A Non-destructive Sampling Technique for Spodoptera mauritia (Boisduval) (Lepidoptera: Noctuidae) and Herpetogramma licarsisalis (Walker) (Lepidoptera: Pyralidae) in Turf

Experiments were conducted using chemical irritants to develop a non-destructive sampling technique for pests of high quality turf and lawn. POW detergent at a concentration of 35 mL in 3.5 L of water applied to an area of 1,600 cm² was the most effective for sampling Spodoptera mauritia and Herpetogramma licarsisalis and resulted in 84% of S. mauritia larvae surfacing within 4 min of application. No larvae surfaced after 4 min. Kendon pyrethrin SF applied at a concentration of 0.46 mL in 3.5 L of water resulted in similar numbers of larvae surfacing relative to 35 mL POW detergent. However, larvae took up to 20 min to surface. Formaldehyde and the pyrethroid, Mavrik® AF did not cause S. mauritia and H. licarsisalis larvae to surface.     

New cell lines from larval fat bodies of Spodoptera exigua: characterization and susceptibility to baculoviruses (Lepidoptera: Noctuidae)

Two new cell lines, designated IOZCAS-Spex-II and IOZCAS-Spex-III, were initiated from the fat bodies of larvae of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. The spherical cells were predominant among the various cell types and measures approximately 15 microm in diameter. The cell lines were mainly composed of tetraploid cells with chromosome numbers ranging from 116 to 131 (n=31). The cell lines were confirmed to have originated from the S. exigua by DAF-PCR technique. They were susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses from S. exigua.     

Occurrence and characterization of a nucleopolyhedrovirus from Spodoptera littoralis (Lepidoptera: Noctuidae) isolated in the azores

A nucleopolyhedrovirus (SpliMNPV-Az) was isolated from diseased larvae of Spodoptera littoralis, collected at the Island of S. Miguel in Azores. The virulence of this isolate was tested against S. littoralis larvae in laboratory. LD₅₀ against 2nd and 3rd instars were not significantly different, 1.44 × 10⁴, 3.89 × 10⁴ OBs per larvae, respectively, but both were significantly different from that against 4th instar, which was 61.3 × 10⁴ OBs per larvae. The complete codons sequence of SpliMNPV-Az Polh gene obtained was 750 bp (NCBI GenBank Accession No. AY600451). This sequence was compared to other 38 polyhedrin genes from NPVs and to 6 granulin genes from GVs and resulted to be identical to the sequence of a SpliMNPV previously published, thus indicating that the natural host of SpliMNPV-Az must be S. littoralis. Genetic distances estimated from restriction enzymes profiles showed SpliMNPV-Az is close to the Egyptian SpliMNPV type B, despite some degree of genetic divergence suggested by slight differences observed on PstI profile.     

Bacillus subtillis RTSBA6 6.00, a new strain isolated from gut of Helicoverpa armigera (Lepidoptera: Noctuidae) produces chymotrypsin-like proteases

Exploring bacterial communities with proteolytic activity from the gut of the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) insect pests was the purpose of this study. As initial efforts to achieve this goal here we report the isolation of new Bacillus subtillis RTSBA6 6.00 strain from the gut of H. armigera and demonstrated as proteases producer. Zymographic analysis revealed 12 proteolytic bands with apparent molecular weights ranging from 20 to 185 kDa. Although some activity was detected at acidic pH, the major activity was observed at slight alkaline pH (7.8). The optimum temperature was found to be 35 °C with complete loss of activity at 70 °C. All proteases were completely inactivated by PMSF (phenylmethylsulfonyl fluoride) and TPCK (N-tosyl-l-phenylalanine chloromethyl ketone), suggesting that proteases secreted by B. subtillis RTSBA6 6.00 belong to serine proteases class with chymotrypsin-like activity. The occurrence of protease producing bacterial community in the gut of the H. armigera advocates its probable assistance to insect in proteinaceous food digestion and adaptation to protease inhibitors of host plants.     

Treatment of larvae with repeated doses of precocene II induces precocious metamorphic changes in Spodoptera mauritia (Lepidoptera: Noctuidae)

In Spodoptera mauritia repeated daily treatments of larvae with 40 μg of precocene II throughout the fifth and sixth instar larval period had no effect on the larval-larval period but prolonged larval-pupal ecdysis. The resulting pupae showed precocious adult differentiation of mouth parts, wings, eyes, legs, and fat body.     

Exposure to herbicides reduces larval sensitivity to insecticides in Spodoptera litura (Lepidoptera: Noctuidae)

Herbicides and insecticides are widely used in modern agriculture. It has been reported in various studies that application of insecticides can increase tolerance of herbivorous insects to insecticides. However, limited information exists on susceptibility to insecticides when insects are exposed to herbicides. This study was conducted to investigate the potential impact of the herbicides trifluralin and 2-methyl-4-chlorophenoxyacetic acid sodium salt (MCPA-Na) on the susceptibility of the nocturnal moth Spodoptera litura to the insecticides X-cyhalothrin, phoxim and bifenthrin. We found that larvae exposed to trifluralin or MCPA-Na became significantly less susceptible to both insecticides than nonexposed control larvae. Herbicide-treated larvae did not show altered growth under the used test conditions. However, heads of herbicide-treated larvae showed increased expression of the acetylcholinesterase genes SI Ace I and SI Ace 2. Moreover, the fat body and midgut of herbicide-treated larvae displayed elevated expression of detoxification genes (the carboxylesterase gene SI CarE;the glutathione S-transferase genes SlGSTe2 and SlGSTe3\ the cytochrome P450 monooxygenase genes CYP6B48, CYP9A40 and CYP321B1). The CYP6B48 gene exhibited highest inducibility. In conclusion, the data of this study suggest that exposure of S. litura larvae to herbicides may stimulate detoxification mechanisms that compromise the efficacy of insecticides.     

Identification of serine protease,serine protease homolog and prophenoloxidase genes in Spodoptera frugiperda (Lepidoptera: Noctuidae)

In insects, proteolytic cascades medicated by serine proteases (SPs), serine protease homologs (SPHs) and prophenoloxidases (PPOs) control several physiological processes, notably the innate immunity. However, no attempts have been made to identify and characterize these genes in Spodoptera frugiperda, one of the most destructive agricultural pests. In this study, 83 SPs, 26 SPHs and four PPOs were respectively identified in S. frugiperda genome based on homology blast against those of other insects. We then analyzed the domain organization of these proteins and assigned them into different groups by phylogenetic reconstruction. Furthermore, the mRNA levels of clip-domain SPs/SPHs (cSPs/cSPHs) and PPOs were quantified in response to a mixed infection of Micrococcus luteus and Escherichia coli, and obvious accumulations were recorded in immune tissues, including hemocytes and fat body. In the latter study, we profiled the expression patterns of highly expressed cSPs and PPOs in different developmental stages, including egg, larva, pupa, female and male adults. It was shown that most cSPs were abundantly expressed in adults, while PPOs were detected at high levels in both egg and larval stages. These current findings substantially add to our understanding of the roles of S. frugiperda SPs, SPHs and PPOs in immune regulation and further lay a solid foundation for uncovering the interaction mechanisms between insects and pathogens.     

A new cell line from Spodoptera exigua (Lepidoptera: Noctuidae) and its differentially expressed genes

A new cell line, designated IOZCAS‐Spex XI, was established from the pupal ovaries of Spodoptera exigua (Lepidoptera: Noctuidae) in TNM‐FH medium containing 10% foetal bovine serum. The spherical cells were predominant among the various cell types. The population‐doubling time during the logarithmic phase of growth was 81.7 h. It was confirmed that the cell line originated from S. exigua by DAF‐PCR technique. Analysis of susceptibility to baculovirus showed that the new cell line was susceptible to S. exigua nucleopolyhedrovirus (SeNPV), Autographa californica multiple NPV (AcMNPV) and slightly susceptible to S. litura NPV (SpltNPV), while not permissive to Helicoverpa armigera NPV and Hyphantria cunea NPV (HcNPV). Real‐Time PCR analysis was carried out to compare some differentially expressed genes between the cell line and the primary culture. The result showed that marked significant differences were observed in the expression of the genes of SUMO‐1 activating enzyme, BCCIP‐like protein, 10 kDa HSP, CypA, receptor for activated PKC, PDI‐like protein ERp57, ALDH, DEAD box ATP‐dependent RNA helicase‐like protein (P < 0.01), while a significant difference was obtained in the expression of GST gene between the cell line and the primary culture (P < 0.05).     

Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10^–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.     

Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells

1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.     

Transcytosis of Junonia coenia densovirus VP4 across the gut epithelium of Spodoptera frugiperda(Lepidoptera:Noctuidae)

The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells.The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel,rather than in the gut.In this study,we used fall armyworm,Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two-dimensional ligand blot analysis.We also assessed the rate of flux of the primary viral structural protein,VP4 fused to eGFP with a proline-rich linker(VP4-P-eGFP)through the gut epithelium ex vivo in an Ussing chamber.The mechanisms involved with transcytosis of VP4-P-eGFP were assessed by use of inhibitors.Bovine serum albumin(BSA)and eGFP were used as positive and negative control proteins,respectively.In contrast to BSA,which binds to multiple proteins on the brush border membrane,VP4-P-eGFP binding was specific to a protein of high molecular mass.Protein flux was significantly higher for VP4-P-eGFP after 2 h than for albumin or eGFP,with rapid transcytosis of VP4-P-eGFP within the first 30 min.In contrast to BSA which transcytosed following clathrin-mediated endocytosis,the movement of VP4-P-eGFP was vesicle-mediated but clathrin-independent.The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S.frugiperda.     

Biological Characterization of a Nucleopolyhedrovirus of Spodoptera litura (Lepidoptera: Noctuidae) Isolated from the Philippines

A Philippine Spodoptera litura nucleopolyhedrovirus (SltMNPV) was isolated by plaque purification from an uncloned NPV population in a formulated product that exhibits high insecticidal activity against the onion-attacking common cutworm, S. litura. Its estimated genome size was approximately 142 kb. Comparison with Autographa californica MNPV and Spodoptera exigua NPV by restriction fragmentation analysis showed that it was a distinct isolate. Light microscopy examination showed that SltMNPV-P7 infected insect cell lines derived from Spodoptera littoralis (CLS-79), Spodoptera frugiperda (Sf9), and S. litura (TUAT-SpLi221). It produced typical cytopathic effects in cell lines established from Bombyx mori and S. exigua and induced apoptotic-like cytopathology in Lymantria dispar-derived cell line; it exerted no observable cytopathic effect on Spilosoma imparilis cell line. Significant increase in budded virus (BV) production was observed in CLS-79, Sf9, and TUAT-SpLi221 cell lines, in which TUAT-SpLi221 cell line supported the highest BV titer. No significant BV production was observed in the remaining four cell lines. Similarly, viral DNA replication and polyhedrin production proceeded only in these three spodopteran cell lines. Results showed that SltMNPV-P7 is a unique isolate which can be propagated in vitro for further studies and thus has a potential for development into a more effective microbial insecticide.     

Castor and camphor essential oils alter hemocyte populations and induce biochemical changes in larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae)

Two plant essential oils; camphor and castor were tested for insecticidal and antifeedant activity against the 4th instar larvae of Spodoptera littoralis, a serious pest on cotton in Egypt. Also the impact of LC₁₀ of both oils on some physiological parameters in larvae was studied by using leaf dipping technique. Analysis of both oils using GC–MS revealed several insecticidal and antifeedant compounds. Our results showed higher insecticidal activity and antifeedant index of camphor oil against S. littoralis. The LC₅₀ and the antifeedant indices were 163.1, 246.8?mg/ml and 12.69, 6.62% for camphor and castor bean oil, respectively. The total hemocyte count (THC) and differential hemocyte count (DHC) were reduced significantly after 48?h of treatment compared to controls. Both oils reduced all types of hemocytes except plasmatocytes which were reduced only by castor oil. Camphor oil decreased total proteins and carbohydrates while castor oil targeted only carbohydrate content. Both oils didn't affect the amount of total lipids. Lipase, α-amylase and glucose-6-phosphate dehydrogenase (G6PD) enzyme activities were increased significantly in larvae treated with camphor oil than other treatments. These results clearly indicate that castor and camphor oils can affect the nutritional status in S. littoralis larvae, thereby changing the internal metabolic processes in the larvae which make them as potential control agents in IPM programs against S. littoralis.     

斜纹夜蛾幼虫肠道不同部位对Zn2+积累作用的比较研究

在人工饲料中添加不同浓度的Zn2+连续胁迫3个世代的斜纹夜蛾Spodoptera litura Fabricius幼虫,采用等离子体原子发射光谱仪检测分析了连续3个世代6龄幼虫肠道不同部位对食物中Zn2+的积累作用.结果表明,Zn2+可在六龄幼虫前、中和后肠中积累,积累量受到食物中Zn2+的浓度和胁迫世代数的双重影响.每个世代6龄幼虫前肠、中肠和后肠中的Zn2+含量均随着饲料中Zn2+浓度增加而增加.六龄幼虫肠道3个不同部位对Zn2+的积累作用依次为前肠＜中肠＜后肠.而随着胁迫世代数的增加,不同世代6龄幼虫前肠、中肠和后肠中的Zn2+含量表现出显著减少的趋势.因此,植食性昆虫消化道不同部位对食物中Zn2+的积累作用有所不同,并随着胁迫世代数的增加而显著减少.     

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。     

Characterisation of a Colombian granulovirus (Baculoviridae: Betabaculovirus) isolated from Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

A strategy for biological control of the fall armyworm, Spodoptera frugiperda, has included the use of baculoviruses principally the nucleopolyhedrovirus SfMNPV, which have been extensively characterised. In contrast, the granulovirus of S. frugiperda (SfGV) has been poorly studied even though it is able to enhance the infectivity and virulence of NPVs. In this work, a Colombian SfGV isolate (VG008) was characterised in comparison with a reference isolate from Brazil (VG014). The viral morphology was characterised by ovoidal-shaped occlusion bodies (OB) that contained one single internal virion. Median lethal concentrations (LC₅₀) and mean times to death (MTD) were 4.5 × 10⁵ OBs/mL and 29 days for VG008 and 1.6 × 10⁵ OBs/mL and 33 days for VG014. Both isolates reduced their insecticidal activity by 94%, after one hour of direct irradiation with ultraviolet light type B. The most prominent protein had an apparent molecular mass of 27 kDa and corresponded with the Granulin. Genomic comparison among isolates from Colombia and Brazil generated by restriction profiles showed differences in the number and size of fragments. Partial sequences of lef-8 and lef-9 genes and complete sequence of gran gene of Colombian SfGV isolate (VG008) showed high similarity values with VG014 and SfGV A12-4 homologous sequences, showing genetic distance lower than 0.015 (Kimura 2-parameter model), which confirmed that the three isolates belong to the same viral species. The characterisation of VG008 isolate demonstrated its high genomic and biological similarity with the Brazilian isolate.     

Purification of a human alternative complement pathway inhibitor from hemolymph of larval fall armyworm (Spodoptera frugiperda)

Larval Spodoptera frugiperda hemolymph contains a specific inhibitor of the alternative pathway of human complement. This inhibitor was purified from larval hemolymph (HL) by 50% (NH4)2SO4 precipitation, DEAE-Sephacel chromatography and sequential gel-filtration on Bio-Gel 1.5m, 0.5m and Sephacryl S-200. Purified HL protein (Mr = 110,000) was composed of two Mr 55,000 polypeptide chains. Addition of purified HL protein to human complement resulted in a dose-dependent inhibition of RaRBC lysis and clumping of cells. The protein inhibitor provides a new tool for investigating the regulation of human alternative complement pathway.