HPTLC determination of vasicine and vasicinone in Adhatoda vasica

A simple high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of the pharmacologically important quinazoline alkaloids vasicine and vasicinone in Adhatoda vasica. The assay combines the separation and quantification of the analytes on silica gel 60 GF254 HPTLC plates with visualisation under UV and scanning at 270 and 281 nm. Using this technique, the alkaloidal content of different parts of the title plant have been determined.     

Antifeedant and toxic activity of Trichilia americana extract against the larvae of Spodoptera litura

The biological activity of a crude methanolic extract of Trichilia americana (Sesse and Mocino) Pennington (Meliaceae) was assessed using the Asian armyworm, Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). The extract exhibited strong antifeedant activity in a choice leaf disc bioassay with 0.18 g cm^–2 extract deterring feeding by 50%. In nutritional assays, the crude extract reduced growth, consumption and the utilisation of ingested and digested food in a dose-dependent manner when fed to larvae, suggesting both antifeedant and toxic activities. When relative growth rates were plotted against relative consumption rates, the growth efficiency of the S. litura fed on diet containing T. americana crude extract was significantly less than that of control larvae. This result further indicates that the extract acts as both an antifeedant and chronic toxin. Toxicity is only seen following ingestion and was not observed following topical application or injection into the hemocoel. Larvae reared initially on extract-containing diet then transferred to control diet showed nutritional indices comparable to those of larvae fed continuously on control diet. This suggests that the extract is not permanently damaging the insect's digestive tract. The mode-of-action of the extract as a chronic toxin remains unknown.     

Antifeedant activity and toxicity of two alkaloids from Adhatoda vasica Nees leaves against diamondback moth Plutella xylostella (Linn.) (Lepidoptera: Plutellidae) larvae

Two natural alkaloids viz., Vasicine acetate and 2-Acetyl benzylamine, isolated from Adhatoda vasica leaves, showed antifeedant, larvicidal and moult inhibiting properties against diamondback moth Plutella xylostella in laboratory experiments. Maximum antifeedant activity of 98.5% was recorded at 1000 ppm concentration of Vasicine acetate treatment, whereas as 2-Acetyl benzyl amine recorded only 71.4% antifeedant activity at 1000 ppm concentration. Azadirachtin treatment presented 82% antifeedant activity at the highest concentration (1000 ppm). Both the active compounds of A. vasica showed lethal toxicity on larvae and pupae. The highest larvicidal and pupicidal activities were recorded in 2-Acetyl benzylamine treatment at 125 ppm concentration. The two A. vasica compounds also affected the normal growth and development and moulting process of P. xylostella. Final moulting of larvae into pupae was disrupted by the treatments, which resulted in larval–pupal intermediates and abnormal pupae. Treatments also produced small-size pupae and malformed adults with poorly developed wings.     

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.     

Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae)

Abstract:  Entomopathogenic fungi are a poorly exploited source of insecticidal proteins, which may be used for the development of new natural insecticides and as alternative molecules for transgenic deployment. The crude soluble protein extracts in Adamek's liquid medium of 25 fungal isolates belonging to the fungal species Metarhizum anisopliae , Beauveria bassiana , Beauveria brongniartii and Scopulariopsis brevicaulis were screened by per os toxicity on Spodoptera littoralis larvae. Whilst extracts from two M. anisopliae and two B. bassiana isolates gave significant mortalities when applied either on alfalfa leaf discs or incorporated into artificial diet, the one from M. anisopliae 01/58-Su isolate was the only most toxic that showed promise for S. littoralis control. In leaf disc assays, this extract exhibited strong dose-related toxic and antifeedant activity against the larvae. At 10, 20 and 40  μ g protein/insect, the extract gave 61.3%, 96.6% and 100.0% mortality, respectively, and average survival time of 5.7, 4.3 and 3.1 days respectively. Not only the antifeeding index was dose-related, but it significantly increased over time in a dose-related manner. Longer exposure times led to a dose-related significant increase in larval mortality. The exposure times for 50% mortality were 91.3 h and 62.1 h for 20 and 40  μ g protein/insect respectively. The crude extract when exposed to higher temperature or protease treatment lost toxicity, indicating that toxicity was protein mediated. In addition, the liquid medium composition did not influence its insecticidal activity. The effects of the protein extract on midgut cells of second instar larvae of S. littoralis were investigated by using both light and electron microscopy. A progressive bleeding of the midgut epithelium into the gut lumen was observed along with lysis of the epithelium and deterioration of the microvilli.     

Antifungal activity of Bacillus thuringiensis strains and their efficacy against the cotton leaf worm Spodoptera littoralis

Bacillus thuringiensis strains that belong to B. thuringiensis, B. kurstaki and soil-isolated B.t. were assessed in the following phytopathogenic: Rhizoctonia solani, that had their mycelial growth decreased after incubation in the presence of the bacterial strains. The bacteria have also pathogenic effect against the insect pest Spodoptera littoralis. The isolate B.t. D-1 and the B.t. kurstaki HD-203 were found to be inhibiting R. solani, the strain B. kurstaki HD-203 displayed the highest percentage of inhibition (64%) and B.t. D-1 showed 49% of inhibition. Antagonistic activity was maintained up to pH 8.5, and the antifungal activity was stable to heat at 70?°C for 1?h. Minimal inhibitory concentrations were 152 and 131?μl/ ml for B.t. D-1 and B. kurstaki HD-203, respectively. The two strains also have high efficacy against S. littoralis larvae, B.t. D-1 gave 70% and the B. kurstaki HD-203 strain gave 80% mortality after seven days of treatment.     

黄皮种子提取物对斜纹夜蛾幼虫的杀虫活性及有效成分鉴定

【目的】测定黄皮Clausena lansium种子提取物对斜纹夜蛾Spodoptera litura幼虫的拒食活性,对有效成分进行分离、鉴定,并进一步测定其拒食活性、生长发育抑制活性及细胞毒性,为筛选斜纹夜蛾生态防治植物源杀虫剂提供依据。【方法】采用叶蝶法测定黄皮种子甲醇提取物及其石油醚、乙酸乙酯、正丁醇和水4种溶剂萃取物对斜纹夜蛾3龄幼虫的非选择性拒食活性和生长发育抑制活性;用硅胶柱层析进行石油醚萃取物和乙酸乙酯萃取物拒食活性成分分离;用核磁共振谱(NMR)进行拒食有效成分结构鉴定;用MTT法测定拒食活性成分对SL细胞毒性。【结果】黄皮种子甲醇提取物对斜纹夜蛾3龄幼虫具有较强的拒食活性,活性成分主要分布于石油醚相和乙酸乙酯相中;石油醚相和乙酸乙酯相经硅胶柱层析分离得10个馏分,以黄色晶体馏分2的拒食活性最强;经分离纯化及核磁共振氢谱和碳谱鉴定,黄皮新肉桂酰胺B(lansiumamide B)为主要的拒食活性成分,对斜纹夜蛾3龄幼虫48 h拒食中浓度为214.95μg/m L,同时具有较强的细胞毒性,对SL细胞48 h抑制中浓度为10.09μg/m L。【结论】黄皮种子提取物对斜纹夜蛾幼虫具有较强的拒食活性及细胞毒性,黄皮新肉桂酰胺B是主要活性成分之一,作为植物源杀虫剂开发具有较好的发展前景。     

Ovicidal activity of Sesamum indicum (L.) oil against Spodoptera littoralis (Boisd.)

In the research for alternative tools and botanical products to control Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). Sesamum indicum (L.) (Lamiales: Pedaliaceae) oil was assayed as an ovicide. The mortality increased with existence of fatty acids. Chemical analysis of S. indicum oil using GLC analysis showed palmitic acid as the major fatty acid (51.27%), while the major hydrocarbon and sterols were found to be heneicosane (58.63%) and β-sitosterol (2.60%), respectively. Generally, the values of LC₅₀s indicated that one-day-old egg masses are more susceptible than three-day-old eggs. Also, the leaf dip technique is more efficient than the spraying one. Results showed several features of chorionic surface deformation treated with sesame and KZ oils than control using scanning electron microscopy. Meanwhile, the tested oils caused significant reduction in both total soluble protein and transaminase enzymes as compared to control. Additionally, the oils elongated the incubation period and larval duration than control.     

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.     

Significance of penetration and metabolism on topical toxicity of diflubenzuron in Spodoptera littoralis and Spodoptera exigua

Toxicity assays indicated that when diflubenzuron (DFB) was topically applied on larvae of the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval), and the beet armyworm, Spodoptera exigua (Hübner), estimated LD₅₀ values were similar, reaching 0.47 and 0.44 ng/larva, respectively. This study evaluated the importance of the rate of uptake and excretion, and of enzymatic metabolism in building up an insecticidal toxicity after topical application on the insect cuticle in both species. In general, penetration of DFB in S. littoralis was about 2-fold higher than in S. exigua, whereas metabolic breakdown was of minor importance in S. littoralis as compared with S. exigua. The current data indicate the importance of cuticle penetration and discuss that extrapolation from oral feeding toxicity results to topical toxicity data should be evaluated with caution, especially for insect growth regulators used in integrated pest management.     

Pathogen-mediated modulation of host metabolism and trophic interactions in Spodoptera littoralis larvae

The success and sustainability of entomopathogens in insect control depend on their stress potential and ability to modulate certain physiological aspects of their insect hosts. In the present study, newly moulted fourth instars of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) were incubated for 24 h with the median lethal dose (LD₅₀) of Steinernema riobrave, Heterorhabditis bacteriophora, or Beauveria bassiana to determine their effects on certain metabolic activities and nutritional physiology compared to those of uninfected (control) larvae. Infection decreased total protein, lipid, and carbohydrate contents. Carbohydrase activity was found to be pathogen and enzyme dependent, with S. riobrave and H. bacteriophora infection increasing amylase, invertase, and trehalase activities. Beauveria bassiana infection decreased amylase and invertase activities but increased that of trehalase. Infection with the three pathogens also enhanced phosphatase activity. All pathogens reduced transaminase activity. Changes in the nutritional indices varied not only with the variation in the candidate entomopathogen and surviving instar but also with the variation in the age of the same instar, with a profound change in late last instars (3-day-old sixth instars). The link between the metabolic activities and nutritional indices was discussed. Overall, the entomopathogen-host interaction appears to be primarily nutritional. The pleiotropic effects of the median lethal infections on the insect pest might eventually help in the biological control potential of the entomopathogens tested.     

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.     

杀菌剂丙环唑对斜纹夜蛾的毒性

在研究比较了11种农药对斜纹夜蛾Spodoptera litura细胞（简称SL细胞）的毒杀活性基础上,选择毒杀活性最高的杀菌剂丙环唑,对其毒理学机理进行进一步研究。结果表明,丙环唑的细胞毒力最高,在100 μg/mL浓度下处理后48 h,SL细胞的死亡率为98.08％。处理后36 h,丙环唑对SL细胞的LC₅₀值为20.31 μg/mL。丙环唑能明显降低SL细胞的蛋白质含量。以0.5 μg/头的丙环唑注射斜纹夜蛾4龄幼虫,处理后72 h,试虫血淋巴总含量及血细胞数分别下降了26.80％和25.26％;在1.0 μg/头的剂量下,则分别下降了37.67%和36.32%。以0.5 μg/头和1.0 μg/头的丙环唑注射处理后,斜纹夜蛾幼虫体重显著降低。此外,丙环唑能降低斜纹夜蛾幼虫血淋巴含糖量及血淋巴蛋白质含量。在注射处理后96 h和120 h,丙环唑对斜纹夜蛾4龄幼虫的LD₅₀值分别为0.59 μg/头和0.45 μg/头。丙环唑对SL细胞和斜纹夜蛾幼虫均具有较好的毒杀活性,显示出丙环唑类似物控制害虫的可能性。     

Virulence potential of some fungal isolates and their control-promise against the Egyptian cotton leaf worm,Spodoptera littoralis

A preliminary virulence test of four fungal isolates, Beauveria bassiana IMI 382302, Beauveria bassiana IMI 386701, Trichoderma harzianum T24 and Aspergillus flavus Link against larvae of Spodoptera littoralis was performed. The most effective isolates against larvae of S. littoralis were B. bassiana 302 and T. harzianum T24, which also showed the lower percentage of pupation compared with the other two isolates under the same conditions of treatments. Three concentrations (1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ ml^?1) of the aqueous conidial suspension of the four tested isolates were carried out against both larval and pupal stages of S. littoralis within five days post-treatment. T. harzianum T24 showed 80% larval mortality only when applied at the highest conidial concentration, while A. flavus showed 100% pupal mortality only, at all of its conidial concentrations. However, B. bassiana IMI 382302 showed relatively high dose-dependant larval and pupal mortalities, while strain IMI 386701 of B. bassiana showed a very weak mortality against pupae at higher concentrations, and no virulence against larvae was recorded. Enzymatic and antibiosis bioassays of the four fungal isolates showed relatively high activities against Fusarium spp. for most of the tested isolates. Clear zone of enzyme activity on agar plates proportionally increased with increasing the concentration of enzyme substrate and prolongation of the incubation period. Mtabolites produced in the agar culture inhibited the growth of Fusarium spp. and the productivity differed greatly among isolates or strains of the same isolate. Volatile and non-volatile compounds produced by A. flavus Link showed a higher inhibition activity against Fusarium spp. compared with the other fungal isolates. The humoral antifungal response of insect host is relatively high compared to the anti-bacterial one. Injection of larvae with the immune sensitive bacteria Micrococcus luteus (5 × 10³ bacteria/larva) showed a detectable humoral response by 2 h, peaked around 12 h and became hardly detectable by 24 h post-injection. Injection of larvae with conidial suspension (5 × 10³ conidia/larva) from each of the fungal isolates showed humoral antifungal activity against B. bassiana IMI 386701 and A. flavus only. This activity was detectable by 12 h, peaked around 36 h and became hardly detectable by 48 h post-injection. Although the humoral antifungal response was started slowly compared to the antibacterial one, it lasted for longer and enabled larvae to withstand the infection with these immune-sensitive fungal strains. No humoral activity was detected against B. bassiana IMI 382302, although however, weak activity was detected against T. harzianum T24 only at the low conidial concentration but not at the higher one (1 × 10⁸ ml^?1). Thus, this study concludes that larvae of S. littoralis showed immune-dependant sensitivity to T. harzianum T24 and B. bassiana IMI 382302. Therefore, this study may recommend these two fungal isolates as mycoinsecticides in the battle against cotton leaf worm in Egypt. Hence, they have been selected for future comprehensive bioassays in the laboratory under conditions similar to that in the field. This, in fact, may help for developing effective mycoinsecticides against this pest. Penetration mechanims of insect cuticle by entomopathogenic fungi will be discussed.     

番荔枝内酯化合物布拉它辛对斜纹夜蛾的杀虫活性及对SL细胞的致凋亡作用

为了明确番荔枝内酯化合物布拉它辛的杀虫活性和探索杀虫作用机理,本研究采用浸叶法测定该化合物对斜纹夜蛾Spodoptera litura幼虫的生物活性,采用MTT检测法和流式细胞术,研究该化合物对斜纹夜蛾离体培养卵巢细胞（SL细胞）的细胞毒力和致细胞凋亡作用,以及对SL细胞线粒体膜电位的影响。结果表明: 布拉它辛不仅对斜纹夜蛾幼虫具有良好的拒食活性,处理后24 h,对2龄和3龄幼虫的AFC₅₀值分别为60.25 μg/mL和86.73 μg/mL,还对幼虫生长有良好的抑制作用。布拉它辛处理SL细胞后24 h和48 h,IC50值分别为22.32 μg/mL和10.03 μg/mL。流式细胞仪检测结果表明,布拉它辛对SL细胞具有明显的致凋亡作用,可导致SL细胞线粒体膜电位显著下降。本研究表明布拉它辛对斜纹夜蛾幼虫具有良好的拒食活性与生长抑制作用, 并且该化合物能明显抑制SL细胞的增殖,诱导细胞凋亡,降低线粒体膜电位。因此,布拉他辛具有广阔的研究应用前景。     

The role of the different fermentation media and infection processes on the infectivity of the bacterial symbionts to Spodoptera littoralis (biosduval) (Lipedoptera: Noctuidae)

Abstract

Bacterial symbionts are one of the most toxic bacteria to insect pests. They have been isolated from the intestine of the entomopathogenic nematodes. Ten strains of the entomopathogenic nematodes, that may be different, have been isolated out of different soil fauna having different crops from different Governorates in Egypt. The bacterial symbionts in these strains have been isolated and tested for production parameters using four different media. Experiments showed that both Loria Broth (LB), and Nutrient Broth (NB) gave good results in laboratory production of bacterial symbionts, concerning stability, cell size, and pigment production during culturing. Experiments using different techniques of introducing the bacterial symbionts to larvae of the cotton leafworm showed that the injection technique was the most effective among all the tested techniques. This is followed by oral and forced feeding which seemed to give equal results. On the other hand, toxicity experiments showed that the four bacterial isolates named, G-NRC-A₃, SH-NRC-A₅, SH-NRC-A₆, and N.Sinai-NRC-A₈, besides the bacterial symbiont of Steinernema abbasi, Flavimonas oryzihabitans, all give 100% mortalities to Spodoptera littoralis larvae after 24 h post-treatment at the higher dose (5×10⁴ cells/10 μl) but at the lower doses 5×10³ and 5×10² cells/10 μl of injection solution, a 100% mortality was reached after 72 h post-treatment.     

Antifeedant and cytotoxic activity of gibberellic acid against Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The present study investigates the effect of gibberellic acid (GA3), a terpenoid and phytohormone, on the digestive physiology and intermediary metabolism of the cotton bollworm Helicoverpa armigera Hübner (Lepidoptera, Noctuidae). Incorporation of GA3 (800 μg g^?1 diet) in an artificial diet results in significant reductions in the rates of diet consumption and the efficiency of conversion of food consumption by by H. armigera larvae. The relative growth rate decreases as the concentration increases. The relative α‐amylase activity in sixth‐instar larvae of H. armigera decreases significantly after ingestion of four concentrations of GA3. Histological studies of the midgut in GA3‐treated larvae (800 μg g^?1 diet) show degeneration of the epithelial cells. The alanine and aspartate aminotransferase activity decreases at the highest concentration. However, acid phosphatase, alkaline phosphatase, γ‐glutamyl transferase and lactate dehydrogenase activity increase significantly compared with the control. The results clearly demonstrate the adverse effects of GA3 on H. armigera via interruption of nutritional physiology and metabolism.     

Bioefficacy of Ceasalpinea bonduc (L.) Roxb. against Spodoptera litura Fab. (Lepidoptera: Noctuidae)

Bioefficacy of hexane, chloroform and ethyl acetate extracts of the seeds of Ceasalpinea bonduc (L.) Roxb. was studied against third instar larvae of Spodoptera litura at 0.25, 0.5, 1, 2.5 and 5.0% concentrations. Significant antifeedant, larvicidal and pupicidal activities and least LC₅₀ values were observed in chloroform extract. The chloroform extract was subjected to fractionation using silica gel column chromatography. Six fractions were obtained; among these, the third fraction showed high antifeedant, larvicidal and pupicidal activities at 1000 ppm concentration. Abnormalities in adults were also observed. All the activities were concentration dependent. C. bonduc could be useful in integrated pest management programme.