Overexpression of CYP6CX4 contributing to field-evolved resistance to flupyradifurone, one novel butenolide insecticide, in Bemisia tabaci from China.

Bemisia tabaci is a formidable insect pest worldwide, and exhibits significant resistance to various insecticides. Flupyradifurone is one novel butenolide insecticide and has emerged as a new weapon against B. tabaci, but field-evolved resistance to this insecticide has become a widespread concern. To unravel the mechanisms of field-evolved flupyradifurone resistance, we conducted a comprehensive investigation into susceptibility of twenty-one field populations within the Beijing-Tianjin-Hebei Region of China. Alarmingly, thirteen of these populations displayed varying degrees of resistance, ranging from low to medium levels, and building upon our prior findings, we meticulously cloned and characterized the CYP6CX4 gene in B. tabaci. Our investigations unequivocally confirmed the association between CYP6CX4 overexpression and flupyradifurone resistance in three of the thirteen resistant strains via RNA interference. To further validate our findings, we introduced CYP6CX4 overexpression into a transgenic Drosophila melanogaster line, resulting in a significant development of resistance to flupyradifurone in D. melanogaster. Additionally, homology modeling and molecular docking analyses showed the stable binding of flupyradifurone to CYP6CX4, with binding free energy of -6.72 kcal mol-1. Collectively, our findings indicate that the induction of CYP6CX4 exerts one important role in detoxification of flupyradifurone, thereby promoting development of resistance in B. tabaci.

Independent parallel pyrolysis kinetics of model components in sewage sludge analyzed by BPM neural network.

Analyzing the kinetic behavior of sewage sludge pyrolysis is essential for the design of efficient reactors to produce biofuel and syngas. To understand the complex pyrolysis process of sewage sludge, we pyrolyzed six model components (i.e., cellulose, hemicellulose, lignin, protein, soluble sugars, and lipid) using a thermogravimetric analyzer. The effects of the heating rate on the pyrolysis process were examined at four different heating rates (5, 15, 25, and 50 °C/min). As temperature increased, the derivative thermogravimetric peaks shifted to higher temperature zones. The temperature ranges of the maximum mass loss rate for cellulose, hemicellulose, lignin, protein, soluble sugars, and lipid were within 326.1-368.0 °C, 288.7-315.5 °C, 375.1-429.4 °C, 291.9-308.0 °C, 251.0-314.1 °C, and 410.8-454.1 °C, respectively. The apparent activation energies of the model components were obtained using non-isothermal kinetic analysis methods (Flynn-Wall-Ozawa and Kissinger-Akahira-Sunose). In addition, a back-propagation artificial neural network with a momentum algorithm (BPM) was developed to predict the relationship between the pyrolysis experiment and the activation value. The best BPM model (BPM5) for predicting the cellulose pyrolysis was identified.

Knockdown of TRPV gene Nanchung decreases resistance to the novel pyropene insecticide, afidopyropen, in Bemisia tabaci.

Insect TRPV is one subfamily of transient receptor potential (TRP) channels, and it is composed of Inactive (Iav) and Nanchung (Nan), the molecular targets of afidopyropen in several sucking insect pests. In this study, we performed successive selection and synergism tests based on previous work. The resistant afidopyropen strain HD-Afi of Bemisia tabaci reached about 86-fold resistance to afidopyropen, and only part of the resistance resulted from detoxification of cytochrome P450 monooxygenases. Then, we cloned and characterized Nan and Iav from B. tabaci (BtNan and BtIav), and found that they were expressed in all stages of development and tissues of adult, and the expression level of BtNan in strains of HD-S and HD-Afi were significantly increased after afidopyropen treatment. Further, expression of BtNan was downregulated after gene silencing, and it resulted in significantly decreased afidopyropen resistance in the strain of HD-Afi. Our data revealed the first evidence that overexpression of the TRPV Nan gene is responsible for causing afidopyropen resistance in B. tabaci, and our results could provide new visions on the function of TRPVs in the development of resistance to pesticide and supply evidence for developing novel strategies of pest management.

Behavioral and genomic divergence between a generalist and a specialist fly.

The evolution of feeding habits leads to speciation in insects. Bactrocera true fruit flies display diverse feeding habits across species. We combine behavioral and functional genomic studies to probe the divergence between the specialist B. minax and the generalist B. dorsalis. We find that both vision and olfaction contribute to their respective host preferences, with a dominant effect of vision over the olfaction in short range. Correspondingly, host location-related genes are significantly enriched in the phototransduction pathway, of which the long-wavelength rhodopsin confers the color preference in both species and has been subject to selection in the specialist. We also find a massive expansion of olfactory receptors in the generalist, along with signatures of conditional expression and positive selection. The phylogenetic context suggests an ancestrally important role of vision in the host location of Bactrocera, as well as the increased performance and plasticity of olfaction alongside the arising of generalism.

Acylsugar protection of Nicotiana benthamiana confers mortality and transgenerational fitness costs in Spodoptera litura.

Acylsugars are secondary metabolites that are produced in the trichomes of some solanaceous species and can help control several herbivorous insect pests. Previously, knockout mutations (asat2 mutants) were shown to significantly reduce the acylsugar content of Nicotiana benthamiana, and significantly improve the fitness of six generalist insect herbivores. The current study compared the significant mortality and fitness costs in Spodoptera litura conferred by acylsugar protection of N. benthamiana (wild-type plants) compared to S. litura strains reared in acylsugar-deficient plants with depleted acylsugar biosynthesis. Acylsugar protection prolonged the developmental duration and decreased viability in the larval stages. Further, the fecundity of females and the hatching rate of eggs significantly decreased under acylsugar protection. For F1 offspring, acylsugar protection still exerted significant negative effects on larval survival rate and fecundity per female. The net reproductive rate and relative fitness of the S. litura strain were strongly affected by acylsugar. Altogether, these results indicate that acylsugar could contribute to plant protection due to toxicity to pests, diffused availability, and low environmental persistence. This could represent a complementary and alternative strategy to control populations of insect pests.

Selection and Evaluation of Reference Genes for miRNA Expression Analysis in Bemisia tabaci Under Insecticide Tolerance.

A growing number of studies have focused on the microRNA (miRNA) expression in Bemisia tabaci, one devastating agricultural insect pest of the tropical and subtropical areas for which the primary means of control are insecticides. In studying the genetic underpinnings of insecticide resistance, the choice of stable reference genes for normalizing data plays a key role to acquire unbiased expression profile results from quantitative real-time PCR (qPCR) analysis. Expression profiles of 11 selected reference genes were determined systematically in B. tabaci exposure to 11 insecticides. Furthermore, we assessed the stability of all the selected candidates in relation to other variables including sex, tissue type, and developmental stage. Candidate reference gene validation was conducted by analyzing the let-7-5p expression under various experimental treatments. Five programs BestKeeper, NormFinder, geNorm, △Ct, and RefFinder were applied to verify the accuracy of the selected candidates. Our results displayed that the best choices of the selected candidates for pymetrozine, sulfoxaflor, flonicamid, cyantraniliprole, afidopyropen, and deltamethrin treatment were miR-1-3p and miR-100-5p, U6 and miR-100-5p were best for chlorpyrifos and imidacloprid treatments, and U6 and miR-1-3p were best for flupyradifurone and ß-cypermethrin treatments. The reference genes miR-624, miR-252, and miR-275 worked best in adult tissues, miR-100-5p and miR-1-3p worked best in either sex, and miR-624 and miR-11 were best to use across developmental stages. Not even one reference gene was found to be suitable for all experimental conditions. Our results contributed to the growing body of the literature on qPCR reference gene selection under various experimental conditions and facilitate further investigation on gene expression changes in B. tabaci, resulting from pesticide exposure.

[Tradeoffs and synergies of ecosystem services in western mountainous China: A case study of the Bailongjiang watershed in Gansu, China]. / è¥¿éƒ¨å±±åŒºæµåŸŸç”Ÿæ€ç³»ç»ŸæœåŠ¡æƒè¡¡ä¸ŽååŒå ³ç³»­­ä»¥ç”˜è‚ƒç™½é¾™æ±ŸæµåŸŸä¸ºä¾‹.

The Bailongjiang watershed of Gansu is an important water conservation and ecological barrier area in the upper reaches of Yangtze River. It is necessary to reveal the tradeoffs and synergies of ecosystem services (ESs) for the "win-win" of watershed ecological system and social eco-nomy development. Based on the InVEST model, four typical ESs including soil conservation (SC), water conservation (WC), food supply (FS), and habitat quality (HQ) were assessed, and the multi-scale tradeoffs and synergies of ESs and its drivers were analyzed by correlation and root mean square deviation (RMSD). The results showed that there were significant synergies among SC, WC, and HQ, and a significant tradeoff between FS and HQ, SC, WC, respectively. The areas with high tradeoff intensity between the three pairs of synergistic services (SC-WC, SC-HQ, WC-HQ), and between FS and HQ were mainly concentrated in the steep forest area of middle-high mountain in Wenxian, Diebu and Zhouqu. The high intensity of tradeoffs between FS-SC, FS-WC were mainly concentrated in the gentle apricus farming and pastoral areas of middle-low mountain in Tanchang and Wudu. The spatial variation of land use/cover caused by human activities was an important factor affecting the degree of ES tradeoffs and its scale effect.

Mutation of Bdpaired induces embryo lethality in the oriental fruit fly, Bactrocera dorsalis.

BACKGROUND: Pair-rule genes were identified and named for their role in segmentation in animal embryos. Paired, a homolog of mammalian PAX3, acts as one of several pair-rule genes and is key in defining the boundaries of future parasegments and segments during insect embryogenesis. RESULTS: We cloned the paired gene from the oriental fruit fly, Bactrocera dorsalis, and then applied CRISPR/Cas9-mediated genome editing to investigate its physiological function in the embryonic stage of this pest. We identified one transcript for a paired homolog in B. dorsalis, which encodes a protein containing a Paired Box domain and a Homeobox domain. Phylogenetic analysis confirmed that the paired gene is highly conserved and the gene was highly expressed at the 12-14 h-old embryonic stage. Knock-out of Bdpaired led to lack of segment boundaries, cuticular deficiency, and embryonic lethality. Sequence analysis of the CRISPR/Cas9 mutants exhibited different insertion and deletions in the Bdpaired locus. In addition, the relative expression of Wingless (Wg) and Abdominal A (Abd-A) genes were significantly down-regulated in the Bdpaired mutant embryos. CONCLUSION: These results indicate that Bdpaired gene is critical for the embryonic development of B. dorsalis, and could be a novel molecular target for genetic-based pest management practices to combat this serious invasive pest. © 2019 Society of Chemical Industry.

[Distribution of 16 Polycyclic Aromatic Hydrocarbons in Dianchi Lake Surface Sediments After the Integrated Water Environment Control Project].

The external pollution of Dianchi Lake has been effectively controlled with the implementation of the integrated water environment control project. However, further attention should be paid to endogenous pollutants, such as surface sediments. To investigate the distribution of 16 polycyclic aromatic hydrocarbons (PAHs) in surface sediments of Dianchi Lake, PAH concentrations in 19 surface sediment samples (collected in December 2016) were quantitatively measured by gas chromatography-mass spectrometry (GC-MS). The spatial and temporal distribution, sources, and ecological risks were also analyzed. The concentration of total PAHs (TPAHs) in the Dianchi Lake surface sediments varied in the range of 92.31-1546.78 ng·g-1 with an average of 496.30 ng·g-1. The average concentration of TPAHs in the surface sediments from Caohai (932.37 ng·g-1) was much greater than that from Waihai (380.02 ng·g-1). With the implement of the integrated water environment control project, the concentration of TPAHs in the surface sediments from Dianchi Lake was significantly lower than those detected in 2012, and was already relatively low level among other key waterbodies in China. The PAH with the highest concentration was fluoranthene (80.65 ng·g-1) and the substance with the highest toxic equivalent quantity (TEQ) was dibenz[a, h] anthracene (42.97 ng·g-1). The PAHs were mainly composed of 4 ring and 5-6 ring compounds (with the concentration ratio of 40.38% and 40.22%, respectively), which indicated that the proportions of middle-ring and high-ring compounds were generally consistent. The results of the molecular diagnostic ratio analysis showed that the primary source of PAHs in Dianchi Lake surface sediments are biomass and coal combustion. Based on the potential ecological risk marker comparison method, the entire lake was classified as having a low ecological risk, while the ecological risk of Caohai was relatively higher, which should be concerned further. The results provide initial data and act as an important reference for the conservation and improvement of water quality in Dianchi Lake.

Symbiotic bacteria motivate the foraging decision and promote fecundity and survival of Bactrocera dorsalis (Diptera: Tephritidae).

BACKGROUND: The gut bacteria of tephritid fruit flies play prominent roles in nutrition, reproduction, maintenance and ecological adaptations of the host. Here, we adopted an approach based on direct observation of symbiotic or axenic flies feeding on dishes seeded with drops of full diet (containing all amino acids) or full diet supplemented with bacteria at similar concentrations to explore the effects of intestinal bacteria on foraging decision and fitness of Bactrocera dorsalis. RESULTS: The results show that intestinal probiotics elicit beneficial foraging decision and enhance the female reproduction fitness and survival of B. dorsalis (symbiotic and axenic), yet preferences for probiotic diets were significantly higher in axenic flies to which they responded faster compared to full diet. Moreover, females fed diet supplemented with Pantoea dispersa and Enterobacter cloacae laid more eggs but had shorter lifespan while female fed Enterococcus faecalis and Klebsiella oxytoca enriched diets lived longer but had lower fecundity compared to the positive control. Conversely, flies fed sugar diet (negative control) were not able to produce eggs, but lived longer than those from the positive control. CONCLUSIONS: These results suggest that intestinal bacteria can drive the foraging decision in a way which promotes the reproduction and survival of B. dorsalis. Our data highlight the potentials of gut bacterial isolates to control the foraging behavior of the fly and empower the sterile insect technique (SIT) program through the mass rearing.

Hierarchical spiral-scan trajectory for efficient scanning ion conductance microscopy.

Scanning ion conductance microscopy (SICM) is an emerging technique for non-contact, high-resolution topography imaging, especially suitable for live cells investigation in a physiological environment. Despite its rapid development, the extended acquisition time issues of its typical hopping/backstep scanning mode still restrict its application for more fields. Herein, we propose a novel SICM scanning approach to effectively reduce the retract distance of existing hopping/backstep mode. In this approach, the SICM probe first gradually descends in the z-direction. Then by using Archimedes spiral trajectory, which has the advantage of higher angular velocity due to its continuous and smooth trajectory, the probe rapidly detects the highest point of the sample in the xy-plane in a layer-by-layer way. Further, the maximum height that decides the retrace distance of pipet in the detected region can be quickly achieved, avoiding a huge retrace distance usually adopted in the existing methods without any prior knowledge (sample height and steepness in the scanning region). Therefore, this new scanning method can greatly reduce the imaging time by minimizing the retrace height of each measurement point. Theoretical analysis is conducted to compare the imaging time of traditional and new method. And various factors in the new method that affect the imaging speed are analyzed. In addition, PDMS (polydimethylsiloxane) and biological samples (C2C12 cells) were imaged by SICM that was operated in the hopping mode, raster-based detecting and developed method with a single-barrel pipet, respectively. The experimental results suggest that the new method has a faster imaging speed than conventional scanning modes but does not sacrifice the imaging quality.

Comparative transcriptome analysis of two races of Heterodera glycines at different developmental stages.

The soybean cyst nematode, Heterodera glycines, is an important pest of soybeans. Although resistance is available against this nematode, selection for virulent races can occur, allowing the nematode to overcome the resistance of cultivars. There are abundant field populations, however, little is known about their genetic diversity. In order to elucidate the differences between races, we investigated the transcriptional diversity within race 3 and race 4 inbred lines during their compatible interactions with the soybean host Zhonghuang 13. Six different race-enriched cDNA libraries were constructed with limited nematode samples collected from the three sedentary stages, parasitic J2, J3 and J4 female, respectively. Among 689 putative race-enriched genes isolated from the six libraries with functional annotations, 92 were validated by quantitative RT-PCR (qRT-PCR), including eight putative effector encoding genes. Further race-enriched genes were validated within race 3 and race 4 during development in soybean roots. Gene Ontology (GO) analysis of all the race-enriched genes at J3 and J4 female stages showed that most of them functioned in metabolic processes. Relative transcript level analysis of 13 selected race-enriched genes at four developmental stages showed that the differences in their expression abundance took place at either one or more developmental stages. This is the first investigation into the transcript diversity of H. glycines races throughout their sedentary stages, increasing the understanding of the genetic diversity of H. glycines.

Exploring the host parasitism of the migratory plant-parasitic nematode Ditylenchus destuctor by expressed sequence tags analysis.

The potato rot nematode, Ditylenchus destructor, is a very destructive nematode pest on many agriculturally important crops worldwide, but the molecular characterization of its parasitism of plant has been limited. The effectors involved in nematode parasitism of plant for several sedentary endo-parasitic nematodes such as Heterodera glycines, Globodera rostochiensis and Meloidogyne incognita have been identified and extensively studied over the past two decades. Ditylenchus destructor, as a migratory plant parasitic nematode, has different feeding behavior, life cycle and host response. Comparing the transcriptome and parasitome among different types of plant-parasitic nematodes is the way to understand more fully the parasitic mechanism of plant nematodes. We undertook the approach of sequencing expressed sequence tags (ESTs) derived from a mixed stage cDNA library of D. destructor. This is the first study of D. destructor ESTs. A total of 9800 ESTs were grouped into 5008 clusters including 3606 singletons and 1402 multi-member contigs, representing a catalog of D. destructor genes. Implementing a bioinformatics' workflow, we found 1391 clusters have no match in the available gene database; 31 clusters only have similarities to genes identified from D. africanus, the most closely related species to D. destructor; 1991 clusters were annotated using Gene Ontology (GO); 1550 clusters were assigned enzyme commission (EC) numbers; and 1211 clusters were mapped to 181 KEGG biochemical pathways. 22 ESTs had similarities to reported nematode effectors. Interestedly, most of the effectors identified in this study are involved in host cell wall degradation or modification, such as 1,4-beta-glucanse, 1,3-beta-glucanse, pectate lyase, chitinases and expansin, or host defense suppression such as calreticulin, annexin and venom allergen-like protein. This result implies that the migratory plant-parasitic nematode D. destructor secrets similar effectors to those of sedentary plant nematodes. Finally we further characterized the two D. destructor expansin proteins.

A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana.

SUMMARY The Hg-SYV46 parasitism gene is expressed exclusively in the dorsal oesophageal gland cell of parasitic stages of the soybean cyst nematode, Heterodera glycines, and it encodes a secretory protein that contains a C-terminal motif of the CLAVATA3/ESR-related (CLE) family in Arabidopsis thaliana. In shoot and floral meristems of Arabidopsis, the stem cells secret CLV3, a founding member of the CLE protein family, that activates the CLV1/CLV2 receptor complex and negatively regulates WUSCHEL expression to restrict the size of the stem cell population. Mis-expression of Hg-SYV46 in Arabidopsis (ecotype Columbia-0) under control of the CaMV35S promoter resulted in a wus-like phenotype including premature termination of the shoot apical meristem and the development of flowers lacking the central gynoecium. The wus-like phenotype observed was similar to reports of over-expression of CLV3 and CLE40 in Arabidopsis, as was down-regulation of WUS expression in the shoot apices of 35S::Hg-SYV46/Col-0 plants. Expression of 35S::Hg-SYV46 in a clv3-1 mutant of Arabidopsis was able partially or fully to rescue the mutant phenotype, probably dependent upon localization and level of transgene expression. A short root phenotype, as reported for over-expression of CLV3, CLE40 and CLE19 in roots, was also produced in primary 35S::Hg-SYV46/Col-0 transgenic plants. The results suggest a functional similarity of HG-SYV46 to plant-secreted CLE ligands that may play a role in the differentiation or division of feeding cells induced in plant roots by parasitic nematodes.

Molecular characterisation and developmental expression of a cellulose-binding protein gene in the soybean cyst nematode Heterodera glycines.

Secretory proteins encoded by 'parasitism genes' expressed in the oesophageal gland cells of plant-parasitic nematodes play key roles in nematode infection and parasitism of host plants. A cellulose-binding protein-encoding cDNA, designated Hg-cbp-1, was cloned from a Heterodera glycines oesophageal gland-cell long-distance PCR cDNA library. The cDNA hybridised to genomic DNA of H. glycines in Southern blots, and the genomic sequence of Hg-cbp-1 contained only one intron. The Hg-cbp-1 cDNA contained an open reading frame encoding 132 amino acids, with a predicted signal peptide sequence for secretion and a cellulose-binding domain. Bacterial expressed recombinant HG-CBP-1, minus the signal peptide sequence, had no hydrolytic activity on carboxymethyl-cellulose but was able to bind to cellulose. The developmental expression of Hg-cbp-1, determined by real-time reverse transcriptase PCR, showed that Hg-cbp-1 is expressed throughout the parasitic cycle of H. glycines, with a relatively higher expression level in developing parasitic stages.

Developmental expression and biochemical properties of a beta-1,4-endoglucanase family in the soybean cyst nematode, Heterodera glycines.

SUMMARY The soybean cyst nematode, Heterodera glycines, produces beta-1,4-endoglucanases (cellulases) that are secreted during infection of soybean. The gene structures of three, hg-eng-4, hg-eng-5 and hg-eng-6, of the six beta-1,4-endoglucanase genes, all family 5 glycosyl hydrolases previously identified from H. glycines, are presented here. Furthermore, we present the detailed expression analyses of beta-1,4-endoglucanase genes as well as the biochemical properties of four H. glycines endoglucanase enzymes. Two of the endoglucanases, HG-ENG-5 and HG-ENG-6, differed significantly in their amino acid sequence of the catalytic domains and their gene structure from that of the other four beta-1,4-endoglucanases. Quantitative real-time RT-PCR revealed distinct developmental expression differences among the hg-eng family members during the early stages of parasitism and relatively low expression levels in late parasitic stages, with the exception of the adult male stage for some eng genes. Recombinant HG-ENGs degraded carboxymethylcellulose and optimum enzyme activity ranged from pH 5.5 for HG-ENG-5 to pH 8 for HG-ENG-6. EDTA, Ca(2+), Co(2+), Mg(2+) and Fe(2+) did not affect enzyme activity of any ENG protein, whereas Zn(2+), Cu(2+) and Mn(2+) inhibited enzyme activity from 23% to 73% in some cases. In tests with 12 different polysaccharide substrates, enzyme activity was restricted to beta-1,4 linkages with all ENG proteins tested. Only HG-ENG-5 and HG-ENG-6 had relatively high activity on xylan and slightly degraded microcrystalline cellulose. Together, these data reveal distinct differences in expression and biochemistry of cyst nematode parasitism genes and proteins, respectively, and cast light on the intricate interactions between a parasitic animal and its plant host.

The parasitome of the phytonematode Heterodera glycines.

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.

A profile of putative parasitism genes expressed in the esophageal gland cells of the root-knot nematode Meloidogyne incognita.

Identifying parasitism genes encoding proteins secreted from a nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Meloidogyne incognita parasitism genes were cloned by microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. Of 2,452 cDNA clones sequenced, deduced protein sequences of 185 cDNAs had a signal peptide for secretion and, thus, could have a role in root-knot nematode parasitism of plants. High-throughput in situ hybridization with cDNA clones encoding signal peptides resulted in probes of 37 unique clones specifically hybridizing to transcripts accumulating within the subventral (13 clones) or dorsal (24 clones) esophageal gland cells of M. incognita. In BLASTP analyses, 73% of the predicted proteins were novel proteins. Those with similarities to known proteins included a pectate lyase, acid phosphatase, and hypothetical proteins from other organisms. Our cell-specific analysis of genes encoding secretory proteins provided, for the first time, a profile of putative parasitism genes expressed in the M. incognita esophageal gland cells throughout the parasitic cycle.

Characterisation and developmental expression of a chitinase gene in Heterodera glycines.

A chitinase full-length cDNA (designated Hg-chi-1) was isolated from a Heterodera glycines oesophageal gland cell-specific long-distance PCR cDNA library. The cDNA hybridised to genomic DNA of H. glycines in Southern blots. The Hg-chi-1 cDNA contained an open reading frame encoding 350 amino acids with the first 23 amino acids being a putative signal peptide for secretion. Hg-CHI-1 contained a chitinase 18 family catalytic domain, and chitinolytic activity of recombinant Hg-CHI-1 was confirmed in glycol-chitin substrate gel electrophoresis. In situ mRNA hybridisation analyses showed that transcripts of Hg-chi-1 accumulated specifically in the subventral oesophageal gland cells of parasitic stages of H. glycines, but Hg-chi-1 expression was not detected in eggs or hatched pre-parasitic second-stage juveniles, suggesting that this chitinase does not have a role in egg hatching of H. glycines. The biological function of Hg-CHI-1 in H. glycines remains to be determined.