Fifth-instar Larvae Research Articles

Effects of Added CeCl3 on Resistance of Fifth-Instar Larvae of Silkworm to Bombyx mori Nucleopolyhedrovirus Infection

One of the most important agents causing lethal disease in the silkworm is the Bombyx mori nucleopolyhedrovirus (BmNPV), while low-dose rare earths are demonstrated to increase immune capacity in animals. However, very little is known about the effects of added CeCl(3) on decreasing BmNPV infection of silkworm. The present study investigated the effects of added CeCl(3) to an artificial diet on resistance of fifth-instar larvae of silkworm to BmNPV infection. Our findings indicated that added CeCl(3) significantly decreased inhibition of growth and mortality of fifth-instar larvae caused by BmNPV infection. Furthermore, the added CeCl(3) obviously decreased lipid peroxidation level and accumulation of reactive oxygen species such as O(2)(-), H(2)O(2), (·)OH, and NO and increased activities of the antioxidant enzymes including superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, ascorbate, and glutathione contents in the BmNPV-infected fifth-instar larvae. In addition, the added CeCl(3) could significantly promote acetylcholine esterase activity and attenuate the activity of inducible nitric oxide synthase in the BmNPV-infected fifth-instar larvae. These findings suggested that added CeCl(3) may relieve oxidative damage and neurotoxicity of silkworm caused by BmNPV infection via increasing antioxidant capacity and acetylcholine esterase activity.

Design, semisynthesis and insecticidal activity of novel podophyllotoxin derivatives against Brontispa longissima in vivo

In an attempt to find the effective phytopesticides, a series of novel podophyllotoxin derivatives were firstly synthesized and preliminarily tested for their antifeedant and insecticidal effects against the fifth-instar larvae of Brontispa longissima. The different antifeedant and insecticidal activity ranges of compounds 3a-l showed that variations of NR 1R 2 groups in the 4-position of podophyllotoxin skeleton markedly affected the activity profiles of this compound class, and some important SAR information has been revealed from it. To clarify their mode of action of insecticidal activity, the docking models as well as tubulin inhibitory effect of representative compound 3i were also investigated, and the result indicated that the insecticidal activity of these compounds was due to the tubulin inhibitory effect of these derivatives, thereby possibly providing some useful information for rational designs of novel podophyllotoxin-based insecticides.

Podophyllotoxin-derived insecticidal agents: Part XIII – Evaluation of insecticidal activity of podophyllotoxin derivatives against Brontispa longissima

In an attempt to find the biorational insecticides for Brontispa longissima control, 12 podophyllotoxin (PPT) analogues were tested for their insecticidal activity against the fifth-instar larvae of B. longissima in vivo for the first time. Among all the tested compounds, especially compounds 6 and 8 showed more promising and pronounced insecticidal activity than toosendanin, a commercial insecticide derived from Melia azedarach. The different insecticidal activity range of compounds 1–12 indicated that the variation of chemical structures in the PPT skeleton markedly affected the activity profiles of this compound class, and some important structure–activity relationship information has been revealed. Together, these preliminary results may be useful in guiding further modification of PPTs in the development of potential new insecticides.

English

The effects of sublethal doses (LD10: 1.00 ng/larva; LD30: 3.75 ng/larva) of chlorfluazuron on the amounts of testicular biochemical constituents of the common cutworm, Spodoptera litura (F.) was described. Chlorfluazuron was applied topically to newly ecdysed fifth-instar larvae under laboratory conditions. Sublethal doses of chlorfluazuron significantly reduced the amount of protein with no effect on carbohydrate and lipid. The relative proportion of the amounts of controltesticular DNA was: 7.52±0.9 µg/mg at the day before adult emergence> 4.54±0.44 µg/mg at newly emerged adults> 3.52±0.49 µg/mg at 1st day after adult emergence.Sublethal doses of chlorfluazuron significantly (p < 0.05) reduced the amounts of testicular, seminal vesicular and aedeagular DNA compared with the controls.Similar reduction was observed in RNA as found in DNA. Three peaks in ecdysteroid titres were observed during 80 to 208 h. Compared with the controls, all peaks were reduced in LD10 or LD30 treated testis as follows: 11.5 or 21%; 12 or 22%; 12.5 or 23%, respectively. Sublethal doses of chlorfluazuron reduced the ecdysteroid titres with no effect on its pattern. Sublethal doses of chlorfluazuron reduced the amounts of biochemical testicular constituents during the development of testes in S. litura.   Key words: Chlorfluazuron, ecdysteroid titre, nucleic acid, protein, Spodoptera litura, sublethal doses, testicular biochemical, seminal vesicle, aedeagus.

Proteomic analysis of peritrophic membrane (PM) from the midgut of fifth-instar larvae, Bombyx mori

The insect peritrophic membrane (PM), separating midgut epithelium and intestinal contents, is protective lining for the epithelium and plays the important role in absorption of nutrients, and also is the first barrier to the pathogens ingested through oral feeding. In order to understand the biological function of silkworm larval PM, shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) approach was applied to investigate its protein composition. Total 47 proteins were identified, of which 51.1% of the proteins had the isoelectric point (pI) within the range of 5-7, and 53.2% had molecular weights within the range 15-45kDa. Most of them were found to be closely related to larval nutrients metabolism and innate immunity. Furthermore, these identified proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process and cell localization. Most of the proteins had catalytic activity, binding activity and transport function. The knowledge obtained from this study will favour us to well understand the role of larval PM in larval physiological activities, and also help us to find the potential target and design better biopesticides to control pest, particularly the Lepidoptera insect.

Effect of culture substrates on virulence of Beauveria bassiana (Ascomycota: Cordycipitaceae) conidia against the browntail moth, Euproctis chrysorrhoea (Lepidoptera: Lymantriidae)

We studied the effect of different solid substrates on virulence of two Beauveria bassiana isolates against the browntail moth, Euproctis chrysorrhoea (L.) (Lep.: Lymantriidae). Conidia produced on wheat grains, wheat flour, wheat bran, rice flour, rice bran, rice paddy, corn flour, millet, and Sabouraud's dextrose agar with 1% yeast extract (SDAY) as control were compared. There were significant differences among these substrates for their effects on the virulence of produced conidia. Applying 107 conidia/mL of B. bassiana EUT105, produced on rice bran caused the highest (84.9%) and on rice flour, the lowest (57.6%) mortalities. Bioassay on fifth-instar larvae using aerial conidia harvested from wheat grains, rice paddy, and SDAY indicated that conidia from wheat grains had the highest virulence while those from rice paddy, the lowest.

Effects of sublethal doses of chlorfluazuron on the biochemical constituents of eggs ofSpodoptera litura(Lepidoptera: Noctuidae)

AbstractThe effects of sublethal doses (LD10: 1.00 ng/larva; LD30: 3.75 ng/larva) of chlorfluazuron on the biochemical constituents of eggs of the tobacco cutworm,Spodoptera litura(F.), are described. Chlorfluazuron was applied topically to fifth-instar larvae and the subsequent adults were allowed to mate according to larval treatment (LD10-treated female × LD10-treated male and LD30-treated female × LD30-treated male). Biochemical constituents of eggs resulting from these pairings were analyzed at various stages of embryonic development. Compared with controls, LD10or LD30reduced egg constituents as follows: protein (min.–max.) by 32.8%–34.5% or 62.0%–67.3%, lipid by 33%–34% or 62%–67%, carbohydrates by 30%–39% or 60%–67%, DNA by 33%–40% or 60%–69%, RNA by 31%–34% or 59%–67%, and ecdysteroid by 22%–83% or 28%–92%, respectively. The relative proportions of constituents in control eggs were as follows: protein > lipid > carbohydrate, and RNA > DNA. Three low and three high peaks in ecdysteroid titres were observed. Compared with controls, all peaks were reduced in LD10or LD30eggs as follows: low peaks: 1st (at 8 h): 32% or 66%; 2nd (at 16 h): 33% or 67%; 3rd (at 52 h): 35% or 65%; high peaks: 1st (at 32 h): 83% or 92%; 2nd (at 64 h): 65% or 82%; 3rd (at 84 h): 84 h, 36% or 63%, respectively. In addition, the first two high peaks were delayed by 4 h in LD10eggs and by 8 h in LD30eggs compared with controls. Sublethal doses of chlorfluazuron reduced the amounts of biochemical constituents of eggs during embryogenesis inS. litura.

Isolation, purification, crystallization and preliminary X-ray studies of two 30 kDa proteins from silkworm haemolymph

Juvenile hormone-binding protein (JHBP) and the low-molecular-mass lipoprotein PBMHP-12 belong to a group of 30 kDa proteins that comprise the major protein component of the haemolymph specific to the fifth-instar larvae stage of the mulberry silkworm Bombyx mori L. Proteins from this group areoften essential for the development of the insect. In a project aimed at crystallographic characterization of B. mori JHBP (BmJHBP), it was copurified together with PBMHP-12. Eventually, the two proteins were isolated and crystallized separately. The BmJHBP crystals were orthorhombic (space group C222(1)) and the PBMHP-12 crystals were triclinic. The crystals diffracted X-rays to 2.9 Å (BmJHBP) and 1.3 Å (PBMHP-12) resolution.

Effect of Organophosphate Phoxim Exposure on Certain Oxidative Stress Biomarkers in the Silkworm

Organophosphorus (OP) insecticides are widely used in agriculture, which are toxic to insect pests and nontarget organisms. The current study mainly assessed the effect of the pesticide phoxim on oxidative stress by certain biomarkers in the fat body and midgut of the silkworm, Bombyx mori (L.), after exposure to 50% lethal concentration (LC50) of phoxim for 2 h. Malondialdehyde (MDA) content, activity of glutathione transferase (GST), and expression of GST at transcriptional level were assayed. LC50 value of phoxim was 2.5 mg/liter at 2-h exposure for the day 3 of the fifth-instar larvae. After exposure of phoxim, MDA content in the fat body significantly increased at 4-20 h posttreatment (p.t.),the highest increase was approximately 4.11-fold from 0.451 +/- 0.053 to 1.854 +/- 0.113 nmol/mg protein compared with corresponding control. In the midgut, significant increase in the MDA content (from 1.40- to 3.16-fold) was observed at 8-42 h p.t. The activity of GSTs increased to 1.48-2.00-fold at 24-42 h p.t. and 1.33-1.48-fold at 20-24 h p.t. in the fat body and midgut, respectively. The peroxidase activity of GSTs also was induced, which increased to 1.46-2.06-fold and 1.31-1.50-fold in the fat body and midgut, respectively. BmGSTe8 showed a late up-regulation of transcripts at 24-42 h after exposure to phoxim, which might contribute to the improved phoxim tolerance of silkworm larvae. These results indicated that phoxim could trigger oxidative stress and that MDA content and GST activity might be used as biomarkers of OP insecticide exposure. In addition, activity of GSTs were more inducible in the fat body than in midgut.

Khayasin and 2'S-methylbutanoylproceranolide: Promising candidate insecticides for the control of the coconut leaf beetle, Brontispa longissima

Brontispa longissima (Gestro) is a serious pest of coconut palms in tropical areas. In our continuing search for potential pesticides from tropical mangrove plants, khayasin (1) and 2′S-methylbutanoylproceranolide (2), two mexicanolides with a Δ8,14 double bond, were isolated from seeds of an Indian mangrove, Xylocarpus moluccensis. The structures of these compounds were established by comparison with data in the literature. Compound 1 was found to exhibit potent insecticidal activities against second- to fifth-instar larvae of B. longissima at a concentration of 50 μg/ml, and compound 2 displayed marked insecticidal activity against fifth-instar larvae of the same pest at the same concentration. LC50 (concentration of 50% mortality) values for compound 1 against fifth-instar larvae of B. longissima at exposure times of 24 and 48 h are 7.27 and 3.39 μg/ml, respectively, and those for compound 2 are 10.57 and 4.03 μg/mL, respectively. The insecticidal activities of compounds 1 and 2 against fifth-instar larvae of B. longissima are more potent than those of azadirachtin and toosendanin, and are similar to those of rotenone; however, compounds 1 and 2 only exhibited moderate antifeedant activities against third-instar larvae of Prodenia litura (Fabricius). These results suggested that compounds 1 and 2 had selectively insecticidal activities against the larvae of B. longissima. They are promising candidate insecticides for the control of B. longissima.

Cloning and expression pattern of 3‐dehydroecdysone 3β‐reductase (3DE 3β‐reductase) from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, reproduction, etc. Ecdysone was inactivated to 3-dehydroecdysone (3DE) under ecdysone oxidase (EO), and followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid under 3DE 3a-reductase. On the other hand, 3-dehydroecdysone undergoes reversible reduction to ecdysone by 3DE 3β-reductase in the hemolymph. In this article, we cloned and characterized 3-dehydroecdysone 3β-reductase (3DE 3β-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L. The B. mori 3DE 3β-reductase cDNA contains an ORF 972 bp and the deduced protein sequence containing 323 amino acid residues. Analysis showed that the deduced 3DE 3β-reductase belongs to the aldo-keto reductase (AKR) superfamily, which has the NAD(P)-binding domain, indicating that the function of 3DE 3β-reductase depends on the existence of NAD(P)H. Using Escherichia coli, a high level expression of a fusion polypeptide band of approx. 40 kDa was observed. The high transcription of 3DE 3β-reductase was mainly observed in the genitalia and fatty bodies in the third day of the fifth-instar larvae, followed next in the head, epidermis, and hemocytes. The expression of 3DE 3β-reductase in the early of every instar was lower than that in the late of instar. When the titer of 3DE is low, higher expression of 3DE 3β-reductase is necessary to maintain the ecdysone titer in body through converting 3DE to ecdysone, while the 3DE titer is high, the expression of 3DE 3β-reductase showed feedback inhibition.

Response of the gypsy moth, Lymantria dispar to transgenic poplar, Populus simonii x P. nigra, expressing fusion protein gene of the spider insecticidal peptide and Bt-toxin C-peptide.

The response of the Asian gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) to a fusion gene consisting of the spider, Atrax robustus Simon (Araneae: Hexanthelidae) ω?-ACTX-Ar1 sequence coding for an ω?-atracotoxin and a sequence coding for the Bt-toxin C-peptide, expressed in transgenic poplar Populus simonii x P. nigra L. (Malphigiales: Salicaceae) was investigated. Individual performance, feeding selection, midgut proteinase activity and nutrition utilization were monitored. The growth and development of L. dispar were significantly affected by continually feeding on the transgenic poplar, with the larval instars displaying significantly shorter developmental times than those fed on nontransgenic poplar, but pupation was delayed. Mortality was higher in populations fed transgenic poplar leaves, than for larvae fed nontransgenic poplar leaves. The cumulative mortality during all stages of larvae fed transgenic leaves was 92% compared to 16.7% of larvae on nontransgenic leaves. The highest mortality observed was 71.7% in the last larval instar stage. A two-choice test showed that fifth-instar larvae preferred to feed on nontransgenic leaves at a ratio of 1:1.4. Feeding on transgenic leaves had highly significant negative effects on relative growth of larvae, and the efficiency of conversion of ingested and digested food. Activity of major midgut proteinases was measured using substrates TAME and BTEE showed significant increases in tryptase and chymotrypsinlike activity (9.2- and 9.0-fold, respectively) in fifth-instar larvae fed on transgenic leaves over control. These results suggest transgenic poplar is resistant to L. dispar, and the mature L. dispar may be weakened by the transgenic plants due to Bt protoxins activated by elevated major midgut proteinase activity. The new transgenic poplar expressing fusion protein genes of Bt and a new spider insecticidal peptide are good candidates for managing gypsy moth.

Evaluation of insecticidal activity of podophyllotoxin derivatives against Brontispa longissima

Twenty podophyllotoxin analogues were first tested for their insecticidal activity against the fifth-instar larvae of Brontispa longissima in vivo. Among them, compounds 6– 9 and 19 showed more promising and pronounced insecticidal activity than toosendanin, a commercial insecticide derived from Melia azedarach. The different insecticidal activity ranges of compounds 1– 20 indicated that variation of chemical structures in the podophyllotoxin skeleton markedly affected the activity profiles of this compound class, and some important SAR information has been revealed from it. The results obtained from SAR analysis show good correlation with the docking models as well as with QSAR studies, which allows for the rational design of more potent podophyllotoxin derivatives in the development of potential new insecticides.

Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda.

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.

Molecular characterization, immunohistochemical localization and expression of a ribosomal protein L17 gene from Apis cerana cerana

Ribosomal protein L17 (RPL17) is a core protein of the large (60S) ribosomal subunit and plays an important role in protein synthesis. In this report, a RPL17 gene was isolated from Apis cerana cerana, designated as AccRPL17. Alignment analysis showed that AccRPL17 exhibits high homology to other known RPL17s. Moreover, genomic sequence analysis revealed that five exons are splitted by four introns, and the position of the first intron is comparatively conservative, being localized in the 5' untranslated region. Partial putative cis-acting elements related to development were also examined. Quantitative real-time PCR showed that the highest mRNA level was detected in larvae on the fifth day. Simultaneously, immunohistochemical localization showed that AccRPL17 is primarily concentrated in muscular tissues, stigma, body wall, and the surrounding of the eye in the fifth-instar larvae. Further studies suggested that AccRPL17 might be involved in responses to abiotic stresses. This is a report attempting to analyze the expression and distribution of RPL17 in A. cerana cerana. These results indicated that AccRPL17 might play an important role in insect development, and the importance of AccRPL17 in participating in abiotic stresses is discussed.

Caracterização bromatológica de cultivares de amoreira, Morus spp., e determinação dos índices nutricionais de Bombyx mori L. (Lepidoptera: Bombycidae)

The objective of this work was to assess, through consumption and utilization of natural food measurements, whether mulberry cultivars, Morus spp., could be recommended to rear the silkworm, Bombyx mori L., in a commercial scale. The mulberry cultivars Miura (standard), Korin and Tailandesa and the hybrids FM 3/3, FM 86, SK 1 and SK 4 were tested. Seventy five fifth-instar commercial hybrid larvae were individualized in gerbox® unities and maintained in a rearing room (25 ± 3ºC and 80 ± 10% RH).The mulberry leaves of each of the five cultivars used to feed the silkworm larvae were submitted to bromatological analysis. The dry weight of larvae at the beginning and at the end of the fifth instar, the food consumed and the feces eliminated were recorded to determine the following indexes: relative consumption rate (RCR), relative metabolic rate (RMR), relative growth rate (RGR), approximate digestibility (AD), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), metabolic cost (MC). The bromatological composition analysis of mulberry leaf cultivars revealed that the hybrid SK 4 presented superior nutritional quality compared to the standard cultivar Miura, due to its higher content in crude protein and ethereal extract, and lower detergent fiber content. The hybrid SK 4 was the most adequate food to the silkworm larvae because it was ingested in small amounts, thus providing good digestibility to the larvae, low metabolic cost, good growth rate and one of the highest efficiencies in the conversion of the ingested food and ingested and digested in biomass.

Podophyllotoxin Derivatives Show Activity against Brontispa Longissima Larvae

In an attempt to find biorational insecticides, eleven podophyllotoxin analogues were tested for their insecticidal activity against the fifth-instar larvae of Brontispa longissima in vivo for the first time. Among all of the tested compounds, deoxypodophyllotoxin (3) and beta-apopicropodophyllin (4) showed more promising and pronounced insecticidal activity than toosendanin, a commercial insecticide derived from Melia toosendan, and important SAR information has been revealed. Together, these preliminary results may be useful in guiding further modification of podophyllotoxins in the development of potential new insecticides.

Visceral-Locomotory Pistoning in Crawling Caterpillars

Animals with an open coelom do not fully constrain internal tissues, and changes in tissue or organ position during body movements cannot be readily discerned from outside of the body. This complicates modeling of soft-bodied locomotion, because it obscures potentially important changes in the center of mass as a result of internal tissue movements. We used phase-contrast synchrotron X-ray imaging and transmission light microscopy to directly visualize internal soft-tissue movements in freely crawling caterpillars. Here we report a novel visceral-locomotory piston in crawling Manduca sexta larvae, in which the gut slides forward in advance of surrounding tissues. The initiation of gut sliding is synchronous with the start of the terminal prolegs' swing phase, suggesting that the animal's center of mass advances forward during the midabdominal prolegs' stance phase and is therefore decoupled from visible translations of the body. Based on synchrotron X-ray data and transmission light microscopy results, we present evidence for a two-body mechanical system with a nonlinear elastic gut that changes size and translates between the anterior and posterior of the animal. The proposed two-body system--the container and the contained--is unlike any form of legged locomotion previously reported and represents a new feature in our emerging understanding of crawling.

Changes in the free amino acid composition of the hemolymph from the fifth-instar larvae, Bombyx mori, infected with Beauveria bassiana

Changes in the free amino acid composition of the hemolymph from the fifth-instar larvae, Bombyx mori, infected with Beauveria bassiana

Digestibility of defense proteins in latex of milkweeds by digestive proteases of Monarch butterflies, Danaus plexippus L.: A potential determinant of plant–herbivore interactions

This study describes the digestive protease activity extracted from the gut of fifth-instar Monarch butterfly larvae, and its proteolytic activity on latex proteins of their host plant, Calotropis procera (the milkweed) and related non-host species from the milkweed family. Gut extracts digested azocasein, BANA and BApNA. Cysteine protease inhibitors such as E-64 and iodoacetamide inhibited proteolytic activity on azocasein; however, the serine protease inhibitors PMSF and leupeptin were more effective. Gut extracts promptly digested LP and were not affected by endogenous latex proteases. Gut extracts, however, did not digest LP from Cryptostegia grandiflora and only slightly digested LP from Plumeria rubra, two plant species that are not consumed by Monarch larvae. The protein profiles of latex proteins extracted from healthy and attacked plants were different. A protein identified as glycoside hydrolase was detected in increased concentrations in latex from damaged plants. Larvae fed on artificial diets containing 1% or 5% latex proteins were not adversely affected and gained weight faster than control larvae. These results provide new information on the resistance of Monarch larvae fed on C. procera and suggest that the ability of Monarch proteolytic enzymes to promptly digest LP can explain (at least in part) how these insects overcome the defensive proteins found in C. procera latex.