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This study explored the bactericidal role of the epidermal mucus (EM) of five freshwater Cyprinid fish species namely Ctenopharyngodon idella, Labeo rohita, Catla catla, Hypophthalmichthys molitrix, and Cirrhinus mrigala after treatment with Aeromonas hydrophila. Extracts of EM (crude and acidic) of each species showed bactericidal activity against various Gram −ve (Pseudomonas aeruginosa, Escherichia coli, Aeromonas hydrophila, Edwardsiella tarda, Salmonella enterica, Klebsiella pneumonia, Serratia marcescens, and Enterobacter cloacae) and Gram +ve (Bacillus wiedmannii and Staphylococcus aureus) bacteria compared with standard antibiotics (Fosfomycin). The zone of inhibition (ZOI) was measured in millimetres against antibiotics (Fosfomycin). Variations in bactericidal activity of EM were observed against bacteria from the same and different fish species. The acidic extract was more effective than the crude extract and showed significantly higher ZOI values against various bacteria and Fosfomycin antibiotics. This result shows that fish EM may perform an important role in fish defence against bacteria. Therefore, this study may hint towards the substitution of synthetic antibiotics with fish EM that may be used as a novel 'bactericidal' in aquaculture as well as in humans against bacterial infections. [ABSTRACT FROM AUTHOR]

Simple Summary: The red palm weevil poses a significant threat to palm species, resulting in substantial economic losses. While multiple methods have been developed to control its infestations, there is an urgent need for eco-friendly insecticides that selectively target its critical systems or pathways. One such target is its digestive system, which is essential for its survival. This review highlights the potential of using the digestive system of the red palm weevil to manage its infestations. Proteomic and transcriptomic data analyses on the weevils have provided a better understanding of the protein and gene compositions in its digestive system. With technological advancements, a more comprehensive approach can be taken to explore the opportunities in manipulating the data on the digestive system of red palm weevil, leading to improved management methods. Rhynchophorus ferrugineus, commonly known as red palm weevil (RPW), is a high-risk insect pest that has become a threat to many important palm species. There are several dominant factors that lead to the successful infestation of RPW, including its stealthy lifestyle, highly chitinized mouthpart, and high fecundity rate. Due to that, millions of dollars of losses have been suffered by many countries invaded by RPW. Several methods have been designed to control its invasion, including the usage of insecticides, but many cause resistance and environmental pollution. Therefore, an environmentally friendly insecticide that targets specific systems or pathways in RPW is urgently needed. One of the potential targets is the digestive system of RPW, as it is the major interface between the insect and its plant host. The related knowledge of RPW's digestive system, such as the anatomy, microflora, transcriptomic analysis, and proteomic analysis, is important to understand its effects on RPW's survival. Several data from different omics regarding the digestive systems of RPW have been published in separate reports. Some of the potential targets have been reported to be inhibited by certain potential insecticides, while other targets have not yet been tested with any inhibitors. Hence, this review may lead to a better understanding on managing infestations of RPW using the system biology approach for its digestive system. [ABSTRACT FROM AUTHOR]

Simple Summary: The increasingly growing demand for palm oil (PO) blankets heavy pressure for sustainable PO production. PO, the world's most efficient oilseed crop, is being used in various applications, including food, cosmetics, and biofuels. However oil palm is always at risk being exposed to Metisa plana infestation, which contributes to the negative impacts on the oil palm plantations and industries. Therefore, conventional pesticides are routinely applied as a control measure in outbreak management. Nevertheless, the perpetual occurrences of M. plana infestation remain, affecting the overall yield productivity. This study uses an integrated bioinformatics approach, including a gene co-expression network and clustering method, to screen, identify, and determine the key genes involved in controlling insect hormone biosynthesis with ecdysone and juvenile hormones among them. The findings provide key regulatory genes (Hnf4, Hr4, MED14, Usp, Tai, and Trr) that could be used as potential targets for gene silencing technologies such as RNA interference (RNAi) desired in understanding M. plana gene function. Metisa plana Walker (Lepidoptera: Psychidae) is a major oil palm pest species distributed across Southeast Asia. M. plana outbreaks are regarded as serious ongoing threats to the oil palm industry due to their ability to significantly reduce fruit yield and subsequent productivity. Currently, conventional pesticide overuses may harm non-target organisms and severely pollute the environment. This study aims to identify key regulatory genes involved in hormone pathways during the third instar larvae stage of M. plana gene co-expression network analysis. A weighted gene co-expression network analysis (WGCNA) was conducted on the M. plana transcriptomes to construct a gene co-expression network. The transcriptome datasets were obtained from different development stages of M. plana, i.e., egg, third instar larvae, pupa, and adult. The network was clustered using the DPClusO algorithm and validated using Fisher's exact test and receiver operating characteristic (ROC) analysis. The clustering analysis was performed on the network and 20 potential regulatory genes (such as MTA1-like, Nub, Grn, and Usp) were identified from ten top-most significant clusters. Pathway enrichment analysis was performed to identify hormone signalling pathways and these pathways were identified, i.e., hormone-mediated signalling, steroid hormone-mediated signalling, and intracellular steroid hormone receptor signalling as well as six regulatory genes Hnf4, Hr4, MED14, Usp, Tai, and Trr. These key regulatory genes have a potential as important targets in future upstream applications and validation studies in the development of biorational pesticides against M. plana and the RNA interference (RNAi) gene silencing method. [ABSTRACT FROM AUTHOR]

In Southeast Asia, Conopomorpha cramerella (Snellen) which is commonly known as the cocoa pod borer (CPB) moth has been identified as the most detrimental pest of Theobroma cacao L. Apart from the various side effects on human health and non‐target organisms, heavily relying on synthetic pyrethroid insecticides to control CPB infestations also increases the environmental contamination risks. Thus, developing biorational insecticides that minimally affect the non‐target organism and environment by targeting the insect growth regulation process is needed to manage the pest population. In insects, juvenile hormones (JH) regulate critical biological events, especially metamorphosis, development and reproduction. Since the physiological roles of JH III vary among different organisms, the biochemical properties, especially substrate specificity and analogue inhibition, may also be different. Therefore, studies on the JH III biosynthetic pathway enzymes in both plants and insects are beneficial to discover more effective analogues. Bioinformatic analysis and biochemical characterization of a NADP+‐dependent farnesol dehydrogenase, an intermediate enzyme of the JH III pathway, from C. cramerella (CcFolDH), were described in this study. In addition, the farnesol analogues that may act as a potent analogue inhibitor for CcFolDH ware determined using in vitro enzymatic study. The phylogenetic analysis indicated that CcFolDH shared a close phylogenetic relationship to the honeybee's short‐chain dehydrogenase/reductase. The 27 kDa CcFolDH has an NADP(H) binding domain with a typical Rossmann fold and is likely a homotetrameric protein in the solution. The enzyme had a greater preference for substrate trans, trans‐farnesol and coenzyme NADP+. In terms of analogue inhibitor inhibition, hexahydroxyfarnesyl acetone showed the highest inhibition (the lowest Ki) compared to other farnesol analogues. Thus, hexahydroxyfarnesyl acetone would serve as the most potent active ingredient for future biorational pesticide management for C. cramerella infestation. Based on the bioinformatic analyses and biochemical characterizations conducted in this research, we proposed that rCcFolDH differs slightly from other reported farnesol dehydrogenases in terms of molecular weight, substrate preference, coenzymes utilization and analogue inhibitors selection. [ABSTRACT FROM AUTHOR]

Juvenile hormone III (JH III) plays an important role in insect reproduction, development, and behavior. The second branch of JH III production includes oxidation of farnesol to farnesal by farnesol dehydrogenase. This study reported the identification and characterization of Plutella xylostella farnesol dehydrogenase (PxFoLDH). Our results showed that PxFoLDH belongs to the short-chain dehydrogenase/reductase superfamily, consisting of a single domain with a structurally conserved Rossman fold, an NAD(P) (H)-binding region and a structurally diverse C-terminal region. The purified enzyme displayed maximum activity at 55 |$\ $| °C with pH 9.5 and was stable in the temperature below 70 |$\ ^\circ $| C. PxFoLDH was determined to be a monomer with a relative molecular weight of 27 kDa and highly specific for trans, trans- farnesol, and NADP+. Among analog inhibitors tested, farnesyl acetate was the most effective inhibitor with the lowest  K i value of 0.02 µ m. Our findings showed this purified enzyme may represent as NADP+-farnesol dehydrogenase. [ABSTRACT FROM AUTHOR]

Citral is a mixture of neral and geranial, which are of great interest to the fragrance industry due to its lemon-scented aroma. A newly characterized nerol dehydrogenase of Persicaria minor (PmNeDH) from our recent findings has shown a capacity to convert citral from nerol. Differential gene expression analysis revealed that the expression level of PmNeDH was highly upregulated during early treatment of several stress-related phytohormones i.e. methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA). SA and ABA were shown to have a prolonged effect on PmNeDH expression level until second day of treatment. The findings were in agreement with the cis-regulatory elements predicted from the gene promoter. The phylogenetic relationship of PmNeDH with its homologs from the medium-chain dehydrogenases/reductases (MDR) superfamily was also mentioned. In this study, we proposed a possible biological function of PmNeDH gene in P. minor, which might play significant roles in plant defence mechanism. [ABSTRACT FROM AUTHOR]

Plants emit semiochemicals as alarm signals upon attack by herbivores or insect pests. Complex insect-plant interaction through alarm pheromones can be manipulated to improve crop protection. Geraniol, citral and geranic acid are monoterpenoid compounds from plants and they play a role as semiochemical alarm pheromones. In plants, the oxidation of geraniol into geranic acid is catalyzed by two oxidoreductases, geraniol dehydrogenase and citral dehydrogenase. In this study, citral dehydrogenase isoenzymes from Persicaria minor ( Polygonum minus) leaves were purified to homogeneity and characterized. Enzyme purification through Toyopearl GigaCap Q-650 M column chromatography at pH 7.5 produced two activity peaks, suggesting the existence of two citral dehydrogenase isoenzymes. Both isoenzymes were different in isoelectric point and kinetic parameters but similar in pH and optimal temperature as well as in substrate specificity. Findings from this study will provide a basic understanding for the development of recombinant production of these particular enzymes. Further studies on molecular structure involved could be exploited in transgenic plant as an integrated pest management strategy. [ABSTRACT FROM AUTHOR]

Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that’s highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant. [ABSTRACT FROM AUTHOR]

Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control. [ABSTRACT FROM AUTHOR]

The article discusses the findings of a study which purified and characterized isolated geraniol dehydrogenase (DH) enzymes. It notes the presence of the monoterpenes, geraniol and citral in aromatic plants. Researchers cited that investigation of geraniol DH will help in understanding the metabolic pathway of the monoterpene in leaves of the Polygonum minus plant.

The red palm weevil (RPW; Rhynchophorus ferrugineus Olivier (Coleoptera Curculionidae)) is an invasive insect pest that is difficult to manage due to its nature of infesting the host palm trees from within. A holistic, molecular-based approach to identify proteins that correlate with RPW infestation could give useful insights into the vital processes that are prevalent to the host's infestation response and identify the potential biomarkers for an early detection technique. Here, a shotgun proteomic analysis was performed on oil palm (Elaeis guineensis; OP) under untreated (control), wounding by drilling (wounded), and artificial larval infestation (infested) conditions at three different time points to characterise the RPW infestation response at three different stages. KEGG pathway enrichment analysis revealed many overlapping pathways between the control, wounded, and infested groups. Further analysis via literature searches narrowed down biologically relevant proteins into categories, which were photosynthesis, growth, and stress response. Overall, the patterns of protein expression suggested abscisic acid (ABA) hormone signalling to be the primary driver of insect herbivory response. Interspecies molecular docking analysis between RPW ligands and OP receptor proteins provided putative interactions that result in ABA signalling activation. Seven proteins were selected as candidate biomarkers for early infestation detection based on their relevance and association with ABA signalling. The MS data are available via ProteomeXchange with identifier PXD028986. This study provided a deeper insight into the mechanism of stress response in OP in order to develop a novel detection method or improve crop management. [ABSTRACT FROM AUTHOR]

The article reports on the results of a study of the purification and characterization of the enzyme, 4-N-trimethylaminobutyraldehyde dehydrogenase from Pseudomonas sp. 13CM. A description of the experimental set-up and measurement methods is presented. The study concluded that the enzyme was inhibited by SH reagents and by heavy metal ions.

The article describes the purification and some properties of the enzyme 4-N-trimethylamino-1-butanol dehydrogenase (TMA-Butanol-DH) from Pseudomonas sp. 13CM. The enzyme had a molecular mass of 45 kDa and appeared to be a monomer enzyme. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and amino acid terminal sequence.

Simple Summary: The diamondback moth, Plutella xylostella, is the most destructive pest for Brassica vegetable crops worldwide. The management of this pest was estimated to cost about United States dollars (USD) 4–5 billion annually. Prolonged and unsupervised insecticide exposures have led to not only the emergence of insecticide resistance in P. xylostella, but also negative impacts on human health, environmental pollution, and nontargeted organisms. Therefore, the development of new safer, environmentally friendly, and target-specific insecticides is vital in order to combat this pest. In this study, we evaluated the potential of selected farnesyl derivative compounds that could act as biorational insecticides targeting the juvenile hormone biosynthesis of P. xylostella. Out of five farnesyl derivatives tested, farnesyl acetate showed the highest mortality percentage of P. xylostella. Then, the sublethal effects of farnesyl acetate on biological characteristics of P. xylostella were determined. The results demonstrated that farnesyl acetate had negative effects on development, pupal weight, pupation, adult emergence, female ratio, fecundity, egg hatching rate, and oviposition period of P. xylostella. Farnesyl acetate also induced abnormalities in pupal and adults of P. xylostella. These findings indicate that farnesyl acetate can reduce the population number and reproductive success of P. xylostella, possibly leading to the effective management of this pest. The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the most important pest of cruciferous vegetables worldwide. In this study, we evaluated the properties of selected farnesyl derivative compounds against P. xylostella. The toxicity and sublethal concentration (LC50) of farnesyl acetate, farnesyl acetone, farnesyl bromide, farnesyl chloride, and hexahydrofarnesyl acetone were investigated for 96 h. The leaf-dip bioassays showed that farnesyl acetate had a high level of toxicity against P. xylostella compared to other tested farnesyl derivatives. The LC50 value was 56.41 mg/L on the second-instar larvae of P. xylostella. Then, the sublethal effects of farnesyl acetate on biological parameters of P. xylostella were assessed. Compared to the control group, the sublethal concentration of farnesyl acetate decreased pupation and emergence rates, pupal weight, fecundity, egg hatching rate, female ratio, and oviposition period. Furthermore, the developmental time of P. xylostella was extended after being exposed to farnesyl acetate. Moreover, the application of farnesyl acetate on P. xylostella induced morphogenetic abnormalities in larval–pupal intermediates, adults that emerged with twisted wings, or complete adults that could not emerge from the cocoon. These results suggested that farnesyl acetate was highly effective against P. xylostella. The sublethal concentration of farnesyl acetate could reduce the population of P. xylostella by increasing abnormal pupal and adults, and by delaying its development period. [ABSTRACT FROM AUTHOR]

Bagworm, Metisa plana (Lepidoptera: Psychidae) is a ubiquitous insect pest in the oil palm plantations. M. plana infestation could reduce the oil palm productivity by 40% if it remains untreated over two consecutive years. Despite the urgency to tackle this issue, the genome and transcriptome of M. plana have not yet been fully elucidated. Here, we report a comprehensive transcriptome dataset from four different developmental stages of M. plana, comprising of egg, third instar larva, pupa and female adult. The de novo transcriptome assembly of the raw data had produced a total of 193,686 transcripts, which were then annotated against UniProt, NCBI non-redundant (NR) database, Gene Ontology, Cluster of Orthologous Group, and Kyoto Encyclopedia of Genes and Genomes databases. From this, 46,534 transcripts were annotated and mapped to 146 known metabolic or signalling KEGG pathways. The paper further identified 41 differentially expressed transcripts encoding seven genes in the chitin biosynthesis pathways, and their expressions across each developmental stage were further analysed. The genetic diversity of M. plana was profiled whereby there were 21,516 microsatellite sequences and 379,895 SNPs loci found in the transcriptome of M. plana. These datasets add valuable transcriptomic resources for further study of developmental gene expression, transcriptional regulations and functional gene activities involved in the development of M. plana. Identification of regulatory genes in the chitin biosynthesis pathway may also help in developing an RNAi-mediated pest control management by targeting certain pathways, and functional studies of the genes in M. plana. [ABSTRACT FROM AUTHOR]

The red palm weevil (RPW) is a stem boring Coleoptera that decimates host palm trees from within. The challenge of managing this pest is due to a lack of physical symptoms during the early stages of infestation. Investigating the physiological changes that occur within RPW-infested palm trees may be useful in establishing a new approach in RPW detection. In this study, the effects of RPW infestation were investigated in Elaeis guineensis by observing changes in physical and physiological parameters during the progress of infestation by visual inspection and the comparison of growth, gas exchange, stomatal conductance, and chlorophyll content between the non-infested control, physically wounded, and RPW-infested E. guineensis groups. During the study period, four distinct levels of physical infestation were observed and recorded. The RPW-infested group displayed significantly lower maximum photosynthesis activity (Amax) starting from the third week post-infestation. However, growth in terms of change in plant height and stem circumference, leaves' stomatal conductance, and chlorophyll content were not significantly different between the three groups during the duration of the study. The significant drop in photosynthesis was observed one week before physical changes appeared. This suggests the promising utilisation of photosynthesis activity as a signal for detecting RPW infestation at the early stage of attacks, which could be useful for integration in integrated pest management (IPM). [ABSTRACT FROM AUTHOR]

The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP+ as coenzyme. The substrate specificity studies showed trans, trans -farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP+-dependent farnesol dehydrogenase. The docking of trans, trans -farnesol and NADP+ into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control. • NADP+ dependent farnesol dehydrogenase enzyme of T. cacao (TcFolDH) was expressed, purified and biochemically characterized. • TcFolDH enzyme was the first homotrimer reported among farnesol dehydrogenases. • The TcFolDH was highly specific to NADP+ as coenzyme and trans, trans-farnesol was the most preferred substrate. • TcFolDH gene can serve as a novel target for pest control based on the JH-dependent regulation. [ABSTRACT FROM AUTHOR]

Bagworm, Metisa plana (Lepidoptera: Psychidae) is a ubiquitous insect pest in the oil palm plantations. M. plana infestation could reduce the oil palm productivity by 40% if it remains untreated over two consecutive years. Despite the urgency to tackle this issue, the genome and transcriptome of M. plana have not yet been fully elucidated. Here, we report a comprehensive transcriptome dataset from four different developmental stages of M. plana , comprising of egg, third instar larva, pupa and female adult. The de novo transcriptome assembly of the raw data had produced a total of 193,686 transcripts, which were then annotated against UniProt, NCBI non-redundant (NR) database, Gene Ontology, Cluster of Orthologous Group, and Kyoto Encyclopedia of Genes and Genomes databases. From this, 46,534 transcripts were annotated and mapped to 146 known metabolic or signalling KEGG pathways. The paper further identified 41 differentially expressed transcripts encoding seven genes in the chitin biosynthesis pathways, and their expressions across each developmental stage were further analysed. The genetic diversity of M. plana was profiled whereby there were 21,516 microsatellite sequences and 379,895 SNPs loci found in the transcriptome of M. plana . These datasets add valuable transcriptomic resources for further study of developmental gene expression, transcriptional regulations and functional gene activities involved in the development of M. plana . Identification of regulatory genes in the chitin biosynthesis pathway may also help in developing an RNAi-mediated pest control management by targeting certain pathways, and functional studies of the genes in M. plana .

Cocoa bean ( Theobroma cacao L.) is part of the global cocoa and chocolate industry valued at 44 billion US dollars in 2019. Cocoa pod borer (CPB), Conopomorpha cramerella is a major pest of cocoa in Malaysia and Indonesia that is responsible for the decline for cocoa production. They have been detected since 1980s. Unfortunately, current control strategies are inefficient for CPB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for successful application of these emerging technologies. We generated a comprehensive RNA-seq dataset (135,915,430 clean reads) for larva and adult stages of CPB by using the Illumina Hiseq TM 4000 system to increase the understanding of CPB in relation to molecular features. The CPB transcriptome was assembled de novo and annotated. The final assembled produced 249,280 unigenes, of which 75,929 unigenes annotated against NCBI NR database and were distributed among 156 KEGG pathways. The raw data were uploaded to SRA database and the BioProject ID is PRJNA553611. The transcriptomic dataset we present are the first reports of transcriptome information in CPB that is valuable for further exploration and understanding of CPB molecular pathways., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Authors.)

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor . Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment., Methods: The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server., Results: Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C 15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β -sesquiphellandrene as a major product and β -farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases., Discussion: The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, K m and k cat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases., Competing Interests: The authors declare there are no competing interests.

Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant., Competing Interests: The authors have declared that no competing interests exist.

Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control.