Fifth-instar Larvae Research Articles

Cloning, Expression, and Cell Localization of a Novel Small Heat Shock Protein Gene: BmHSP25.4

Using molecular approaches, a new member of the Bombyx mori small heat shock protein family was cloned and characterized. The isolated gene contains an open reading frame of 672 bp, encodes a polypeptide of 223 amino acid residues with a predicted molecular mass of 25.4 kDa, and is therefore named BmHSP25.4. The gene codes for a protein homologous to the previously characterized HSP20.4 and HSP19.9. Western blotting analysis revealed that BmHSP25.4 existed in the fifth-instar larva's fatty body and blood tissues. Immunohistochemistry assay also showed that BmHSP25.4 was located in the fifth-instar larva's fatty body. The results of above studies have indicated constitutive expression of BmHSP25.4 in fatty body, blood tissues, and Bm5 cells. Finally, we examined the effect of heat stress on localization of BmHSP25.4 using anti-BmHSP25.4 polyclonal antibody by immunofluorescence. Under normal conditions, BmHSP25.4 was mostly found in the cytoplasm. However, after heat treatment, most of BmHSP25.4 distributed in the cell membrane. After 3 h of recovery following the heat shock treatment, the localization of BmHSP25.4 was the same as that under normal conditions.

INDEKS NUTRISI DAN KESINTASAN LARVA SPODOPTERA LITURA F. YANG DIBERI PAKAN MENGANDUNG EKSTRAK KULIT JENGKOL (PITHECELLOBIUM LOBATUM BENTH.)

Nutrition indices and survivorship of Spodoptera Litura F. larvae is fed nutrition including jengkol bark extract (Pithecellobium Lobatum Benth). The effect of P. lobatum barks extract on nutrition indices and survivorship S. litura larvae has been investigated. Nutrition indices were observed by giving fifth-instar larvae one of the three diets, every group of diet was added various concentration of barks extract, containing either 1.10; 2.20; or 4.40%. Survivorship were observed by giving first-instar larvae one of the three diets, every group of diet was added various concentration of barks extract, containing either 0.6; 1.2; or 2.4%. The result showed that, the Relative Consumption Rate (RCR), Relative Growth Rate (RGR), Efficiencies of food use (ECD and ECI) value of larvae treated were significantly lower in the case of treated larvae than that of the controls, Approximate Digestibility (AD) value of larvae treated were significantly higher in the case of treated larvae than that of the controls and decrease of survivorship tested larvae.

Expressed sequence tags from larval gut of the European corn borer (Ostrinia nubilalis): Exploring candidate genes potentially involved in Bacillus thuringiensis toxicity and resistance

BackgroundLepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST) libraries from the guts of lepidopteran larvae. Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxins, and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis), one of the most destructive pests of corn in North America and the western world. Our goals were to establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB.ResultsWe constructed two cDNA libraries from the guts of the fifth-instar larvae of ECB and sequenced a total of 15,000 ESTs from these libraries. A total of 12,519 ESTs (83.4%) appeared to be high quality with an average length of 656 bp. These ESTs represented 2,895 unique sequences, including 1,738 singletons and 1,157 contigs. Among the unique sequences, 62.7% encoded putative proteins that shared significant sequence similarities (E-value ≤ 10-3)with the sequences available in GenBank. Our EST analysis revealed 52 candidate genes that potentially have roles in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 18 chymotrypsin-like proteases, 13 aminopeptidases, 2 alkaline phosphatases and 1 cadherin-like protein. Comparisons of expression profiles of 41 selected candidate genes between Cry1Ab-susceptible and resistant strains of ECB by RT-PCR showed apparently decreased expressions in 2 trypsin-like and 2 chymotrypsin-like protease genes, and 1 aminopeptidase genes in the resistant strain as compared with the susceptible strain. In contrast, the expression of 3 trypsin- like and 3 chymotrypsin-like protease genes, 2 aminopeptidase genes, and 2 alkaline phosphatase genes were increased in the resistant strain. Such differential expressions of the candidate genes may suggest their involvement in Cry1Ab resistance. Indeed, certain trypsin-like and chymotrypsin-like proteases have previously been found to activate or degrade Bt protoxins and toxins, whereas several aminopeptidases, cadherin-like proteins and alkaline phosphatases have been demonstrated to serve as Bt receptor proteins in other insect species.ConclusionWe developed a relatively large EST database consisting of 12,519 high-quality sequences from a total of 15,000 cDNAs from the larval gut of ECB. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Our work provides a foundation for future research to develop an ECB gut-specific DNA microarray which can be used to analyze the global changes of gene expression in response to Bt protoxins/toxins and the genetic difference(s) between Bt- resistant and susceptible strains. Furthermore, we identified 52 candidate genes that may potentially be involved in Bt toxicity and resistance. Differential expressions of 15 out of the 41 selected candidate genes examined by RT-PCR, including 5 genes with apparently decreased expression and 10 with increased expression in Cry1Ab-resistant strain, may help us conclusively identify the candidate genes involved in Bt resistance and provide us with new insights into the mechanism of Cry1Ab resistance in ECB.

Higher survival of aposematic prey in close encounters with predators: an experimental study of detection distance

Aposematic animals are often conspicuous. It has been hypothesized that one function of conspicuousness in such prey is to be detected from afar by potential predators: the ‘detection distance hypothesis’. The hypothesis states that predators are less prone to attack at long detection range because more time is allowed for making the ‘correct’ decision not to attack the unprofitable prey. The detection distance hypothesis has gained some experimental support in that time-limited predators make more mistakes. To investigate effects of prey presentation distance we performed two experiments. First, in experiment 1, we investigated at what distance chicks, Gallus gallus domesticus, could see the difference in colour between aposematic and plain mealworms. Birds chose the correct track in a two-way choice when prey were at 20, 40 and 60 cm distance but not at 80 cm. Second, in experiment 2, fifth-instar larvae of the aposematic bug Lygaeus equestris were presented to experienced chicks at 2, 20 or 60 cm distance. We found no difference in attack probability between distances. However, prey mortality was significantly lower for the shortest presentation distance. In conclusion, we found no support for the hypothesis that aposematic prey benefit from long-range detection; in fact they benefit from shortdistance detection. This result, and others, suggests that the conspicuousness of aposematic prey at a distance may simply be a by-product of an efficient signalling function after detection.

Pathogenesis of Autographa californica multiple nucleopolyhedrovirus in fifth-instar Anticarsia gemmatalis larvae

We have investigated infection and pathogenesis of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Anticarsia gemmatalis (velvetbean caterpillar) larvae using a lacZ recombinant virus (AcMNPV-hsp70/lacZ) to track the temporal progression of infection in the midgut intestine and haemocoel. A. gemmatalis was highly resistant to fatal infection by occlusion bodies (OBs; LD(50)>5.5 x 10(5) OB) and budded virus (BV; LD(50)>3 x 10(5) BV) administered via oral and systemic routes, respectively. Orally administered occlusion-derived virus (ODV) efficiently attached and fused to midgut cells; however, high levels of infection-induced apoptosis limited infection in the midgut. Transcriptional analysis of AcMNPV genes expressed in the midgut of OB-inoculated A. gemmatalis larvae showed high levels of mRNA encoding the major capsid protein VP39 in the absence of immediate-early transactivator 1 (ie-1) expression. In the midgut, virus was efficiently transferred from infected midgut epithelial cells to nearby tracheolar cells and circulating haemocytes to initiate systemic infection in the haemocoel. However, haemocoelic BV did not efficiently disseminate infection and only cuticular epidermal cells displayed high levels of viral infection. Flow cytometry analysis of haemocytes isolated from BV-inoculated A. gemmatalis larvae showed low-level expression of the BV envelope protein GP64 on the cell surface, suggesting that A. gemmatalis haemocytes have a limited capacity for amplifying virus. These results show that AcMNPV is not an effective biological control agent for limiting crop damage caused by A. gemmatalis larvae.

Toxicity and disruption of midgut physiology in larvae of the European corn borer, Ostrinia nubilalis, by anion transporter blockers

In this study, four blockers of anion transporters (ATs) belonging to four different classes of organic acids, including DIDS (4, 4'-diisothiocyanatostilbene-2, 2'- disulfonic acid; a stilbene disulfonic acid), NPPB [(5-nitro-2-(3-phenylpropylamino) benzoic acid; an anthranilic acid)], 9-AC (anthracene-9-carboxylic acid; an aromatic carboxylic acid), and IAA-94 (indanyloxy acetic acid; an indanyloxy alkanoic acid), were tested for their toxicity against the European corn borer (ECB), Ostrinia nubilalis. All the AT blockers inhibited the growth of larvae, increased the developmental time, and decreased survival compared to controls, when second-instar ECB larvae were fed for seven days on treated diet. In general, DIDS and NPPB were the most active compounds, with the rank order of activity being DIDS>NPPB>IAA-94>9-AC. All the AT blockers decreased the midgut alkalinity in fifth-instar larvae when fed for 3 h on treated diet. Effective concentrations required for 50% decrease in midgut alkalinity (EC(50)) ranged between 29.1 and 41.2 ppm and the rank order of activity was NPPB>DIDS>IAA-94>9-AC. Similarly, all the tested AT blockers inhibited (36)Cl(-) uptake from the midgut lumen in fifth-instar larvae when fed for 3 h on treated diet. Concentrations required for 50% inhibition of (36)Cl(-) uptake (IC(50)) ranged between 7.4 and 11.0 ppm and the rank order of activity was DIDS>NPPB>9-AC >IAA-94. Modest to highly strong positive correlations observed among growth, midgut alkalinity, and midgut Cl(-) ion transport in AT blocker-fed larvae suggested that these effects are causally related to each other. Finally, AT blockers have the potential to become good candidates for development of insecticides with a unique mode of action.

Proteome analysis of silkworm 1. Fat body

Proteome analysis of silkworm (Bombyx mori L.) fat bodies was performed on the fifth-instar day-3 larva stage and the expression of proteins separated on two-dimensional polyacrylamide gels was tracked from the fourth-instar day-1 larva stage to the stage before adult moth. Proteins on the 2D-gels were excised manually and treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry after proteolysis using trypsin. In the previous report1), the amino acid sequence database obtained from the Drosophila genome was used for the protein identification. This analysis used the amino acid sequence data elucidated from the silkworm genome. Additionally, protein expression in fat bodies was compared with that at other stages and in other tissues described in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi). Several unidentified proteins from the previous report1) were identified with high Mascot scores. Induction and reduction of proteins during metamorphosis were observed as changes in spot concentration on sequential 2D-gels on the web.

Proteome analysis of silkworm 2. Hemolymph

Proteomic analysis of silkworm hemolymph at the fifth-instar day-3 larva stage was performed by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Prominent spots on 2D-gel were analyzed after trypsinization and proteins were identified by Mascot and Sequest using the amino acid sequence data obtained from the genome sequence of silkworm. The gel images were tracked from the fourth-instar day-1 larva stage to the stage before adult moth for twenty-one days. The results of mass spectrometry analysis and changes in gel images are summarized in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi).

Gamma irradiation effects on the induction of three heat shock protein genes ( piac25, hsc70 and hsp90) in the Indian meal moth, Plodia interpunctella

Effects of gamma irradiation on expression of heat shock protein genes and larval mortality were demonstrated in Plodia interpunctella, a serious pest of stored grains and food products. We obtained partial clones of three heat shock protein ( hsp) genes ( piac25, hsc70 and hsp90) and confirmed, by comparing deduced amino acid sequences, gene homology with corresponding hsps of other lepidopteran insects. Using Northern blot hybridization, expression levels of each gene were compared in fifth-instar larvae which were irradiated with gamma rays at doses from 0.1 to 2.0 kGy. The transcript level of hsc70 was increased at 0.5 kGy and higher doses, but the piac25 and hsp90 levels did not change. The hsc70 level also increased in irradiated pupae, but needed a 2.0 kGy dose. Mortality of irradiated larvae was also dose-dependent, and all larvae were killed by ≥1 kGy irradiation. Our results show that hsc70, but not piac25 and hsp90, was highly responsive to irradiation and suggest that specific hsp gene(s) may have a potential as molecular markers for the detection of irradiated insects.

Juvenile hormone acid methyl transferase is a key regulatory enzyme for juvenile hormone synthesis in the Eri silkworm, Samia cynthica ricini

During the period of adult emergence in the Eri silkworm, Samia cynthia ricini, the corpora allata (CA) are apparently reactivated in females, but not males. This creates a significant sexual dimorphism in juvenile hormone (JH) synthesis by CA. To determine the underlying molecular mechanisms in this process, we cloned cDNAs of two enzymes involved in the JH synthesis pathway: 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and juvenile hormone acid methyl transferase (JHAMT). Both Samcri-HMGR and -JHAMT mRNAs were detected in CA almost exclusively. However, their expression patterns were different from each other. During the period of adult emergence, Samcri-HMGR was expressed in CA at a constantly high level suggesting it plays little role for the regulation of JH synthesis. In contrast, the patterns of both Samcri-JHAMT mRNA level and enzyme activity were closely correlated with the patterns of JH synthesis, CA reactivation, and sexual dimorphism of JH synthesis. In addition, JHAMT mRNA levels were paralleled JH synthesis in the fifth-instar larvae of S. cynthia ricini and the pharate adults of the silkworm Bombyx mori. We infer from these results that JHAMT is a key regulatory enzyme for JH synthesis in the Eri silkworm.

Evaluation of two endemic and five commercial entomopathogenic nematode species (Rhabditida: Heterorhabditidae and Steinernematidae) against annual bluegrass weevil (Coleoptera: Curculionidae) larvae and adults

The annual bluegrass weevil, Listronotus maculicollis, is a highly destructive pest of golf course turfgrass in the northeastern United States. We assessed the virulence of entomopathogenic nematodes to larvae and adults of L. maculicollis as an initial step in developing a biological control program for this pest. Two endemic and five commercially available nematode species were tested in soil and field infested turf core assays. Adult susceptibility was generally low to moderate (11–65% mortality) and was not affected by the age or overwintering condition of the insect. A mixture of two nematode species with different foraging behaviors ( Steinernema carpocapsae and Heterorhabditis bacteriophora) achieved the highest levels of adult control, but required 6 days to obtain 50% mortality at five times standard field application rate (125 nematodes/cm 2). Conversely, fourth- and fifth-instar larvae were highly susceptible to nematodes. Steinernema feltiae, S. carpocapsae, and S. kraussei significantly reduced fourth instars in both years of the study. The same three species along with the commercial strain of H. bacteriophora significantly reduced high densities of fifth instars to below field damage thresholds. In general, Steinernema spp. trended towards greater control than Heterorhabditis spp. for both fourth and fifth instars. No difference was observed among the virulence of endemic and commercial nematode strains to any L. maculicollis stage tested. The data indicate that Steinernema spp. could provide high levels of control when applied curatively against L. maculicollis larvae.

Toxoneuron nigriceps parasitization delays midgut replacement in fifth-instar Heliothis virescens larvae

We have analyzed the effects of Toxoneuron nigriceps parasitization on the midgut development of its host Heliothis virescens. In parasitized H. virescens larvae, the midgut epithelium undergoes a complete replacement, which is qualitatively not different to that observed in synchronous unparasitized larvae, with similar temporal profiles of cell death and metabolic activity. However, the whole gut replacement process is significantly delayed in parasitized larvae, with complete differentiation of the new gut epithelium being observed 4 days later than in unparasitized controls. The administration of juvenile hormone before commitment and of 20-hydroxyecdysone (20E) after commitment delays and fosters, respectively, the replacement process of the midgut epithelium; moreover, the injection of 20E into developmentally arrested and 20E-deficient host last-instar larvae parasitized by T. nigriceps immediately triggers regular gut development. These hormone-based experiments suggest that endocrine alterations in the larval host, induced by T. nigriceps parasitism, are responsible for the temporal alterations in the gut replacement process. The role of this parasitoid-induced developmental change in the host regulation process is discussed.

Does larval aggregation pheromone of codling moth,Cydia pomonella, induce attraction or arrestment of receivers?

Cocoon-spinning larvae of the codling moth, Cydia pomonella, emit a pheromone that mediates aggregation by pupation site-seeking fifth-instar larvae. It was unknown and, thus, we tested whether the aggregation pheromone induces arrestment or attraction responses. In paired straight-tube experiment 1, fifth-instars moved faster and farther upwind toward cospecific cocoons compared to blank controls. In still-air cage experiment 2, fifth-instars selected more often as first and final choices of pupation sites those with cocooning conspecifics than those without. Finally, in Y-tube olfactometer experiment 3, fifth-instars anemotactically responded to, and preferred, side arms with cocooning conspecifics to those without. Our data provide evidence that codling moth larvae are attracted to, rather than merely arrested by, larval aggregation pheromone. These results help explain reported aggregations or clumped distributions of larvae on tree trunks, which would likely not occur if they were based merely on chance encounter of cocoon-spinning larvae by foraging larvae.

Phosphodiesterase Activity of CvfA Is Required for Virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.

Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm <italic>Bombyx mori</italic>

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein (egfp) gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part (up to 14 amino acid residues) of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide (mutant 13 aa), the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue(s) in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue(s). Furthermore, the deletion of Arg2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.

Cloning of a novel protease required for the molting of Locusta migratoria manilensis

Molting is required for progression between larval stages in the life cycle of an insect. The essence of insect molting is the laying down of new cuticle followed by shedding of the old cuticle. Degradation and recycling of old cuticle are brought about by enzymes present in the molting fluid, which fills the space between the old and new cuticle. Here, we describe the cloning of a novel protease gene from Locusta migratoria manilensis, designated as Lm-TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). Sequence analysis indicated that Lm-TSP belongs to the trypsin-like serine protease family. We show, by RNA interference (RNAi), that silencing of Lm-TSP leads to dramatic reductions in protease and cuticle-degrading activity of a molting fluid, which leads to molting defects from fourth-instar larvae (L4) to fifth-instar larvae (L5), and between L5 and adult stages. These observations suggest that Lm-TSP plays a critical role in L. migratoria manilensis ecdysis.

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2−3 days post-injection, followed by a decrease in expression until pupation, which occurred 2−4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.

Eicosanoids mediate melanotic nodulation reactions to viral infection in larvae of the parasitic wasp, Pimpla turionellae

Nodulation is the quantitatively predominant insect cellular immune function activated in response to bacterial, fungal and some viral infections. We posed the hypothesis that parasitoid insects express melanotic nodulation reactions to viral challenge and that eicosanoids mediate these reactions. Treating fifth-instar larvae of the ichneumonid endoparasitoid Pimpla turionellae with Bovine Herpes Simplex Virus-1 (BHSV-1) induced nodulation reactions in a challenge dose-dependent manner. Experimental larvae treated with the cyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitor, esculetin, and the phospholipase A 2 inhibitor, dexamethasone, resulted in severely impaired nodulation reactions to our standard BHSV-1 challenge dose. The immunoinhibitory influence of dexamethasone was reversed in larvae reared on culture medium amended with arachidonic acid, the fatty acid precursor of eicosanoid biosynthesis. Larvae that had been reared on media amended with indomethacin, esculetin, or dexamethasone were also compromised in their nodulation reactions to viral challenge. The influence of the orally administered pharmaceutical was expressed in a dose-dependent manner. Finally, wasp larvae reared in the presence of indomethacin and dexamethasone expressed significantly decreased levels of phenoloxidase activity in response to viral challenge. These findings draw attention to the idea that endoparasitoid insects express cellular immune reactions to viral challenge; they also support our hypothesis that eicosanoids mediate nodulation reactions to viral challenge in these highly specialized insects.

Synthesis and insecticidal activities of pyridine ring derivatives of podophyllotoxin

In an attempt to find the biorational pesticides, we synthesized 12 pyridinyl derivatives of podophyllotoxin (PPT) and 4′-demthylepipodophylltoxin (4′-DMEP) in this study. Their structures and the α/β substitution at C-4 were confirmed by 1H NMR, IR, MS spectral analyses and elemental analysis. The insecticidal activities were tested against fifth-instar larvae of Pieris rapae and the third-instar larvae of Cullex pipiens pallens at concentrations of 250 and 10 μg ml −1. Four derivatives of PPT, 4.1, 4.2, 4.3 and 4.5, showed higher insecticidal activities against P. rapae than PPT, while three derivatives of PPT, 4.4, 4.5 and 4.6, displayed higher mosquito larvicidal activity than PPT, with LC 50 values of 1.66, 3.96 and 1.54 mg l −1, respectively. Interestingly, we also found that the pyridine ring derivatives of PPT showed delayed insecticidal activity, which is different from traditional neurotic insecticides. The results suggest that 4′-OCH 3 in the PPT derivatives is essential to keep the insecticidal activity and the insecticidal activities of pyridine ring derivatives of PPT are higher than that of the derivatives of 4′-DMEP, supporting PPT has the potential to be a lead structure of semi-synthetic insecticides.

Industrial-scale radio frequency treatments for insect control in walnuts: II: Insect mortality and product quality

This paper reports on the second part of a scaling-up study investigating the technical feasibility of using radio frequency (RF) energy in commercial postharvest insect control in in-shell walnuts as an alternative to chemical fumigation. A large-scale treatment for conveyorized walnuts was designed based on a 25 kW, 27 MHz RF system to achieve an average walnut surface temperature of 60 °C, and minimum temperature of 52 °C, for 5 min. The treatment caused 100% mortality of fifth-instar navel orangeworm larvae, the most heat tolerant target pest, in both unwashed and air-dried walnuts, and was effective over a relatively wide range of walnut moisture contents (3–7.5%). Walnut quality was not affected by the RF treatments; kernel color, peroxide values and fatty acid values of treated walnuts were similar to untreated controls after 20 days at 35 °C simulating 2 years of storage under commercial conditions at 4 °C. The RF treatment slightly reduced the moisture content of the walnuts, especially the shells. The RF treatment developed in this study should also control codling moth, Indianmeal moth and red flour beetle in in-shell walnuts. This treatment will provide an effective and environmentally friendly phytosanitary treatment technology for the walnut industry.