Ferritin acts as a target site for the snowdrop lectin （GNA） in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA （Galanthus nivalis agglutinin） has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor（s） responsible for binding of GNA in the larval midgut of a major lepidopteran pest （the cotton leafworm, Spodoptera littoralis） to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA-binding proteins and BLAST analyses revealed that the N-terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta （76% sequence identity）, Calpodes ethlius （80% sequence identity） and Bombyx mori （61% sequence identity）. Furthermore, the N-terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta （88% sequence identity）.     

Susceptibility of Spodoptera littoralis caterpillars to entomopathogenic nematode Steinernema feltiae after consumption of non-specific transgenic plants

Spodoptera littoralis caterpillars were transferred from an artificial diet to potato leaves at the start of the third or fifth instar and exposed to the infective juveniles of the nematode Steinernema feltiae since the beginning of the sixth instar until the start of pupation. Leaves were taken from the control potatoes or from genetically modified potatoes expressing either Cry3Aa toxin of Bacillus thuringiensis (Bt) or Galanthus nivalis agglutinin (GNA) which are mainly non-specific to S. littoralis larvae. The nematodes killed all the caterpillars within seven days compared with the starved larvae in the same period of exposure. The average time to death and the number of nematodes successfully invaded the larvae were affected by the period of feeding on potato leaves. In the non-starved caterpillars, which received potato leaves throughout the whole period of exposure to the nematodes, the type of potato leaves had no effect on the number of nematodes inside cadavers (p = 0.352 and F = 1.070) and also on the effect on the length of survival after exposure to the nematodes (p = 0.7892 and F = 0.596). No hazardous effect on the development and survival of entomopathogenic nematode S. feltiae which successfully invaded larvae fed on modified potato (Bt or GNA) was reported.     

Distribution and metabolism of exogenous ecdysteroids in the egyptian cotton leafworm Spodoptera littoralis (lepidoptera: noctuidae)

After ingestion of various amounts of either [³H]ecdysone or [³H]20-hydroxyecdysone (0.8 ng to 10 μg) by sixth instar larvae of the Egyptian cotton leafworm Spodoptera littoralis, apolar metabolites are rapidly detected in the gut and frass. Hydrolysis of the apolar products with Helix hydrolases releases solely [³H]ecdysone or [³H]20-hydroxyecdysone, respectively. This, coupled with the formation of chemical derivatives (acetonide and acetate) which cochromatograph with authentic reference compounds on hptlc and hplc demonstrates that these apolar metabolites consist of ecdysone or 20-hydroxyecdysone esterified at C-22 with common long-chain fatty acids. The major fatty acids have been identified by RP-hplc and their contribution to the mixture determined. In contrast, [³H]ecdysone injected into the haemolymph of S. littoralis is metabolized to yield 20-hydroxyecdysone, ecdysonoic acid, and 20-hydroxyecdysonoic acid. Thus, two different pathways exist for the metabolism of ecdysteroids in this species. In addition to an essentially polar pathway operating on injected and endogenous ecdysteroids, exogenous ecdysteroids entering the gut of S. littoralis are detoxified, yielding apolar ecdysteroid 22-fatty acyl esters which are rapidly excreted. The significance of these results in relation to the effects of ingested ecdysteroids on S. littoralis is discussed. Arch. Insect Biochem. Physiol. 34:329–346, 1997. © 1997 Wiley-Liss, Inc.     

The snowdrop lectin Galanthus nivalis agglutinin (GNA) and a fusion protein ButaIT/GNA have a differential affect on a pest noctuid Lacanobia oleracea and the ectoparasitoid Eulophus pennicornis

Fusion proteins have considerable potential as novel insect control agents because they enable the oral delivery of insecticidal peptides to the haemolymph of pests. Transport is achieved via fusion of the toxin to a carrier protein Galanthus nivalis agglutinin (GNA) that, after ingestion, binds to and crosses the insect gut epithelia. A fusion protein comprising a toxin from the South Indian red scorpion (Mesobuthus tamulus) that is fused to a GNA polypeptide (ButaIT/GNA) has a detrimental effect on the development of tomato moth Lacanobia oleracea (L.) (Lepidoptera: Noctuidae) larvae. The present study examines the effects of ButaIT/GNA and GNA, delivered orally or by injection, on the development of L. oleracea larvae, and the subsequent effects on the gregarious ectoparasitoid Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae) developing on ButaIT/GNA‐ and GNA‐treated hosts. The fusion protein, but not GNA, reduces the growth of fifth stadium L. oleracea larvae. The development of E. pennicornis is not affected by the presence of ButaIT/GNA in hosts that ingest the protein, although it is affected when hosts are injected with the protein. This difference is considered to be a result of higher levels of fusion protein being present when the fusion protein is injected. Intact ButaIT/GNA is detected by immunoassay in the haemolymph of L. oleracea larvae after ingestion of the fusion protein. More unexpectedly, negative effects are observed for the growth of E. pennicornis larvae developing on hosts that have either ingested, or been injected with GNA.     

Effect of constant light on male sterility in the cotton leafworm Spodoptera littoralis

Females of the cotton leafworm (Spodoptera littoralis) reared in long day conditions (LD 16:8 h) and mated to males kept throughout the whole period of development in continuous light (LL) oviposit very small numbers of mostly sterile eggs. It was found that in control males reared from the first larval instar in long day conditions there was a large accumulation of euperene sperm bundles in their testes on day 1 after imaginal moult. On day 10 of adult life the number of the sperm bundles was very small. In males kept from the first instar in continuous light there was also high number of sperm bundles on day 1 after imaginal moult but it did not decrease significantly on day 10 as was observed in controls. Transfer of different developmental stages of S. littoralis from long day conditions to continuous light resulted in a big difference in the density of eupyrene sperm bundles in their testes. In control insects reared through the whole of their development in long day conditions there was a significant decrease in the density of eupyrene sperm bundles on day 10 of adult life. By contrast, in males in continuous light, regardless of their developmental stage when transferred, there was either no change in density of sperm bundles in day 10 adults or there was a significant increase in comparison with day 1 adults. The highest density of eupyrene sperm bundles was observed in day 10 adults when they were transferred to continuous light shortly before moulting to the last instar (as day 4 larvae in the last stadium or day 1 pupae). Generally, the density of eupyrene sperm bundles on day 10 of adult development was about 2–2.5 times higher in males in continuous light then those under long day conditions. The results presented here indicate that the last larval instar and the pupa are the stages most sensitive to constant light treatment, which greatly reduces the amount of eupyrene sperm bundles released from the testes.     

Potency of Bacillus thuringiensis and Bacillus subtilis against the cotton leafworm,Spodoptera littoralis (Bosid.) Larvae

The biological activities of two species of bacteria isolated from soil of cotton fields identified as Bacillus subtilis strain NRC313 (BS NRC313) and Bacillus thuringiensis strain NRC335 (BT NRC335) were evaluated against the third larval instar of the cotton leafworm, Spodoptera littoralis (Boisd.). The different entomopathogenic bacteria of BS NRC313and BT NRC335 contained 10 × 10⁸ cell/ml, and caused mortality of 100 and 97.3% for the above mentioned strains, respectively. Concentrations of 2.5 × 10⁸ to 10 × 10⁸ cell/ml of strains BS NRC313 and BT NRC335 were applied to the larvae: LC₅₀ were 3.3 × 10⁸ and 3.9 × 10⁸ cell/ml respectively. The influence of exposure to toxin concentrations manifested in terms of decreasing the adult emergence and prolongation of the generation period. The percentage of larvae that survived and succeeded to pupate increased by decreasing the concentration. The longevity of adult emergence that resulted from larvae treated with Bacillus subtilis were 6.0 ± 0.51 and 9.0 ± 0.63 days at 5 × 10⁸ and 2.5 × 10⁸ cell/ml, respectively compared with 9.8 ± 0.47 in control. The results indicated that Bacillus subtilis was more potent than Bacillus thuringiensis. Field applications of B. thuringiensis, B. subtilis and Reldan achieved 55.6, 67.4 and 89.4% reduction of the cotton leafworm larvae Spodoptera littoralis in clover plants under field conditions.     

Some physiological,biochemical and histopathological effects of Artemisia monosperma against the cotton leafworm,Spodoptera littoralis

The effect of Artemisia monosperma wild plant hexane extract on some biological and physiological aspects of Spodoptera littoralis, as well as some biochemical and histopathological changes after treatment with this extract have been studied. The results indicated that treatment with larvae for two days revealed satisfactory effects in larval mortality (50%) and in larval weight and prolonged the effect in the pupal stage. Also the treatment reduced the food consumption and the amount of faeces of the tested larvae which led to a significant reduction in the final body weight with 76.4%. Treatment also extended the time of food passage through the midgut to 3.2 h compared to 1.38 h in control, and the larvae had a smaller midgut. The enzymatic activity of the digestive enzymes amylase and invertase revealed a significant inhibition in the treated larvae. Also transaminase enzyme activity (AST and ALT) reduced after treatment. The results of phosphatase enzyme activity (ACP and ALP) showed a potent inhibitory effect on both enzymes which more was pronounced in ALP than ACP activity, the percent inhibition being 73.8% and 30.6%, respectively. The histopathological effects of A. monosperma treatment on the midgut of Spodoptera littoralis revealed that the epithelium membrane was exfoliated and completely destroyed. Meanwhile, epithelia cells were strongly vacuolated and considerably elongated. Boundary lines between the cells disappeared.     

The analysis of flight paths of male Egyptian cotton leafworm moths, Spodoptera littoralis, to a sex pheromone source in the field

ABSTRACT. The flight of male Spodoptera littoralis (Boisd.) (Noctuidae) towards a pheromone source was recorded during the early part of the night using a cine camera and an image intensifier. The cine films were analysed frame by frame to produce flight tracks from which it was possible to calculate the mean advance rate of moths towards the pheromone source and their projected ground speed, for a series of positions downwind of the source. As wind speed was measured the moth's air speed was also estimated. The moths compensated for changes in wind speed by varying their air speed, hence maintaining a ground speed independent of wind speed. The ground speed itself was found to decrease as moths flew closer to the pheromone source.     

The role of bacterial symbionts in suppressing the defence reaction in larval hemolymph of the cotton leafworm Spodoptera littoralis (Biosd.)

Abstract

Spodoptera littoralis hemolymph exhibited a decrease in phenoloxidase activity during the first hour of exposure to alive or dead Xenorhabdus nematophilus and Photorhabdus luminescens even in the presence or absence of laminarin and α-chymotrypsin. Also, as the bacterial numbers in the hemolymph of the larvae of S. littoralis increased, the suppression of the enzyme, phenoloxidase, activity in vivo increased. On the other hand, in the in vitro incubation of the infected hemolymph with X. nematophilus for 30 min with laminarin, the decrease in phenoloxidase activity reached 92% in the infection with the highest dose (1 × 10¹⁰ cells/ml) live bacteria, and reached 100% at the same dose in the infection with live bacteria in the absence of laminarin. A two-fold decrease in enzyme activity was recorded in the case of injection of (1 × 10⁶ cells/ml) dead X. nematophilus and the absence of laminarin compared with injection of the same dose in the presence of laminarin. The same trend was also observed by the end of incubation of X. nematophilus-treated hemolymph without α-chymotrypsin. The decrease in phenoloxidase activity was highly significantly different in injection of dead P. luminescens and the absence of laminarin during incubation. In the case of injection of (1 × 10⁸ cells/ml) live P. luminescens a higher degree in the reduction of enzyme activity was recorded in the absence of α-chymotrypsin, where it reached to nearly a two-fold decrease. The results of these studies indicated that both X. nematophilus and P. luminescens alive or dead suppress the phenoloxidase activity in the presence or absence of both laminarin and α-chymotrypsin but, the suppression in absence of both during the in vitro incubation was highly comparable.     

Quadriflagellar primary spermatocytes in the cotton leafworm,Spodoptera littoralis (Boisd.) (Noctuidae,Lepidoptera)

Summary The surface morphology of primary spermatocytes from testicular cysts of the last instar larvae of Spodoptera littoralis was examined by scanning electron microscopy (SEM). At this stage, each primary spermatocyte possesses four developing flagella, directed towards the lumen of the cyst. The identical length of flagella in all primary spermatocytes from a single cyst indicates that the initiation and rate of flagellar growth are synchronized. Some asynchrony can, however, be observed in the translocation of flagella to secondary spermatocytes during the first meiotic division.     

Role of Challenger pesticide and plant extracts on some physiological parameters of the cotton leafworm,Spodoptera littoralis (Boisd.)

Fourth instar larvae of cotton leafworm, Spodoptera littoralis (Boisd.), fed on castor bean leaves treated with sub-lethal concentrations of both alcohol and hexane extracts of Egyptian conyza and Challenger 36% SC insecticide as well to study their effects on mortality, food consumption and utilisation of food. The tested insecticides exhibited relatively high mortality in alcoholic extract of Egyptian conyza (5.0% concentration) followed by Challenger (1.0% concentration). Results showed a slight reduction in the consumption index for larvae treated with both alcoholic extract and Challenger at low concentrations, while hexane extract (5%) recorded significant increase. The approximate digestibility (AD) was reduced in all treatments of alcoholic extract and the relative growth rate insignificantly decreased at 5% concentration, whereas AD increased for larvae fed on Challenger 0.25% concentration. The lower concentration of alcoholic extract of conyza and all treatments of Challenger pesticides induced insignificant decrease in the efficiency of conversion of the ingested food, while hexane extract exhibited insignificant increase. Challenger at all tested concentrations achieved insignificant decrease in the efficiency of the conversion of digested food with respect to the control.     

Ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens)

The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA-binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects.     

Viral-enhancing activity of an optical brightener for Spodoptera littoralis (Lepidoptera: Noctuidae) nucleopolyhedrovirus

The effect of an optical brightener on the insecticidal activity of a Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) nucleopolyhedrovirus (SpliNPV) were examined in three instars of S. littoralis. LD₅₀ values of the SpliNPV were reduced from 33 to <5, 73 to 5.7 and 342 321 to 288 412 occlusion bodies for second, third and fourth instars, respectively, by the addition of 1% Tinopal UNPA-GX. Relative potencies were >66, 12.8, and 1.2 for second-. third- and fourth-instar S. littoralis larvae, respectively. Relative mortality between the treatments with and without the brightener decreased from third- to fourth-stage larvae. In terms of speed of kill, the ST₅₀ values of the baculovirus-infected larva were reduced from 210 to 159, 213 to 147, and 207 to 165 h for second-, third- and fourth-instar larvae, respectively, by the addition of the optical brightener at biologically equivalent doses.     

Direct effects of snowdrop lectin (GNA) on larvae of three aphid predators and fate of GNA after ingestion

Plants genetically modified to express Galanthus nivalis agglutinin (GNA) have been found to confer partial resistance to homopteran pests. Laboratory experiments were conducted to investigate direct effects of GNA on larvae of three species of aphid predators that differ in their feeding and digestive physiology, i.e. Chrysoperla carnea, Adalia bipunctata and Coccinella septempunctata. Longevity of all three predator species was directly affected by GNA, when they were fed a sucrose solution containing 1% GNA. However, a difference in sensitivity towards GNA was observed when comparing the first and last larval stage of the three species. In vitro studies revealed that gut enzymes from none of the three species were able to break down GNA. In vivo feed-chase studies demonstrated accumulation of GNA in the larvae. After the larvae had been transferred to a diet devoid of GNA, the protein stayed present in the body of C. carnea, but decreased over time in both ladybirds. Binding studies showed that GNA binds to glycoproteins that can be found in the guts of larvae of all three predator species. Immunoassay by Western blotting of haemolymph samples only occasionally showed the presence of GNA. Fluorescence microscopy confirmed GNA accumulation in the midgut of C. carnea larvae. Implications of these findings for non-target risk assessment of GNA-transgenic crops are discussed.     

GNA成熟蛋白基因亚克隆及其原核表达载体构建

雪花莲外源凝集素（GNA）对刺吸式昆虫和某些咀嚼式昆虫以及多种线虫均有毒性,从含GNA前体蛋白基因的质粒中亚克隆出GNA的成熟蛋白基因MGNA,将MGNA基因插入大肠杆菌表达载体pET22b的不同位点,再经测序验证,得到了三种不同表达形式的GNA原核表达载体;22bG1(分泌型融合GNA蛋白）,22bG2(包涵体型GNA蛋白）,22bG3(分泌型天然GNA蛋白）,这为进一步在大肠杆菌中表达GNA和将GNA制成生物农药奠定了基础。     

雪花莲凝集素基因转化小麦及转基因小麦抗蚜性的研究

雪花莲凝集素对具有刺吸式口器的同翅目害虫具有毒杀作用。用基因枪法将1个新的雪花莲凝集素(GNA)基因转入普通春小麦品种中-60634和生产上正在推广的冬小麦高产品种——豫麦66中,分别获得了转基因小麦植株。抗蚜实验证明,转化gna基因的小麦植株对我国北方冬麦区的主要麦蚜——麦长管蚜和禾谷缢管蚜的抗性效果不尽相同。对禾谷缢管蚜,在接种当代即表现出明显的毒杀作用。对麦长管蚜,则表现为虫体发育减缓并且降低了其所生产的若蚜成活率。在自然放养条件下,gna基因则对这两种麦蚜的取食均起到了一定的抑制作用。     

Comparative study of entomopathogenic nematode isolation using Galleria mellonella (Pyralidae) and Spodoptera littoralis (Noctuidae) as baits

Entomopathogenic nematode (EPN) occurrence in soil from natural areas and crop field edges from La Rioja (northern Spain) was compared using two insects as baits: Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). Both insects trapped Steinernema feltiae, S. kraussei and S. carpocapsae, with G. mellonella being more efficient than S. littoralis recording 5.4 and 2.6% of positive soil samples, respectively. EPN recovery frequency and abundance obtained with G. mellonella were not statistically different between natural and crop field edges values; however, S. littoralis was more successful trapping EPNs from crop field edges. Statistical differences were observed for recovery frequency recorded by both hosts in natural areas. Significant differences in larval mortality between both insects were not observed. The use of S. littoralis in entomopathogenic nematode surveys is discussed.     

Secretion of Spodoptera littoralis female reproductive system on deposited egg masses that possibly acts as oviposition deterring substance for conspecific females

Egg masses laid by Spodoptera littoralis mated female moths were extracted by petroleum ether (PE), ethanol (E) and Ringer's solution (RS). Egg-wash extracts were evaporated and the weights of crude materials were obtained. Different aqueous concentrations were made. The amount of extracted material increased as the weight of eggs used increased and vice versa. Coating Nerium oleander leaves with aqueous egg-wash extracts prepared from S. littoralis egg-masses deterred the mated conspecific female moths from ovipositing their eggs on treated leaves, as well as causing a decrease in the total number of deposited eggs per female during the moth's life span. The highest deterrent effect on conspecific female moths to oviposit their eggs was obtained after treatment of N. oleander leaves with PE or E egg-wash extract. The deterrent effects of the tested egg-wash extracts was concentration dependant; an increase in the concentration of any extract caused an obvious decrease in the number of deposited egg-masses and the total number of laid eggs on the treated N. oleander leaves.     

Molecular characterization of an inhibitor of apoptosis in the Egyptian armyworm, Spodoptera littoralis, and midgut cell death during metamorphosis

The cDNA corresponding to an inhibitor of apoptosis (IAP) from the Egyptian armyworm, Spodoptera littoralis, was cloned by RT-PCR. Sequence analysis showed that the IAP of S. littoralis (SlIAP) contains two baculoviral IAP repeat (BIR) motifs, followed by a RING finger, an organization which is very similar to that of other lepidopteran IAPs. SlIAP mRNA was detected in ovary, testis, salivary gland, fat body, epidermis, brain and midgut of S. littoralis. During the last larval instar, prepupal and pupal stages, brain mRNA levels remained approximately constant, whereas those of midgut showed a large peak centred in the prepupal stage. Midgut morphology changed during metamorphosis from a semi-transparent, cylindrical structure in last instar larvae to a brownish globular mass in pupae. TUNEL assays, LysoTracker staining and caspase-3 immunohistochemistry, indicated that programmed cell death in midgut starts actively at the onset of pupation process, coinciding with the dramatic decrease of SlIAP mRNA levels observed at the same time.     

Inhibitory effect of 10,11-methyl-enetetradec-10-enoic acid on a Z9-desaturase in the sex pheromone biosynthesis of Spodoptera littoralis

The effect of 10,11-methylenetetradec-10-enoic acid on the sex pheromone biosynthetic pathway of Spodoptera littoralis is reported. This new cyclopropene fatty acid inhibited the biosynthesis of the main pheromone component from labeled myristicacid. The study of each Z desaturation step revealed that the Z9-desaturase of E11–14:Acid was inhibited, whereas the Z11-desaturase of 16:Acid was not affected. The results presented in this article agree with our hypothesis that the methylenehexadecenoic acids are beta-oxidized in the pheromone gland to the corresponding methylenetetradecenoic acids. © 1994 Wiley-Liss, Inc.