Cui Hu

Analysis of Cry1Ab toxin bioaccumulation in a food chain of Bt rice, an herbivore and a predator.

Lessons from organophosphorus pesticides, which could be bioaccumulated in non-target organisms at different trophic levels and caused unexpected negative impacts, necessitate a study of the possibility of biotransfer and bioaccumulation of Bacillusthuringiensis (Bt) insecticidal toxin(s) expressed in Bt plants. Using ELISA, we evaluated the transfer of Cry1Ab toxin in a food chain of Bt rice (KMD1 and KMD2), the target insect, Cnaphalocrocismedinalis, and its predator, Piratasubpiraticus. Cry1Ab was detected in C. medinalis and P. subpiraticus. However, the concentration of Cry1Ab detected from C. medinalis and P. subpiraticus did not increase as feeding or preying time increased. A binding study of Cry1Ab to the brush border membrane vesicle of C. medinalis and P. subpiraticus indicated that P. subpiraticus does not have binding receptors in its midgut to Cry1Ab, while C. medinalis does. Survivorship and fecundity of P. subpiraticus preying on Bt rice-fed C. medinalis were not significantly different from those preying on non-Bt rice-fed C. medinalis. Developmental time of P. subpiraticus was significantly longer when it preyed on Bt rice-fed C. medinalis than on non-Bt rice-fed prey. However, a 3-year field trial indicated that Bt rice did not significantly affect the density of P. subpiraticus.

Field Assessment of the Effects of Transgenic Rice Expressing a Fused Gene of cry1Ab and cry1Ac from Bacillus thuringiensis Berliner on Nontarget Planthopper and Leafhopper Populations

Abstract In 2003 and 2004, field studies were conducted at three sites in Zhejiang Province in China to assess the impacts of Bacillus thuringiensis (Bt) Berliner rice expressing a fused gene of cry1Ab and cry1Ac on nontarget planthoppers and leafhoppers. Populations in Bt plots were sampled with yellow sticky card traps, Malaise traps, and a vacuum-suction machine, and compared with samples from non-Bt control (IR72) plots. The results from yellow sticky card trap samplings indicated no significant differences between Bt and non-Bt plots in the species composition or densities of each species of planthopper and leafhopper. Three species of planthoppers, Sogatella furcifera (Horvath), Nilaparvata lugens (Stål), and Laodelphax striatellus (Fallén), were collected at all sites, and three species of leafhoppers, Nephotettix cincticeps (Uhler), Thaia subrufa (Motschulsky), and Recilia dorsalis (Motschulsky), were collected at Hangzhou. Another species of leafhopper, N. virescens (Distant), was collected at Anji and Jiande instead of T. subrufa. The results from the Malaise trap and vacuum-suction samples revealed no significant differences between Bt and non-Bt plots in species structure of planthoppers and leafhoppers or in population changes of the predominant planthopper species, S. furcifera, the predominant leafhopper species, N. cincticeps, or N. virescens throughout most sampling dates. Densities of planthoppers and leafhoppers were significantly affected by year and site but not by Bt rice. In general, our results suggest that the Bt rice line tested did not adversely affect nontarget planthopper and leafhopper populations and will not lead to higher populations or damage by planthoppers and leafhoppers.

Proteomic analysis of the venom from the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Parasitoid venom is a complex mixture of active substances with diversified biological functions. Because of its range of activities, venom is an important resource with respect to potential application in agriculture and medicine. Only a limited number of peptides, proteins, and enzymes have been identified and characterized from parasitoid venom. Here we describe a proteomic analysis of the venom from the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae). Venom resolved by two-dimensional electrophoresis yielded 56 protein spots with major proteins in the pI range 4-7 and molecular mass range of 25-66.2 kDa. The amino acid sequences of the proteins were identified by mass spectrometry. Several venom proteins such as calreticulin, venom acid phosphatase, serine protease, arginine kinase, serine protease homolog, aminotransferase-like venom protein, and heat shock protein 70, were identified in silico based on their amino acid sequences. The full-length cDNAs of calreticulin and arginine kinase were cloned. Calreticulin showed 62% identity with calreticulin in the venom of Cotesia rubecula. Arginine kinase showed a high level of sequence identity (92%) with its counterpart in the venom of Cyphononyx dorsalis. RT-PCR analysis revealed that the transcript levels of calreticulin and arginine kinase were developmentally changed, suggesting a possible correlation with the oviposition process. This study contributes to our appreciation of a parasitoid wasp venom composition.

Cuticular Hydrocarbon Composition in Pupal Exuviae for Taxonomic Differentiation of Six Necrophagous Flies

Abstract Gas chromatography-mass spectrometry was used to analyze the cuticular hydrocarbons extracted from the pupal exuviae of six necrophagous flies: Aldrichina grahami (Aldrich), Chrysomya megacephala (F.), Lucilia sericata (Meigen), Achoetandrus rufifacies (Macquart), Boettcherisca peregrina (Robineau-Desvoidy), and Parasarcophaga crassipalpis (Macquart). A discriminant model including the variables of peak 1 (tricosane), peak 7 (9-,11-,13-methyl-pentacosane), peak 21 (11,12-;9,13-dimethyl-hexacosane), peak 24 (octocosane), peak 41 (7,11-dimethyl-nonacosane), peak 42 (3-methyl-nonacosane), peak 46 (2-methyl-hentriacontane), and peak 51 (unknown) was constructed, which allowed a complete separation of the pupal exuviae of the six species. These results indicate that cuticular hydrocarbons as chemotaxonomic characters for insects of forensic importance are of high value and feasibility.

Isolation and characterization of an immunosuppressive protein from venom of the pupa-specific endoparasitoid Pteromalus puparum

In hymenopteran parasitoids devoid of symbiotic viruses, venom proteins appear to play a major role in host immune suppression and host regulation. Not much is known about the active components of venom proteins in these parasitoids, especially those that have the functions involved in the suppression of host cellular immunity. Here, we report the isolation and characterization of a venom protein Vn.11 with 24.1 kDa in size from Pteromalus puparum, a pupa-specific endoparasitoid of Pieris rapae. The Vn.11 venom protein is isolated with the combination of ammonium sulfate precipitation and anion exchange chromatography, and its purity is verified using SDS-PAGE analysis. Like crude venom, the Vn.11 venom protein significantly inhibits the spreading behavior and encapsulation ability of host hemocytes in vitro. It is suggested that this protein is an actual component of P. puparum crude venom as host cellular-immune suppressive factor.

Development changes of cuticular hydrocarbons in Chrysomya rufifacies larvae: potential for determining larval age.

Abstract Age determination is the basis of determining the postmortem interval using necrophagous fly larvae. To explore the potential of using cuticular hydrocarbons for determining the ages of fly larvae, changes of cuticular hydrocarbons in developing larvae of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) were investigated using gas chromatography with flame‐ionization detection and gas chromatography‐mass spectrometry. This study showed that the larvae produced cuticular hydrocarbons typical of insects. Most of the hydrocarbons identified were alkanes with the carbon chain length of 21–31, plus six kinds of alkenes. The hydrocarbon composition of the larvae correlated with age. The statistical results showed that simple peak ratios of n‐C29 divided by another eight selected peaks increased significantly with age; their relationships with age could be modelled using exponential or power functions with R2 close to or > 0.80. These results suggest that cuticular hydrocarbon composition is a useful indicator for determining the age of larval C. rufifacies, especially for post‐feeding larvae, which are difficult to differentiate by morphology.

Accumulation of cadmium and its effects on growth, development and hemolymph biochemical compositions in Boettcherisca peregrina larvae (Diptera: Sarcophagidae)

Abstract Newly emerged larvae of the fleshfly, Boettcherisca peregrina were exposed to two different CdCI2 concentrations of 100 μg/g and 400 mg/g diet fresh weight (DFW). They were administered in the diets until the end of larval stage. Cd‐exposed larvae accumulated significant amounts of Cd and this accumulation increased with the exposure dose and time. The body weights were lightened and lengths of larvae were shortened considerably after Cd exposure, especially at the higher Cd concentration. The total larval duration was also extremely affected due to Cd exposure. The average duration was prolonged significantly by 14 h at the lower Cd concentration, while it was increased by 33.7 h over controls at the higher Cd concentration. A significant decrease in contents of either soluble proteins, total lipids or caloric values in the hemolymph occurred due to Cd exposure throughout the entire tested period but after 120 h of Cd exposure. In contrast, when exposed to Cd with its higher concentration, total sugar contents in the hemolymph were increased strikingly over the whole tested time, except after 96 h of Cd exposure, while they were not apparently altered except after 24 h of Cd exposure at the lower concentration. Thus, it is suggested that Cd exposure shows significant adverse impact on the growth and development, as well as metabolism, in larvae of this fleshfly, depending on its exposure time and dose.

Venom of parasitoid, Pteromalus puparum, suppresses host, Pieris rapae, immune promotion by decreasing host C-type lectin gene expression.

Background Insect hosts have evolved immunity against invasion by parasitoids, and in co-evolutionary response parasitoids have also developed strategies to overcome host immune systems. The mechanisms through which parasitoid venoms disrupt the promotion of host immunity are still unclear. We report here a new mechanism evolved by parasitoid Pteromalus puparum, whose venom inhibited the promotion of immunity in its host Pieris rapae (cabbage white butterfly). Methodology/Principal Findings A full-length cDNA encoding a C-type lectin (Pr-CTL) was isolated from P. rapae. Quantitative PCR and immunoblotting showed that injection of bacterial and inert beads induced expression of Pr-CTL, with peaks of mRNA and Pr-CTL protein levels at 4 and 8 h post beads challenge, respectively. In contrast, parasitoid venom suppressed Pr-CTL expression when co-injected with beads, in a time and dose-dependent manner. Immunolocalization and immunoblotting results showed that Pr-CTL was first detectable in vesicles present in cytoplasm of granulocytes in host hemolymph, and was then secreted from cells into circulatory fluid. Finally, the secreted Pr-CTL bound to cellular membranes of both granulocytes and plasmatocytes. Injection of double-stranded RNA specific for target gene decreased expression of Pr-CTL, and a few other host immune-related genes. Suppression of Pr-CTL expression also down-regulated antimicrobial and phenoloxidase activities, and reducing phagocytotic and encapsulation rates in host. The inhibitory effect of parasitoid venom on host encapsulation is consistent with its effect in suppressing Pr-CTL expression. Binding assay results showed that recombinant Pr-CTL directly attached to the surface of P. puparum egges. We infer that Pr-CTL may serve as an immune signalling co-effector, first binding to parasitoid eggs, regulating expression of a set of immune-related genes and promoting host immunity. Conclusions/Significance P. puparum venom inhibits promotion of host immune responses by silencing expression of host C-type lectin gene Pr-CTL, whose expression affected transcription of other host immune-related genes.

Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host, Pieris rapae (Lepidoptera: Pieridae)

Pteromalus puparum is a predominant endoparasitoid wasp of Pieris rapae. Its venom is the only active factor injected into host associated with oviposition. In this report, we explored whether the venom alone from this wasp affects the endocrine system of its host or not. We monitored the changes of hemolymph juvenile hormone (JH; only JH III detected), ecdysteroid, and juvenile hormone esterase activity (JHE) over 72 h in parasitized and venom-microinjected P. rapae pupae. Non-parasitized and PBS-microinjected P. rapae served as controls. Results showed that JH titers were significantly higher in parasitized and venom-microinjected pupae than that in control pupae during 24 to 72 h. After 12 h, JH titers were significantly promoted by parasitization and venom microinjection. JHE activities of non-parasitized and PBS-microinjected pupae were significantly higher than that of parasitized and venom-microinjected pupae, which was with a peak at 12 h (parasitized pupae) or 24 h (venom-microinjected pupae) during 6 to 48 and 12 to 36 h, respectively. The hemolymph titers of ecdysteroid in non-parasitized and PBS-microinjected pupae increased rapidly during 12 to 36 h with a peak at 36 h, and were higher than treatments before 48 h, while presenting a significant difference at 24 to 48 h between the treatments and controls. The results demonstrate that venom alone of this parasitoid wasp can disrupt its hosts endocrine system.

Morphology and ultrastructure of the venom apparatus in the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

The venom apparatus of the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) was studied with light and electron microscope and was subjected to the electrophoretic and immunohistochemical analyses. Typically its venom apparatus consists of an unbranched venom gland and a venom reservoir, which is associated with a Dufour gland. The venom gland is lined by a series of secretory units. Each secretory unit comprises a secretory cell and a duct cell. The secretory cell is associated with an end apparatus to collect its secretions into the gland lumen. Secretory cells in the venom gland are characterized by extensive rough endoplasmic reticulum and numerous electron-dense vesicles in the distal and middle parts. They also exhibit several secretory granules and vacuoles. The venom reservoir presents three distinct regions: an external layer, composed by numerous fine muscle fibers; an internal layer, represented by epithelial cell with large nucleus; and an intima portion, represented by thin and uniform organization. The morphological aspect of numerous well-developed organelles responsible for protein generation observed is in agreement with the electrophoretic and immunohistochemical results which reveal that the rich proteinaceous components are present in the venom gland and venom reservoir. The venom proteins are first mainly produced in the secretory unit of venom gland, then drained to the lumen through the end apparatus, and are finally collected and stored in the venom reservoir.