Recombinant Helicoverpa armigera nucleopolyhedrovirus with arthropod‐specific neurotoxin gene RjAa17f from Rhopalurus junceus enhances the virulence against the host larvae

A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV) expressing the insect‐selective neurotoxin (RjAa17f) from Cuban scorpion Rhopalurus junceus was constructed by replacing the UDP‐glucosyltransferase gene (egt) using λ‐red homologous recombination system. Another egt deleted control HearNPV was constructed in a similar way by inserting egfp gene into the egt locus. One‐step viral growth curve and viral DNA replication curve analysis confirmed that the recombination did not affect the viral growth and DNA replication in host cells. There is no discernable difference in occlusion‐body morphogenesis between RjAa17f‐HearNPV, Egfp‐HearNPV and HZ8‐HearNPV, which was confirmed by transmission electron microscopy analysis. However, the insecticidal activity of RjAa17f‐HearNPV is enhanced against the third instar H. armigera larvae according to the bioassay on virulence comparison. There is a dramatic reduction (56.9%) in median lethal dose (LD₅₀) and also a reduction (13.4%) in median survival time (ST₅₀) for the recombinant RjAa17f‐HearNPV compared to the HZ8‐HearNPV, but only a 27.5% reduction in LD₅₀ and 10.1% reduction in ST₅₀ value when Egfp‐HearNPV is compared with HZ8‐HearNPV. The daily diet consumption analysis showed that the RjAa17f‐HearNPV was able to inhibit the infected larvae feeding compared with the egt minus HearNPV. These results demonstrated that this novel recombinant RjAa17f‐HearNPV could improve the insecticidal effect against its host insects and RjAa17f could be a considerable candidate for other recombinant baculovirus constructions.     

Timely inoculation of Helicoverpa armigera nucleopolyhedrovirus improves its efficiency in combination with benzoylphenylurea compounds

Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is highly virulent against cotton bollworm, and its combination with peritrophic membrane affecting chemicals, especially benzoylphenylurea compounds, can increase the relative speed of kill, leading to early and timely pest control. The biological activity of three HearNPV isolates (IR03, IR18, and IR20) was primarily evaluated on the second instar larvae of H. armigera. The highly virulent isolate (IR18, LC₅₀: 8.43 × 10³ OB/ml) and four benzoylphenylurea compounds (diflubenzuron, teflubenzuron, flufenoxuron, and lufenuron) were assessed on the pest larvae according to four per orally diet inoculation schedules. The highest mortality values were obtained when the virus was inoculated on the second (schedule 1) and third (schedule 2) days after flufenoxuron contamination with 98.67% and 97.30%, respectively, followed by teflubenzuron. All the benzoylphenylureas showed the highest mortalities using these two schedules. Flufenoxuron had a higher relative efficiency percent than the other compounds in all the schedules. The second schedule yielded the lowest LT₅₀ value using flufenoxuron with the highest relative speed of kill (38.40%), followed by the first schedule (30.02%). The second schedule can be relatively considered the most appropriate method for the combined use of benzoylphenylurea and HearNPV.     

Density‐dependent polyphenism and baculovirus resistance in teak defoliator,Hyblaea puera (Cramer)

1. Field populations of the teak defoliator larvae, Hyblaea puera Cramer exhibit colour polyphenism under different population densities: greyish‐green with black‐ and orange‐coloured dorsal bands in low‐density endemic populations and uniformly black or intermediate colour during high‐density population. 2. The density dependence of colour polyphenism was confirmed by field monitoring of H. puera populations during 2008–2010. 3. The above findings were later substantiated by rearing H. puera larvae under different densities (i.e. solitary and crowded in the laboratory). Ninety one per cent of the solitary reared laboratory population developed bright coloration whereas, 92% of the group reared larvae turned to black. Eight per cent of larvae from both the rearing densities were of intermediate colour. 4. Density‐dependent resistance build‐up against H. puera nucleopolyhedrovirus by H. puera were tested using the fifth instar larvae. The results showed three‐fold increase of median lethal dose (LD₅₀) value for the group reared larvae (5332 polyhedral occlusion bodies/larvae) compared to the solitary reared ones (1727 polyhedral occlusion bodies/larva) and also significant difference for the mean time to death (3.6 and 3.3 days, respectively). 5. The study revealed the strong influence of larval density on H. puera larval melanism and resistance build‐up against H. puera nucleopolyhedrovirus.     

Geographic variations and their impact on bioefficacy amongst <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> Nuclear Polyhedrosis Virus isolates from India

Helicoverpa armigera Nuclear Polyhedrosis Virus (HearNPV) was isolated from the larva, obtained from four different geographic locations in India. All the HearNPV isolates appeared clear, irregular, six sided in outline with rounded edges; phase bright under phase contrast microscope; but varied in size, bioefficacy and restriction profiles. Anand isolate had largest mean diameter, highest frequency of large polyhedral inclusion bodies and gave highest total percent mortality with lowest LC₅₀ and LT₅₀ followed by Surat, Patancheru and Junagadh isolates. Mortality rate of Anand and Surat isolates projected two peaks at two different time intervals pointing at the presence of more than one genotype in the same host origin. Restriction digestion analysis produced many unique fragments for each isolates proving that variation existed between the four isolates. These studies have clearly demonstrated that baculoviruses are very diverse, and that this diversity occurs at a range of scales from between isolates collected from the same host species, to between caterpillars collected at the same site and, from the current study, within variants collected from within an individual larva. The efficacy of such geographic isolates can be greatly improved by a deeper knowledge of their physical, chemical and biological properties. The knowledge can be applied for strain selection of the viruses from wild populations which would prove useful to develop NPVs desirable for biopesticides.     

Systematic differences in the population density of green spruce aphid, Elatobium abietinum in a provenance trial of Sitka spruce, Picea sitchensis

Quantitative bioassay techniques were used to measure the susceptibility of Heliothis armigera to three nuclear polyhedrosis viruses (NPVs): H. armigera singly-enveloped NPV (HaSNPV), H. zea SNPV (HzSNPV) and H. armigera multiply-enveloped NPV (HaMNPV). Viruses were identified by EcoRI restriction endonuclease analysis. Electrophoretic profiles of DNA fragments revealed that the HaSNPV isolate was a previously undescribed genotypic variant. Bioassays with neonate and 6-day-old larvae measured small but significant differences in virulence between the three viruses. HzSNPV was the most virulent for neonate larvae with a median lethal dose (LD₅₀) of five polyhedra. HaMNPV was least virulent for 6-day-old larvae, with a LD₅₀ of 1400 polyhedra compared with 640–670 polyhedra for HaSNPV and HzSNPV. In addition, the median lethal time (LT₅₀) for infection with HaMNPV in neonate larvae was approximately 1·7 days longer than for the other viruses. Although they varied in virulence, each of the three viruses was sufficiently virulent to have considerable potential as a microbial control agent of H. armigera.     

Inductive and synergistic interactions between plant allelochemical flavone and Bt toxin Cry1Ac in Helicoverpa armigera

Genetically engineered crops simultaneously produce defensive allelochemicals and Bacillus thuringiensis (Bt) toxin proteins to kill some of the world's most devastating insect pests. How the two types of toxins, when ingested sequentially or simultaneously, interact at both lethal and sublethal doses in these pests remains underexplored. Here, we examined the toxicological interactions between the Bt toxin Cry1Ac and the flavonoid allelochemical flavone in Helicoverpa armigera. Simultaneous exposure of H. armigera neonates to lethal doses (LC₂₅) of Cry1Ac and flavone caused a mortality significantly higher than that of either toxin alone and their expected additive mortality. Preexposure for 24 h to a sublethal dose (LC₁₀) of Cry1Ac followed by 6-d simultaneous exposure to the same dose of Cry1Ac plus a lethal dose (1.6 mg/g diets, LC₅₀) of flavone resulted in a mortality significantly higher than that of the LC₅₀ dose of flavone alone and the expected additive mortality of the LC₅₀ dose of flavone plus the LC₁₀ dose of Cry1Ac. One-day preexposure to the sublethal dose (LC₁₀) of flavone followed by 6-d simultaneous exposure to the LC₅₀ dose (6 ng/cm²) of Cry1Ac plus the LC₁₀ dose of flavone yielded a mortality significantly higher than that of the LC₅₀ dose of Cry1Ac but similar to the expected additive mortality of the LC₅₀ dose of Cry1Ac plus the LC₁₀ dose of flavone. The results suggest that Cry1Ac induces and synergizes the toxicity of flavone against H. armigera larvae.     

Phytochemical variation mediates the susceptibility of insect herbivores to entomoviruses

It has been reported that the susceptibility of insect herbivores to entomoviruses is affected by phytochemicals ingested during the acquisition of viral inoculum on the foliage of host plants. However, the relationship between this susceptibility and phytochemicals is poorly understood. To test this hypothesis of plant‐mediated effects on this susceptibility, we measured the effects of foliage from three plants, soybeans (Glycine max), collards (Brassica oleracea) and water convolvuluses (Ipomoea aquatica), on the susceptibility of larval beet armyworm (Spodoptera exigua) to nucleopolyhedrovirus (NPV), and analysed six foliar chemicals (total phenolics, peroxidase [POD], catalase [CAT], superoxide dismutase [SOD], endochitinase and exochitinase) in the three plants, respectively. The results of exponential modelling indicated that the LD_50s (median lethal dose) of NPV to larvae increased with the increase in both phenolics and POD but declined with the increase in four other foliar chemicals, while the opposite trend was found between median lethal time (LT₅₀) of NPV and the six foliar chemicals. This study reveals that phenolics and POD decrease host susceptibility to the entomoviruses and that CAT, SOD, endochitinase and exochitinase increase this susceptibility.     

Resurgence of the cotton bollworm Helicoverpa armigera in northern Greece associated with insecticide resistance

Helicoverpa armigera has been controlled effectively with chemical insecticides in the major cotton crop production areas of northern Greece for many years. However, a resurgence of the pest was observed in 2010, which significantly affected crop production. During a 4‐year survey (2007 – 2010), we examined the insecticide resistance status of H. armigera populations from two major and representative cotton production areas in northern Greece against seven insecticides (chlorpyrifos, diazinon, methomyl, alpha‐cypermethrin, cypermethrin, gamma‐cyhalothrin and endosulfan). Full dose‐response bioassays on third instar larvae were performed by topical application. Lethal doses at 50% were estimated by probit analysis and resistance factors (RF) were calculated, compared to a susceptible laboratory reference strain. Resistance levels were relatively moderate until 2009, with resistance ratios below 10‐fold for organophosphates and carbamates and up to 16‐fold for the pyrethroid alpha‐cypermethrin. However, resistance rose to 46‐ and 81‐fold for chlorpyrifos and alpha‐cypermethrin, respectively in 2010, when the resurgence of the pest was observed. None of the known pyrethroid resistance mutations were found in the pyrethroid‐resistant insects. The possible association between resistance and H. armigera resurgence in Greece is discussed.     

EFFECT OF FEEDING STIMULANT ON THE FEEDING BEHAVIOR AND MORTALITY OF HELICOVERPA ARMIGERA ON DIETS WITH BACILLUS THURINGIENSIS*

Abstract Bacillus thuringiensis (Bt) was noticed to be highly deterrent to the feeding of Helicoverpa armigera larva when incorporated into diets, even at the non‐lethal dosage. As the dosage of Bt increased, the deterrence showed a tendency of elevation. Feeding stimulant, FS‐1 could attract larvae for feeding, and increase larval feeding time and consumption. Addition of 1% (vlv) FS1 could alleviate the deterrent effect of Bt, especialy in low concentration. In no‐choice test, it was observed that while 1% (v/v) FS1 was added, LC₅₀, of Bt underwent a 41% decrease, but in the choice test, the mortality of larvae was not significantly enhanced.     

Baculovirus <Emphasis Type="Italic">per os</Emphasis> infectivity factors are involved in HearNPV ODVs infection of HzAM1 cells <Emphasis Type="Italic">in vitro</Emphasis>

Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry. Foundation items: National Nature Science Foundations of China (30325002, 30470075); National Basic Research Priorities Program of China (2003CB1140).     

Manifold passages in an assorted infection in a host could improve virulence of Helicoverpa armigera Nucleopolyhedrovirus (HaNPV)

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is serious pests of cotton and several other crops. Helicoverpa armigera Nucleopolyhedrovirus (HaNPV) can be important alternative to synthetic insecticides for the management of H. armigera. However, the efficacy of HaNPV can vary in horizontal and vertical transmission. In the current study, we evaluated the efficacy of HaNPV of a virulent strain (vertically transmitted up to six generations) and wild strains (used after isolation from the field infected larvae). Both strains were applied to the 2nd instar larvae of H. armigera @ 1 × 10⁹ polyhedral inclusion bodies (PIB)/ml. There were six replications of each strain (strains). The results indicated higher mortalities in larvae exposed to virulent strains (68.33 ± 6.07%) as compared to wild strain (45 ± 2.24%). Virulent strains killed the larvae quite faster than wild strain. The lethal time (LT₅₀) to kill 50% of the larvae by virulent strain was 7.15 days and for wild strain it was 19.47 days. The results showed that multiple passage of HaNPV through several generations enhances its efficacy to kill H. armigera larvae faster. The results of this study will be helpful to manage H. armigera and other related lepidopoterous pests.     

Molecular and Morphological characterization of multiple nucleopolyhedrovirus from Mexico and their insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae)

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is the most important pest of maize in many countries. Entomopathogenic viruses mainly Baculoviruses family are excellent biological control agents and therefore a viable alternative for managing this pest. The aim of this study was to determine the biological activity of eight native nucleopolyhedrovirus (NPVs) against FAW larvae. Additionally, two of the most virulent isolates (SfCH32 and SfCH15) were characterized biologically by bioassays to estimate their median lethal dose (LD₅₀) and median lethal time (LT₅₀), morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and molecularly by restriction enzymes. Three (SfCH15, SfCH18 and SfCH32) of the eight tested native nucleopolyhedrovirus isolates caused mortalities ˃98% at 168-hr post-inoculation (hpi) with a dose of 9.2 × 10⁴ OBs/larva. SfCH15 and SfCH32 isolates showed occlusion bodies (OBs) of irregular shape and size (1.02–2.24 μm). The SfCH15 and SfCH32 isolates showed similar median lethal dose (5.6 × 10²–6.4 × 10² OBs/larva). The lowest median lethal time (114.5 hpi) was observed with the SfCH15 isolate at the highest concentration (2.5 × 10⁶ OBs/larva). The DNA restriction profiles for SfCH15 and SfCH32 were different, with their genome size being ~128,000 bp and 132,000 bp, respectively. SfCH15 and SfCH32 isolates showed similar morphological characteristics and the highest virulence against fall armyworm. This study showed that native isolates were highly virulent against S. frugiperda larvae, being similar to other reported strains; however, field studies are required to confirm their insecticidal effect.     

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT₅₀ of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60–75 g/kg rice), faster sedimentation time (ST₅₀) (2.3–2.65 h in 0.1% (w/v) Tween 80) and lower LC₅₀ (1.4–5.7×10³ conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).     

Comparative selectivity of pesticides used in greenhouses,on the aphid parasitoid Aphidius colemani (Hymenoptera: Braconidae)

A series of bioassays were conducted under laboratory conditions to determine the relative toxicities of various pesticides (acetamiprid, cypermethrin, chlorantraniliprole and emamectin benzoate, Bacillus thuringiensis var. kurstaki and Helicoverpa armigera nucleopolyhedrovirus, copper oxychloride, iprodione, mandipropamid, a mixture of propamocarb?+?fluopicolide and mixture of fludioxonil?+?cyprodinil) on Aphidius colemani adults and mummies, as well as sublethal effects on female fecundity. Cypermethrin was highly toxic to pupa of A. colemani within host mummies. Acetamiprid, cypermethrin, emamectin benzoate, a mixture of propamocarb?+?fluopicolide and mixture of fludioxonil?+?cyprodinil were also highly toxic to A. colemani adults (92–100% mortality at 48?h post treatment). Mandipropamid, iprodione and copper oxycloride treatments significantly reduced fecundity of the female parasitoids. In contrast B. thuringiensis var. kurstaki, H. armigera nucleopolyhedrovirus and chlorantraniliprole were harmless (<30% mortality) to the parasitoid species tested according to International Organisation for Biological Control toxicity classification and are likely to be compatible with integrated pest management programmes.     

Chemical composition,insecticidal and biochemical effects of Melaleuca alternifolia essential oil on the Helicoverpa armigera

Pesticide resistance has developed as a result of long‐term and extensive use of chemical pesticides. Essential oils from aromatic plants may provide a new and safe alternative to conventional insecticides. In this study, the insecticidal activities of the essential oil of Melaleuca alternifolia and their chemical constituents against Helicoverpa armigera Hubner were investigated, and the underlying mechanisms were studied. The essential oil showed distinct antifeedant (AFC₅₀ = 8.93 mg/ml) and good contact (LD₅₀ = 50.28 μg/larva) activities against H. armigera at 24 hr. Ten chemical components were identified using a gas chromatograph/mass spectrometer, and mainly included terpinen‐4‐ol (40.09%), γ‐terpinene (21.85%), α‐terpinene (11.34%), α‐terpineol (6.91%), α‐pinene (5.86%), terpinolene (3.24%) and 1,8‐cineole (1.83%). Among them, five components were determined and results showed that these constituents possessed obvious antifeedant activities. The activities of acetylcholinesterase and glutathione S‐transferase were notably inhibited by the essential oil, as compared with the control, with strong dose‐ and time‐dependent effects. The results provide a basis for their development and utilization in the control of insects in the future.     

Effect of repeated in vitro sub-culturing on the virulence of Metarhizium anisopliae against Helicoverpa armigera (Lepidoptera: Noctuidae)

The effect of repeated conidial sub-culturing of Metarhizium anisopliae on its virulence against Helicoverpa armigera (Hübner) was studied. The LT₅₀ observed against third instar larvae of H. armigera for the first sub-culture was 3.4 days; it increased to 4.5 and 5.6 days for the 20th and the 40th sub-cultures, respectively. The LT₅₀ values after passage of the 40th sub-culture on H. armigera decreased to 4.4 and 3.7 days for the 40th (first in vivo) and the 40th (fifth in vivo) passages, respectively. Similarly, the LC₅₀ of M. anisopliae towards third instar larvae of H. armigera increased from the first sub-culture (0.17×10⁴) to (3.0×10⁴) for the 40th conidial transfers on potato dextrose agar and again decreased to 0.74×10⁴ and 0.23×10⁴ in the 40th (first in vivo) and the 40th (fifth in vivo) passage, respectively. Similar trends for LC₅₀ and LT₅₀ values were seen when sugarcane woolly aphid, Ceratovacuna lanigera Zehntner was used as a host. Significant variation in appressorium formation and cuticle-degrading enzyme production such as chitinase, chitin deacetylase, chitosanase and protease during subsequent sub-culturing and passage through H. armigera was observed. Though there was no effect on internal transcribed spacer (ITS) sequence pattern, interestingly, in randomly amplified polymorphic DNA (RAPD), significant differences in the band intensities and in the banding pattern for different sub-cultures of M. anisopliae were observed. As stable virulence towards the insect pest is desirable for commercialisation of a mycoinsecticide, such changes in virulence due to repeated in vitro transfer need to be monitored and minimised.     

棉铃虫核型多角体病毒与化学杀虫剂和卵磷脂混用的增效作用

棉铃虫核型多角体病毒(HaNPV)分别与三氟氯氰菊酯、溴氰菊酯、氰戊菊酯、灭净菊酯、灭多威、辛硫磷、甲基对硫磷和乙酰甲胺磷等化学杀虫剂混合饲喂棉铃虫幼虫,统计致死中浓度LC₅₀,计算增效比,测定虫体内与抗性有关的三种重要酶：多功能氧化酶(MFO)、羧酸酯酶(CarE)、乙酰胆碱酯酶(AChE)的活性。研究大豆卵磷脂对HaNPV致病性的影响。结果表明：HaNPV与化学杀虫剂混合饲喂抗性棉铃虫,生测统计增效比均大于1.0,特别是病毒与甲基对硫磷混用,增效比更是达到3.53,表现出良好的增效作用。混剂感染抗性棉铃虫,虫体内MFO的活性比化学杀虫剂单用时降低3～12倍,CarE和AChE的活性也比化学杀虫剂单用时低,HaNPV明显抑制了化学杀虫剂对MFO和CarE的诱导作用。HaNPV与大豆卵磷脂混用,提高了HaNPV对棉铃虫的感染致死率,缩短了致死中时间(LT₅₀)。