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.     

Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera:Noctuidae)

AIMS: To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains. METHODS AND RESULTS: The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet. CONCLUSIONS: It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.     

Juvenile hormone biosynthesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera, Noctuidae)

The in vitro production of juvenile hormones (JH) was investigated by using corpora allata (CA) of larvae and corpora cardiaca-corpora allata (CC-CA) complexes of adult females of the fall armyworm Spodoptera frugiperda. In female moths, JH release was high compared to that in 5th and 6th instar larvae. Concentrations of 0.11-0.12 mM methionine, 180-200 mM Na(+), 5.8-8.3 mM K(+), 10-50 mM Ca(2+) and a pH range of 5.7-6.3 yielded optimal incorporation of L-[methyl-(3)H] methionine in vitro by CC-CA complexes. The highest hourly incorporation occurred during a 9-h incubation period following a 1.5-h lag-phase. JH release from CC-CA complexes of adult females was shown to be age-dependent with a peak value on day 2 (approx. 4 pmol h(-1) CA(-1)). By a combination of reversed phase (RP)- and normal phase (NP)-high performance liquid chromatography (HPLC), two major labelled products released by the complex were separated. One compound co-migrated with chemically synthesized JH II diol, the second compound with JH III diol. Only traces of JH II and III could be detected in some samples. Gland extracts also contained both the major radiolabelled products. Double labelling experiments using [3H]methionine and [14C]acetate confirmed their de novo synthesis in CC-CA complexes of female moths. The nature of chemically synthesized reference JH III diol was proved by LC-MS (ESI mass spectrometry) and 1H-NMR (nuclear magnetic resonance spectroscopy).     

Molecular variability of Spodoptera frugiperda (Lepidoptera: Noctuidae) populations associated to maize and cotton crops in Brazil

The molecular variability among 10 populations of Spodoptera frugiperda (J.E. Smith), collected from maize, Zea mays L., or cotton Gossypium hirsutum L. crops located at distinctive geographical regions in Brazil, was assessed through random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR). In total, 208 RAPD markers were evaluated, and 98% of them were polymorphic. The mean genetic similarity was 0.6621 and 0.2499 by the Simple Matching and Jaccard matrices, respectively. In general, the unweighted pair-group method with arithmetic average dendrograms separated the populations into clusters related to the geographical origin of the samples. No branch of the dendrograms underpinning a molecular association of S. frugiperda has been identified to either of the two host plants. The molecular variance analysis showed that 18 and 82% of the genetic variation was distributed among and within the groups of populations, respectively. The principal coordinate analysis reinforced the pattern of population clustering found with the unweighted pair-group method with arithmetic average method. These results suggest the occurrence of considerable gene flow between S. frugiperda populations from maize and cotton fields located in the same region in Brazil. Therefore, for an effective management of this pest, there is an urgent need for a better understanding of the gene flow of S. frugiperda populations associated to different host plants along the distribution range of this pest over time in a specific cropping system.     

交配和温度对草地贪夜蛾性信息素通讯的影响

【目的】明确交配和温度对草地贪夜蛾Spodopterafrugiperda性信息素通讯的影响,为田间草地贪夜蛾的性信息素防治提供参考。【方法】采用溶剂浸提法提取草地贪夜蛾雌蛾性信息素腺体中的化学组分;利用气相色谱-质谱联用（GC-MS）技术对提取物中的组分进行化学鉴定;利用气相色谱（GC）技术分析雌蛾腺体中各组分的含量;利用触角电位（EAG）技术测定雄蛾对腺体各组分及性信息素类似物的电生理反应。在不同温度（20-35）℃下饲养草地贪夜蛾,以研究温度对其性信息素通讯的影响。【结果】GC-MS分析发现,草地贪夜蛾雌蛾腺体内含有Z7-12:Ac等7种组分。交配实验表明,雌蛾在1个暗期内只交配1次,在羽化后7 d内可交配多次,其中交配2次及以上的占44%（单对放置）和67%(30对放置)。交配显著抑制雌蛾在次日暗期（交配后第1个暗期）的求偶活动,但在交配后第2个暗期基本恢复正常;在交配后第1个暗期,雌蛾腺体中主要组分Z9-14:Ac的含量和比例较对照雌蛾显著升高,其他组分无显著变化。在20-35℃的饲养温度范围内,雌蛾腺体内16:Ac的含量在35℃时最高,在20℃时未能检测出;各测试温度下Z11-16:Ac的含量相差不显著;其余5种组分均在25℃时最高;主要活性组分Z7-12:Ac和Z9-14:Ac在35℃时显著降低。饲养温度对腺体内各组分的相对比例也有较大影响,12:Ac、Z11-14:Ac、Z11-16:Ac和16:Ac所占比例均在30-35℃时较高,而Z9-14:Ac则在20-25℃时较高。不同饲养温度所得雄蛾对腺体主要组分的触角电位反应无显著差异。【结论】交配显著抑制草地贪夜蛾雌蛾在次日的求偶行为;25℃饲养条件最适宜雌蛾腺体内性信息素等组分的合成。     

Demographic parameters of Spodoptera exigua (Lepidoptera: Noctuidae) on different soybean cultivars

Life table, reproduction, and population growth parameters of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), were determined on nine soybean (Glycine max L.) cultivars ('032', '033', 'Hill', 'M4', 'M7', 'M9', 'M11', 'TMS', and 'Zane') at 25°C. The age-specific survival rate of eggs and immature stages was higher on 033 in comparison with other cultivars. The gross fecundity rate ranged from 911 on M9 to 1,296 on Zane, but the differences were not statistically significant. The net fecundity rate was significantly higher on 033 (1,082 eggs per female) than on other cultivars. The gross fertility rate significantly differed on tested cultivars, and was the highest on Zane (1,257 eggs per female) and lowest on Hill (813 eggs per female). The net fertility rate varied from 532 on M11 to 1,082 eggs per female on 033. Both daily number of eggs and daily number of fertile eggs laid per female varied significantly among the different cultivars, and were the highest on TMS (90 for both parameter) and lowest on M7 (47 and 43), respectively. The values of net reproductive rate, intrinsic rate of increase, and finite rate of increase were significantly higher on 033. The shortest generation time (23 d), shortest doubling time (1.66 d), and highest percentage of female offspring (56%) also were obtained on 033. According to population growth parameters, 033 and Hill partially were susceptible and resistant, respectively, to S. exigua. The results of this study provide direction to design a more comprehensive integrated pest management program for this pest.     

Ultralow rates of spinosad in phagostimulant granules provide control of Spodoptera frugiperda (Lepidoptera: Noctuidae) in maize

Field trails in 2002 and 2003 were performed to determine the efficacy of maize flour-based granular formulations with ultralow rates of the naturally derived insecticide spinosad (0.1, 0.3, and 1.0 g [AI]/ha), for control of Spodoptera frugiperda (J.E. Smith) in maize, Zea mays L., in southern Mexico. Spinosad formulations were compared with a chemical standard, a commercial granular formulation of chlorpyrifos (150 g [AI]/ha). In both years, application of spinosad resulted in excellent levels of control, indicated by the number of living S. frugiperda larvae recovered from experimental plots. The efficacy of spinosad applied at 0.3 and 1.0 g (AI)/ha was very similar to that of chlorpyrifos. Natural reinfestation caused S. frugiperda numbers in insecticide treated plots to return to values similar to the control treatmentby 10-15d postapplication. Many spinosad-intoxicated larvae collected in the field died later in the laboratory in 2002, but not in 2003. Percentage mortality due to parasitoid emergence did not differ in any treatment in either field trial. The number of parasitoids that emerged from S. frugiperda collected in each treatment was significantly reduced after application of spinosad (all rates) or chlorpyrifos due to a reduction in the number of host larvae. Parasitoid numbers returned to control values by 9-15 d postapplication in all treatments. The most prevalent parasitoid was the braconid Chelonus insularis Cresson, which represented approximately 80% of emerging parasitoids in both years. We conclude that appropriate formulation technology can greatly enhance the performance of this naturally derived, biorational insecticide.     

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.     

Purification and properties of a beta-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae

Two beta-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl beta-D-glucoside (NPbetaGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl beta-D-galactoside (NPbetaGal) better than NPbetaGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0. The Mr 47,000 BG N-terminal sequence has high identity to plant beta-glycosidases and to mammalian lactase-phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids (galactosidase site) and di- and oligosaccharides (cellobiase site) derived from hemicelluloses, thus resembling mammalian lactase-phlorizin hydrolase.     

Bioenergy crops Miscanthus x giganteus and Panicum virgatum reduce growth and survivorship of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Large-scale cultivation of plants used as biofuels is likely to alter the ecological interactions of current agricultural crops and their insect pests in a myriad of ways. Recent evidence suggests many contemporary maize pests will be able to use potential biofuel crops such as switchgrass, Panicum virgatum L., and miscanthus as hosts. To determine how suitable these biofuels are to the maize, Zea mays L., pest and generalist graminivore, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), we examined host plant preference and larval performance on foliage grown for commercial biofuel production. Larvae fed leaf tissue from both field- and greenhouse-grown switchgrass and miscanthus were monitored for survival, development, and food use relative to field-grown maize. Survivorship on biofuel crops was high on greenhouse-grown leaf tissue but severely reduced for field-grown switchgrass, and no larvae survived on field-grown miscanthus. Larvae fed field-grown tissue had larger head capsules yet achieved lower pupal weights because the increased toughness of the leaf tissue prevented the assimilation of nitrogen. Given that larvae overwhelmingly preferred maize to other biofuel crop species and that survival and performance were dramatically reduced on biofuel crop species, it is likely that biofuel crops, as grown for field cultivation, will suffer reduced damage from maize pests such as S. frugiperda because of reduced suitability.     

Genetic structure and molecular variability of Spodoptera frugiperda (Lepidoptera: Noctuidae) collected in maize and cotton fields in Brazil

The purpose of this research was to evaluate the genetic similarity and structure of the fall armyworm, Spodoptera frugiperda (J.E. Smith), populations associated with maize and cotton crops in Brazil using amplified fragment length polymorphisms. Mean genetic similarity among populations was 0.45. The unweighted pair group method with arithmetic mean analysis dendrograms did not separate populations of S. frugiperda into clusters related to the host plant in which the insects were collected. No genetic variation was observed among maize and cotton populations of S. frugiperda, suggesting that the same populations are injuring both crops in Brazil. This research validates the need for stewardship of crop-protection methods for managing S. frugiperda to reduce the incidence of pesticide resistance, due to the spatial and temporal overlapping of maize and cotton crops in some regions in Brazil.     

RFLP analysis of genetic variation in North American populations of the fall armyworm moth Spodoptera frugiperda (Lepidoptera: Noctuidae)

A high level of genetic variability was detected in North American fall armyworm (FA W), Spodoptera frugiperda (J. E. Smith), populations by restriction fragment length polymorphism (RFLP) analysis of genomic DNA. In nearly all cases individual larvae could be differentiated using 22 probe-enzyme combinations. Laboratory colonies formed distinct groups based on RFLP analysis. Individuals from a colony of the previously determined ‘rice strain’ formed a group showing obvious deviation in RFLP patterns from the other five populations, which included a population from a ‘com strain’ colony. The results were consistent with previous studies using allozymes which indicated that there are two genetically differentiated forms of the fall armyworm. Diagnostic markers were also found that distinguish these two groups.     

Host association of Spodoptera frugiperda (Lepidoptera: Noctuidae) corn and rice strains in Argentina, Brazil, and Paraguay

Spodoptera frugiperda (J.E. Smith) is composed of two genetically distinct strains, the so-called corn strain and the rice strain. Whether the two strains differ in their host use is unclear, because laboratory experiments have not been able to show consistent host performance or preference differences between them, and field studies showed high rates of hybridization, as well as some degree asymmetric host use. To determine the distribution of the two strains and their association with host plants, we collected fall armyworm larvae from different crops (corn, rice, alfalfa, and sorghum) and grasses in 15 different localities over 4 yr in Argentina, Brazil, and Paraguay. The strain identity was analyzed using two polymorphisms in the mitochondrial cytochrome oxidase subunit I gene. We identified the corn and rice haplotypes and three types of populations were characterized based on the frequencies of the individuals that belonged to any of these haplotypes: in 44% of populations the corn haplotype predominated, in 44% of populations the rice haplotype was the most frequent, and 11% of populations showed both haplotypes at similar proportions. In total, eight populations (47%) showed the expected pattern, two populations (12%) were polymorphic within the same field, and seven populations (41%) showed the inverse pattern. Taken together, there was no consistent pattern of host association between the two sympatric genotypes and their respective host plants. This investigation supports the need for additional studies to determine which other forces keep the genotypes separate, and what is the degree of genetic differentiation between these populations.     

Frequency of Cry1F Non-Recessive Resistance Alleles in North Carolina Field Populations of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a target species of transgenic corn (Zea mays L.) that expresses single and pyramided Bacillus thuringiensis (Bt) toxin. In 2014, S. frugiperda were collected from a light trap in North Carolina, and a total of 212 F₁/F₂ isofemale lines of S. frugiperda were screened for resistance to Bt and non-Bt corn. All of the 212 isolines were susceptible to corn tissue expressing Cry1A.105 + Cry2Ab, Cry1F + Cry1A.105 + Cry2Ab, and Cry1F + Cry1Ab + Vip3Aa20. Growth rate bioassays were performed to isolate non-recessive Bt resistance alleles. Seven individuals out of the 212 isofemale lines carried major non-recessive alleles conferring resistance to Cry1F. A pooled colony was created from the seven individuals. This colony was 151.21 times more resistant to Cry1F than a known-susceptible population and was also resistant to Cry1A.105, but was not resistant to Cry2Ab and Vip3Aa20. The results demonstrate that field populations of S. frugiperda collected from North Carolina are generally susceptible to Cry1F, but that some individuals carry resistant alleles. The data generated in this study can be used as baseline data for resistance monitoring.     

Evaluation of substituted oxime ethers for growth regulatory activity against Spodoptera litura (Lepidoptera: Noctuidae)

Insect growth regulator (IGR) activity of 52 substituted oxime ethers was evaluated against an important polyphagous lepidopteran pest, Spodoptera litura (F.) (Lepidoptera: Noctuidae). Several compounds produced symptoms comparable to exogenously applied juvenile hormone. Maximum ICR activity was exhibited by 4'-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3'-buten-2'(E)-ketoxime-N-O-alkyl ether with an ED50 (morphological) of 40 microg g(-1) body weight, compared with 20 microg g(-1) of juvenile hormone (JH) III. Two more compounds, namely, 4'-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3'-buten-2'(Z)-ketoxime-N-O-methyl propyl ether (ED50 of 192 microg g(-1)) and 4'-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3'-buten-2'(E)-ketoxime-N-O-pentyl ether (ED50 of 380 microg g(-1)) showed considerable IGR activity, whereas 4'-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3'-buten-2'(E)-ketoxime-N-O-pentyl ether was found to be acutely toxic to larvae (ED50 of 268 microg g(-1)). Three compounds used in this study also were synergized by piperonyl butoxide. Synergistic ratios ranged from 1.330 to 4.605. No significant ovicidal activity was obtained.     

Biochemical Analysis of a Castor Bean Leaf Extract and its Insecticidal Effects Against Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae)

Plant extracts represent a great source of molecules, with insecticidal activity, which are used for pest control in several crop production systems. This work aimed to evaluate the toxicity of an aqueous extract of leaves of castor bean against larvae of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) in search for different classes of molecules with insecticidal activities by using in vitro assays. The effects of the castor bean leaf extract on the food utilization, development, and survival of S. frugiperda larvae was evaluated by feeding the larvae an artificial diet supplemented with different concentrations of the extract (0%, 1%, 2.5%, 5%, and 10% w/v). The effects observed were dose-dependent, and the highest concentration evaluated (10% w/v) was the one the most affected food utilization by altering the nutritional indices, as well as larval weight gain, development time, and survivorship. In vitro assays to detect saponins, lectins, and trypsin inhibitors in the castor bean leaf extract were performed, but only trypsin inhibitors were detected. No preference for the diet source was detected in S. frugiperda by feeding the larvae in choice experiments with diets containing different concentrations of the castor bean extract tested. The data obtained indicate the existence of a potential molecule in the tested extract of castor bean to be used as an alternative insecticide to be integrated in the management of S. frugiperda.