Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor gene in Spodoptera littoralis larvae

In noctuid moths cuticular pigmentation is regulated by the pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family, which also mediates a variety of other functions in moths and other insects. Numerous studies have shown that these neuropeptides exert their functions through activation of the PBAN receptor (PBAN-R), with subsequent Ca(2+) influx, followed by either activation of cAMP or direct activation of downstream kinases. Recently, several PBAN-Rs have been identified, all of which are from the pheromone gland of adult female moths, but evidence shows that functional PK/PBAN-Rs can also be expressed in insect larvae, where they mediate melanization and possibly other functions (e.g., diapause). Here, we identified a gene encoding a G-protein-coupled receptor from the 5th instar larval tissue of the moth Spodoptera littoralis. The cDNA of this gene contains an open reading frame with a length of 1050 nucleotides, which translates to a 350-amino acid, 42-kDa protein that shares 92% amino acid identity with Helicoverpa zea and Helicoverpa armigera PBAN-R, 81% with Bombyx mori PBAN-R and 72% with Plutella xylostella PBAN-R. The S. littoralis PBAN-R gene was stably expressed in NIH3T3 cells and transiently in HEK293 cells. We show that it mediates the dose-dependent PBAN-induced intracellular Ca(2+) response and activation of the MAP kinase via a PKC-dependent but Galphai-independent signaling mechanism. Other PK/PBAN family peptides (pheromonotropin and a C-terminally PBAN-derived peptide PBAN(28-33)NH(2)) also triggered MAP kinase activation. This receptor, together with the previously cloned PBAN-R, may facilitate our understanding of the cell-specific responses and functional diversities of this diverse neuropeptide family.     

Ovipositional discrimination by Microplitis rufiventris females between healthy and granulosis virus-infected Spodoptera littoralis larvae

Ovipositional choice tests by Microplitis rufiventris females (Hym., Braconidae) between granulosis virus‐infected (GVI) and non‐infected (NI) Spodoptera littoralis larvae (Lep., Noctuidae), were assessed using discriminatory methods for re‐isolating the NI and virus‐infected hosts after removing the female parasitoid. When M. rufiventris females were given a choice between NI and GVI S. littoralis hosts, the adult females exhibited marked preference (P < 0.01) for the NI (i.e. higher quality) hosts. In this case, M. rufiventris females and S. littoralis GV (SlGV) did not significantly compete for the same type of host larvae and are, generally, compatible. However, when the choice was given between two low qualities of S. littoralis hosts, i.e. virus‐free previously parasitized hosts and viral‐infected hosts a significant preference (P < 0.01) of the parasitoid females for the GVI larvae was observed. In this case, the parasitoid would be at a disadvantage when competing with GV for the same host. However, the parasitoid could be used as an additional tool for the dissemination of biocontrol viruses within different pest populations, i.e. hosts other than S. littoralis. Importantly, the results showed different strategies of parasitoid female in egg‐laying management. When M. rufiventris female was given a choice between healthy and SlGVI hosts, the female deposited more eggs than when she was given a choice between two low qualities of host larvae. The results of the study may have implications in pest management strategies using M. rufiventris and SlGV against S. littoralis larvae.     

Ascorbic acid, ascorbates and analogs in Spodoptera littoralis diet: Stability and effect on larvae

The dietary effect of L-ascorbic acid, its ascorbates and analogs at equimolar concentrations on larvae and pupae of the Egyptian Cotton Leafworm, Spodoptera littoralis (Boisd.) was studied. The daily activity loss of these compounds in the diet was determined with a 2,4 dinitrophenylhydrazine-thin layer chromatography method. This determination of ascorbates and analogs showed a rapid loss of activity down to about 50% after 4 days. However, ascorbic acid was significantly more stable. Storing ascorbic acid, its ascorbates and analogs at 6° only preserved their activity at 10–25% more than at 25°; sodium ascorbate, calcium ascorbate and L-dehydroascorbic acid equalled the activity of L-ascorbic acid in larval and pupal weight, as well as insect development. D-araboascorbic acid (=D-isoascorbic acid) did not support the normal insect development. Stability of ascorbic acid ascorbates and analogs in the diet in relation to their dietary effects is discussed.

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Résumé Ce travail étudie les effets sur les larves et les nymphes de Spodoptera littoralis (The Egyptian Cotton Leafworm), d'adjonction à l'alimentation d'acide L-ascorbique et de ses ascorbates et analogues à des concentrations équimoléculaires. Les pertes journalières d'activité de ces composés ont été déterminées par une méthode de chromatographie sur couches minces avec le 2–4 dinitrophenylhydrazine. Cette détermination des ascorbats et analogues a révélé une perte rapide d'activité jusqu'à environ 50% après 4 jours. Cependant l'acide ascorbíque est relativement plus stable. Le stockage à 6° de l'acide ascorbique, de ses ascorbates et analogues préservait leúr activité de seulement 10–25% de plus qu'à une température de 25°. L'ascorbate de sodium, l'ascorbate de calcium et l'acide L-dehydroascorbique montraient une activité égale à celle de l'acide L-ascorbique, quant à leur effet sur le poids des larves et pupes, aussi bien que sur le développement de l'insecte. L'acide D-ascorbique ne permet pas le développement normal de l'insecte. La stabilité de l'acide ascorbique, des ascorbates et des analogues dans l'aliment est discutée en rapport avec les effets observés.

     

The calyx fluid of Microplitis rufiventris parasitoid and growth of its host Spodoptera littoralis larvae

The morphology of the female reproductive system of pupal and adult stages of Microplitis rufiventris and the ultrastructure of the ovaries are described and illustrated. Two morphologically distinct types of particles of nuclear origin, i.e., polydnavirus (PDV) and a virus-like filamentous particle (VLFP) were detected in the ovarian calyx fluid of the female wasp. It is likely that these particles are injected into the host during oviposition. PDV initiated replication in the calyx of mid-aged pupae and in pharate adults and were present throughout adult life. VLFP were only seen in the calyx fluid of newly emerged adults, and therefore observed after the PDV. Feulgen and methyl-green pyronin staining revealed the presence of DNA in both types of particles. The effects of injection of Spodoptera littoralis larvae with a combination of parasitoid viruses and venom of M. rufiventris females (CxFV) were investigated and the results were compared with two control groups, i.e., larvae injected with Pringle's saline (PS) and naturally parasitized larvae. CxFV-larvae showed significant declines (P<0.05) in food consumption, weight of ejected faeces and weight gain when compared with PS-larvae. However, naturally parasitized larvae (parasitoid egg+CxFV+ovarian protein) displayed a high significance score (P<0.01) in comparison with those of PS-larvae. The approximate digestibility (AD) values of S. littoralis larvae were positively affected as early as day 2 post-treatment by either injection of CxFV or parasitization. However, a reduction in AD was observed in both PS- and CxFV-larvae on day 3-7 in comparison with naturally parasitized larvae. Other indices of food utilization were unchanged in CxFV-larvae when compared to saline treated or parasitized controls.     

Bioactivities of seven chrysanthemum species flowers powder on Spodoptera littoralis (Boiduval) larvae

We have screened seven Chrysanthemum species, collected from different biotope in Tunisia for the antifeeding and growth regulatory activity against S. littoralis (Lepidoptera: Noctuidae) an insect pest of tomato. The flowers powder was added to the Poitout and Bues (1974) artificial diet at three concentrations: 4, 8, and 16%. After ten days of treatment, the consumed quantity determination revelled that these species have an antifeeding activity proportional to the concentration, more considerable for the C. fuscatum and C. Myconis flowers powder. At the concentration of 16% the larvae weight was significantly delayed in the average time to the control, these results were compared to the effect of Cestrum parquii on S. littoralis and Pieris brassicae The moulting date determination showed an elongation of the third, fourth and fifth stages at the high concentration probably due to the presence of juvenile hormones Analogues.     

Intermediates of stigmasterol metabolism in Spodoptera littoralis

Stigmasterol-24,28-epoxide, 22E-stigmasta-5,22,24(28E)-trien-3 beta-ol, and 22E-cholesta-5,22,24-trien-3 beta-ol were identified as normal metabolites of [3H]stigmasterol in Spodoptera littoralis larvae. Relative concentrations of all three of these metabolites increased when a diazasterol inhibitor was fed in combination with stigmasterol in the artificial diet. Identification of these sterols as intermediates in the conversion of stigmasterol to cholesterol in this insect indicates that intermediates analogous to fucosterol and fucosterol-24,28-epoxide in the conversion of sitosterol to cholesterol are produced in the metabolism of stigmasterol. This is the first published identification of stigmasterol-24,28-epoxide and 22E-stigmasta-5,22,24(28E)-trien-3 beta-ol as intermediates in this pathway in an insect.     

Bioefficacy of cadmium and lead on cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae) larvae

The sensitivity of Spodoptera littoralis second and fourth instar larvae to the heavy metals cadmium (Cd) and lead (Pb) was investigated under laboratory conditions. Both Cd and Pb, even, at very low concentrations, had relatively high toxic effects on S. littoralis larvae. Cd was more toxic to S. littoralis larvae than Pb. In addition, the mean percentage feeding deterrence (FDI%) of Cd and Pb was concentration-dependent. The nutritional indices (consumption index, growth rate, efficiency of converted ingested and digested food, and approximate digestibility) of S. littoralis second and fourth instar larvae were more affected in Cd-treatments compared with those of Pb-treatments. Therefore, the presence of such heavy metals in the environment would exert an adverse impact on S. littoralis larvae and their development.     

Wound-induced changes in the acceptability of tomato to larvae of Spodoptera littoralis: a laboratory bioassay

Abstract. 1. Leaves of tomato (cv. Moneymaker) were artificially damaged and offered to Spodoptera larvae at a range of intervals following damage. Grazing levels on these leaves were compared with those on undamaged leaves on the same or different plants.
2. In separate experiments, three leaves in a middle position on the main stem were clipped and after 48 h grazing levels on undamaged leaves above and below those damaged were compared with similar leaves from control plants.
3. Within 8 h, grazing levels on damaged leaves were significantly lower than those on control leaves, and within 24 h, leaves adjacent to damaged ones were similarly affected. These effects persisted for at least 7 days and leaves above and below those damaged were affected. There was up to nine-fold reduction in area consumed.
4. The possible ecological consequences of reduced palatability at these levels are discussed.     

Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae.

In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.     

Dose-dependent influence of Microplitis rufiventris factors on developmental rate and growth of last-instar Spodoptera littoralis larvae

The solitary endoparasitoid, Microplitis rufiventris, attacks and can develop in earlier instars of Spodoptera littoralis larvae with preference to third‐instar larvae. We used the last stadium (sixth instar), a stage which is not naturally parasitized. The newly moulted larvae (0–3 h old) of this stadium were more acceptable for parasitization by the wasp females than the older ones (24 h old). Parasitization by M. rufiventris wasp of last instar S. littoralis larvae leads to dose (no. of eggs + parasitoid factors)‐dependent effects which were more pronounced at 20°C than at 27°C. A single oviposition into a sixth instar host larva resulted in normal development of the host. However, superparasitization increased the proportions of developmentally arrested hosts and number of live wasp larvae. Development of supernumerary individuals of the parasitoid in the host larva leads to dose‐related adverse effects on host growth and development. The present study may provide interesting opportunities for studying the physiological bases of host–parasitoid interactions and parasitoid intra‐specific competition in the biological system considered.     

The role of temperature in enhancing the insecticidal activity of Bacillus thuringiensis against Spodoptera littoralis larvae

The present study scrutinised how far temperature would affect the velocity of the insecticidal activity of Bacillus thuringiensis, as the rapidity of pest control achievements is of a great concern. Third instar Spodoptera littoralis larvae were treated with Bt at three concentration levels under five different temperatures (15°C, 20°C, 25°C, 30°C and 35°C). LT₅₀s were evaluated in each case. The LT₅₀ values showed various levels of reductions as temperature and/or Bt concentration increased, indicating that the velocity of mortality (1/LT₅₀) and/or the rapidity of Bt activity was almost temperature dependant. However, relatively high and low reduction percentages in the LT₅₀ values on the elevation of 5°C were obtained at lower and higher temperature ranges, respectively. The temperature coefficient, Q ₁₀ values, determined within narrow ranges (5°C) showed great reductions when temperature increased from 15°C to 20°C at all Bt concentrations. Raising temperature by 5°C above 20°C or 25°C almost caused similar Q ₁₀ values indicating constant increase in the response of Bt activity within 20–30°C temperature range. Q ₁₀ values over 30°C were comparatively very low. This proved that decrease in Q ₁₀ values due to the rise of temperature was dependant on the starting temperature.     

Daily changes in the responsiveness of taste receptors correlate with feeding behaviour in larvae of Spodoptera littoralis

Larvae of Spodoptera littoralis were monitored through the final larval stadium (6 days) for weight gain, amount eaten in bioassays and responsiveness of gustatory receptors to stimulation with sodium chloride and four concentrations of sucrose. The age of larvae within the stadium, time of day (am or pm) and interactions between these factors influenced the electrophysiological response to 0.05 M sucrose. Overall, the responsiveness of taste receptors increased in the first half of the stadium, then declined. Neural input correlated with weight gain on days 1 to 4, and with amount eaten in bioassays on days 1 to 5. This correlation between neural input and feeding behaviour indicates that the observed modulation in neural sensitivity has a causal relationship with feeding behaviour. The ways in which the modulation might occur are discussed.     

A porin-like protein from oral secretions of Spodoptera littoralis larvae induces defense-related early events in plant leaves

Insect herbivory on plants is a complex incident consisting of at least two different aspects, namely mechanical damage and chemical challenge, as feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Mechanical wounding alone is sufficient to induce a set of defense-related reactions in host plants, but some early events such as membrane potential (Vm) changes and cytosolic Ca²⁺-elevations can be triggered only by herbivores suggesting that OS-derived molecules are involved in those processes. Following an assay-guided purification based on planar lipid bilayer membrane technique in combination with proteomic analysis, a porin-like protein (PLP) of most likely bacterial origin was determined from collected OS of Spodoptera littoralis larvae. PLP exhibited channel-forming activity. Further, early defense-related events in plant–insect interaction were evaluated by using a purified fraction and α-hemolysin (α-HL) as a commercial pore-forming compound. Both up-regulated the calmodulin-like CML42 in Arabidopsis thaliana, which only responds to oral secretion and not to wounding. An elevation of in vivo [Ca²⁺]_cyt was not observed. Because membrane channel formation is a widespread phenomenon in plant–insect interactions, this PLP might represent an example for microbial compounds from the insect gut which are initially involved in plant–insect interactions.     

Mechanism of entomotoxicity of the plant lectin from Hippeastrum hybrid (Amaryllis) in Spodoptera littoralis larvae

Plant lectins have received a lot of attention because of their insecticidal properties. When orally administered in artificial diet or in transgenic plants, lectins provoke a wide range of detrimental effects, including alteration of the digestive enzyme machinery, fecundity drop, reduced feeding, changes in oviposition behavior, growth and development inhibition and mortality. Although many studies reported the entomotoxicity of lectins, only a few of them investigated the mode of action by which lectins exert toxicity. In the present paper we have studied for the first time the insecticidal potential of the plant lectin from Hippeastrum hybrid (Amaryllis) (HHA) bulbs against the larvae of the cotton leafworm (Spodoptera littoralis). Bioassays on neonate larvae showed that this mannose-specific lectin affected larval growth, causing a development retardation and larval weight decrease. Using primary cell cultures from S. littoralis midguts and confocal microscopy we have elucidated FITC-HHA binding and internalization mechanisms. We found that HHA did not exert a toxic effect on S. littoralis midgut cells, but HHA interaction with the brush border of midgut cells interfered with normal nutrient absorption in the S. littoralis midgut, thereby affecting normal larval growth in vivo. This study thus confirms the potential of mannose-specific lectins as pest control agents and sheds light on the mechanism underlying lectin entomotoxicity.     

Lack of absorption and rapid elimination of Rifampicin as factors precluding tests against nuclear polyhedrosis in Spodoptera littoralis larvae

Rifampicin administered per os in artificial medium to first-instar larvae of Spodoptera littoralis together with nuclear polyhedrosis virus did not affect virus infection and resulted in total larval mortality. A pharmacokinetic study of this antibiotic was carried out by means of a microbiological assay. Using Bacillus thuringiensis var. thuringiensis as the test organism for assaying the antibiotic, as little as 0.5 μg of Rifampicin could be detected in the presence of hemolymph, and no such detectable amounts could be found in the hemolymph of sixth instar larvae which had been fed the antibiotic. Most if not all of the antibiotic was in the feces, excreted in active form. When given parenterally, Rifampicin was detected in the hemolymph 30 min after injection but not 24 hr thereafter, when active antibiotic was found concentrated in the first fecal pellets evacuated after injection. This disappearance is ascribed to excretion via the Malpighian tubules. The larval responses detailed above preclude the testing of Rifampicin as an antiviral drug against nuclear polyhedrosis.     

Seasonal and interspecific variation in the biochemical composition of some British fern species and their effects on Spodoptera littoralis larvae

Fern-feeding insects in Britain are mainly found on mature fronds in late summer. Six fern species (Dtyopteris filix-mas, D. dilatata, D. borreri, Phyllitis scolopendrium, Polyslkhum setiferum and Polypodium vulgare) were analysed for fibre, lignin, cellulose, tannins, cyanogenesis and thiaminase activity in an attempt to determine the biochemical basis for this seasonal pattern of attack. A bioassay was also carried out, using frond material incorporated in the diet of Spodoptera littoralis, to determine the effects of seasonal changes in fern toxins on a non-adapted, insect herbivore. The young fronds of all six species had a high protein content and low fibre-lignocellulose; protein levels decreased and cell wall materials increased as the fronds matured. Tannin concentrations and thiaminase activity showed a less consistent seasonal pattern. Tannins were present in the highest concentration in the young fronds of D. borreri, Polystichum and Polypodium and decreased with season; D. dilatata, Polystichum and Polypodium showed a similar pattern of thiaminase activity. The other species showed no strong seasonality in the levels of either tannins or thiaminase. No cyanogenic activity was detected in any of the fern species. Frond material from all six species decreased survivorship and growth rates of Spodoptera larvae. Larval performance and growth on diets containing fern material from young immature fronds was generally poorer than on diets containing material from mature fronds, collected later in the season. Neither tannin concentrations nor thiaminase activity levels closely corresponded to the observed interspecific and seasonal patterns of larval development and mortality, and other toxins must be involved. It is concluded that ferns are highly toxic to non-adapted herbivorous insects but the effects on adapted species are unknown. The restricted seasonal occurrence of a small number of insect species exhibiting specialized feeding habits suggests, however, that biochemical properties of the ferns determine this pattern of attack and limit the number of species which are able to exploit ferns as food resources.