Heliothis Larvae Research Articles

Analysis of Inducible Terpenoids in Cotton Leaves to Test for Indirect Plant–Endophyte-Herbivore Interactions

Analysis of Inducible Terpenoids in Cotton Leaves to Test for Indirect Plant–Endophyte-Herbivore Interactions

Development of transgenic cotton lines harboring a pesticidal gene (cry1Ab)

Each transgenic plant has its unique genetic architecture and level of expression. The present study was conducted to study each transgenic plant separately in order to develop pure transgenic lines to be further used in efficient cotton breeding programme. Earlier MNH-93 cultivar was transformed with the Bacillus thuringiensis (Bt) gene cry1Ab through Agrobacterium-mediated transformation method using mature cotton embryos as explants. Transformed plants obtained out of this transformation event were used to develop pure lines for five consecutive generations. The selection criteria contained confirmation of transgene through PCR and western dot blot, boll damage percentage, insect mortality percentage in lab bioassay with heliothis larvae, seed cotton yield (g), plant height (cm), number of monopodial branches per plant and number of sympodial branches per plant. All the characters were stably inherited in further generations. The expression of introduced genes was found to be variable; however it conferred protection against targeted insect pests. It was concluded that the transgenic pure lines are an excellent source of germplasm to be used in conventional breeding programme.

Oral insecticidal activity of plant‐associated pseudomonads

Biocontrol pseudomonads are most known to protect plants from fungal diseases and to increase plant yield, while intriguing aspects on insecticidal activity have been discovered only recently. Here, we demonstrate that Fit toxin producing pseudomonads, in contrast to a naturally Fit-deficient strain, exhibit potent oral activity against larvae of Spodoptera littoralis, Heliothis virescens and Plutella xylostella, all major insect pests of agricultural crops. Spraying plant leaves with suspensions containing only 1000 Pseudomonas cells per ml was sufficient to kill 70-80% of Spodoptera and Heliothis larvae. Monitoring survival kinetics and bacterial titres in parallel, we demonstrate that Pseudomonas fluorescens CHA0 and Pseudomonas chlororaphis PCL1391, two bacteria harbouring the Fit gene cluster colonize and kill insects via oral infection. Using Fit mutants of CHA0 and PCL1391, we show that production of the Fit toxin contributes substantially to oral insecticidal activity. Furthermore, the global regulator GacA is required for full insecticidal activity. Our findings demonstrate the lethal oral activity of two root-colonizing pseudomonads so far known as potent antagonists of fungal plant pathogens. This adds insecticidal activity to the existing biocontrol repertoire of these bacteria and opens new perspectives for applications in crop pest control and in research on their ecological behaviour.

Inheritance of an insecticidal gene (cry1Ab) in genetically modified cotton

The present study was conducted to evaluate inheritance pattern of insecticidal gene (Cry1Ab) in F1 and F2 generations derived from six combinations of crosses made between two transgenic lines (CEMB-3 and CEMB-11) and two non transgenic lines (MNH-93 and CIM-482). PCR, southern blot, western dot blot assay and lab biotoxicity assay were used to confirm the gene integration and expression of insecticidal gene in successive generations. The insecticidal gene was stably integrated in the genome of all F1 plants showing the dominant nature of introduced gene (cry1Ab). Furthermore, heterosis, heterobeltiosis, heritability and genetic advance studies of Bt gene were also conducted. Heterosis and heterobeltiosis were estimated for five characters i.e. cotton yield per plant, no. of bolls per plant, boll weight, ginning out turn % age and laboratory bioassay results. The heterosis and heterobeltiosis ranged from—15.19 to 107.07% and 18.58 to 98.79%, respectively for yield per plant; from −20.34 to 81.36% and −20.34 to 81.36%, respectively for number of bolls per plant; from -6.96 to 21.38% and −9.30 to 9.99%, respectively for boll weight; from 13.02 to 26.44% and −0.52 to 26.17%, respectively for ginning outturn; and from −8.11 to 36.23% and −5.56 to 23.68%, respectively for mortality % age of Heliothis larvae in laboratory bioassays. The Broad Sense Heritability and Genetic Advance for insect resistance in Bt versus non-Bt crosses were calculated. Both of these were high in four out of six hybrids. Our data recorded showed that these transgenic lines are an excellent source of germplasm to be used in conventional breeding programme.

Iron Levels Change in Larval Heliothis virescens Tissues Following Baculovirus Infection

Inductively coupled plasma mass spectrometry and (59)Fe radiotracers were used to investigate changes in levels of Fe in the tissues of 4th instar Heliothis virescens larvae following infection with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) or with Autographa californica multiple nucleopolyhedrovirus. Baculovirus infection led to significant changes in hemolymph Fe levels late in infection. (24)Na radiotracer ingested by 4th instar larvae was rapidly cleared to nearly undetectable levels 6 h post-ingestion. In contrast, (59)Fe radiotracer fed to 4th instar larvae declined within the first few hours of ingestion and then remained constant at approximately 60% of the initial tracer activity. While Fe radiotracer levels among larval tissues changed, whole insect tracer levels did not decline from 6 to 60 h post-ingestion. Tissues from HzSNPV larvae had higher radiotracer levels in the hemolymph and midgut 36 and 60 h post-infection. The protein-bound/free ratio of (59)Fe was significantly higher in baculovirus infected hemolymph than in uninfected hemolymph at 60 h post-infection, indicating that Fe released from damaged cells is protein-bound. In both studies, hemolymph Fe levels were higher in HzSNPV infected larvae. This first study of tissue Fe levels during viral infection of an insect clearly demonstrates that Fe homeostasis is substantially disrupted.

Changes in Trace Metals in Hemolymph of Baculovirus-Infected Noctuid Larvae

We studied how biologically relevant trace metals (i.e., micronutrients) in the hemolymph of larval Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) changed in response to per os baculovirus infection, larval development, and injection of heat-killed bacteria. Concentrations of hemolymph Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, and Zn were measured using inductively coupled plasma-mass spectrometry. H. virescens larvae exhibited greater fluctuations in hemolymph trace metal levels in response to baculovirus infection and development than did H. zea larvae. H. zea single nucleopolyhedrosis virus infection significantly altered the levels of Cu, Fe, Mg, Mn, Mo, and Zn in fourth instar H. virescens larvae. Conversely, in fifth instar H. virescens and both H. zea instar infections, no metal levels were significantly different between infected and uninfected larvae. In fourth instar H. virescens hemolymph, Cu, Fe, Mo, and Zn increased during development. Cu, Fe, Mg, Mn, Mo, and Zn levels changed significantly during development in fifth instar H. virescens as well as both H. zea instars. Based on this analysis, metals were identified whose levels changed during development in both species and during the immune response of H. virescens larvae.

Ascorbic acid influences the development and immunocompetence of larval Heliothis virescens

AbstractAscorbic acid, known to be a free radical scavenger and vital to insect development, is important in larval resistance to baculovirus infection. We sequentially elevated the ascorbic acid content in an ascorbic acid‐depleted diet and evaluated the effect on larval Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) development and immunocompetence. Dietary ascorbic acid levels lower than 0.7 g l−1 slowed the growth rate of larvae, reduced pupal weights significantly, and severely inhibited adult emergence. Larvae developing on ascorbic acid‐free diet experienced far higher levels of mortality following per os infection with virus. Additionally, viral infection in larvae fed an ascorbic acid‐free diet, as monitored by epifluorescence microscopy, showed signs of infection much earlier than larvae fed control levels of ascorbic acid. These results demonstrate an indirect correlation between the level of ascorbic acid in the food stream of larval H. virescens and the susceptibility of the insect to baculoviral infection.

Analysis of ESTs generated from immune-stimulated hemocytes of larval Heliothis virescens

Heliothis virescens immunome components responding to baculoviral and bacterial infection were identified from expressed sequence tags (ESTs) generated from an immune-stimulated larval hemocyte cDNA library. A total of 5548 ESTs were generated comprising 448 contigs and 1114 singletons, totaling 1606 putative transcripts 1101 of which had BLAST scores, including many known orthologs from other insect species. Orthologs of known or putative immune function were identified among them melanization pathway components, proteases, antibacterial proteins, lectins, bacteria-binding proteins, ferritins, scavenger receptors, cell surface receptors, signaling pathway components, and stress response enzymes. Additionally, many enzymes of central metabolism, cytoskeletal, mitochondrial, and ribosomal components, as well as transcriptional and translational regulators were identified. The effect of bacterial and baculoviral infection upon transcript levels of three identified immunome targets from among the ESTs was quantitated using real time PCR. Scolexin-B, C-type lectin and growth-blocking peptide binding protein transcripts were significantly elevated by bacterial infection. Per os infection with the baculovirus Helicoverpa zea single nucleopolyhedrovirus however did not significantly alter transcript levels of these three genes. The ESTs reported here are the first large scale report of the H. virescens immunome responding to entomopathogens, and represent a first step to a more complete transcriptome for this pest moth.

A Polymerase Chain Reaction Screen of Field Populations of <I>Heliothis virescens</I> for a Retrotransposon Insertion Conferring Resistance to <I>Bacillus thuringiensis</I> Toxin

The evolution of pest resistance to transgenic crop plants producing insecticidal toxins from Bacillus thuringiensis (Bt) Berliner poses a continuing threat to their sustainable use in agriculture. One component of the U.S.-wide resistance management plan for Bt cotton, Gossypium hirsutum L., involves monitoring the frequency of resistance alleles in field populations. However, existing methods are expensive and may not detect recessive resistance alleles until their frequencies are too high for countermeasures to be effective; therefore, more sensitive methods are needed. The first Bt resistance-causing mutation described at the molecular level was a retrotransposon insertion into the gene encoding a 12-cadherin-domain protein expressed in the midgut of larval Heliothis virescens (F.). We report the first large-scale screen for this mutation using a polymerase chain reaction (PCR)-based approach on >7,000 field-collected individuals. The specific insertion was not detected in any of these samples, nor was it detected in three progeny-tested, field-caught males thought to carry a Bt resistance gene. Unlike the targets of many chemical insecticides where a limited number of resistance-causing mutations compatible with viability can occur; a very large number of such mutations seem possible for the 12-cadherin-domain gene. However, even if these mutations are viable in the laboratory, they may not threaten the effectiveness of transgenic crops because of a high fitness cost in the field. The challenge remains to detect the subset of possible resistance-conferring alleles that are still rare but are viable in the field and increasing due to selection by Bt cotton. This situation will complicate PCR-based Bt resistance monitoring strategies.

Remarkable susceptibility of the diamondback moth (Plutella xylostella) to ingestion of Pir toxins from Photorhabdus luminescens

AbstractGenes encoding Pir toxins were cloned and sequenced from Photorhabdus luminescens (Enterobacteriaceae) strain Hm. Cultures of Escherichia coli expressing the Pir A and B proteins were highly toxic when fed to larvae of Plutella xylostella L. (Lepidoptera: Plutellidae), as had been reported previously. Histological examination of P. xylostella larvae fed with recombinant E. coli revealed gross abnormalities of the midgut epithelium, with profound swelling and shedding of the apical membranes. However, the recombinant E. coli had no effect on the growth or mortality of larval Heliothis virescens F. (Lepidoptera: Noctuidae), Manduca sexta L. (Lepidoptera: Sphingidae), Lymantria dispar L. (Lepidoptera: Lymantriidae), or Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Based on these results, P. xylostella is at least 300‐fold more susceptible to Pir toxins than other insect species tested, suggesting that they may not be broadly useful as insecticidal proteins. Because Pir B has sequence similarities with N‐terminal portions of Cry proteins from Bacillus thuringiensis, we also tested the recombinant E. coli against a strain of P. xylostella that is resistant to the Cry 1A toxin, but found no difference in mortality between resistant and susceptible strains.

Uptake of dietary micronutrients from artificial diets by larval Heliothis virescens

Micronutrient assimilation from artificial diet by larvae of Heliothis virescens during selenium (Se) supplementation was studied. The metal content of pupae and plugs of the artificial diet on which they had developed from hatching was analyzed by inductively coupled plasma-mass spectrometry. Levels of the metals Cr, Co, Fe, Mg, Mn, Ni, Se, Na, and Zn were not bioaccumulated from the diet regardless of the amount of Se added to the diet. Only pupal Cu and Mo bioaccumulation were found to be altered significantly by dietary Se supplementation. Larvae fed Zn, which was found in higher levels in pupae than diet, had a deleterious response to increasing levels of dietary Zn. Larvae fed Cr, found in higher levels in diet than in pupae, were not adversely affected when increasing levels of Cr were added to the diet. Based on this analysis, metals were identified that might well impact the fitness of a given colony of insects in relation to their diet.

Potent virucidal activity in larval Heliothis virescens plasma against Helicoverpa zea single capsid nucleopolyhedrovirus.

Lepidopteran larvae resist baculovirus infection by selective apoptosis of infected midgut epithelial cells and by sloughing off infected cells from the midgut. Once the infection breaches the midgut epithelial barrier and propagates from infective foci to the haemocoel, however, there are few mechanisms known to account for the resistance and clearance of infection observed in some virus-host combinations. The hypothesis that factors present in the plasma of infected pest larvae act to limit the spread of virus from initial infective foci within the haemocoel was tested. An in vitro bioassay was developed in which Helicoverpa zea single capsid nucleopolyhedrovirus (HzSNPV) was incubated with plasma collected from uninfected Heliothis virescens larvae. Infectious HzSNPV particles were then titrated on HzAM1 cells. Diluted plasma from larval Heliothis virescens exhibited a virucidal effect against HzSNPV in vitro, reducing the TCID(50) ml(-1) by more than 64-fold (from 4.3+/-3.6x10(5) to 6.7+/-0.6x10(3)). The antiviral activity was heat-labile but was unaffected by freezing. In addition, protease inhibitors and specific chemical inhibitors of phenol oxidase or prophenol oxidase activation added to diluted plasma eliminated the virucidal activity. Thus, in the plasma of larval lepidopterans, the enzyme phenol oxidase may act as a constitutive, humoral innate antiviral immune response.

Expression and characterization of a novel teratocyte protein of the braconid, Microplitis croceipes (cresson)

Microplitis croceipes wasps overcome host immunity by inducing changes in host physiology using factors derived from the embryo and/or larva. Teratocytes of some parasitic wasps circulate in the host hemolymph after egg hatch and synthesize proteins (TSPs), some of which are secreted to alter host physiology in support of endoparasitoid development. TSPs appear to alter host physiology, at least in part, by inhibiting synthesis of certain proteins. M. croceipes teratocytes synthesize a 13.9 kDa protein (TSP14), which inhibits synthesis of host proteins that are linked to larval growth and development. A cDNA encoding TSP14 was generated by RT-PCR from teratocyte RNA, and cloned into yeast expression vectors to produce sufficient recombinant protein for functional analyses. RecTSP14 was produced using the yeast expression system at a concentration of more than 300 μg/L. The recTSP14 inhibited in vitro translation of larval Heliothis virescens RNA, with the activity sensitive to boiling, protein concentration, incubation time, and storage temperatures. Although recTSP14 inhibited translation of some cellular RNAs in vitro, the in vivo incorporation of [ 35S]-methionine into proteins of selected insect and mammalian cell lines was not inhibited. These findings suggest that recTSP14 entry is cell type-specific and required to inhibit synthesis of target protein(s).

Cockroach midgut peptides that regulate cell proliferation, differentiation, and death in vitro.

The number of insect midgut cells is maintained homeostatically in vivo and in vitro. However, during starvation, the midgut shrinks and the rate of cell replacement appears to be suppressed. When they undergo metamorphosis, the internal organs of insects are drastically remodeled by cell proliferation, differentiation, and apoptotic processes, and the net number of cells usually increases. An extract of 1650 midguts of Periplaneta americana was fractionated by high-performance liquid chromatography (HPLC) to obtain the peptides that regulate these processes. The HPLC fractions were tested for myotropic activity in the foregut and for effects on cell proliferation or loss in primary cultures of larval Heliothis virescens midgut cells and in a cell line derived from the last-instar larval fat body of Mamestra brassicae. Some fractions stimulated midgut stem cell proliferation and differentiation, while others caused loss of differentiated columnar and goblet cells. Other fractions stimulated cell proliferation in the larval fat body cells.

Introduction and establishment of <i>Microplitis croceipes</i> a larval parasitoid of heliothis in North Island pine forests

Heliothis (Helicoverpa armigera) is a periodic pest of young pine trees (Pinus radiata) In response to a recent outbreak of this pest Microplitis croceipes a parasitoid of heliothis larvae was introduced into forests of the North Island volcanic plateau to augment biological control of heliothis A total of 13600 M croceipes pupae were released at monthly intervals over three seasons (199598) Successful overwintering of M croceipes populations confirmed establishment in the forests Mean monthly parasitism reached 107 in the third season Surveys at points radiating out from the release sites indicated that M croceipes was spreading at approximately 1 km per year

Dicranolaius bellulus (Guérin‐Méneville) (Coleoptera: Melyridae: Malachiinae), a possible biological control agent of lepidopterous pests in inland Australia

AbstractCarrot and potato crops grown in northern Victoria, along the Murray River, were sampled for macroinvertebrates. Dicranolaius bellulus was abundant in both crops. Feeding trials in the laboratory demonstrated that this species is predaceous and scavenging, and feeds on a variety of food sources. It readily accepts as food the eggs and first‐instar larvae of potato moth (Phthorimaea operculella) and heliothis (Helicoverpa punctigera), and also dead first‐instar crickets (Teleogryllus commodus).

Soil Inactivation of the Bacillus thuringiensis Subsp. kurstaki CryIIA Insecticidal Protein within Transgenic Cotton Tissue: Laboratory Microcosm and Field Studies

The environmental fate of Bacillus thuringiensis subsp. kurstaki CryIIA insecticidal protein expressed within transgenic cotton plant tissue (= Bt-cotton) was evaluated by determining reduction in the biological activity of the protein incubated in soil for 120 days. Studies were conducted in a laboratory microcosm and under field conditions during the fall and winter of 1995−1996 in St. Louis, MO. An insect bioassay, based on growth inhibition of larval Heliothis virescens (F.), was used to estimate DT50 values (50% dissipation time = “half-life” of bioactivity) of the CryIIA protein. DT50 values were 15.5 and 31.7 days for the laboratory and field, respectively. The percentages of initial CryIIA protein bioactivity remaining after 40 days of incubation were similar for the laboratory and field samples. In both environments, <25% of the initial bioactivity remained after 120 days. These results indicate that the biological activity of CryIIA protein, as a component of postharvest Bt-cotton plants, readil...

Preparation of cyanobacterial peptide toxin as a biopesticide against cotton pests

Glycine-rich peptide toxin of cyanobacterium Scytonema MKU 106 was purified. UV spectral analysis showed an absorption maximum at 228 nm and the molecular mass was less than 12 kDa. The mortality rate of American boll worms (Helicoverpa armigera) was about 80% and 40% 84 h after treatment with 0.001% crude and purified peptide toxins respectively; 100% mortality was observed after 108 h treatment with both purified and crude peptide toxins. The LC(50) (lethal concentration to 50% of the population) for Heliothis larvae after 96 h was 8.3 mu g/ml purified peptide toxin and 6.2 mu g/ml crude peptide toxin. Observations also show that the peptide toxin at 0.01% concentration acts as a biopesticide and at high (0.1%) concentrations it will act as an anti-feeding compound for Stylepta derogata (leaf-roller) larvae of the cotton crop.

Nerve Insensitivity tocis-Cypermethrin is Expressed in AdultHeliothis virescens

Adult and third-instar larval Heliothis virescens were examined for nerve insensitivity to cis-cypermethrin using a rapid and robust neurophysiological method. Three laboratory strains, BRC susceptible, PEG-87 resistant and MS2 metabolic resistant, were compared with a series of pyrethroid-resistant strains collected from a field site in Louisiana, USA, during the 1992 cotton season. Nerve insensitivity was present in a proportion of the individuals in both the PEG-87 and the field strains, at both life stages, minimal in the MS2 strain and absent in the BRC strain. Frequencies of nerve-sensitive and nerve-insensitive individuals in each strain were not significantly different at the two life stages examined.

Bacterial Entomopathogens from the Drosophila paulistorum Semispecies Complex

Bacteria which are infectious by inoculation in lepidoptera have been isolated and characterized from semispecies comprising the Drosophila paulistorum complex. These microorganisms are pathogenic toward lepidopteran hosts such as Heliothis virescens when introduced by injection of Drosophila tissue extracts and have been given the trivial name DpLE ( D. paulistorum lepidopteran entomopathogen). The DpLE from two of the semispecies, Transitional and Andean, were determined to be related to Proteus vulgaris based upon nucleotide sequence comparisons of 16S rDNA genes. Infectivity and 16S rDNA-based PCR assays showed the bacterium to be localized in a number of drosophilid tissues except adult heads and thoraces. Based upon similar experiments, the DpLE in transinfected Heliothis larvae were found in all tissues assayed prior to the onset of mortality. Stocks of Drosophila which had spontaneously lost DpLE continued to produce sterile sons when crossed with incompatible semispecies' females, confirming that the bacilliform DpLE is not the causative agent of the Drosophila paulistorum intersemispecific hybrid male sterility. Acquisition of the sequences of the 16S rDNA molecules of DpLE from all six semispecies permitted the construction of a phylogenetic tree in which the groupings were found not to be congruent with the phylogenies of their insect hosts.