Geographic variation in diapause induction and termination of the cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

Overwintering diapause in Helicoverpa armigera, a multivoltine species, is controlled by response to photoperiod and temperature. Photoperiodic responses from 5 different geographical populations showed that the variation in critical photoperiod for diapause induction was positively related to the latitudinal origin of the populations at 20, 22 and 25 °C. Diapause response to photoperiod and temperature was quite different between northern and southern populations, being highly sensitive to photoperiod in northern populations and temperature dependence in southern populations. Diapause pupae from southern population showed a significantly shorter diapause duration than from northern-most populations when they were cultured at 20, 22, 25, 28 and 31 °C; by contrast, overwintering pupae from southern populations emerged significantly later than from northern populations when they were maintained in natural conditions, showing a clinal latitudinal variation in diapause termination. Diapause-inducing temperature had a significant effect on diapause duration, but with a significant difference between southern and northern populations. The higher rearing temperature of 22 °C evoked a more intense diapause than did 20 °C in northern populations; but a less intense diapause in southern population. Cold exposure (chilling) is not necessary to break the pupal diapause. The higher the temperature, the quicker the diapause terminated. Response of diapause termination to chilling showed that northern populations were more sensitive to chilling than southern population.     

Analysis of pupal head proteome and its alteration in diapausing pupae of Helicoverpa armigera

The proteomic approach has proven to be an useful tool for understanding insect diapause processes. Using 2D gel electrophoresis and matrix assisted laser/desorption ionization (MALDI) time of flight (TOF), we identified 24 proteins in the head of Helicoverpa armigera pupae with diverse functional characteristics, including cytoskeleton proteins, heat-shock proteins, insect development regulation factors, ATPases, proteins regulating signal pathway and enzymes involved in metabolism, etc. A proteomic comparison between nondiapausing and diapausing pupae revealed three proteins that were present only in nondiapausing pupae, and six proteins represented ≥2.0-fold or ≤0.5-fold changes. The differentially expressed proteins, including heat-shock protein 90, chitin deacetylase, alpha-tubulin and transitional endoplasmic reticulum ATPase, etc. were reported for the first time in H. armigera. Identification of these proteins will enable us to further characterize the regulated functions of diapause in this important species.     

Effects of Bacillus thuringiensis toxin Cry1Ac and cytoplasmic polyhedrosis virus of Helicoverpa armigera (Hübner) (HaCPV) on cotton bollworm (Lepidoptera: Noctuidae)

In this study, interactions on the mortality and debilitating effects between Cry1Ac, a toxic protein produced by Bacillus thuringiensis (Berliner) and HaCPV (Chinese strain) on first and third instars larvae of Helicoverpa armigera were evaluated in laboratory. When first instar was exposed to combination of Bt cotton leaf discs containing HaCPV (6 × 10⁶, 1 × 10⁷, and 3 × 10⁷ PIB ml⁻¹) the effect on mortality was additive, when such instar larvae exposed to combination of Cry1Ac (0.9, 2.7, or 8.1 μg g⁻¹) and the same concentrations of HaCPV the effect on mortality was additive except for the combination of Cry1Ac (0.3 μg g⁻¹) and HaCPV concentrations that showed synergism. When third instars of H. armigera were infected using a suspension containing both HaCPV and Cry1Ac, most combinations of them showed additive effect except for the combination of Cry1Ac (0.3 μg g⁻¹) and HaCPV (3 × 10⁷ PIB ml⁻¹) that showed synergism. However, when they exposed to Bt cotton leaf discs and HaCPV the effect on mortality was synergism except combination of Bt cotton leaf discs and HaCPV (6 × 10⁶ PIB ml⁻¹) that showed additive. Most of the combinations are showed additive effect in the toxicity and in combinations of Cry1Ac at lowest and HaCPV at highest concentrations synergism is observed. Not only were larval growth and development delayed, but pupation and pupal weight also decreased when larvae were fed on artificial diet containing Cry1Ac and HaCPV or transgenic Bt cotton leaf discs specially in first instar.     

Effects of heat shock on survival and reproduction of Helicoverpa armigera (Lepidoptera: Noctuidae) adults

1.

Under natural conditions insects are occasionally subjected to thermal stresses. Data concerning the effects of these temperature extremes on tolerance and on life history parameters of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) have been lacking.     

Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates

To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated. Respiration rates, measured as the rate of CO₂ production (VCO₂), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD₂₅ or LD₇₅ survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO₂ values of larvae that survived inoculation (0.0288 ml h⁻¹), the uninoculated controls (0.0250 ml h⁻¹), and the larvae that did not survive inoculation (0.0199 ml h⁻¹) differed significantly from one another. At 4 dpi, the VCO₂ of the uninoculated controls were significantly lower than the VCO₂ of inoculation survivors, but significantly higher than the VCO₂ of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4 dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.     

Inheritance of Cry1Ac resistance and associated biological traits in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

The analysis of reciprocal genetic crosses between resistant Helicoverpa armigera strain (BH-R) (227.9-fold) with susceptible Vadodara (VA-S) strain showed dominance (h) of 0.65-0.89 and degree of dominance (D) of 0.299-0.782 suggesting Cry1Ac resistance as a semi-dominant trait. The D and h values of F₁ hybrids of female resistant parent were higher than female susceptible parent, showing maternally enhanced dominance of Cry1Ac resistance. The progeny of F₂ crosses, backcrosses of F₁ hybrid with resistant BH-R parent did not differ significantly in respect of mortality response with resistant parent except for backcross with female BH-R and male of F₁ (BH-R × VA-S) cross, suggesting dominant inheritance of Cry1Ac resistance. Evaluation of some biological attributes showed that larval and pupal periods of progenies of reciprocal F₁ crosses, backcrosses and F₂ crosses were either at par with resistant parent or lower than susceptible parent on treated diet (0.01 μg/g). The susceptible strain performed better in terms of pupation and adult formation than the resistant strain on untreated diet. In many backcrosses and F₂ crosses, Cry1Ac resistance favored emergence of more females than males on untreated diet. The normal larval period and the body weight (normal larval growth) were the dominant traits associated with susceptible strain as contrast to longer larval period and the lower body weight (slow growth) associated with resistance trait. Further, inheritance of larval period in F₂ and backcross progeny suggested existence of a major resistant gene or a set of tightly linked loci associated with Cry1Ac sensitivity.     

Characterization of a Cry1Ac toxin-binding alkaline phosphatase in the midgut from Helicoverpa armigera (Hübner) larvae

Midgut membrane-bound alkaline phosphatases (mALP) tethered to the brush border membrane surface by a glycosylphosphatidylinositol (GPI) anchor have been proposed as crucial for Cry1Ac intoxication. In the present work, two full-length cDNAs-encoding alkaline phosphatases in the midgut of Helicoverpa armigera larvae were cloned and named HaALP1 (GenBank accession no. EU729322) and HaALP2 (GenBank accession no. EU729323), respectively. These two clones displayed high identity (above 94%) at the amino acid sequence, indicating that they may represent allelic variants, and were predicted to contain a GPI anchor. Protein sequence alignment revealed that HaALPs were grouped with mALP from the Heliothis virescens midgut. The HaALP1 and HaALP2 (∼68 kDa) proteins were heterologously expressed in Sf9 cells using a baculovirus expression system and purified to homogeneity. Ligand blot and dot blot analysis revealed that the Cry1Ac bound to both denatured and native purified HaALPs. Data from lectin blots, competition assays with soybean agglutinin (SBA) lectin and GalNAc binding inhibition assays were indicative of the presence of GalNAc on HaALPs and binding of Cry1Ac toxin to this residue. This observation was further confirmed through N-glycosidase digestion of HaALPs, which resulted in reduced Cry1Ac binding. Our data represent the first report on HaALPs and their putative role as receptors for Cry1Ac toxin in H. armigera.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants

Midgut digestive amylases and proteinases of Helicoverpa armigera, a polyphagous and devastating insect pest of economic importance have been studied. We also identified the potential of a sorghum amylase inhibitor against H. armigera midgut amylase. Amylase activities were detected in all the larval instars, pupae, moths and eggs; early instars had lower amylase levels which steadily increased up to the sixth larval instar. Qualitative and quantitative differences in midgut amylases of H. armigera upon feeding on natural and artificial diets were evident. Natural diets were categorized as one or more members of legumes, vegetables, flowers and cereals belonging to different plant families. Amylase activity and isoform patterns varied depending on host plant and/or artificial diet. Artificial diet-fed H. armigera larvae had comparatively high amylase activity and several unique amylase isoforms. Correlation of amylase and proteinase activities of H. armigera with the protein and carbohydrate content of various diets suggested that H. armigera regulates the levels of these digestive enzymes in response to macromolecular composition of the diet. These adjustments in the digestive enzymes of H. armigera may be to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. H. armigera, a generalist feeder experiences a great degree of nutritional heterogeneity in its diet. An investigation of the differences in enzyme levels in response to macronutrient balance and imbalance highlight their importance in insect nutrition.     

In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors

We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro- and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera.     

A true summer diapause induced by high temperatures in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Summer diapause in the cotton bollworm, Helicoverpa armigera (Hübner), which prolongs the pupal stage, particularly in males, is induced by high temperatures. In the laboratory, summer-diapausing pupae of H. armigera were induced at high temperatures (33-39 degrees C) with a photoperiod of LD8:16; winter-diapausing and non-diapausing pupae, cultured at 20 degrees C with a photoperiod of LD8:16 and at 27 degrees C, LD16:8, respectively, acted as a control. Retention time of eye spots, weight, and lipid and glycogen levels were compared. At high temperatures, both body weight and energy storage capacity were much higher in summer-diapausing pupae than in non-diapausing pupae reared at 33-39 degrees C. At temperatures (>33 degrees C) high enough to maintain summer diapause, the eye spots of summer-diapausing pupae did not move during the 30-day experiment. However, eye spots of summer-diapausing pupae placed at 30 degrees C began to move about 10 days after they were transferred, significantly later than in non-diapausing pupae reared at 33-39 degrees C or non-diapausing pupae reared at 27 degrees C, which initiated eye spot movement 2 days after pupation. The differences in retention time of eye spots between summer- and winter-diapausing pupae shows that winter diapause is more intense than summer diapause in this insect. The weight loss, and lipid and glycogen metabolism curves indicate that the summer-diapausing pupae's metabolism is very low. We conclude that summer diapause in the cotton bollworm is a true diapause and that the summer diapause enables the cotton bollworm to withstand the high temperatures of summer.     

Oviposition deterrents in larval frass of the cotton boll worm, Helicoverpa armigera (Lepidoptera: Noctuidae): chemical identification and electroantennography analysis

Oviposition deterrents in the frass of cotton bollworm (CBW), Helicoverpa armigera larvae fed on an artificial diet (FA) and on cotton Gossypium hirsutum leaves (FC) were investigated by behavioral bioassays and electroantennography analyses in the laboratory. It was found that a water suspension or a hexane extract of the frass FA or FC, in contrast to the corresponding foods, significantly deterred oviposition of conspecifics. When hexane extracts of the frass FA and FC were further partitioned into polar and neutral lipid fractions, two polar fractions significantly reduced oviposition. The neutral fraction from frass FC also exhibited significant deterrence, although the activity was much lower than that of the corresponding polar fraction. The polar lipid fractions contained several fatty acids, mainly palmitic and oleic acid at the ratio nearly 1:1. A blend of authentic fatty acids of the same composition found in frass FA or FC mimicked the deterring effect. Moreover, these fatty acids and their blend at the ratio found in frass FA or FC elicited significant electroantennogram responses and typical dose-response curves. Thus, it is suggested that CBW larvae may deploy two types of oviposition deterrents: a non-specific and a specific one. The former is a blend of fatty acids, independent of food and plays an important role in oviposition deterrence, whereas the latter may be produced only when the larvae feed on cotton leaves. The possible explanations of this deployment have also been discussed.     

Differential expression, phosphorylation of COX subunit 1 and COX activity during diapause phase in the cotton bollworm, Helicoverpa armigera

Helicoverpa armigera (Lepidoptera, Noctuidae) is an important agricultural pest with a pupal diapause. Cytochrome c oxidase (COX) is a key speed-limited enzyme of oxidative phosphorylation in mitochondria for ATP production. A differentially expressed cDNA fragment encoding COX subunit 1 (cox1) was cloned by differential display-PCR from the pupal brain at diapause termination with an injection of ecdysone. We then obtained the full length of H. armigera cox1 (Hea-cox1) cDNA which has an open reading frame of 1530 nucleotides encoding a putative protein of 510 amino acid residues, with CGA as a start codon. To evaluate the response to different energy demands during pupal development and at diapause termination, we assessed the expression of Hea-cox1 mRNA and protein, COX activity and its phosphorylation. The results show that Hea-cox1 expression at the mRNA and protein levels is associated with COX activity, and high levels of Hea-cox1 expression and COX activity are present in nondiapause pupae, suggesting that low energy metabolism provided by oxidative phosphorylation in mitochondria in diapause individuals is necessary. After diapause is broken by injection of 20-hydroxyecdysone, expression of Hea-cox1 mRNA and protein increases gradually and COX activity increases significantly. Furthermore, Hea-cox1 phosphorylation is closely correlated with COX activity, suggesting that reversible protein phosphorylation may play a key role in insect diapause by suppressing the rate of energy production.     

Relationship between oviposition preference and offspring performance in Australian Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Abstract We investigated the oviposition preference and larval performance of Helicoverpa armigera under laboratory conditions to determine if the oviposition preference of individual females on maize, cowpea and cotton correlates with offspring performance on the leaves of the same host plants. The host-plant preference hierarchy of females did not correlate with their offspring performance. Female moths chose host plants that contributed less to their offspring fitness. Plant effects accounted for the largest amount of variation in offspring performance, while the effects of female (family) was low. The offspring of most females (80%, n  = 10) were broadly similar, but 20% (two out of 10), showed marked difference in their offspring performance across the host-plant species. Similarly, there was no relation between larval feeding preference and performance. However, like most laboratory experiments, our experi-mental design does not allow the evaluation of ecological factors (for example, natural enemies, host abundance, etc.) that can play an important role in larval performance in the field. Overall, the results highlight the importance of carrying out preference performance analysis on the individual or family level, rather than pooling individuals to obtain average population data.     

A proteomic analysis of Helicoverpa armigera adults after exposure to UV light irradiation

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. To gain a better understanding of the response of Helicoverpa armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1 h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in phototactic insects under UV light irradiation conditions and gives new insights into the adaptation mechanisms responsive to UV light irradiation stress.     

Ultraviolet light-induced oxidative stress: Effects on antioxidant response of Helicoverpa armigera adults

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. In this article, we test the hypothesis that one of the effects of UV light irradiation is to increase oxidative stress on insects. The effects of UV light irradiation on total antioxidant capacity, malondialdehyde (MDA) and protein carbonyl contents and the activities of superoxide dismutase (SOD), catalase (CAT), peroxidases (POX) and glutathione-S-transferase (GST) were investigated in Helicoverpa armigera adults. The adults were exposed to UV light for various time periods (0, 30, 60 and 90 min). We found that exposure to UV light for 30 min resulted in increased total antioxidant capacity, protein carbonyl content and activities of SOD, CAT, POX and GST. When the exposure time lasted for 60 and 90 min, the protein carbonyl content and activities of CAT and GST remained significantly higher than the control. However, the antioxidant capacity and SOD activity returned to control levels, and POX activity decreased at 60 and 90 min. Our results confirm the hypothesis that UV light irradiation increases the level of oxidative stress in H. armigera adults.     

Histopathology and ultrastructure of midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) fed Bt-cotton

The interaction of Cry toxins from Bacillus thuringiensis in the midgut of some insect larvae determines their efficacies as insecticides, due to the expression and availability of sites of action of the toxin in the midgut. Researches point out cases of resistance to Cry toxin due to alterations in the binding sites in columnar cell membrane. We analyzed the effects of Cry1Ac toxin expressed by Bt-cotton plants on Alabama argillacea midgut morphophysiology clarifying in levels of morphological and ultrastructural. Larvae in the 4th instar of A. argillacea after 20 min from ingesting Bt-cotton leaves expressing 0.183 ng of Cry1Ac exhibited ultrastructural and morphological modifications in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolization, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells are not the only target of the Cry1Ac toxin. The regenerative cells did not modify their structures and exhibited decrease in regeneration capacity. In conclusion, the ingestion of 0.183 ± 0.077 ng of Cry1Ac was enough to promote alterations in the columnar and goblet cells, besides reducing significantly the number of regenerative cells, which may have contributed to larval death. Nevertheless, further studies are necessary to determine the true cause of death.     

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 °C under a 60 ± 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C and 25 °C under a 60 ± 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 °C, and 12.2, 48.8 and 39.0% at 25 °C, respectively. On the other hand, when mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C, 18 °C and 25 °C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 °C, 14.5, 26.9 and 58.6% at 18 °C, and 8.3, 21.2 and 70.5% at 25 °C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.