solanacearum cysteine synthesis; CysB positively regulated expression of these genes. Moreover, CysB positively regulated expression of the T3SS genes both in vitro and in planta through the PrhG to HrpB pathway, whilst impaired expression of the T3SS genes in cysB mutants was independent of growth deficiency under nutrient-limited conditions. CysB was also demonstrated to be required for exopolysaccharide production and swimming motility, which contribute jointly to the host colonization and infection process of R. solanacearum. Thus, CysB was identified here as a novel regulator on the T3SS expression in R. solanacearum. These results provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3SS in R. solanacearum.     

An Altered Method of Feeding RNAi That Knocks Down Multiple Genes Simultaneously in the Nematode Caenorhabditis elegans

In reverse genetics, RNA interference (RNAi) which is substitutable for gene-disruption, is an outstanding method for knockdown of a gene’s function. In Caenorhabditis elegans, feeding RNAi is most convenient, but this RNAi is not suitable for knockdown of multiple genes. Hence, we attempted to establish an efficient method of feeding RNAi for multiple knockdown. We produced bacteria yielding three distinct double-stranded RNAs bound to one another, and fed those bacteria to C. elegans. Quantitative RT-PCR and observation of phenotypes indicated that our method is much more efficient than the traditional one. Our method is useful for investigating genes’ functions in C. elegans.     

Biochemical characteristics of immune-associated phospholipase A<Subscript>2</Subscript> and its inhibition by an entomopathogenic bacterium, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A₂ (PLA₂) activity. Recently, an immune-associated PLA₂ gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA₂ gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA₂ (TcPLA₂) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA₂ (sPLA₂) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA₂ inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA₂). The X. nematophila culture broth contained PLA₂ inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA₂ inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA₂-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA₂ gene expression by RNA interference.