The mortality of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) neonate larvae in relation to drop-off and soil surface temperature: The dangers of bungy jumping

The first larval instar has been identified as a critical stage for population mortality in Lepidoptera, yet due to the body size of these larvae, the factors that contribute to mortality under field conditions are still not clear. Dispersal behaviour has been suggested as a significant, but ignored factor contributing to mortality in first-instar lepidopteran larvae. The impact that leaving the host plant has on the mortality rate of Helicoverpa armigera neonates was examined in field crops and laboratory trials. In this study the following are examined: (1) the effects of soil surface temperature, and the level of shade within the crop, on the mortality of neonates on the soil after dropping off from the host plant; (2) the percentage of neonates that dropped off from a host plant and landed on the soil; and (3) the effects of exposure to different soil surface temperatures on the development and mortality of neonates. The findings of this study showed that: (1) on the soil, surface temperatures above 43°C were lethal for neonates, and exposure to these temperatures contributed greatly to the overall mortality rate observed; however, the fate of neonates on the soil varied significantly depending on canopy closure within the crop; (2) at least 15% of neonates dropped off from the host plant and landed on the soil, meaning that the proportion of neonates exposed to these condition is not trivial; and (3) 30 min exposure to soil surface temperatures approaching the lethal level (>43°C) has no significant negative effects on the development and mortality of larvae through to the second instar. Overall leaving the plant through drop-off contributes to first-instar mortality in crops with open canopies; however, survival of neonates that have lost contact with a host plant is possible, and becomes more likely later in the crop growing season.

Predator presence moves Helicoverpa armigera larvae to distraction

Displacement of herbivorous insects by the presence of predators on whole plants has rarely been studied. By semi-continuous observations of an externally feeding insect herbivore and a predator, we show how the mere presence of the predator, Geocoris lubra Kirkaldy (Hemiptera: Geocoridae), on a plant can have a strong influence on the movement and behaviors of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. The presence of predators, as opposed to mortality by predators, influenced the proportion of larvae feeding, resting and implementing avoidance activities. In addition, the proportion of time individual larvae allocated to feeding, resting and dropping off plants was affected when predators were present with and without contact between the two. Predators do more than just reduce numbers of herbivores; they influence feeding, displacement and subsequently the distribution of plant damage.

Pest-damage relationships for Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on soybean (Glycine max) and dry bean (Phaseolus vulgaris) during pod-fill.

The response of soybean (Glycine max) and dry bean (Phaseolus vulgaris) to feeding by Helicoverpa armigera during the pod-fill stage was studied in irrigated field cages over three seasons to determine the relationship between larval density and yield loss, and to develop economic injury levels. H. armigera intensity was calculated in Helicoverpa injury equivalent (HIE) units, where 1 HIE was the consumption of one larva from the start of the infestation period to pupation. In the dry bean experiment, yield loss occurred at a rate 6.00 ± 1.29 g/HIE while the rates of loss in the three soybean experiments were 4.39 ± 0.96 g/HIE, 3.70 ± 1.21 g/HIE and 2.12 ± 0.71 g/HIE. These three slopes were not statistically different (P > 0.05) and the pooled estimate of the rate of yield loss was 3.21 ± 0.55 g/HIE. The first soybean experiment also showed a split-line form of damage curve with a rate of yield loss of 26.27 ± 2.92 g/HIE beyond 8.0 HIE and a rapid decline to zero yield. In dry bean, H. armigera feeding reduced total and undamaged pod numbers by 4.10 ± 1.18 pods/HIE and 12.88 ± 1.57 pods/HIE respectively, while undamaged seed numbers were reduced by 35.64 ± 7.25 seeds/HIE. In soybean, total pod numbers were not affected by H. armigera infestation (out to 8.23 HIE in Experiment 1) but seed numbers (in Experiments 1 and 2) and the number of seeds/pod (in all experiments) were adversely affected. Seed size increased with increases in H. armigera density in two of the three soybean experiments, indicating plant compensatory responses to H. armigera feeding. Analysis of canopy pod profiles indicated that loss of pods occurred from the top of the plant downwards, but with an increase in pod numbers close to the ground at higher pest densities as the plant attempted to compensate for damage. Based on these results, the economic injury levels for H. armigera on dry bean and soybean are approximately 0.74 HIE and 2.31 HIE/m2, respectively (0.67 and 2.1 HIE/row-m for 91 cm rows).

Pest-damage relationships for Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on vegetative soybean.

The response of vegetative soybean (Glycine max) to Helicoverpa armigera feeding was studied in irrigated field cages over three years in eastern Australia to determine the relationship between larval density and yield loss, and to develop economic injury levels. Rather than using artificial defoliation techniques, plants were infested with either eggs or larvae of H. armigera, and larvae allowed to feed until death or pupation. Larvae were counted and sized regularly and infestation intensity was calculated in Helicoverpa injury equivalent (HIE) units, where 1 HIE was the consumption of one larva from the start of the infestation period to pupation. In the two experiments where yield loss occurred, the upper threshold for zero yield loss was 7.51 ± 0.21 HIEs and 6.43 ± 1.08 HIEs respectively. In the third experiment, infestation intensity was lower and no loss of seed yield was detected up to 7.0 HIEs. The rate of yield loss/HIE beyond the zero yield loss threshold varied between Experiments 1 and 2 (-9.44 ± 0.80 g and -23.17 ± 3.18 g, respectively). H. armigera infestation also affected plant height and various yield components (including pod and seed numbers and seeds/pod) but did not affect seed size in any experiment. Leaf area loss of plants averaged 841 and 1025 cm2/larva in the two experiments compared to 214 and 302 cm2/larva for cohort larvae feeding on detached leaves at the same time, making clear that artificial defoliation techniques are unsuitable for determining H. armigera economic injury levels on vegetative soybean. Analysis of canopy leaf area and pod profiles indicated that leaf and pod loss occurred from the top of the plant downwards. However, there was an increase in pod numbers closer to the ground at higher pest densities as the plant attempted to compensate for damage. Defoliation at the damage threshold was 18.6 and 28.0% in Experiments 1 and 2, indicating that yield loss from H. armigera feeding occurred at much lower levels of defoliation than previously indicated by artificial defoliation studies. Based on these results, the economic injury level for H. armigera on vegetative soybean is approximately 7.3 HIEs/row-metre in 91 cm rows or 8.0 HIEs/m2.

Development of a synthetic plant volatile-based attracticide for female noctuid moths. I. Potential sources of volatiles attractive to Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae).

This paper is the first of a series which will describe the development of a synthetic plant volatile-based attracticide for noctuid moths. It discusses potential sources of volatiles attractive to the cotton bollworm, Helicoverpa armigera (Hubner), and an approach to the combination of these volatiles in synthetic blends. We screened a number of known host and non-host (for larval development) plants for attractiveness to unmated male and female moths of this species, using a two-choice olfactometer system. Out of 38 plants tested, 33 were significantly attractive to both sexes. There was a strong correlation between attractiveness of plants to males and females. The Australian natives, Angophora floribunda and several Eucalyptus species were the most attractive plants. These plants have not been recorded either as larval or oviposition hosts of Helicoverpa spp., suggesting that attraction in the olfactometer might have been as nectar foraging rather than as oviposition sources. To identify potential compounds that might be useful in developing moth attractants, especially for females, collections of volatiles were made from plants that were attractive to moths in the olfactometer. Green leaf volatiles, floral volatiles, aromatic compounds, monoterpenes and sesquiterpenes were found. We propose an approach to developing synthetic attractants, here termed 'super-blending', in which compounds from all these classes, which are in common between attractive plants, might be combined in blends which do not mimic any particular attractive plant.

Complete genome sequences of seven Helicoverpa armigera SNPV-AC53-derived strains

Wild-type baculovirus isolates typically consist of multiple strains. We report the full genome sequences of seven alphabaculovirus strains derived by passage through tissue culture from Helicoverpa armigera SNPV-AC53 (KJ909666).

Purification and characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera

Phenoloxidases are oxidative enzymes, which play an important role in both cell mediated and humoral immunity. Purification and biochemical characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera (Hubner) were carried out to study its biochemical properties. Prophenoloxidase consists of a single polypeptide chain with a relative molecular weight of 85 kDa as determined by SDSPAGE, MALDITOF MS and LCESI MS. After the final step, the enzyme showed 71.7 fold of purification with a recovery of 49.2%. Purified prophenoloxidase showed high specific activity and homology with phenoloxidase subunit-1 of Bombyx mori and the conserved regions of copper binding (B) site of phenoloxidase. Purified prophenoloxidase has pH optima of 6.8 and has high catalytic efficiency towards the dopamine as a substrate in comparison to catechol and L-Dopa. The PO activity was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and kojic acid.

A Kunitz-type serine protease inhibitor from Butea monosperma seed and its influence on developmental physiology of Helicoverpa armigera

A protease inhibitor from the seeds of Butea monosperma (BmPI) was purified, characterized and studied for its influence on developmental physiology of Helicover-pa armigera. BmPI on two-dimensional separations indicated the presence of a 14 kDa protein with an isoelectric point in the acidic region (pl 5.6). Multiple Sequence Analysis data suggested that the BmPI contains a sequence motif which is conserved in various trypsin and chymotrypsin inhibitors of Kunitz-type. The inhibitor exhibited trypsin inhibitory activity in a broad range of pH (4-10) and temperature (10-80 degrees C). The enzyme kinetic studies revealed BmPI as a competitive inhibitor with a K-i value of 1.2 x 10(-9) M. In vitro studies with BmPI indicated measurable inhibitory activity on total gut proteolytic enzymes of H. armigera (IC(50)2.0 mu g/ml) and bovine trypsin. BmPI supplemented artificial diet caused dose dependent mortality and reduction in growth and weight. The fertility and fecundity of H. armigera, declined whereas the larval-pupal duration of the insect life cycle extended. These detrimental effects on H. armigera suggest the usefulness of BmPl in insect pest management of food crops. (C) 2014 Elsevier Ltd. All rights reserved.

Functional analysis of FP25K of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

The fp25k gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was studied. HearNPV fp25k gene transcription was found starting from about 18 h post-infection, and protein could be detected from the same time with antiserum against FP25K. To study the function of HearNPV fp25k, a recombinant HearNPV (HaBacWD11) with an enhanced green fluorescent protein (GFP) gene replacing the fp25k was constructed using HaBacHZ8, a bacmid of HearNPV that lacks the polyhedrin gene. Growth curve analysis showed that HaBacWD11 produced higher titres of budded viruses (BVs) than its wild-type counterpart HaBacHZ8-GFP. Electron microscopic analysis indicated that at the late stage of infection, the number of intranuclear enveloped nucleocapsids in HaBacWD11-infected cells was much less than that of HaBacHZ8-GFP. A rescue recombinant virus HaBacWD14 was constructed by reintroducing fp25k gene into HaBacWD11. The growth curve and electron microscopic analysis of the rescued recombinant confirmed that the increase of BV yield and the decrease of the virion production in infected cells were the result of fp25k deletion. The expression of membrane fusion protein (Ha133) and ODV-E66 were studied using the FP25K mutants HaBacWD11 and HaBacHZ8-GFP. Unlike FP25K mutants in Autographa californica multicapsid NPV (AcMNPV), which caused an increase in the expression of membrane fusion protein GP64 and a decrease of ODV-E66, no obvious changes at the expression level of Ha133 and ODV-E66 were observed in HearNPV FP25K mutant.

Characterization and phylogenetic analysis of the chitinase gene from the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

A putative chitinase gene was identified within the fragment EcoRI-K of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV, also called HaSNPIV) genome. The open reading frame (ORF) contains 1713 nucleotides (nt) and encodes a protein of 570 amino acids (aa) with a predicted molecular weight of 63.6 kDa. Transcription started at about 18 h post infection (p.i.) and the protein was first detected at 20 h p.i. The times of transcription and expression are characteristic of a late baculovirus gene. 5' and 3' RACE indicated that transcription was initiated from the adenine residue located at -246 nt upstream from the ATG start site and the poly (A) tail was added at 267 nt downstream from the stop codon. This is the first report on the molecular characterization of a chitinase from a single nucleocapsid NPV. The phylogeny of baculoviral chitinase genes were extensively examined in comparison with chitinases derived from bacteria, fungi, nematode, actinomycetes, viruses, insects and mammals. Neighbor-joining and most parsimony analyses showed that the baculoviral chitinases were clustered exclusively within gamma-proteobacteria. Our results strongly suggest that baculoviruses acquired their chitinase genes from bacteria. (C) 2004 Elsevier B.V. All rights reserved.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Selección de un aislado del nucleopoliedrovirus de Helicoverpa armigera (HearNPV) para su uso como base de un bioinsecticida

Helicoverpa armigera (Lepidoptera; Noctuidae), conocida como el taladro del tomate, es una especie polífaga y de amplia distribución, responsable de grandes pérdidas económicas en más de 60 cultivos a lo largo de las regiones tropicales y subtropicales del mundo. Estas plagas se controlan mayoritariamente con plaguicidas químicos, aunque existe un gran interés por desarrollar otros agentes de control biológico. Entre estos, se encuentra el nucleopoliedrovirus de Helicoverpa armigera (HearNPV, Baculoviridae), que por sus características de seguridad y eficacia, sería útil para impulsar los programas de gestión integrada de plagas que se fomentan desde la Directiva 2009/128/CEE. El objetivo de este trabajo fue realizar una caracterización bioquímica y biológica de varios aislados de HearNPV : un aislado silvestre español (Badajoz) HearNPV-SP1, un aislado chino HearSNPV-G4, tres aislados sudafricanos (HearNPV-Whl, HearNPV-Kzn, HearNPV-Alb) y la materia activa de un producto comercial en uso en Europa (HearNPV-Hx). El análisis con las enzimas de restricción determinó que la enzima BglII generaba perfiles similares pero con fragmentos característicos en todos los casos a excepción de los aislados HearNPV-Kzn y HearNPVAlb, que no pudieron ser diferenciados entre sí con ninguna de las enzimas probadas. El análisis filogenético, basado en las secuencias parciales de los genes poliedrina (polh), lef-8 y lef-9, donde se incluyeron las secuencias correspondientes a 18 genomas mostró que el aislado HearNPV-Whl es filogenéticamente próximo a las cepas de origen ibérico, mientras que los aislados HearNPV-Hx y HearNPV-Alb comparten la misma rama que los aislados asiáticos y australiano. La caracterización insecticida de los aislados HearNPV-SP1, HearNPV-Hx y HearNPV-G4 reveló que la virulencia (TMM) del aislado HearNPV-SP1 (104 h) fue significativamente menor que la de los aislados HearNPV-G4 (109 h) y HearNPV-Hx (111 h). En este trabajo, se determinó que el tiempo de acción del HearNPV-SP1 es menor al de otros bioinsecticidas en uso en Europa, por lo que se confirma la posibilidad de mejorar los productos activos en uno de los aspectos más sensibles de cara a su comercialización como es su tiempo de actuación.

Quantitative genetics of preference and performance on chickpeas in the noctuid moth, Helicoverpa armigera

If a novel, resistant host-plant genotype arises in the environment, insect populations utilising that host must be able to overcome that resistance in order that they can maintain their ability to feed on that host. The ability to evolve resistance to host-plant defences depends upon additive genetic variation in larval performance and adult host-choice preference. To investigate the potential of a generalist herbivore to respond to a novel resistant host, we estimated the heritability of larval performance in the noctuid moth, Helicoverpa armigera, on a resistant and a susceptible variety of the chickpea, Cicer arietinum, at two different life stages. Heritability estimates were higher for neonates than for third-instar larvae, suggesting that their ability to establish on plants could be key to the evolution of resistance in this species; however, further information regarding the nature of selection in the field would be required to confirm this prediction. There was no genetic correlation between larval performance and oviposition preference, indicating that female moths do not choose the most suitable plant for their offspring. We also found significant genotype by environment interactions for neonates (but not third-instar larvae), suggesting that the larval response to different plant genotypes is stage-specific in this species.

Mitochondrial DNA analysis of field populations of Helicoverpa armigera (Lepidoptera : Noctuidae) and of its relationship to H. zea

Background
Helicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea.

Results
We obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017 – 0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago.

Conclusion
Our mitochondrial DNA sequence data supports the single species status of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either.