Biological and genetic characterization of a Pakistani isolate of Spodoptera litura nucleopolyhedrovirus

Spodoptera litura is an emerging insect pest in a wide range of crops worldwide. The insect is difficult to control because of resistance development to synthetic insecticides and emerging resistance to Bacillus thuringiensis toxins. Therefore, there is a need to develop biological control agents, preferably from an indigenous source to avoid risks associated with the importation of exotic natural antagonists. A Pakistani isolate of S. litura nucleopolyhedrovirus (SpltNPV, Baculoviridae), SpltNPV-Pak-BNG, was obtained from the field and characterized biologically and genetically, and compared to a SpltNPV reference isolate, SpltNPV-G1, thought to be of Chinese origin. The dose–mortality response (LD₅₀) of SpltNPV-Pak-BNG was not significantly different from that of the reference isolate SpltNPV-G1, but the time-to-death (LT₅₀) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1. DNA restriction enzyme profiling indicated that SpltNPV-Pak-BNG and SpltNPV-G1 are different viruses. Sequence analysis of ‘ORF24’, specific for SpltNPV (and S. littoralis NPV as ORF21), and the conserved baculovirus core genes polyhedrin, DNApol, pif-2 and lef-8 confirmed that this was indeed the case and that SpltNPV-Pak-BNG is a genuine SpltNPV variant, whereas the SpltNPV-G1 isolate we used is, in fact, a SpliNPV variant, renamed to SpliNPV-G1. The newly isolated SpltNPV-Pak-BNG has the potential for development as a biocontrol agent of S. litura in Pakistan.     

Development of Microplitis bicoloratus on Spodoptera litura and implications for biological control

Microplitis bicoloratus Chen (Hymenoptera:Braconidae:Microgastrinae), a new species of Microplitis Förster from China, is a solitary endoparasitoid of the larvae of the cotton leafworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). This parasitoid is the first to be successfully reared and evaluated in the laboratory as a potential agent for the biological control of S. litura in China. Oviposition, immature development, and the effects of parasitism on the development of S. litura were studied. In long-term oviposition trials, females laid eggs on S. litura larvae for up to 10 days; oviposition was heavily skewed toward the first few days, with approximately one third of the eggs laid on day 1 and over 50% laid by day 3. This rapid oviposition rate increases the potential for biological suppression of host populations because the likelihood of mortality for the parasites from exposure to detrimental environmental factors or generalist predators increases with time. Immature development of the parasitoid in its host only required 7 days: eggs hatched within 24 h, the first instar larva required 2 days, the second instar larva needed 3 days, and the third instar larvae exited the host and pupated in 1 day, at 27±1°C, 60–80% relative humidity and a 12:12-h (long day) photoperiod. The development of the parasitized hosts was disrupted. When the parasitoid larvae finished development, the body weights of host larvae were significantly reduced regardless of which host instar was parasitized. Our results suggest that M. bicoloratus has considerable potential as a biological control agent for S. litura.     

Flavonoid glycosides in Malabar spinach Basella alba inhibit the growth of Spodoptera litura larvae

Basella alba is a perennial plant of the Basellaceae and is known by various common names including Malabar spinach. There are few insects that cause damage to B. alba. In this study, we examined the effect of B. alba leaves on the growth of Spodoptera litura larvae. B. alba leaves and a methanolic extract of the leaves inhibited the growth of S. litura larvae. Half of the larvae reared on the leaves died within 1 week. We found that two flavonoids, vitexin, and vitexin-2″-O-arabinofuranoside, were abundant in the methanol extract of leaves. When larvae were reared on purified vitexin or vitexin-2″-O-arabinofuranoside, their growth was significantly impaired compared with larvae reared on control spinach leaves. These results suggested that the flavonoid glycosides in B. alba leaves act as deterrents to S. litura larvae.     

Enhanced bioefficacy of Bacillus thuringiensis var. kurstaki against Spodoptera litura (Lepidoptera: Noctuidae) through particle size reduction and formulation as a suspension concentrate

Technical powder of DOR Bt-127, a local isolate of Bacillus thuringiensis (Bt) var. kurstaki, containing 105?µm particles was subjected to milling in a planetary ball mill. Dynamic light scattering of samples drawn at 30?min intervals revealed reduced particle sizes ranging 1171–210?nm. Alkali-soluble protein was higher at 153–175?mg/g in milled Bt powders over 140?mg/g in unmilled Bt powder. All milled powders gave higher mortality of Spodoptera litura (Fabricius) larvae in comparison to the unmilled Bt powder. Bt powder from 60?min milling containing 559?nm particles with a low polydispersity index of 0.338 gave a lower LC₅₀ value of 1.35?mg/ml against third instar S. litura larvae in comparison to 2.04?mg/ml for unmilled Bt powder. LC₅₀ value of the suspension concentrate (SC) formulation developed with 559?nm Bt particles was 2.84?µl/ml containing only 0.95?mg Bt. Field evaluation of the SC formulation against S. litura on castor revealed highest per cent larval reduction of 92.4 and 96.2 at concentrations of 2.5 and 3.0?ml/l, respectively.     

Induction of 2-cyanoethyl-isoxazolin-5-one as an antifeedant against the tobacco cutworm (Spodoptera litura) by jasmonic acid in sweet pea leaf

ABSTRACT

Although sweet pea (Lathyrus odoratus) beans contain toxic β-aminopropionitrile, the plant itself is readily attacked by insects and is, therefore, protected through the use of pesticides. Consequently, the induction of L. odoratus resistance to insect attack via exogenous treatment is promising for pest control development. Screening of inducible elicitor effects showed that treatment of sweet pea foliage with jasmonic acid (JA) can induce antifeeding-based resistance to tobacco cutworm (Spodoptera litura) larvae. Spectroscopic analysis identified 2-cyanoethyl-isoxazolin-5-one (2-CEIX) as the antifeedant with a half-maximal effective concentration of 33.6 µmol/g fr. wt., i.e., exogenous JA treatment induced antifeeding activity due to the accumulation of 2-CEIX. Moreover, 2-CEIX-induced mortality of S. litura larvae was evaluated by a dipping test and the half-maximal lethal dose was determined to be 5.9 mg/mL. Therefore, 2-CEIX was concluded to be a suitable induced resistance target for elicitors or a lead compound for insecticide development.     

保幼激素类似物对斜纹夜蛾核型多角体病毒在宿主血淋巴中增殖动态的影响

通过研究保幼激素类似物(juvenile hormone analogues, JHA) methoprene对斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus, SpltNPV）在宿主血淋巴中增殖的影响,以探明JHA促进 SpltNPV增殖的初步机制,为阐明JHA促进病毒增殖提供更全面的理论依据。应用SDS PAGE及Western blot法,分析了methoprene对SpltNPV多角体蛋白(polyhedron, POLH)在宿主斜纹夜蛾6龄幼虫血淋巴中合成的影响。结果表明：经methoprene处理后2～3天可明显促进幼虫血淋巴液中POLH的合成。在此基础上,通过荧光定量PCR检测,发现methoprene对SpltNPV在幼虫血淋巴液复制的影响主要发生在处理后的第4和第5天,该期间polh基因的拷贝数比对照显著增加,拷贝数的峰值达1.22×10¹⁰/mL。     

Time-of-day specific changes in pesticide detoxification ability of Spodoptera litura (Lepidoptera: Noctuidae)

Circadian clocks govern daily physiological and molecular rhythms, and putative rhythms in the expression of metabolizing xenobiotics have been described in insects. Such rhythms could have important consequences for outcomes of chemical exposures at different times of the day. The proportion of photophase (light) and scotophase (dark) also influence the enzyme activities. Several studies have been done on the mechanism of insecticide resistance in Spodoptera litura exposed to chemical insecticides. This study is aimed at understanding the circadian variations of cypermethrin detoxification mechanisms in S. litura. The toxicity of insecticide, cypermethrin exposed to three different photoperiods in 3rd instar larvae of S. litura has been investigated. Detoxification enzyme profiles of α- and β-esterases, glutathione S-transferase (GST), and cytochrome P450 (Cyt P450) were assessed. The results showed that larvae were more tolerant to cypermethrin treated larvae at 8 h L: 16 h D photoperiod as compared with two other photoperiods tested. We observed significant increases in α- and β-esterases and cyt P450 activities in 4 and 8 h at different photoperiods. GST activity was significantly changed at different photoperiods at different timings. Activities of specific detoxification enzymes fluctuated during the time, and for specific insecticides, the concentration resulting in 50% mortality varied significantly during the different photoperiods. The time of the day when chemical exposure is imposed should be an important consideration in the experimental design, and use of pesticides.     

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.     

闹羊花素Ⅲ对斜纹夜蛾细胞系的活性及作用机理

研究了闹羊花素Ⅲ对斜纹夜蛾Spodoptera litura离体培养细胞（SL细胞）的活性,并测定了对SL细胞Na⁺、K⁺和葡萄糖吸收以及对4龄幼虫血细胞数量的影响。结果表明：以400 µg/mL 和200µg/mL闹羊花素Ⅲ处理SL细胞,24 h后细胞的相对死亡率为79.00% 和56.69%,处理后8 h,16 h,24 h和48 h的LC₅₀分别为240.09 µg/mL,173.45 µg/mL,113.56 µg/mL和73.40 µg/mL;闹羊花素Ⅲ处理SL细胞后10 min,细胞对离子的吸收迅速增加,30 min后吸收作用逐渐减弱;处理后3天内细胞对葡萄糖的吸收迅速增加,4～5 天后,细胞对葡萄糖的吸收基本停止。以叶碟法和注射法处理4龄幼虫,8 h后幼虫血细胞数量显著降低,随处理时间增加,幼虫血细胞数量又逐渐增加。     

金龟子绿僵菌对斜纹夜蛾幼虫的生防效果、抗氧化酶活性和肠道细菌群落的影响

【目的】测定金龟子绿僵菌(Metarhizium anisopliae)对斜纹夜蛾(Spodoptera litura) 2龄幼虫的毒力,研究金龟子绿僵菌侵染后寄主体内抗氧化酶活性和肠道内细菌群落的变化,探讨斜纹夜蛾对金龟子绿僵菌侵染的防御机制。【方法】采用浸渍法测定不同浓度金龟子绿僵菌对斜纹夜蛾2龄幼虫的毒力;应用IlluminaMiSeq高通量测序技术测定肠道细菌群落。【结果】不同浓度的孢悬液对斜纹夜蛾2龄幼虫均有一定的毒力,处理7 d时半致死浓度(LC₅₀)为3.944 107个孢子/mL;浓度为1.0×10⁹个孢子/mL时,半致死时间最短(LT₅₀)为4.6 d,校正后的死亡率为81.03%。处理后未致死的斜纹夜蛾幼虫体内抗氧化酶活性显著高于对照组。处理后致死的斜纹夜蛾幼虫肠道细菌群落多样性显著高于对照组;且处理后致死的斜纹夜蛾幼虫肠道细菌群落组成与对照组差异显著。【结论】金龟子绿僵菌对斜纹夜蛾幼虫的致死率和致死效率与金龟子绿僵菌的浓度呈正相关;斜纹夜蛾幼虫体内的抗氧化酶可能在抵抗金龟子绿僵菌侵染的过程中起重要作...     

Parasitism of <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae) by <Emphasis Type="Italic">Microplitis prodeniae</Emphasis> (Hymenoptera: Braconidae)

Spodoptera litura Fabricius is a major vegetable pest that is widely distributed throughout tropical, subtropical and temperate regions. Microplitis prodeniae Rao and Chandry is a solitary endoparasitoid of S. litura. To assess the potential use of this parasitoid as a biological control agent, the reproductive schedule, fecundity and functional response of M. prodeniae were investigated under conditions of 28 ± 1°C and 70 ± 10% relative humidity with a 14:10-h L:D photoperiod. The parasitoid’s average lifetime fecundity was 171.0 ± 10.4 eggs, of which approximately 50% were laid within the first 3 days. Additionally, M. prodeniae exhibited a Holling type II functional response, and the estimated maximum numbers of the 1st, 2nd and 3rd instar larvae that were parasitized by a single M. prodeniae female were 71.6, 78.4 and 41.5 larvae over a 24-h period, respectively. The results of this study suggest that M. prodeniae has great potential as a candidate for controlling S. litura and can guide efforts in its mass production.     

Effect of Host Plant on the Infectivity of Sl MNPV to Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Larvae

The susceptibility of Spodoptera litura to SlMNPV infection was markedly affected by phyto-chemicals ingested during the acquisition of viral inoculum on foliage of tomato and cauliflower. The LD₅₀ values computed for second, third and fourth instar larvae assayed on tomato leaves were 254, 819 and 23395 PIBs/larva, respectively whereas, it was 326, 1719 and 43843 PIBs/larva for respective instars when assayed on cauliflower leaves. Thus LD₅₀ values for second, third and fourth instar larvae were 1.28-, 2.09- and 1.87- fold lower, respectively in tomato leaves. Similarly, LT₅₀ values for second, third and fourth instar larvae assayed on tomato leaves were 7.1 and 7.5 days, respectively at inoculum dose of 2.7×10⁴ PIBs/larva whereas, it was 7.7 and 8.0 days for respective instars when assayed on cauliflower leaves at same inoculum. This result also showed that the S. litura were more susceptible on tomato leaves in comparison to cauliflower leaves as the time required for mortality was lower in tomato leaves. The possible biochemical bases for differential level of mortality of S. litura larvae on tomato and cauliflower crops needs to be investigated.     

Feeding deterrence,larvicidal and haemolymph protein profiles of an Indian traditional medicinal plant Alangium salviifolium (L.F.) Wangerin on cluster caterpillar,Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Different organic solvent crude extracts from the leaves of Alangium salviifolium (L.F.) Wangerin, were tested for their feeding deterrence, larvicidal activity and protein concentrations on the fourth instar larvae of Spodoptera litura L. under laboratory conditions. Treatments were given through two different host plant leaves such as brinjal and castor. The bioassay was carried out at different concentrations viz., 0.625, 1.25, 2.5 and 5%. The maximum antifeedant activity in brinjal (52.0%) and castor (29.97%) leaf discs was recorded in ethyl acetate extract as compared with hexane and chloroform extracts at 5% concentration. The result of larvicidal activity revealed that the maximum larval mortality was registered in brinjal leaves (65.81%) and castor with (57.48%) in ethyl acetate extract as compared to hexane and chloroform extracts at 5% concentration. The ethyl acetate extract of A. salviifolium contained alkaloids, diterpenoids, and saponins. The treatment also reduced haemolymph protein concentration after 48 h.     

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura

Juvenile hormone (JH) is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA (cDNA) was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate‐binding motifs were predicted in the protein. Modeling of the 3‐D structure showed that the protein consisted of eight α‐helixes linked with loops, with no β‐sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura.     

Growth inhibitory activity of Millettia pachycarpa (Bentham) extracts against Tobacco Caterpillar,Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Abstract

Millettia pachycarpa (Bentham), a perennial climbing tree, found endemic in north-east India used by tribes for treating various ailments. To ascertain the potential of M. pachycarpa for pest management, the present investigation was formulated against a polyphagous pest, Spodoptera litura (Fabricius). Growth inhibitory activity of various extracts and extract fractions was evaluated against third instar larvae of S. litura through topical as well as diet incorporation method. It was observed that dichloromethane extract showed very good growth inhibition in both topical (GI₅₀ 7.77?ppm) and diet incorporation methods (GI₅₀< 5?ppm). The growth inhibitory potential of this plant is also demonstrated by the presence of various abnormalities and reduction in larval and pupal weight.     

Feeding‐induced phenol production in Capsicum annuum L. influences Spodoptera litura F. larval growth and physiology

We studied the role of induced plant phenols as a defense response to insect herbivory. Phenolic compounds were induced in Capsicum annuum L., the source of many culinary peppers, after feeding by different stages of the insect pest, Spodoptera litura F. The phenols were identified and quantified using high performance liquid chromatography (HPLC) and effects produced by these phenols on larval development were studied. Vanillic acid was identified in plants challenged by second, fourth, and fifth instar larvae, but not in plants challenged by third instar nor unchallenged plants. Syringic acid production was induced in chili plants infested with second (0.429 ± 0.003 μg/g fresh weight, fourth (0.396 ± 0.01 μg/g fresh weight), and fifth instar (5.5 ± 0.06 μg/g fresh weight) larvae, compared to untreated plants (0.303 ± 0.01 μg/g fresh weight) plants. Leaves surface treated with the rutin deterred oviposition. Dietary exposure to chlorogenic acid, vanillic acid, syringic acid, sinapic acid, and rutin led to enhanced activities of detoxifying enzymes, β‐glucosidase, carboxyl esterase, glutathione S‐transferase, and glutathione reductase in the midgut tissues of all the larval instars, indicating the toxic nature of these compounds. Protein carbonyl content and acetylcholinesterase activity was analyzed to appreciate the role of induced plant phenols in insect protein oxidation and terminating nerve impulses.     

Effect of climate change on the potential distribution of the common cutworm (Spodoptera litura) in South Korea

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), known as common cutworm, is a worldwide pest that causes severe damage to various crops and vegetables in South Korea. In this study, we predicted the potential distribution of S. litura in South Korea in a climate change scenario by applying species distribution modeling. We used the CLIMEX software as a main tool and determined optimal parameter values to simulate the current distribution of S. litura in Asia. We then used these parameter values to predict the species' future distribution in South Korea. As a result, we prepared maps indicating areas with suitable climate for S. litura and showed that these areas gradually increased as a result of climate change. Approximately 98% of the areas in South Korea were predicted to have a favorable climate for S. litura in 2100; 63.2% of the area in South Korea is currently favorable. To the best of our knowledge, this is the first study to predict the potential distribution of S. litura in South Korea, and it provides the basic data necessary to establish an optimal control strategy of this species.     

A modified semi-synthetic diet for bioassay of non-sporeforming entomopathogenic bacteria against Spodoptera litura

Standard semi-synthetic diet used for rearing Spodoptera litura (Fab) larvae contains many antimicrobials. This diet is not suitable for evaluating non-sporeforming bacteria. We determined the effect of antimicrobials present in S. litura semi-synthetic diet on growth of entomopathogenic Serratia marcescens. Nutrient broth supplemented with four and two times reduced doses of sorbic acid (0.15 g) and methyl parahydroxybenzoate (0.05 g) supported bacterial growth comparable to control medium. No growth could be observed even at five times reduced concentrations of formaldehyde and streptomycin sulphate. Diet for rearing S. litura was modified accordingly and validated using two S. marcescens strains. Larval mortality ranged between 20% and 30% on the standard diet and more than 90% in the modified diet.     

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。