In silico secretome analyses of the polyphagous root-knot nematode Meloidogyne javanica: a resource for studying M. javanica secreted proteins.

BACKGROUND: Plant-parasitic nematodes (PPNs) that cause most damage include root-knot nematodes (RKNs) which are a major impediment to crop production. Root-knot nematodes, like other parasites, secrete proteins which are required for parasite proliferation and survival within the host during the infection process. RESULTS: Here, we used various computational tools to predict and identify classically and non-classically secreted proteins encoded in the Meloidogyne javanica genome. Furthermore, functional annotation analysis was performed using various integrated bioinformatic tools to determine the biological significance of the predicted secretome. In total, 7,458 proteins were identified as secreted ones. A large percentage of this secretome is comprised of small proteins of ≤ 300 aa sequence length. Functional analyses showed that M. javanica secretome comprises cell wall degrading enzymes for facilitating nematode invasion, and migration by disintegrating the complex plant cell wall components. In addition, peptidases and peptidase inhibitors are an important category of M. javanica secretome involved in compatible host-nematode interactions. CONCLUSION: This study identifies the putative secretome encoded in the M. javanica genome. Future experimental validation analyses can greatly benefit from this global analysis of M. javanica secretome. Equally, our analyses will advance knowledge of the interaction between plants and nematodes.

Functional analysis of 3 genes in xenobiotic detoxification pathway of Bursaphelenchus xylophilus against matrine.

Bursaphelenchus xylophilus is the causative agent of pine wilt disease. It has caused devastating damage to ecosystems worldwide, owing to the characteristic of being widely spread and uncontrollable. However, the current methods of control are mainly based on pesticides, which can cause irreversible damage to the ecosystem. Therefore, the search for new drug targets and the development of environmentally friendly nematicides is especially valuable. In this study, three key genes of the xenobiotic detoxification pathways were cloned from B. xylophilus, which were subsequently subjected to bioinformatic analysis. The bioassay experiment was carried out to determine the concentration of matrine required for further tests. Subsequently, enzyme activity detection and three gene expression pattern analysis were performed on matrine treated nematodes. Finally, RNA interference was conducted to verify the functions carried out by the three genes in combating matrine. The results indicated that cytochrome P450 and glutathione S-transferase of B. xylophilus were activated by matrine, which induced high expression of BxCYP33C4, BxGST1, and BxGST3. After RNA interference of three genes of B. xylophilus, the sensitivity of B. xylophilus to matrine was increased and the survival rate of nematodes was reduced to various degrees in comparison to the control group. Overall, the results fully demonstrated that BxCYP33C4, BxGST1, and BxGST3 are valuable drug targets for B. xylophilus. Furthermore, the results suggested that matrine has value for development and exploitation in the prevention and treatment of B. xylophilus.

Toxicity of Bacillus thuringiensis Strains Derived from the Novel Crystal Protein Cry31Aa with High Nematicidal Activity against Rice Parasitic Nematode Aphelenchoides besseyi.

The plant parasitic nematode, Aphelenchoides besseyi, is a serious pest causing severe damage to various crop plants and vegetables. The Bacillus thuringiensis (Bt) strains, GBAC46 and NMTD81, and the biological strain, FZB42, showed higher nematicidal activity against A. besseyi, by up to 88.80, 82.65, and 75.87%, respectively, in a 96-well plate experiment. We screened the whole genomes of the selected strains by protein-nucleic acid alignment. It was found that the Bt strain GBAC46 showed three novel crystal proteins, namely, Cry31Aa, Cry73Aa, and Cry40ORF, which likely provide for the safe control of nematodes. The Cry31Aa protein was composed of 802 amino acids with a molecular weight of 90.257 kDa and contained a conserved delta-endotoxin insecticidal domain. The Cry31Aa exhibited significant nematicidal activity against A. besseyi with a lethal concentration (LC50) value of 131.80 µg/mL. Furthermore, the results of in vitro experiments (i.e., rhodamine and propidium iodide (PI) experiments) revealed that the Cry31Aa protein was taken up by A. besseyi, which caused damage to the nematode's intestinal cell membrane, indicating that the Cry31Aa produced a pore-formation toxin. In pot experiments, the selected strains GBAC46, NMTD81, and FZB42 significantly reduced the lesions on leaves by up to 33.56%, 45.66, and 30.34% and also enhanced physiological growth parameters such as root length (65.10, 50.65, and 55.60%), shoot length (68.10, 55.60, and 59.45%), and plant fresh weight (60.71, 56.45, and 55.65%), respectively. The number of nematodes obtained from the plants treated with the selected strains (i.e., GBAC46, NMTD81, and FZB42) and A. besseyi was significantly reduced, with 0.56, 0.83., 1.11, and 5.04 seedling mL-1 nematodes were achieved, respectively. Moreover, the qRT-PCR analysis showed that the defense-related genes were upregulated, and the activity of hydrogen peroxide (H2O2) increased while malondialdehyde (MDA) decreased in rice leaves compared to the control. Therefore, it was concluded that the Bt strains GBAC46 and NMTD81 can promote rice growth, induce high expression of rice defense-related genes, and activate systemic resistance in rice. More importantly, the application of the novel Cry31Aa protein has high potential for the efficient and safe prevention and green control of plant parasitic nematodes.

A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode Aphelenchoides besseyi.

RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and in planta methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes (Ab-far-1, Ab-far-4, and combinatorial Ab-far-1 and Ab-far-4) of the rice white tip nematode (RWTN), Aphelenchoides besseyi, were used as target genes to construct a fungal RNAi vector, and the Ab-far-n dsRNA transgenic Botrytis cinerea (ARTBn) were generated using Agrobacterium-mediated transformation technology. After RWTN feeding on ARTBn, the expression of Ab-far-1 and Ab-far-4 in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The Ab-far-1 and Ab-far-4 co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB1 were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs.Abbreviations: FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, Aphelenchoides besseyi); Ab-far-n (Fatty acid and retinol binding protein gene of A. besseyi); ARTB1 (Ab-far-1 hpRNA transgenic Botrytis cinerea); ARTB4 (Ab-far-4 hpRNA transgenic Botrytis cinerea); ARTB1/4 (combinatorial Ab-far-1 and Ab-far-4 hpRNA transgenic B. cinerea); EVTB (Empty vector transgenic B. cinerea); GRTB (eGFP hpRNA transgenic B. cinerea); WTB (Wild-type B. cinerea).

Potential Molecular Mimicry Proteins Responsive to α-pinene in Bursaphelenchus xylophilus.

: Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic analysis of B. xylophilus. The expression levels of three genes encoding secreted B. xylophilus proteins were influenced by α-pinene. We cloned one gene encoding a thaumatin-like protein, Bx-tlp-2 (accession number MK000287), and another gene encoding a cysteine proteinase inhibitor, Bx-cpi (accession number MK000288). Additionally, α-pinene appeared to induce Bx-tlp-1 expression, but had the opposite effect on Bx-cpi expression. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees revealed that the α-pinene content was consistent with the expression levels of Bx-tlp-1 (Bx-cpi) and Pm-tlp (Pm-cpi) over time. Thus, these genes likely have important roles contributing to the infection of pine species by B. xylophilus. The results of this study may be relevant for future investigations of the functions of Bx-tlp-1, Bx-tlp-2 and Bx-cpi, which may provide a point to explore the relationship between B. xylophilus and host pines.

Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

Immune response of insect vectors to transmitted pathogens or insect hosts against parasites are well studied, whereas the mechanism of tripartite interactions remains elusive. In this study, we investigated the immune interactions of the vector beetle Monochamus alternatus ( Ma) to the devastating plant parasitic nematode Bursaphelenchus xylophilus ( Bx) and the insect parasitic nematode Howardula phyllotretae ( Hp). We report the unique immune mechanism by which the vector beetle tolerates many devastating Bx in its trachea, yet that immune tolerance is compromised by the parasitic nematode Hp. Contact with either nematode species triggers epithelial reactive oxygen species (ROS) production in Ma. Only the entry of Bx, not Hp, infection, induces increased expression of antioxidative genes, through which the ROS levels are balanced in the trachea of beetles. Furthermore, we found that up-regulation of antioxidative genes was induced by the interaction of Toll receptors. In contrast, beetles infected by Hp retain high levels of oxidative stress and melanization in trachea, and as a result, decrease Bx loading. This study highlights the role of Toll receptors in mediating the activation of antioxidative genes in immune tolerance to plant parasitic nematodes, and suggests the use of insect parasites as a biologic control.-Zhou, J., Zhao, L.-L., Yu, H.-Y., Wang, Y.-H., Zhang, W., Hu, S.-N., Zou, Z., Sun, J.-H. Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

STATAWAARS: a promoter motif associated with spatial expression in the major effector-producing tissues of the plant-parasitic nematode Bursaphelenchus xylophilus.

BACKGROUND: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. RESULTS: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel "pioneer" effectors. CONCLUSIONS: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda.

Trehalose metabolism genes render rice white tip nematode Aphelenchoides besseyi (Nematoda: Aphelenchoididae) resistant to an anaerobic environment.

After experiencing anaerobic environments, Aphelenchoides besseyi will enter a state of suspended animation known as anoxybiosis, during which it may use trehalose as an energy supply to survive. To explore the function of trehalose metabolism, two trehalose-6-phosphate synthase (TPS) genes (Ab-tps1 and Ab-tps2) encoding enzymes catalysing trehalose synthesis, and three trehalase (TRE) genes (Ab-ntre1, Ab-ntre2 and Ab-atre) encoding enzymes catalysing the hydrolysis of trehalose, were identified and investigated. Ab-tps1 and Ab-tps2 were active during certain periods of anoxybiosis for A. besseyi, and Ab-tps2, Ab-ntre1, Ab-ntre2 and Ab-atre were active during certain periods of recovery. The results of RNA interference experiments suggested that TRE genes regulated each other and both TPS genes, while a single TPS gene only regulated the other TPS gene. However, two TPS genes together could regulate TRE genes, which indicated a feedback mechanism between these genes. All these genes also positively regulated the survival and resumption of active metabolism of the nematode. Genes functioning at re-aeration have a greater impact on nematode survival, suggesting that these genes could play roles in anoxybiosis regulation, but may function within restricted time frames. Changes in trehalose levels matched changes in TRE activity during the anoxybiosis-re-aeration process, suggesting that trehalose may act as an energy supply source. The observation of up-regulation of TPS genes during anoxybiosis suggested a possible signal role of trehalose. Trehalose metabolism genes could also work together to control trehalose levels at a certain level when the nematode is under anaerobic conditions.

Effects of the plant volatile trans­2-hexenal on the dispersal ability, nutrient metabolism and enzymatic activities of Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus causes pine wilt disease (PWD), which severely damages pine species. The plant volatile trans­2-hexenal has strong activity against nematodes, although the precise mechanism of this inhibitory action remains unclear. In this paper, the fumigant effects of the LC10 and LC30 of trans­2-hexenal on B. xylophilus were demonstrated. The trans­2-hexenal treatments significantly inhibited the dispersal ability of nematodes. The results also indicated that trans­2-hexenal affects the metabolism of nutrients and the activity of digestive enzymes. Among detoxifying enzymes, after treatment with trans­2-hexenal, glutathione S-transferase activity increased significantly and general esterase activity decreased significantly. Based on these results, trans­2-hexenal disturbs the normal physiological and biochemical activities of this nematode. These results provide valuable insight into the nematicidal mechanisms of trans­2-hexenal.

Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus.

Bursaphelenchus mucronatus (B. mucronatus) isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while ß-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus' pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future.

Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea).

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

Screening and functional analysis of the peroxiredoxin specifically expressed in Bursaphelenchus xylophilus--the causative agent of pine wilt disease.

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode.

Catalytic and chaperone-like functions in an intrinsically disordered protein associated with desiccation tolerance.

Intrinsically disordered proteins (IDPs) lack well-defined structure but are widely represented in eukaryotic proteomes. Although the functions of most IDPs are not understood, some have been shown to have molecular recognition and/or regulatory roles where their disordered nature might be advantageous. Anhydrin is an uncharacterized IDP induced by dehydration in an anhydrobiotic nematode, Aphelenchus avenae. We show here that anhydrin is a moonlighting protein with two novel, independent functions relating to desiccation tolerance. First, it has a chaperone-like activity that can reduce desiccation-induced enzyme aggregation and inactivation in vitro. When expressed in a human cell line, anhydrin localizes to the nucleus and reduces the propensity of a polyalanine expansion protein associated with oculopharyngeal muscular dystrophy to form aggregates. This in vivo activity is distinguished by a loose association of anhydrin with its client protein, consistent with a role as a molecular shield. In addition, anhydrin exhibits a second function as an endonuclease whose substrates include supercoiled, linear, and chromatin linker DNA. This nuclease activity could be involved in either repair of desiccation-induced DNA damage incurred during anhydrobiosis or in apoptotic or necrotic processes, for example, but it is particularly unexpected for anhydrin because IDP functions defined to date anticorrelate with enzyme activity. Enzymes usually require precise three-dimensional positioning of residues at the active site, but our results suggest this need not be the case. Anhydrin therefore extends the range of IDP functional categories to include catalysis and highlights the potential for the discovery of new functions in disordered proteomes.

Nematicidal activity of furanoeremophilenes against Meloidogyne incognita and Nacobbus aberrans.

BACKGROUND: While searching for novel small molecules for new organic pesticide agents against plant-parasitic nematodes, we found that the hexane extract from the roots of Senecio sinuatos and its main secondary metabolite, 3ß-angeloyloxy-6ß-hydroxyfuranoeremophil-1(10)-ene (1), possess nematicidal activity against the second stage juvenile (J2) of Meloidogyne incognita and Nacobbus aberrans. Both species reduce yield of various vegetable crops. These results encouraged us to synthesize esters 3-9 formed by diol 2, obtained by alkaline hydrolysis of 1 and acetic anhydride, benzoic acid, 2-nitrobenzoic acid, 2-bromobenzoic acid, 4-nitrobenzoic acid, 4-bromobenzoic acid, and 4-methoxybenzoic acid, respectively. The nematicidal activity of these esters was evaluated and compared with that of the free benzoic acids. RESULTS: Natural product 1 and derivatives 2-9 were obtained and characterized by their physical and spectroscopic properties, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) experiments; X-ray diffraction analysis established their absolute configuration. The nematicidal activity of compounds 1-9 was assessed in vitro against M. incognita and N. aberrans J2 and was compared to activity shown by benzoic acid, 2-nitrobenzoic acid, 2-bromobenzoic acid, 4-nitrobenzoic acid, 4-bromobenzoic acid, and 4-methoxybenzoic acid. The esters suppressed nematodes more than free benzoic acid. Nacobbus aberrans J2 were suppressed, with compounds 5, 6, and 8 being the most active. CONCLUSION: Esters formed by 3ß,6ß-dihydroxyfuranoeremophil-1(10)-ene and ortho- or para-substituted benzoic acids containing electron acceptor groups had nematicidal activity against N. aberrans. These compound can potentially serve as a model for the development of new organic nematicidal agents. © 2022 Society of Chemical Industry.

Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

Development of a Redox Dye-Based Rapid Colorimetric Assay for the Quantitation of Viability/Mortality of Pine Wilt Nematode.

Control of pine wilt disease, which is caused by pine wilt nematode, Bursaphelenchus xylophilus, is heavily dependent on the use of chemicals such as abamectin. Although such chemicals are highly effective, demands for alternatives that are derived preferentially from natural sources, are increasing out of environmental concerns. One of the challenges to discovery of alternative control agents is lack of fast and efficient screening method that can be used in high-throughput manner. Here we described the development of colorimetric assay for the rapid and accurate screening of candidate nematicidal compounds/biologics targeting B. xylophilus. Contrary to the conventional method, which relies on laborious visual inspection and counting of nematode population under microscope, our method utilizes a redox dye that changes its color in response to metabolic activity of nematode population in a given sample. In this work, we optimized parameters of our colorimetric assay including number of nematodes and amount of redox dye, and tested applicability of our assay for screening of chemicals and biologics. We demonstrated that our colorimetric assay can applied to rapid and accurate quantification of nematode viability/mortality in a nematode population treated with candidate chemicals/biologics. Application of our method would facilitate high-throughput endeavors aiming at finding environment-friendly control agents for deadly disease of pine trees.

The efficiency of RNA interference in Bursaphelenchus xylophilus.

RNA interference (RNAi) was performed on several essential genes in the pinewood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. Double-stranded RNA (dsRNA) was delivered to larvae or adult worms by soaking, electroporation, or microinjection. Soaking and electroporation of L2-L3 stage worms in solutions containing dsRNA for essential genes induced over 25% lethality after 5 days, and gene-specific phenotypes were observed. This lethality agreed with significant reductions of the targeted transcripts, as assayed by reverse-transcription coupled with real time PCR. Microinjection was the most efficient route as measured by the hatching rate of F1 embryos, which was reduced by 46%. When adult worms were soaked in dsRNA, lethality was induced in the F1 larvae, revealing the persistence of knockdown phenotypes. The penetrance of the RNAi phenotypes for essential genes was relatively low but consistent, indicating that RNAi should be useful for studying the in vivo functions of B. xylophilus gene products.

Assessment of the behaviour and survival of nematodes under low oxygen concentrations.

Oxygen is required for the completion of almost all known metazoan lifecycles, but many metazoans harbour abilities to withstand varying degrees and periods of hypoxia. Caenorhabditis elegans, one of the most popular model organism is extensively used as a model for the study of hypoxia and anoxia biology and it has been found that this nematode is capable of tolerance to varying degrees of hypoxia. Considering the extremely high diversity of nematodes, the effects of low oxygen concentration and mechanisms of adaptation to oxygen depletion differ among species. In this study, we used a simple assay to examine anoxia tolerance in four nematode species, including three free-living and one plant parasitic nematode. We found that the plant parasitic nematode Bursaphelenchus xylophilus can survive more than 14 days under anoxic conditions. Comparisons of behaviour during anoxia induction and the repertoire of oxygen sensation genes among the tested species suggested the existence of different oxygen sensation systems between B. xylophilus and C. elegans, which quickly introduce suspended animation in response to oxygen depletion to survive long-term anoxia.

A decline in transcript abundance for Heterodera glycines homologs of Caenorhabditis elegans uncoordinated genes accompanies its sedentary parasitic phase.

BACKGROUND: Heterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin) during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia. RESULTS: We developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of C. elegans and H. glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97. CONCLUSION: The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.

Effects of trans-2-hexenal on reproduction, growth and behaviour and efficacy against the pinewood nematode, Bursaphelenchus xylophilus.

BACKGROUND: Bursaphelenchus xylophilus is a serious quarantined pest that causes severe damage and major economic losses to pine forests. Because of the adverse effects of some traditional nematicides on humans and the environment, the search for new plant toxicants against these nematodes has intensified. Nematicidal activity of trans-2-hexenal, which is a six-carbon aldehyde present in many plants, was tested against the nematode. RESULTS: trans-2-Hexenal showed significant efficacy against B. xylophilus in a dose range of 349.5-699 g m-3 by fumigation of pinewood logs. Additionally, it had significant nematicidal activity against different life stages of B. xylophilus in an in vitro test, with second-stage larvae (L2s) being the most sensitive, with an LC50 value of 9.87 µg mL-1 at 48 h. Egg hatch was also significantly inhibited. Further studies revealed that trans-2-hexenal inhibited the reproductive activity of B. xylophilus, with negative effects on reproduction rate and egg numbers. Moreover, trans-2-hexenal reduced the body length of B. xylophilus. Respiratory rate and thrashing behaviour of B. xylophilus also decreased following treatment with this compound. CONCLUSION: trans-2-Hexenal had significant nematicidal activity against B. xylophilus, providing a basis for elucidation of the mode of action of trans-2-hexenal against plant-parasitic nematodes in future studies. © 2016 Society of Chemical Industry.