Nejvíce citovaný článek - PubMed ID 12063017
BACKGROUND: Leishmaniasis is a group of neglected vector-borne diseases transmitted by phlebotomine sand flies. Leishmania parasites must overcome various defenses in the sand fly midgut, including the insects's immune response. Insect immunity is regulated by the ecdysone hormone, which binds to its nuclear receptor (EcR) and activates the transcription of genes involved in insect immunity. However, the role of ecdysone in sand fly immunity has never been studied. Phlebotomus perniciosus is a natural vector of Leishmania infantum; here, we manipulated its neuroendocrine system using azadirachtin (Aza), a natural compound known to affect ecdysone synthesis. METHODS: Phlebotomus perniciosus larvae and adult females were fed on food containing either Aza alone or Aza plus ecdysone, and the effects on mortality and ecdysis were evaluated. Genes related to ecdysone signaling and immunity were identified in P. perniciosus, and the expression of antimicrobial peptides (AMPs), EcR, the ecdysone-induced genes Eip74EF and Eip75B, and the transcription factor serpent were analyzed using quantitative polymerase chain reaction (PCR). RESULTS: Aza treatment inhibited molting of first-instar (L1) larvae to L2, with only 10% of larvae molting compared to 95% in the control group. Serpent and Eip74EF, attacin, defensin 1, and defensin 2 genes were downregulated by Aza treatment in larvae. Similarly, Aza-treated adult females also presented suppression of ecdysone signaling-related genes and the AMPs attacin and defensin 2. Notably, all gene repression caused by Aza was reversed by adding ecdysone concomitantly with Aza to the larval or female food, indicating that these genes are effective markers for ecdysone repression. CONCLUSIONS: These results highlight the critical role of ecdysone in regulating the development and immunity of P. perniciosus, which potentially could interfere with Leishmania infection.
- Klíčová slova
- Phlebotomus perniciosus, Antimicrobial peptides, Azadirachtin, Ecdysone,
- MeSH
- antimikrobiální peptidy genetika farmakologie MeSH
- ekdyson * MeSH
- hmyz - vektory účinky léků genetika parazitologie imunologie MeSH
- hmyzí proteiny genetika metabolismus MeSH
- larva * účinky léků imunologie genetika MeSH
- limoniny * farmakologie MeSH
- Phlebotomus * účinky léků genetika parazitologie imunologie MeSH
- shazování tělního pokryvu účinky léků MeSH
- signální transdukce * účinky léků MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antimikrobiální peptidy MeSH
- azadirachtin MeSH Prohlížeč
- ekdyson * MeSH
- hmyzí proteiny MeSH
- limoniny * MeSH
Eurasian spruce bark beetle (Ips typographus [L.]) causes substantial damage to spruce forests worldwide. Undoubtedly, more aggressive measures are necessary to restrict the enduring loss. Finishing genome sequencing is a landmark achievement for deploying molecular techniques (i.e., RNA interference) to manage this pest. Gene expression studies assist in understanding insect physiology and deployment of molecular approaches for pest management. RT-qPCR is a valuable technique for such studies. However, accuracy and reliability depend on suitable reference genes. With the genome sequence available and the growing requirement of molecular tools for aggressive forest pest management, it is crucial to find suitable reference genes in Ips typographus under different experimental conditions. Hence, we evaluated the stability of twelve candidate reference genes under diverse experimental conditions such as biotic (developmental, sex and tissues) and abiotic factors (i.e., temperature and juvenile hormone treatment) to identify the reference genes. Our results revealed that ribosomal protein 3a (RPS3-a) was the best reference gene across all the experimental conditions, with minor exceptions. However, the stability of the reference gene can differ based on experiments. Nevertheless, present study provides a comprehensive list of reference genes under different experimental conditions for Ips typographus and contributes to "future genomic and functional genomic research".
- MeSH
- brouci * genetika MeSH
- exprese genu MeSH
- kůra rostlin MeSH
- nosatcovití * genetika MeSH
- reprodukovatelnost výsledků MeSH
- smrk * genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.
Gene expression analysis through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) depends on correct data normalization by reference genes with stable expression. Although Clostridium beijerinckii NRRL B-598 is a promising Gram-positive bacterium for the industrial production of biobutanol, validated reference genes have not yet been reported. In this study, we selected 160 genes with stable expression based on an RNA sequencing (RNA-Seq) data analysis, and among them, seven genes (zmp, rpoB1, rsmB, greA, rpoB2, topB2, and rimO) were selected for experimental validation by RT-qPCR and gene ontology (GO) enrichment analysis. According to statistical analyses, zmp and greA were the most stable and suitable reference genes for RT-qPCR normalization. Furthermore, our methodology can be useful for selection of the reference genes in other strains of C. beijerinckii and it also suggests that the RNA-Seq data can be used for the initial selection of novel reference genes, however, their validation is required.
- Klíčová slova
- Clostridium, HKG, biofuel, housekeeping genes, non-model organisms,
- Publikační typ
- časopisecké články MeSH
