Rapid antibody-based field test to distinguish between Helicoverpa armigera (Lepidoptera: Noctuidae) and Helicoverpa punctigera (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are the two most important insect pests of cotton production in Australia and require application of insecticides to control them. H. armigera has developed resistance to several insecticides but H. punctigera has not. Cost-effective management of insecticide resistance requires that growers be able to determine the proportion of H. armigera eggs or young larvae present on their crop before applying insecticides. This is impossible visually. We generated two monoclonal antibodies that reacted with the insect protein "lipophorin" and were capable of discriminating individuals of the two species at all life-stages. The antibodies were incorporated into a rapid test kit that was tested under field conditions over two growing seasons. Results obtained with the kit agreed closely with those obtained by rearing larvae through to second instar.     

Exon-primed intron-crossing (EPIC) PCR markers of Helicoverpa armigera (Lepidoptera: Noctuidae)

Applying microsatellite DNA markers in population genetic studies of the pest moth Helicoverpa armigera is subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers for H. armigera based on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele 'dropouts'. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species, H. zea, H. assulta and H. punctigera.     

A simple and reliable method for discriminating between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae)

Abstract The cotton bollworm Helicoverpa armigera and the oriental tobacco budworm H. assulta are sibling species, both being important agricultural pests. Morphologically, the two insects are almost indistinguishable at the egg, larval and pupal stages. One of the big challenges in the study of these insects, in particular in integrated pest management, is a timely and dependable identification of these insects at their early stages of development. Here, we report a H. armigera‐specific nuclear DNA marker, and demonstrate that it can be employed to reliably discriminate between H. armigera and H. assulta by simple polymerase chain reaction amplification experiment.     

Mating compatibility among helicoverpa armigera (Lepidoptera: Noctuidae) occurring on selected host plants and Bt cotton survivors

Helicoverpa armigera (Hiibner) (Lepidoptera: Noctuidae) is a well-known polyphagous insect pest. Mating compatibility among the insects occurring on different host plants is essential for free gene flow among populations. We tested the extent of crossability and fecundity of the insects that survived on Bacillus thuringiensis (Bt) cotton with those occurring on pigeon pea, Cajanus cajun (L.) Millsp., non-Bt cotton, Gossypium hirsutum L.; sunflower, Helianthus annuus; sorghum, Sorghum bicolor L. Moench.; okra, Abelmoschus moschatus Medikus; chickpea, Cicer arietinum L.; marigold, Tagetes spp.; and tomato, Lycopersicum esculentum L., crops. The insects from different crops were freely crossable with those collected from Bt cotton and among themselves. The average fecundity across different crosses ranged from 314.1 to 426.3 in direct and from 305.8 to 421.7 eggs per female in reciprocal crosses. In any given cross, a minimum of 85.89% egg hatch was recorded. Furthermore, the F1 individuals of different cross combinations were found to cross freely with their parents (BC1) and among themselves with similar fecundity and egg hatch. High crossability among H. armigera occurring on different host plants suggests that crop mosaics that may exist in countries such as India could play an important role as natural, nonstructured refugia and prolong the durability of the genes deployed for controlling this insect.     

Effects of age and mated status on flight potential of Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract The effects of ageing and female mated status on the flight potential of Helicoverpa armigera (Hübner), collected as larvae from a pigeonpea crop in southern India, were investigated using a tethered-flight technique. In non-mated moths fed sugar solution, from the first night after adult eclosion, the durations of both total and longest continuous flight per night increased up to night 4 and remained at this level until at least night 6. Ovarian maturation was rapid with 77% of unmated moths having commenced oviposition by the third night. On the basis of field evidence it is likely that most females would be mated by the third or fourth night, provided plants with nectar or sugary exudates were locally available. In successfully-mated females a 15-fold decrease in total flight duration and a 28-fold decrease in longest continuous flight duration was observed in contrast to non-mated females of similar age. As host plants suitable for adult feeding and oviposition were locally available during the time of feral adult emergence, synchronous pre-reproductive migration was unlikely to occur in the population studied.     

Return migration of Helicoverpa armigera (Lepidoptera: Noctuidae) during autumn in northern China

The autumn migration of Helicoverpa armigera (Hübner) was observed with radar and two types of light-trap at Langfang, Hebei province, China in 2001 and 2002. The sudden increase in the proportion of H. armigera moths in the searchlight trap indicated migration into the area and catches increased 10-fold during the second half of the night due to the landing of migrants before dawn. The moths' migratory flights took place at up to 2000 m above the ground, and moths flew differentially at times, and heights, when favourable (i.e. northerly) winds occurred. This facilitated the maximum displacement of moths towards the south during these 'return' migrations. The moths flew over the radar site at consistently high densities through the night, and the resulting flight durations of c. 10 h, at displacement speeds of 30-33 km h-1, would allow moths emerging in the far northeast of China (i.e. Liaoning and Jilin provinces and the Inner Mongolia autonomous region) to migrate into northern China (Hebei, Shandong and Henan provinces). The association of the seasonal migratory movements of H. armigera with crops in northern China is briefly discussed.     

Effect of maysin on wild-type,deltamethrin-resistant,and Bt-resistant Helicoverpa armigera (Lepidoptera: Noctuidae)

Larvae of the Old World corn earworm, Helicoverpa armigera (Hübner), were fed diets containing lyophilized silks from maize genotypes expressing varying levels of maysin, a flavone glycoside known to be toxic to the New World corn earworm, Helicoverpa zea Boddie. Three different H. armigera colonies were tested: a wild-type colony (96-S), a colony selected for resistance to deltamethrin (Del-R), and a colony selected for resistance to the Cry1Ac protoxin of Bacillus thuringiensis (Bt-R). A colony of H. zea was also tested as a control. High-maysin silk diets significantly slowed the growth and arrested the development of larvae from all H. armigera colonies compared with low-maysin silk diets, maysin-lacking silk diets, and no-silk control diets. The effects on the H. armigera and H. zea colonies were similar across maysin levels, although H. zea is a larger insect than H. armigera and this overall size difference was observed. Among the H. armigera colonies, maysin effects were generally similar, although 7-d-old Del-R larvae were significantly smaller than 7-d-old Bt-R and 96-S larvae for one no-silk control and two maysin-containing silk treatments. The toxic effect of maysin on the Bt-R and Del-R colonies suggests that physiological mechanisms of H. armigera resistance to Cry1Ac and deltamethrin do not confer cross-resistance to maysin.     

Overwintering survival and spring emergence of Helicoverpa armigera (Lepidoptera: Noctuidae) in northern Greece

Overwintering survival of Helicoverpa armigera (Hübner) was studied under field conditions during the winter of 2004-2005 and 2005-2006 to clarify whether a local population of this insect is established in northern Greece. Additionally, the postdiapause eclosion times of the overwintering generation was compared with adult male dynamics using pheromone traps. Our study supplies strong evidence that part, if not all, of the late-season generation of H. armigera overwinters as diapausing pupa in northern Greece, suggesting that a local population exists in this area. Pupae enter diapause during September and October in response to declining temperatures and photoperiod. A limited number of degree-days in autumn prevented part of the population from developing to the diapausing pupal stage. Larvae of H. armigera that were placed in field conditions late in October were not able to pupate and died. The termination of diapause and the eclosion of adults in the following spring were determined by the local soil temperatures. Diapause terminated between mid-April and early May, and adult emergence occurred within a 4- to 6-wk period from late April to early June. Females emerged slightly earlier than the males. The emergence of 10, 25, 50, 75, and 90% of adults required 153, 199, 252, 303, and 347 DD, respectively. Differences in timing and degree of overlap between adult emergence from the experimental population and pheromone trap catches suggests that the spring population of this species also includes immigrants.     

Effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Lepidoptera: Noctuidae)

The ability to store different insect stadia for prolonged periods provides considerable flexibility and ability to conduct experiments properly. Therefore, studies were undertaken to determine the effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Hübner). The percentage egg hatch and incubation period were significantly (P=0.01) influenced by egg age, storage temperature, and storage duration. Egg hatch ranged from 0.0 to 96.8% across temperatures and storage durations. None of the eggs hatched when stored at -20 and 0 degrees C. The regression model with the optimum Mallow Cp statistic for any of the identified linear and quadratic terms did not improve the precision of prediction in egg hatch beyond 67.0%. Forecasting of incubation period based on egg age, storage duration, and durationxtemperature was quite effective (R2=84.2%). Day degrees required for egg hatching decreased with an increase in temperature from 10 to 27 degrees C, and egg age from 0 to 3 days. The day degree requirements were highest for 0-day-old eggs at 10 degrees C, and lowest at 27 degrees C. Although the incubation period was higher, the hatchability was lower for 0- and 1-day-old eggs stored at constant 10 degrees C, these eggs can be stored for 10 days at 10 degrees C, with a hatchability of >75.0%. It was safer to store the H. armigera eggs for 10 days at 10 degrees C, which will hatch within 1.6 to 2.0 days after restoration at 27 degrees C with a hatchability of >75.0%. This information will be useful in planning and execution of experiments involving H. armigera on various aspects of research in entomology.     

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%).     

Hybridization between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae): development and morphological characterization of F1 hybrids

Reciprocal hybridizations between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) were studied. The cross between females of H. armigera and males of H. assulta yielded only fertile males and sterile individuals lacking an aedeagus, valva or ostium bursae. A total of 492 larvae of the F1 generation were obtained and 374 of these completed larval development and pupated. Only 203 pupae were morphologically normal males, the remaining 171 pupae were malformed. Larvae and pupae that gave rise to morphologically abnormal adults exhibited longer development times. Sterility was not only associated with malformed external sex organs, but also a range of abnormalities of the internal reproductive system: (i) loss of internal reproductive organs, (ii) with one to three copies of an undeveloped bursa copulatrix; or (iii) with one or two undeveloped testes. Normal male hybrid adults showed higher flight activity in comparison with males of both species. In contrast, the cross between females of H. assulta and males of H. armigera yielded morphologically normal offspring (80 males and 83 females). The interaction of the Z-chromosome from H. assulta with autosomes from H. armigera might result in morphological abnormalities found in hybrids and backcrosses, and maternal-zygotic incompatibilities might contribute to sex bias attributed to hybrid inviability.     

Arthropod Natural Enemies of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India

Helicoverpa armigera Hubner Lepidoptera: Noctuidae is one of the most serious insect pests in the Old World. In India, it causes substantial losses to legume, fibre, cereal oilseed and vegetable crops. This paper reviews the literature on the biology, ecology, efficacy, rearing and augmentation of endemic parasitoids and predators, as well as exotic parasitoids introduced and released in India. It also provides updated lists of H. armigera natural enemies native to India. In addition, reports of augmentative releases of Trichogramma spp., the most extensively studied natural enemy of H. armigera are summarized.     

Pyrethroid resistance and mechanisms of resistance in field strains of Helicoverpa armigera (Lepidoptera: Noctuidae)

Pyrethroid resistance was found in 54 field strains of Helicoverpa armigera collected between 1995 and 1999 from 23 districts in seven states of India. LD50 values of the field strains ranged from 0.06 to 72.2 microg/larva with slopes of 0.5-3.1. Resistance was highest in regions where pyrethroid use was frequent (four to eight applications per season). Resistance to deltamethrin was exceptionally high with resistance ratios of 13,570 and 27,160 in two strains collected during February 1998 in central India. Resistance to cypermethrin, fenvalerate and cyhalothrin also was high with resistance ratios of >1,000 in four strains collected from central and southern India. Resistance ratios were below 100 in >50% of the strains tested. Pyrethroid resistance was high in strains collected from the districts in Andhra Pradesh where a majority of the cotton farmer suicide cases in India were reported. Resistance to pyrethroids appeared to have increased over 1995-1998 in most of the areas surveyed. Studies carried out through estimation of detoxification enzyme activity and synergists indicated that enhanced cytochrome p450 and esterase activities were probably important mechanisms for pyrethroid resistance in field strains. Pyrethroid nerve insensitivity also was found to be a major mechanism in some parts of the country where the use of pyrethroids was high. The information presented illustrates the importance of proper insect management programs to avoid the consequences associated with improper insecticide use.     

Effects of constant and changing temperature conditions on diapause induction in Helicoverpa armigera (Lepidoptera: Noctuidae)

The effects of photoperiod and temperature on the induction and termination of facultative pupal diapause in Helicoverpa armigera (Lepidoptera: Noctuidae) were investigated under laboratory conditions. Exposing H. armigera larvae to both constant and fluctuating temperature regimes with a mean of 25°C and 20°C resulted in a type-III photoperiodic response curve of a short-long day insect. The long-day critical daylengths for diapause induction were ten hours and 12 hours at the constant temperatures of 25°C and 20°C, respectively. Higher incidences of diapause and higher values both for the longer and the shorter critical photoperiods for diapause induction were observed at fluctuating regimes compared with the corresponding constant ones. At alternating temperatures, the incidence of diapause ranged from 4.2% to 33.3% and was determined by the temperature amplitude of the thermoperiod and by the interaction of cryophase or thermophase with the photoperiod. Helicoverpa armigera larvae seem to respond to photoperiodic stimuli at temperatures >15°C and <30°C; all insects entered diapause at a constant temperature of 15°C, whereas none did so at a constant temperature of 30°C under all the photoperiodic regimes examined. Although chilling was not a prerequisite for diapause termination, exposure of diapausing pupae to chilling conditions significantly accelerated diapause development and the time of adult emergence. Therefore, temperature may be the primary factor controlling the termination of diapause in H. armigera.     

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of ～ 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50–60 °C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca^+ 2, Mn^+ 2 and Mg^+ 2 somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.     

Baseline toxicity of emamectin and spinosad to Helicoverpa armigera (Lepidoptera: Noctuidae) for resistance monitoring

Acute toxicity studies of emamectin and spinosad against Helicoverpa armigera revealed that the pest is highly susceptible to both the insecticides. The median lethal dose (LD₅₀) of emamectin is 3.86 × 10⁻³ µg per larva. The median lethal concentrations (LC₅₀) of emamectin and spinosad were found to be 0.09 and 2.94 ppm, respectively. The discriminating doses were fixed based on the LC₉₅ of the susceptible population of H. armigera as 0.80 ppm for emamectin and 10 ppm for spinosad. Resistance was not observed when the discriminating doses of emamectin and spinosad were applied on field-collected populations of H. armigera from two intensive cotton growing areas, Coimbatore and Madurai, India.     

In-season changes in resistance to insecticides in Helicoverpa armigera (Lepidoptera: Noctuidae) in India

Discriminating doses of fenvalerate, cypermethrin, quinalphos, and endosulfan were determined with an insecticide-susceptible Helicoverpa armigera (Hübner) strain. In-season changes in insecticide resistance were monitored with discriminating dose assays at weekly intervals throughout the cropping season for 6 yr from 1993 to 1999 in central India. Resistance to pyrethroids was high throughout all seasons. Resistance to 0.75 microg of quinalphos was consistent, with seasonal averages ranging from 23 to 27% survival over the 6 yr. Resistance to 10.0 microg of endosulfan was moderately high at an average of 40-47% survival during 1993-1994 and in 1997-1998. It was lower in 1996-1997 at 27%, and in 1998-1999 at 33%. The weekly monitoring data for all seasons were pooled and the consolidated 6-yr seasonal average profile indicated that resistance to quinalphos and endosulfan was low during September at 21 and 27% survival, respectively, but increased to 28 and 37% by the end of November. Resistance levels to organophosphates and endosulfan increased during the season, depending on the use of these compounds. At almost all monitoring sites, the within-season changes in quinalphos resistance for all seasons through the study period followed a trend similar to that of endosulfan. The results suggest the possibility of cross-resistance between these compounds. Based on this study and the existing information on cotton pest management, we have developed a "window strategy" for cotton pest management with specific emphasis on the management of insecticides for effective control of H. armigera. This strategy has contributed to improved control at reduced costs in extensive trials.