Improved tolerance against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> in transgenic tomato over-expressing multi-domain proteinase inhibitor gene from <Emphasis Type="Italic">Capsicum annuum</Emphasis>

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae.     

Avidin expressed in transgenic tobacco leaves confers resistance to two noctuid pests,Helicoverpa armigera and Spodoptera litura

Fertile transgenic tobacco plants with leaves expressing avidin in the vacuole have been produced and shown to halt growth and cause mortality in larvae of two noctuid lepidopterans, Helicoverpa armigera and Spodoptera litura. Late first instar H. armigera larvae and neonate (<12-h-old) S. litura larvae placed on leaves excised from T₀ tobacco expressing avidin at 3.1–4.6M (moles/kg of fresh leaf tissue) had very poor growth over their first 8 days on the leaves, significant numbers had died by days 11 or 12 and all were dead by day 22 (H. armigera) or day 25 (S. litura). Similar results were obtained when late first instar H. armigera larvae were placed on leaves from T₁ plants expressing avidin at six different average concentrations, ranging from 3.7 to 17.3M. Two larvae on the lowest expressing leaves survived to pupation, but there was total mortality among the other groups and no relationship between avidin concentration and the effects on the larvae. Synergistic effects between avidin-expressing tobacco plants and a purified Bt toxin, Cry1Ba, were demonstrated. Late instar H. armigera larvae fed with leaves from T₂ plants expressing avidin at average concentrations of either <5.3 or >12.9M, and painted with Cry1Ba protein at a rate equivalent to an expression level of 0.5% of total leaf protein, died significantly faster than larvae given either of the two treatments alone. Larvae fed with avidin-expressing leaves painted with the protease inhibitor, aprotinin, at a rate equivalent to 1% of total leaf protein had mortality similar to those given avidin-leaves alone. There was no evidence of antagonism between these two proteins.     

A novel alphabaculovirus isolated from the cotton bollworm, <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hubner) (Lepidoptera: Noctuidae): characterization and pathogenicity

The cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous pest that exist all over the world. It is also very destructive in Turkey on various agricultural products. In this study, we have detected an alphabaculovirus from Helicoverpa armigera larvae collected from cotton field. The virus isolate was named as Helicoverpa armigera nucleopolyhedrovirus (HearNPV-O1) based on morphological and molecular properties. This is the first record of baculovirus from H. armigera in Eurasia region. Scanning electron microscopy examinations of polyhedral inclusion bodies (PIBs) showed their irregular morphology with average diameter of 0.85 to 1.25 μm. Transmission electron microscopy studies revealed that the nucleocapsids were multiple enveloped and bacilliform shaped. The size of nucleocapsid was found as 279 nm with a width of 56 nm. Digestion of viral genome by PstI generated 8 fragments with total size of 130.9 kbp. According to the phylogenetic analysis of the partial polyhedrin (polh), late expression factor 8 (lef8) and late expression factor 9 (lef9) genes, HearNPV-O1 isolate is very close to H. armigera MNPV Indian isolate-443. Five different concentrations of HearNPV-O1 between 10³ and 10⁷ were used in dose-response test on neonate, 3rd and 5th instars larvae of H. armigera. The highest dose (10⁷) showed 92%, 88% and 57% mortality, respectively within 14 days. LC₅₀ values of HearNPV-O1 isolate were calculated as 6?×?10⁴, 7?×?10⁴ and 8?×?10⁶ PIBs/mL^?1 against neonate, 3rd and 5th instars larval stages, respectively. These results demonstrate that HearNPV-O1 isolate may be a potential biocontrol agent to be utilized against H. armigera.     

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for pest resistance management in China.     

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.     

Activity of glutathione S‐transferase and esterase enzymes in Helicoverpa armigera (Hübner) after exposure to entomopathogenic fungi

Helicoverpa armigera, a polyphagous insect of crops and vegetables, is acquiring resistance against many commercial insecticides. The present study shows variations in the activity of two detoxification enzymes, namely esterase and glutathione S‐transferase (GST), in H. armigera after exposure to different isolates of entomopathogenic fungi. After treatment of larvae with the different isolates (Day 0), samples were collected on three days (Days 3, 5 and 7) for enzyme analysis. High GST activity was found in samples of hemolymph, intestine and fat bodies of H. armigera following treatment with Beauveria bassiana (isolate Bb‐08), Metarhizium anisopliae (isolates Ma‐11.1 and Ma‐4.1), and Isaria fumosorosea (isolates If‐02 and If‐2.3). High esterase activity was recorded in samples of the intestine and fat bodies on various days after treatment, whereas increased esterase activity in hemolymph was noted only in samples from Day 5 after treatment with M. anisopliae (Ma‐4.1). The detection of high GST and esterase activity demonstrates the possibility of the development of resistance against these microbial control agents in H. armigera.     

Evaluation of flubendiamide‐induced mitochondrial dysfunction and metabolic changes in Helicoverpa armigera (Hubner)

Phthalic acid diamide insecticides are the most effective insecticides used against most of the lepidopteran pests including Helicoverpa armigera, a polyphagous pest posing threat to several crops worldwide. The present studies were undertaken to understand different target sites and their interaction with insect ryanodine receptors (RyR). Bioassays indicated that flubendiamide inhibited the larval growth in dose‐dependent manner with LD₅₀ value of 0.72 μM, and at 0.8 μM larval growth decreased by about 88%. Flubendiamide accelerated the Ca²⁺‐ATPase activity in dose‐dependent trend, and at 0.8 μM, the activity was increased by 77.47%. Flubendiamide impedes mitochondrial function by interfering with complex I and F₀F₁‐ATPase activity, and at 0.8 μM the inhibition was found to be about 92% and 50%, respectively. In vitro incubation of larval mitochondria with flubendiamide induced the efflux of cytochrome c, indicating the mitochondrial toxicity of the insecticide. Flubendiamide inhibited lactate dehydrogenase and the accumulation of H₂O₂, thereby preventing the cells from lipid peroxidation compared to control larvae. At 0.8 μM the LDH, H₂O₂ content and lipid peroxidation was inhibited by 98.44, 70.81, and 70.81%, respectively. Cytochrome P450, general esterases, AChE, and antioxidant enzymes (catalase and superoxide dismutase) exhibited a dose‐dependent increasing trend, whereas alkaline phosphatase and the midgut proteases, except amino peptidase, exhibited dose‐dependent inhibition in insecticide‐fed larvae. The results suggest that flubendiamide induced the harmful effects on the growth and development of H. armigera larvae by inducing mitochondrial dysfunction and inhibition of midgut proteases, along with its interaction with RyR.     

Insecticide resistance and detoxifying enzyme activity in the principal bancroftian filariasis vector, Culex quinquefasciatus, in northeastern India

The insecticide resistance status of Culex quinquefasciatus Say (Diptera: Culicidae) to DDT and deltamethrin across army cantonments and neighbouring villages in northeastern India was investigated. In India, DDT is still the insecticide of choice for public health programmes. In military stations, pyrethroids, especially deltamethrins, are used for insecticide‐treated nets (ITNs). Recent information on the levels of resistance to DDT and deltamethrin in mosquito populations of northeastern India is scare. Continued monitoring of insecticide resistance status, identification of the underlying mechanisms of resistance in local mosquito populations and the establishment of a baseline data bank of this information are of prime importance. Insecticide susceptibility assays were performed on wild‐caught adult female Cx. quinquefasciatus mosquitoes to the discriminating doses recommended by the World Health Organisation (WHO) to DDT (4%) and deltamethrin (0.05%). Across all study sites, mortality as a result of DDT varied from 11.9 to 50.0%, as compared with 91.2% in the susceptible laboratory strain (S‐Lab), indicating that Cx. quinquefasciatus is resistant to DDT. The species was found to be 100% susceptible to deltamethrin in all study sites except Benganajuli and Rikamari. Knock‐down times (KDT) in response to deltamethrin varied significantly between study sites (P < 0.01) from 8.3 to 17.8 min for KDT₅₀ and 37.4 to 69.5 min for KDT₉₀. All populations exceeded the threshold level of alpha‐esterase, beta‐esterase and glutathion S‐transferase (GST) established for the S‐Lab susceptible strain, and all populations had 100% elevated esterase and GST activity, except Missamari and Solmara. Beta‐esterase activity in Field Unit II (96.9%) was less than in any of the other populations. Benganajuli had the highest activity level for all the enzymes tested. There was a significant correlation between all enzyme activity levels and insecticide resistance phenotype by populations (P < 0.05). The results presented here provide the first report and baseline information of the insecticide resistance status of Cx. quinquefasciatus in northeastern India, and associated information about biochemical mechanisms that are essential for monitoring the development of insecticide resistance in the area.     

The entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera

In this study, we investigated the effect of the entomopathogenic fungus Nomuraea rileyi on Helicoverpa armigera cellular immune responses. Nomuraea rileyi infection had no effect on total hemocyte count (THC), but impaired hemocyte‐mediated phagocytosis, nodulation, and encapsulation responses. Nomuraea rileyi infection led to a significant reduction in hemocyte spreading. An in vitro assay revealed that plasma from N. rileyi infected H. armigera larvae suppressed the spreading ability of hemocytes from naïve larvae. We infer that N. rileyi suppresses the cellular immune response of its host, possibly by secreting exogenous, cytotoxic compounds into the host's hemolymph.     

Partial purification of Cytochrome P450 from Helicoverpa armigera (Hübner)

Abstract The cytochrome P450 (Cyt‐P450) proteins from the fat body and midgut of the cotton bollworm, Helicoverpa armigera, were respectively partially purified by a set of purification procedures including differential centrifugation, solubilization of CHAPS, protein precipitation by PEG precipitation and DE‐32 column chromatography. The Cyt‐P450 was detected by methods of CO difference spectrum and SDS‐PAGE. Fraction of detergent solubilized microsomes from the fat body of H. armigera was purified more than 17‐fold. Three protein bands were detected by SDS‐PAGE with molecular masses of 70 600, 63 300 and 571 200Da. It is possible that the proteins with molecular mass of 63 300 and 571 200Da were the isozymes of Cyt‐P450.     

Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

Stay or move: how Bt‐susceptible Helicoverpa armigera neonates behave on Bt cotton plants

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae occasionally have been reported to survive at management threshold levels in fields of Bollgard II^® cotton, Gossypium hirsutum L. (Malvaceae). The pattern and degree of larval survival is not easily predicted but depends on the ability of first instars to establish on host plants. Experiments were conducted with Bacillus thuringiensis Berliner (Bt)‐susceptible and Bt‐resistant larvae of H. armigera to understand how physiologically Bt‐susceptible H. armigera survive on Bt cotton plants, and examine how their first meal influences survival rates. In assays using cotton plant parts, both strains of larvae displayed similar tendencies to drop‐off specific plant parts of Bt and non‐Bt cotton. However, significantly more Bt‐susceptible larvae dropped off young leaves, mature leaves, and squares of Bt cotton compared to non‐Bt cotton plants. Egg cannibalism significantly improved the survival of Bt‐susceptible H. armigera larvae on Bt cotton plants. Larvae were more likely to eat live aged eggs, than newly laid or dead eggs. Survival significantly improved when larvae cannibalized eggs before feeding on Bt leaves. The behavior of Bt‐susceptible larvae with respect to drop‐off and egg cannibalism may help enhance their survival on Bt cotton plants.     

Mitochondrial P-Glycoprotein ATPase Contributes to Insecticide Resistance in the Cotton Bollworm,Helicoverpa armigera (Noctuidae: Lepidoptera)

Cotton bollworm, Helicoverpa armigera, is one of the most damaging polyphagous pests worldwide, which has developed high levels of resistance to commonly applied insecticides. Mitochondrial P-glycoprotein (Pgp) was detected in the insecticide-resistant strain of H. armigera using C219 antibodies, and its possible role was demonstrated in the efflux of xenobiotic compounds using spectrofluorometer. The TMR accumulated in mitochondria in the absence of ATP, and effluxed out in presence of ATP; the process of efflux was inhibited in the presence of ortho-vandate, an inhibitor of Pgp, in insecticide-resistant larvae of H. armigera. The mitochondria isolated from insecticide-resistant larvae were resistant to insecticide-induced inhibition of oxygen consumption and cytochrome c release. Membrane potential decreased in a dose-dependent manner in the presence of higher concentration of insecticides (>50 µM) in mitochondria of insecticide-resistant larvae. In conclusion, mitochondrial Pgp ATPase detected in the insecticide-resistant larvae influenced the efflux of xenobiotic compounds. Pgp might be involved in protecting the mitochondrial DNA and the components of the electron transport chain from damage due to insecticides, and contributing to the resistance to the deleterious effects of insecticides on the growth of insecticide-resistant H. armigera larvae.     

Antifeedant and cytotoxic activity of gibberellic acid against Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The present study investigates the effect of gibberellic acid (GA3), a terpenoid and phytohormone, on the digestive physiology and intermediary metabolism of the cotton bollworm Helicoverpa armigera Hübner (Lepidoptera, Noctuidae). Incorporation of GA3 (800 μg g^?1 diet) in an artificial diet results in significant reductions in the rates of diet consumption and the efficiency of conversion of food consumption by by H. armigera larvae. The relative growth rate decreases as the concentration increases. The relative α‐amylase activity in sixth‐instar larvae of H. armigera decreases significantly after ingestion of four concentrations of GA3. Histological studies of the midgut in GA3‐treated larvae (800 μg g^?1 diet) show degeneration of the epithelial cells. The alanine and aspartate aminotransferase activity decreases at the highest concentration. However, acid phosphatase, alkaline phosphatase, γ‐glutamyl transferase and lactate dehydrogenase activity increase significantly compared with the control. The results clearly demonstrate the adverse effects of GA3 on H. armigera via interruption of nutritional physiology and metabolism.     

A Kunitz trypsin inhibitor from chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) that exerts anti-metabolic effect on podborer (<Emphasis Type="Italic">Helicoverpa armigera</Emphasis>) larvae

Chickpea (Cicer arietinum L.) seeds contain Bowman–Birk proteinase inhibitors, which are ineffective against the digestive proteinases of larvae of the insect pest Helicoverpa armigera. We have identified and purified a low expressing proteinase inhibitor (PI), distinct from the Bowman–Birk Inhibitors and active against H. armigera gut proteinases (HGP), from chickpea seeds. N-terminal sequencing of this HGP inhibitor revealed a sequence similar to reported pea (Pisum sativum) and chickpea -l-fucosidases and also homologous to legume Kunitz inhibitors. The identity was confirmed by matrix assisted laser desorption ionization – time of flight analysis of tryptic peptides and isolation of DNA sequence coding for the mature protein. Available sequence data showed that this protein forms a distinct phylogenetic cluster with Kunitz inhibitors from Glycine max, Medicago truncatula, P. sativum and Canavalia lineata. The isolated coding sequence was cloned into a yeast expression vector and produced as a recombinant protein in Pichia pastoris. -l-fucosidase activity was not detectable in purified or recombinant protein, by solution assays. The recombinant protein did not inhibit chymotrypsin or subtilisin activity but did exhibit stoichiometric inhibition of trypsin, comparable to soybean Kunitz trypsin inhibitor. The recombinant protein exhibited higher inhibition of total HGP activity as compared to soybean kunitz inhibitor, even though it preferentially inhibited HGP-trypsins. H. armigera larvae fed on inhibitor-incorporated artificial diet showed significant reduction in average larval weight after 18 days of feeding demonstrating potent antimetabolic activity. The over-expression of this gene in chickpea could act as an endogenous source of resistance to H. armigera.     

Cysteine protease enhances plant-mediated bollworm RNA interference

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665–673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.     

A mixture of herbivore-induced plant volatiles from multiple host plant species enhances the attraction of a predatory bug under field-cage conditions

Plants respond to herbivore attack by emitting a blend of volatiles called herbivore-induced plant volatiles (HIPVs), which attract arthropod natural enemies. Under natural conditions and multiple cropping agriculture systems, natural enemies are thought to encounter a mixture of HIPVs emanating from multiple plant species. The effect of such a mixture of HIPVs on the responses of natural enemies under field conditions has not been explored. Our study assessed whether a mixture of HIPVs from multiple host plant species influenced predator responses in field-cage conditions. We investigated (1) foraging behaviors of a predatory bug, Orius strigicollis, on cotton bollworm (Helicoverpa armigera) larvae-infested multiple host plant species, and (2) the attractiveness of a mixture of reconstituted HIPVs from multiple plant species to O. strigicollis in outdoor cages. Significantly, greater numbers of predators were attracted to H. armigera-infested multiple plant species. The predators exterminated significantly greater numbers of H. armigera larvae with the multiple versus single plant species treatments. Significantly, greater numbers of O. strigicollis were captured on traps baited with the mixture of reconstituted HIPVs from multiple versus single plant species. The enhanced attractiveness of a mixture of HIPVs from multiple plant species to O. strigicollis might be the result of an additive effect of HIPVs from the three plant species when combined in a mixture.