scholarly journals Immunological analysis of developmental changes in ecdysone 20-mono-oxygenase expression in the cotton leafworm, Spodoptera littoralis

1994 ◽  
Vol 299 (3) ◽  
pp. 711-717 ◽  
Author(s):  
J H Chen ◽  
T Hara ◽  
M J Fisher ◽  
H H Rees
Keyword(s):  
Spodoptera Littoralis ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Close Correlation ◽  
Developmental Changes ◽  
Cotton Leafworm ◽  
Oxygenase Activity ◽  
Adrenodoxin Reductase ◽  
Cytochrome P 450

The developmental changes in ecdysone 20-mono-oxygenase during the sixth larval instar of the cotton leafworm, Spodoptera littoralis, were investigated. The specific activity of mitochondrial ecdysone 20-mono-oxygenase in the fat-body exhibited a distinct peak at 72 h, at which time the larvae stop feeding. Immunoblot analyses, using antibodies raised against components of vertebrate mitochondrial steroidogenic enzyme systems [anti-(cytochrome P-450scc), anti-(cytochrome P-450(11) beta), anti-adrenodoxin and anti-(adrenodoxin reductase) antibodies], revealed the presence of specific immunoreactive polypeptides in fat-body mitochondrial extracts. In addition, these antibodies effectively inhibited fat-body mitochondrial ecdysone 20-mono-oxygenase activity. This suggests that the S. littoralis steroid-hydroxylating system(s) may contain polypeptide components analogous to those present in vertebrates. A close correlation between developmental changes in mitochondrial ecdysone 20-mono-oxygenase activity and the abundance of polypeptides (approx. 66 kDa and 50 kDa) recognized by the anti-(cytochrome P-450(11) beta) antibody and a polypeptide (approx. 52 kDa) recognized by the anti-(adrenodoxin reductase) antibody were observed in both fat-body and midgut. These results suggest that developmental changes in the abundance of components of the ecdysone 20-mono-oxygenase system may play an important role in the developmental regulation of the enzyme expression and, hence, of 20-hydroxyecdysone titre.

scholarly journals EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm)

1995 ◽  
Vol 307 (3) ◽  
pp. 719-728 ◽  
Author(s):  
J K Shergill ◽  
R Cammack ◽  
J H Chen ◽  
M J Fisher ◽  
S Madden ◽  
...  
Keyword(s):  
High Spin ◽  
Spodoptera Littoralis ◽  
Fat Body ◽  
Spectroscopic Characterization ◽  
Mitochondrial Fraction ◽  
Complex Iii ◽  
Cotton Leafworm ◽  
Steroid Hydroxylase ◽  
High Concentrations ◽  
Cytochrome P 450

EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Purification and characterization of fat body lipase from the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Author(s):  
Rahma R.Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M.S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

AbstractLipid mobilization and transport in insects is under investigation, especially lipases and lipophorin because of their roles in energy production and transport of lipids at flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from last larval instar of Galleria mellonella. Purification methods by combination of ammonium sulfate precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633±0.000551 mg/ml and 1.5754±0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore biochemical characterization of fat body lipase was carried out through testing its activities against several factors such as; different temperatures, pH ranges, metal ions and inhibitors ending by determination of their kinetic parameters with the use of p-Nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35-37°C and 37-40°C and pH ranges of 7-9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+ and Na+ metal ions indicating that fat body lipase is metalloproteinase. Additionally, lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfony fluoride (PMSF), ethylene-diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) providing an evidence of presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 301.95mM Km and 0.316 Umg−1 Vmax. By considering the purification of fat body lipase from larvae and using some inhibitors especially ion chelating agents, it is suggested to develop this study by using lipase inhibitors to reach a successful control of Galleria mellonella in the near future.

scholarly journals Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

2004 ◽  
Vol 382 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Hajime TAKEUCHI ◽  
Jian-Hua CHEN ◽  
John R. JENKINS ◽  
Masanori BUN-YA ◽  
Philip C. TURNER ◽  
...  
Keyword(s):  
Spodoptera Littoralis ◽  
De Novo ◽  
Larval Instar ◽  
Constitutive Expression ◽  
Northern Blotting ◽  
Malpighian Tubules ◽  
Carrier Protein ◽  
Sterol Carrier Protein ◽  
Cotton Leafworm ◽  
Sterol Carrier Protein 2

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.9 kb), which encodes a 57 kDa protein. Northern blotting detected two mRNA transcripts, one of 1.9 kb, encoding SCPx, and one of 0.95 kb, presumably encoding SCP2 (sterol carrier protein 2). The former mRNA was highly expressed in midgut and Malpighian tubules during the last larval instar. Furthermore, constitutive expression of the gene was detected in the prothoracic glands, which are the main tissue producing the insect moulting hormone. There was no significant change in the 1.9 kb mRNA in midgut throughout development, but slightly higher expression in the early stages. Conceptual translation of the cDNA and a database search revealed that the gene includes the SCP2 sequence and a putative peroxisomal targeting signal in the C-terminal region. Also a cysteine residue at the putative active site for the 3-oxoacyl-CoA thiolase is conserved. Southern blotting showed that SCPx is likely to be encoded by a single-copy gene. The mRNA expression pattern and the gene structure suggest that SCPx from S. littoralis (a lepidopteran) is evolutionarily closer to that of mammals than to that of dipterans.

scholarly journals 3′-phosphoadenosine-5′-phosphosulphate synthesis and involvement in sulphotransferase reactions in the insect, Spodoptera littoralis

1982 ◽  
Vol 204 (1) ◽  
pp. 127-133 ◽  
Author(s):  
R E Isaac ◽  
K K Phua ◽  
H H Rees
Keyword(s):  
Spodoptera Littoralis ◽  
Fat Body ◽  
Specific Activity ◽  
Enzyme System ◽  
Instar Larva ◽  
Malpighian Tubules ◽  
Prepupal Stage ◽  
Cytosol Fraction ◽  
Rate Limiting ◽  
Generating System

1. Synthesis of 3′-phosphoadenosine-5′-phosphosulphate from ATP and 35SO4(-2) was demonstrated by homogenates of gut. Malpighian tubules and fat body of Spodoptera littoralis. 2. The enzyme system was most active in the gut tissue, and was primarily located in the cytosol fraction of the cell. Gut cytosol preparations were used as a source of the 3′-phosphoadenosine-5′-phosphosulphate generating system for more detailed studies. 3. Maximum synthesis required an incubation mixture containing Tris/HCl buffer (pH 7.5), ATP (20 mM), MgCl2 (13.0 mM) and K2SO4 (3 mM). 4. The specific activity of 3′-phosphoadenosine-5′-phosphosulphate synthesizing activity in gut cytosol increased during development of the sixth instar larva, reaching a peak at day 4. A sudden fall in specific activity was observed in the prepupal stage. 5. 3′-Phosphoadenosine-5′-phosphosulphate formation is the rate limiting process in the overall sulphation of p-nitrophenol in the gut cytosol preparations from S. littoralis. 6. It is concluded that the properties of the sulphate-activating system in this insect are similar to those reported for vertebrates.

scholarly journals Enzymes of ecdysteroid transformation and inactivation in the midgut of the cotton leafworm, Spodoptera littoralis: properties and developmental profiles

1995 ◽  
Vol 312 (2) ◽  
pp. 561-568 ◽  
Author(s):  
T J Webb ◽  
R Powls ◽  
H H Rees
Keyword(s):  
Spodoptera Littoralis ◽  
Larval Instar ◽  
Physiological Significance ◽  
Peak Activity ◽  
Cotton Leafworm ◽  
Clear Preference ◽  
Prothoracic Glands ◽  
Developmental Profiles

In the midgut cytosol of Lepidoptera, ecdysteroids undergo inactivation by transformation via the 3-dehydro derivative to the corresponding 3-epiecdysteroid (3 alpha-hydroxy) and by phosphate conjugation. The oxygen-dependent oxidase catalyses formation of 3-dehydroecdysteroid, which can be reduced either irreversibly by 3-dehydroecdysone 3 alpha-reductase to 3-epiecdysteroid, or by 3-dehydroecdysone 3 beta-reductase back to the initial ecdysteroid. Furthermore, these ecdysteroids undergo further inactivation by phosphorylation. These ecdysteroid transformations have been investigated in last instar larvae of the cotton leafworm, Spodoptera littoralis. The products of the phosphorylation have been characterized as predominantly ecdysteroid 2-phosphate accompanied by smaller amounts of the corresponding 22-phosphate. The phosphotransferases require Mg2+ and ATP. Whereas the 3-dehydroecdysone 3 alpha-reductase has a clear preference for NADPH rather than NADH, the corresponding 3 beta-reductase markedly favours NADH. The physiological significance of the latter enzyme is unclear. The profiles of the various enzymic activities in dialysed midgut cytosol supplemented with appropriate cofactors were determined throughout the last larval instar. All activities were detectable throughout the instar, but the respective enzymes exhibited maxima at different times. Ecdysone oxidase showed a peak early in the instar, with 3-dehydroecdysone 3 alpha-reductase increasing to a peak as the former activity declined. The 3-dehydroecdysone 3 beta-reductase exhibited peak activity late in the instar, a profile similar to that observed for the corresponding haemolymph enzyme involved in reduction of the 3-dehydroecdysone product of the prothoracic glands to ecdysone. Thus, the significance of the midgut 3 beta-reductase may be related to production of active hormone. Both ecydsteroid 22- and 2-phosphotransferases showed high activities early in the instar and then declined. The physiological significance of the profiles for the ecdysone oxidase, the 3-dehydroecdysone 3 alpha-reductase and phosphotransferases is unclear.

scholarly journals Induction of an inactivation pathway for ecdysteroids in larvae of the cotton leafworm, Spodoptera littoralis

1994 ◽  
Vol 301 (1) ◽  
pp. 89-95 ◽  
Author(s):  
J H Chen ◽  
M Kabbouh ◽  
M J Fisher ◽  
H H Rees
Keyword(s):  
Spodoptera Littoralis ◽  
Actinomycin D ◽  
Hydroxylase Activity ◽  
Free System ◽  
Cotton Leafworm ◽  
Oxygenase Activity ◽  
Rna And Protein Synthesis ◽  
A Cell ◽  
Moulting Hormones ◽  
Aldehyde Derivative

Treatment of the last-instar larvae of the cotton leafworm (Spodoptera littoralis) with ecdysteroids (moulting hormones) results in the induction of an ecdysteroid-inactivation pathway. Administration of ecdysone, 20-hydroxyecdysone or an ecdysteroid agonist, RH 5849, leads to induction of an ecdysteroid 26-hydroxylase activity. This induction occurred in both early sixth-instar larvae and in older larvae which had been head-ligated to prevent the normal developmental increase in ecdysone 20-mono-oxygenase activity. The induction of 26-hydroxylase activity requires both RNA and protein synthesis, as demonstrated by experiments involving actinomycin D and cycloheximide. The 26-aldehyde derivative of ecdysone and ecdyson-26-oic acid were also formed from ecdysone in the RH 5849-induced systems. Formation of the aldehyde and the corresponding 26-oic acid (ecdysonoic acid) from 26-hydroxyecdysone was directly demonstrated in a cell-free system, thus establishing the following inactivation pathway: Ecdysteroid-->26-hydroxyecdysteroid-->ecdysteroid 26-aldehyde-->ecdysteroid 26-oic acid.

scholarly journals Sublethal effects of emamectin benzoate on life table parameters of the cotton leafworm, Spodoptera littoralis (Boisd.)

2020 ◽  
Vol 44 (1) ◽  
Author(s):  
El-Sayed Mokbel ◽  
Amal Huesien
Keyword(s):  
Life Table ◽  
Spodoptera Littoralis ◽  
Sublethal Effects ◽  
Lethal Effect ◽  
Reproductive Rate ◽  
Relative Fitness ◽  
Emamectin Benzoate ◽  
Cotton Leafworm ◽  
Sublethal Concentrations ◽  
Preovipositional Period

Abstract Background The cotton leafworm, Spodoptera littoralis (Boisd.), is a serious economic pest in Egypt. Pest control depends mainly on chemical control with several pesticides include conventional and modern insecticides. Comprehensive analysis of pesticides impacts needs to investigate sublethal effects in addition to lethal effect. Results In the current study, the leaf-dip bioassay method was used to evaluate emamectin benzoate (EMB) sublethal concentrations. Results showed that EMB proved high toxicity against S. littorals with LC50 value of 0.019 mg liter−1. Life table analysis showed that treatments with LC5 and LC15 prolonged larval period, mean longevity of males and females, mean generation time (T), doubling time (DT), adult preovipositional period (APOP), and total preovipositional period (TPOP) compared with control. On the contrary, net reproduction rates (R0), intrinsic rates of increase (r), finite rate (λ), fecundity, gross reproductive rate (GRR), and relative fitness were decreased compared to control. Conclusions The current study clarified that sublethal concentrations of EMB induce adverse effects and suppress the population growth of S. littorals. Our results would be useful to assess the overall effects of EMB on S. littorals and can contribute effectively in pest management.

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