Variation in the time‐mortality responses of laboratory and field populations of Helicoverpa armigera infected with nucleopolyhedrovirus

The virulence of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) to South African laboratory and field populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was evaluated. After determining the volume imbibed by each population, bioassays were performed using the droplet feeding method and gradient‐purified HearNPV occlusion bodies. Evaluation of dose‐ and time‐mortality data showed that the median lethal doses (LD₅₀s) did not differ significantly between the populations but that the median lethal time (LT₅₀) of the laboratory population was significantly shorter than the LT₅₀s of the field populations. The study shows the importance of considering both the dose‐ and time‐mortality responses when comparing the response variability of populations to a nucleopolyhedrovirus infection.     

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus‐killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild‐type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild‐type and one of the recombinant viruses. An egt‐negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt‐deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT‐(Androctonus australis Hector) insect‐selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT‐expressing recombinant and the wild‐type virus died at positions significantly lower, compared to infection with the pure wild‐type viral strain. The results indicate that transmission of egt‐negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild‐type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.     

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.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

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.     

Toxicity and Sublethal Effects of Phthalides Analogs to Rhyzopertha dominica

Phthalides and their precursors have demonstrated a large variety of biological activities. Eighteen phthalides were synthesized and tested on the stored grain pest Rhyzopertha dominica. In the screening bioassay, compounds rac‐(2R,2aS,4R,4aS,6aR,6bS,7R)‐7‐bromohexahydro‐2,4‐methano‐1,6‐dioxacyclopenta[cd]pentalen‐5(2H)‐one ( 15 ) and rac‐(3R,3aR,4R,7S,7aS)‐3‐(propan‐2‐yloxy)hexahydro‐4,7‐methano‐2‐benzofuran‐1(3H)‐one ( 17 ) showed mortality similar to the commercial insecticide, Bifenthrin® (≥90 %). The time (LT₅₀) and dose (LD₅₀) necessary to kill 50 % of the R. dominica population were determined for the most efficacious phthalides 15 and 17 . Compound 15 presented the lowest LD₅₀ (1.97 μg g^?1), being four times more toxic than Bifenthrin® (LD₅₀=9.11 μg g^?1). Both compounds presented an LT₅₀ value equal to 24 h. When applied at a sublethal dose, both phthalides (especially compound 15 ), reduced the emergence of the first progeny of R. dominica. These findings highlight the potential of phthalides 15 and 17 as precursors for the development of insecticides for R. dominica control.     

Bioefficacy and mode of action of rocaglamide from Aglaia elaeagnoidea (syn. A. roxburghiana) against gram pod borer,Helicoverpa armigera (Hübner)

Abstract: Rocaglamide, a highly substituted benzofuran, was isolated and identified as the main biologically active component in Aglaia elaeagnoidea (syn. A. roxburghiana) for gram pod borer Helicoverpa armigera (Hübner). Addition of rocaglamide to an artificial diet retarded the growth of neonate larvae in a dose‐dependent manner with EC₅₀ values of 0.76 p.p.m. These values compared favourably with azadirachtin (EC₅₀ = 0.23 p.p.m.). However, azadirachtin was apparently more potent than rocaglamide in inducing growth inhibition via oral administration to these first stadium larvae. The candidate compound was found to have LD₅₀ and LD₉₅ values of 0.40 and 1.02 μg per larva, respectively, in topical application against third instar larvae 96 h post‐treatment. However, these values for azadirachtin were 8.16 and 25.8 μg per larva for the same period. This shows that azadirachtin was less effective against third instar H. armigera larvae in inducing acute toxicity via topical treatment in comparison with rocaglamide. However, severe morphological larval deformities were observed in such azadirachtin‐treated larvae during the process of ecdysis. The cytotoxic nature of rocaglamide was established by evaluating dietary utilization and the results did not implicate any antifeedant effect but the toxicity‐mediated effect due to reduced efficiency of conversion of ingested food. It was obvious that feeding deterrence is not the primary mode of action but a centrally mediated effect, which could be due to the induced cytotoxicity at non‐specific cellular levels.     

Insecticidal and antifeeding activity of Perilla frutescens‐derived material against the diamondback moth,Plutella xylostella L

Insecticidal and antifeeding activities against Plutella xylostella were observed using whole‐plant‐derived Perilla frutescens material. The active ingredient in P. frutescens was identified by spectroscopic analysis as the sesquiterpenoid α‐farnesene, which showed insecticidal activity against third‐instar larva of P. xylostella in a leaf‐dipping bioassay based on 24‐h LD₅₀ values (LD₅₀ = 53.7 ppm). The feeding inhibition rate of α‐farnesene was 82.98% against P. xylostella at 10 ppm, and the antifeeding responses were determined using an oscilloscope to detect electrophysiological responses. The electrophysiological responses of the medial styloconic sensillum (MSS) were approximately 7‐fold more sensitive at 100 ppm than those of the lateral styloconic sensillum (LSS). These results suggest that the insecticidal and antifeeding effect of α‐farnesene, which is a P. frutescens‐derived material, can be used as a potential control agent for P. xylostella.     

Characterization of dihydrolipoamide dehydrogenase from the mitochondria of Helicoverpa armigera,a pest resistant to insecticides

Dihydrolipoamide dehydrogenase (DHLDH) was isolated from the mitochondria of Helicoverpa armigera, a destructive pest which has developed resistance to commonly used insecticides. The flavoenzyme was purified 17.98‐fold to homogeneity with an overall yield of 10.53% by employing ammonium sulfate precipitation, hydroxylapatite chromatography and CM‐Sephadex chromatography. The purified enzyme exhibited the specific activity of 18.7 U/mg and was characterized as a dimer with a subunit mass of 66 kDa. The enzyme showed specificity for nicotinamide adenine dinucleotide – hydrogen (NADH) and lipoamide, as substrates, with Michaelis‐Menten constants (K_m) of 0.083 mmol/L and 0.4 mmol/L, respectively. The reduction reaction of lipoamide by the enzyme could be explained by ping‐pong mechanism. The spectra of DHLDH showed the maximum absorbance at 420 nm, 455 nm and 475 nm. The enzyme activity was strongly inhibited by mercurial and arsenical compounds. The N‐terminal sequence of Ha‐DHLDH showed homology with those of mammalian and arthropod DHLDH. Since H. armigera has developed high levels of resistance to commonly used insecticides, biochemical properties of the metabolic enzymes such as DHLDH, could be helpful to develop insecticidal molecules for the control of H. armigera, with a different mode of action.     

Expression and activity of a probable toxin from Photorhabdus luminescens

As an insect pathogen, Photorhabdus luminescens possesses an arsenal of toxins. Here we cloned and expressed a probable toxin from P. luminescens subsp. akhurstii YNd185, designated as Photorhabdus insecticidal toxin (Pit). The pit gene shares 94% nucleotide and 98% predicted amino acid sequence identity with plu1537, a predicted ORF from P. luminescens subsp. laumondii TT01 and 30% predicted amino acid sequence similarity to a fragment of a 13.6 kDa insecticidal crystal protein gene of Bacillus thuringiensis (Bt). The pit was expressed as a GST-Pit fusion protein in E. coli, most of which was insoluble and sequestered into inclusion bodies. The inclusion bodies were harvested and dissolved. The resultant protein was purified and the Pit was cleaved from the fusion protein by thrombin and purified from GST then used for bioassay. Pit killed Galleria mellonella (LD₅₀, 30 ng/larva) and Spodoptera litura (LD₅₀, 191 ng/larva) via hemocoel injection. Relative to a control that lacked toxin, Pit did not significantly increase mortality of S. litura and Helicoverpa armigera when introduced orally, but the treatment did inhibit growth of the insects. The present study demonstrated that Pit possessed insecticidal activity.     

Chemical Constituents and Insecticidal Activities of Ajania fruticulosa Essential Oil

The insecticidal activity and chemical constituents of the essential oil from Ajania fruticulosa were investigated. Twelve constituents representing 91.0% of the essential oil were identified, and the main constituents were 1,8‐cineole ( 41.40% ), (+)‐camphor ( 32.10% ), and myrtenol (8.15%). The essential oil exhibited contact toxicity against Tribolium castaneum and Liposcelis bostrychophila adults with LD₅₀ values of 105.67 μg/adult and 89.85 μg/cm², respectively. The essential oil also showed fumigant toxicity against two species of insect with LC₅₀ values of 11.52 and 0.65 mg/l, respectively. 1,8‐Cineole exhibited excellent fumigant toxicity (LC₅₀ = 5.47 mg/l) against T. castaneum. (+)‐Camphor showed obvious fumigant toxicity (LC₅₀ = 0.43 mg/l) against L. bostrychophila. Myrtenol showed contact toxicity (LD₅₀ = 29.40 μg/cm²) and fumigant toxicity (LC₅₀ = 0.50 mg/l) against L. bostrychophila. 1,8‐Cineole and (+)‐camphor showed strong insecticidal activity to some important insects, and they are main constituents of A. fruticulosa essential oil. The two compounds may be related to insecticidal activity of A. fruticulosa essential oil against T. castaneum and L. bostrychophila.     

Effects of stilbene optical brighteners on the insecticidal activity of Bacillus thuringiensis and a single nucleopolyhedrovirus on Helicoverpa armigera

The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC₅₀ values) but their effect on Bacillus thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and occlusion bodies (OBs) of a Spanish isolate of H. armigera single nucleocapsid nucleopolyhedrovirus (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD-1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its insecticidal activity. Mixtures of HearNPV-SP1 OBs with Tinopal UNPA-GX or Leucophor UO resulted in 31.4- and 11.4-fold increases in pathogenicity, respectively, at 1%, and 11.4- and 6.3-fold increases in pathogenicity, respectively, at 0.1%, compared to the OBs alone. However, none of these brighteners increased Bt HD-1 activity. These results appear consistent with the hypothesis that the enhancement of HearNPV-SP1 pathogenicity and the null or antagonistic effects observed in Bt HD-1 against H. armigera were due to optical brightener-mediated degradation of the peritrophic membrane, but additional systematic studies involving a broad range of brighteners and electron microscope observations are required to confirm this premise.     

Effects of Bacillus thuringiensisδ-endotoxin-fed Helicoverpa armigera on the survival and development of the parasitoid Campoletis chlorideae

With the deployment of transgenic crops expressing δ‐endotoxins from Bacillus thuringiensis (Bt) for pest management, there is a need to generate information on the interaction of crop pests with their natural enemies that are important for regulation of pest populations. Therefore, we studied the effects of the Bt δ‐endotoxins Cry1Ab and Cry1Ac on the survival and development of the parasitoid Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) reared on Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae fed on Bt toxin‐intoxicated artificial diet. The H. armigera larvae fed on artificial diet impregnated with Cry1Ab and Cry1Ac at LC₅₀ (effective concentration to kill 50% of the neonate H. armigera larvae) and ED₅₀ (effective concentration to cause a 50% reduction in larval weight) levels before and after parasitization resulted in a significant reduction in cocoon formation and adult emergence of C. chlorideae. Larval period of the parasitoid was prolonged by 2 days when fed on Bt‐intoxicated larvae. No adverse effects were observed on female fecundity. The observed effects appeared to be indirect in nature, because no Bt proteins were detected through enzyme‐linked immunosorbent assay in the C. chlorideae larvae, cocoons, or adults fed on Cry1Ab‐ or Cry1Ac‐treated H. armigera larvae. The effects of Bt toxin proteins on C. chlorideae were due to early mortality of H. armigera larvae, that is, before completion of parasitoid larval development.     

Extracts from the bark of Fagara heitzii (Aubr. et Pel.) (Rutaceae) tree are toxic to two weevils and the American cockroach

The bark of the Fagara heitzii tree in the west‐central African Republic of Congo (Congo–Brazzaville) is known anecdotally to provide protection for human residents against fleas and to be of use as a narcotic in fishing (similar to rotenone). We found that powder and hexane extracts from the bark of the Fagara heitzii tree contain insecticidal compounds. Dried powder (14.5 mg) on the bottom of a Petri dish is the residue at 14.494 mg per dish (LD₅₀) for 20 adult maize weevils Sitophilus zeamays after four days. The LD₅₀ of the same material to 20 adult cowpea weevils Callosobruchus maculatus was slightly higher at 16.144 mg/dish (after 48 h). These weevils cause significant economic damage to cowpea and maize in Congo–Brazzaville. Hexane extracts of the bark of Fagara heitzii were also toxic to these weevils. Symptoms of toxicity suggested the extracts were slow‐acting nerve poisons or respiratory inhibitors or both. The time needed to develop full toxicity suggested a respiratory poison. The hexane extract was also toxic to the American cockroach Periplaneta americana. The symptoms in the cockroach were similar, but cockroaches also exhibited apparent escape attempts suggesting they could detect the compounds. Signs of apparent irritation or exaggerated locomotion in all three insects also suggested irritation or nerve effects.     

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.     

Baculovirus‐induced tree‐top disease: how extended is the role of egt as a gene for the extended phenotype?

Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP‐glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree‐top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree‐top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree‐top disease. Here, we studied tree‐top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree‐top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting‐dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree‐top disease in lepidopteran larvae. The conclusion that egt is a ‘gene for an extended phenotype’ is therefore not generally applicable for all baculovirus–host interactions. We hypothesize that in some baculovirus–host systems (including LdMNPV in L. dispar), an effect of egt on tree‐top disease can be observed through indirect effects of egt on moulting‐related climbing behaviour.     

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%).     

Feeding inhibition of Helicoverpa armigera （HiJbner） by Eucalyptus camaldulensis and Tylophora indica extracts

Crude leaf extracts of Eucalyptus camaldulensis and Tylophora indica were evaluated for their antifeedancy against Helicoverpa armigera larvae in no-choice bioassays. Cabbage leaf-disc dipped in different concentrations of leaf extracts was used for feeding fifth instars of H. armigera for 24 hours. Ethanol extracts from both the plant species exhibited significant feeding inhibition, activity. The effective concentrations of ethanol extracts that caused 50% reduction in larval feeding （EI50） were 6.9% for E. camaldulensis and 2.8% for T. indica, while these values of hexane extracts were 9.3% and 5.2%, respectively. Compared to the crude leaf extracts, crude alkaloids from T. indica and crude tannins from E. camaldulensis were more potent in reducing larval feedings.