Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (β-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, β-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, β-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.
Biological control through the use of antagonistic micro-organisms is a potential and non-chemical means of managing plant diseases. Alternaria porri causes purple blotch disease mostly in onion. In this study, three different entomopathogenic fungi, viz. Beauveria bassiana, Metarhizium anisopliae and Verticillium lecanii, were tested against A. porri. All the three entomopathogenic fungi showed inhibitory effect against A. porri subjected to in vitro studies under dual culture technique, spore (conidial) germination, food poisoning (mycelia germination) and seed germination. The percentage inhibition of mycelial growth (PIMG) of A. porri (69.24, 56.17, and 45.81%) and the percentage inhibition of conidial germination (CG) (97.81, 42.11, and 67.69%) were observed for B. bassiana, V. lecanii and M. anisopliae, respectively. Seed germination index was found to be maximum with M. anisopliae (5557 ± 107.7) and B. bassiana (4390 ± 129.9). B. bassiana exhibited efficient antagonism against A. porri showing the highest PIMG and CG with moderate seed germination index. Microscopic examination showed the disintegrated edge of conidia which had turned black and also stopped the growth of germ tube. Hence, these entomopathogenic fungi were found to be more effective antagonistic organisms and also moderately promote seed vigour index (4390 ± 129.9) as well as plant growth. The present study indicated that the entomopathogenic fungi evaluated have shown significant inhibition of A. porri. Thus, there is a possibility of exploiting them in the management of the disease.
Plant defence mechanisms, including physical barriers like toughened bark and chemical defences like allelochemicals, are essential for protecting them against pests. Trees allocate non-structural carbohydrates (NSCs) to produce secondary metabolites like monoterpenes, which increase during biotic stress to fend off pests like the Eurasian spruce bark beetle, ESBB (
Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes.
The bark beetle, Ips typographus (L.), is a major pest of Norway spruce, Picea abies (L.), causing enormous economic losses globally. The adult stage of the I. typographus has a complex life cycle (callow and sclerotized); the callow beetles feed ferociously, whereas sclerotized male beetles are more aggressive and pioneers in establishing new colonies. We conducted a comparative proteomics study to understand male and female digestion and detoxification processes in callow and sclerotized beetles. Proteome profiling was performed using high-throughput liquid chromatography-mass spectrometry. A total of >3000 proteins were identified from the bark beetle gut, and among them, 539 were differentially abundant (fold change ±2, FDR <0.05) between callow and sclerotized beetles. The differentially abundant proteins (DAPs) mainly engage with binding, catalytic activity, anatomical activity, hydrolase activity, metabolic process, and carbohydrate metabolism, and hence may be crucial for growth, digestion, detoxification, and signalling. We validated selected DAPs with RT-qPCR. Gut enzymes such as NADPH-cytochrome P450 reductase (CYC), glutathione S-transferase (GST), and esterase (EST) play a crucial role in the I. typographus for detoxification and digesting of host allelochemicals. We conducted enzyme activity assays with them and observed a positive correlation of CYC and GST activities with the proteomic results, whereas EST activity was not fully correlated. Furthermore, our investigation revealed that callow beetles had an upregulation of proteins associated with juvenile hormone (JH) biosynthesis and chitin metabolism, whereas sclerotized beetles exhibited an upregulation of proteins linked to fatty acid metabolism and the TCA cycle. These distinctive patterns of protein regulation in metabolic and functional processes are specific to each developmental stage, underscoring the adaptive responses of I. typographicus in overcoming conifer defences and facilitating their survival. Taken together, it is the first gut proteomic study comparing males and females of callow and sclerotized I. typographus, shedding light on the adaptive ecology at the molecular level. Furthermore, the information about bark beetle handling of nutritionally limiting and defence-rich spruce phloem diet can be utilized to formulate RNAi-mediated beetle management.
Abstract 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”.
Plants are associated with microbial communities in their natural environments, causing biotic stress or mutual benefit. Understanding plant defence responses to biotic stresses is fundamental for developing resistant crop varieties to increase productivity. However, ongoing climate change has resulted in emerging plant diseases, unpredictably influencing existing beneficial microbial relationships. Plants respond to pathogen attacks through extracellular and intracellular immune receptors decoding pathogen-associated molecular patterns and pathogen-derived effectors. Understanding plant–pathogen interaction using a multi-omics approach facilitates the generation of resistant crop varieties against biotic stress in a shorter amount of time. The multi-omics approach gathers DNA, RNA, protein, and metabolome level information that leads to better understanding of the molecular underpinnings of plant–pathogen interaction. This chapter discusses the advancement of omics methodologies in exploring complex metabolic networks and regulatory mechanisms during plant–pathogen interaction. Omics technologies can also help understand the beneficial endophytic and epiphytic microbial communities and their interaction with the host in different environmental conditions. Furthermore, genome- and transcriptome- level analysis of plants after pathogen attack facilitates the identification of defence mechanisms and resistance genes. Hence, sustainable agricultural productions in the future heavily rely on omics methodologies.
Host shift is ecologically advantageous and a crucial driver for herbivore insect speciation. Insects on the non-native host obtain enemy-free space and face reduced competition, but they must adapt to survive. Such signatures of adaptations can often be detected at the gene expression level. It is astonishing how bark beetles cope with distinct chemical environments while feeding on various conifers. Hence, we aim to disentangle the six-toothed bark beetle (Ips sexdentatus) response against two different conifer defences upon host shift (Scots pine to Norway spruce). We conducted a bioassay followed by RNA-seq experiments to comprehend the beetle's ability to surpass two different terpene-based conifer defence systems, as documented in our metabolomic analysis. Beetle growth rate and fecundity were increased when reared exclusively on spruce logs (alternative host) compared to pine logs (native host). Comparative gene expression analysis identified differentially expressed genes (DEGs) related to digestion, detoxification, transporter activity, growth, signalling, and stress response in the spruce-feeding beetle gut. Transporter genes were highly abundant during spruce feeding, suggesting they could play a role in pumping a wide variety of endogenous and xenobiotic compounds or allelochemicals out. Trehalose transporter (TRET) is also up-regulated in the spruce-fed beetle gut to maintain homeostasis and stress tolerance. RT-qPCR and enzymatic assays further corroborated some of our findings. Taken together, the transcriptional plasticity of key physiological genes plays a crucial role after the host shift and provides vital clues for the adaptive potential of bark beetles on different conifer hosts.
Species of wild rice (Oryza spp.) possess a wide range of stress tolerance traits that can be potentially utilized in breeding climate-resilient cultivated rice cultivars (Oryza sativa) thereby aiding global food security. In this study, we conducted a greenhouse trial to evaluate the salinity tolerance of six wild rice species, one cultivated rice cultivar (IR64) and one landrace (Pokkali) using a range of electrophysiological, imaging, and whole-plant physiological techniques. Three wild species (O. latifolia, O. officinalis and O. coarctata) were found to possess superior salinity stress tolerance. The underlying mechanisms, however, were strikingly different. Na+ accumulation in leaves of O. latifolia, O. officinalis and O. coarctata were significantly higher than the tolerant landrace, Pokkali. Na+ accumulation in mesophyll cells was only observed in O. coarctata, suggesting that O. officinalis and O. latifolia avoid Na+ accumulation in mesophyll by allocating Na+ to other parts of the leaf. The finding also suggests that O. coarctata might be able to employ Na+ as osmolyte without affecting its growth. Further study of Na+ allocation in leaves will be helpful to understand the mechanisms of Na+ accumulation in these species. In addition, O. coarctata showed Proto Kranz-like leaf anatomy (enlarged bundle sheath cells and lower numbers of mesophyll cells), and higher expression of C4-related genes (e.g., NADPME, PPDK) and was a clear outlier with respect to salinity tolerance among the studied wild and cultivated Oryza species. The unique phylogenetic relationship of O. coarctata with C4 grasses suggests the potential of this species for breeding rice with high photosynthetic rate under salinity stress in the future.
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