EVALUATION OF IN VITRO AND IN VIVO EFFECTS OF SEMIPURIFIED PROTEINASE INHIBITORS FROM THEOBROMA SEEDS ON MIDGUT PROTEASE ACTIVITY OF LEPIDOPTERAN PEST INSECTS

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.

Movement of Heterorhabditis amazonensis and Steinernema arenarium in search of corn fall armyworm larvae in artificial conditions

Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is considered to be the main pest of maize crops in Brazil. Entomopathogenic nematodes (EPN) may be used to control this pest and exhibit different, unique abilities to search for their hosts. The movement of EPN in relation to S. frugiperda was evaluated. To test for horizontal movement, a styrofoam enclosure filled with sand was divided into segments, nematodes were placed at the entrance to the enclosure and a larva was placed at the end of each division. The same approach was used to evaluate vertical movement; however, PVC pipes were used in this case. In general, the mortality was inversely proportional to the initial distance between host and nematodes. In the vertical displacement test, both nematodes were able to kill the larvae up to a distance of 25 cm. Therefore, the infective juveniles of H. amazonensis and S. arenarium can search out, infect and kill larvae of S. frugiperda at distances of up to 60 cm and 25 cm of horizontal and vertical displacement, respectively.

Oviposition by a moth suppresses constitutive and herbivore-induced plant volatiles in maize

Plant volatiles function as important signals for herbivores, parasitoids, predators, and neighboring plants. Herbivore attack can dramatically increase plant volatile emissions in many species. However, plants do not only react to herbivore-inflicted damage, but also already start adjusting their metabolism upon egg deposition by insects. Several studies have found evidence that egg deposition itself can induce the release of volatiles, but little is known about the effects of oviposition on the volatiles released in response to subsequent herbivory. To study this we measured the effect of oviposition by Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) moths on constitutive and herbivore-induced volatiles in maize (Zea mays L.). Results demonstrate that egg deposition reduces the constitutive emission of volatiles and suppresses the typical burst of inducible volatiles following mechanical damage and application of caterpillar regurgitant, a treatment that mimics herbivory. We discuss the possible mechanisms responsible for reducing the plant's signaling capacity triggered by S. frugiperda oviposition and how suppression of volatile organic compounds can influence the interaction between the plant, the herbivore, and other organisms in its environment. Future studies should consider oviposition as a potential modulator of plant responses to insect herbivores. © 2011 Springer-Verlag.

Sequence of arrival determines plant-mediated interactions between herbivores

Induced changes in plant quality can mediate indirect interactions between herbivores. Although the sequence of attack by different herbivores has been shown to influence plant responses, little is known about how this affects the herbivores themselves. We therefore investigated how induction by the leaf herbivore Spodoptera frugiperda influences resistance of teosinte (Zea mays mexicana) and cultivated maize (Zea mays mays) against root-feeding larvae of Diabrotica virgifera virgifera. The importance of the sequence of arrival was tested in the field and laboratory. Spodoptera frugiperda infestation had a significant negative effect on colonization by D. virgifera larvae in the field and weight gain in the laboratory, but only when S. frugiperda arrived on the plant before the root herbivore. When S. frugiperda arrived after the root herbivore had established, no negative effects on larval performance were detected. Yet, adult emergence of D. virgifera was reduced even when the root feeder had established first, indicating that the negative effects were not entirely absent in this treatment. The defoliation of the plants was not a decisive factor for the negative effects on root herbivore development, as both minor and major leaf damage resulted in an increase in root resistance and the extent of biomass removal was not correlated with root-herbivore growth. We propose that leaf-herbivore-induced increases in feeding-deterrent and/or toxic secondary metabolites may account for the sequence-specific reduction in root-herbivore performance. Synthesis. Our results demonstrate that the sequence of arrival can be an important determinant of plant-mediated interactions between insect herbivores in both wild and cultivated plants. Arriving early on a plant may be an important strategy of insects to avoid competition with other herbivores. To fully understand plant-mediated interactions between insect herbivores, the sequence of arrival should be taken into account. © 2011 The Authors. Journal of Ecology © 2011 British Ecological Society.

Within-plant distribution of 1,4-benzoxazin-3-ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore-foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within-plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4-benzoxazin-3-one derivatives (BXDs), affects the foraging behaviour of two leaf-chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well-adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD-free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non-adapted herbivores and thereby likely contribute to feeding niche differentiation

3-β-d-Glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc) is an insect detoxification product of maize 1,4-benzoxazin-3-ones

In order to defend themselves against arthropod herbivores, maize plants produce 1,4-benzoxazin-3-ones (BXs), which are stored as weakly active glucosides in the vacuole. Upon tissue disruption, BXs come into contact with β-glucosidases, resulting in the release of active aglycones and their breakdown products. While some aglycones can be reglucosylated by specialist herbivores, little is known about how they detoxify BX breakdown products. Here we report on the structure of an N-glucoside, 3-β-d-glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc), purified from Spodoptera frugiperda faeces. In vitro assays showed that MBOA-N-Glc is formed enzymatically in the insect gut using the BX breakdown product 6-methoxy-2-benzoxazolinone (MBOA) as precursor. While Spodoptera littoralis and S. frugiperda caterpillars readily glucosylated MBOA, larvae of the European corn borer Ostrinia nubilalis were hardly able to process the molecule. Accordingly, Spodoptera caterpillar growth was unaffected by the presence of MBOA, while O. nubilalis growth was reduced. We conclude that glucosylation of MBOA is an important detoxification mechanism that helps insects tolerate maize BXs.

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores

In monocotyledonous plants, 1,4-benzoxazin-3-ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4-benzoxazin-3-ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4-benzoxazin-3-ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4-benzoxazin-3-ones and active extracts from mutant plants lacking 1,4-benzoxazin-3-ones. Our data show that maize plants possess a two-step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2-β-d-glucopyranosyloxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA-Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA) is released by plant-derived β-glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4-benzoxazin-3-ones such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.

Herbivore-Induced Plant Volatiles Can Serve as Host Location Cues for a Generalist and a Specialist Egg Parasitoid

Herbivore-induced plant volatiles are important host finding cues for larval parasitoids, and similarly, insect oviposition might elicit the release of plant volatiles functioning as host finding cues for egg parasitoids. We hypothesized that egg parasitoids also might utilize HIPVs of emerging larvae to locate plants with host eggs. We, therefore, assessed the olfactory response of two egg parasitoids, a generalist, Trichogramma pretiosum (Tricogrammatidae), and a specialist, Telenomus remus (Scelionidae) to HIPVs. We used a Y-tube olfactometer to tests the wasps’ responses to volatiles released by young maize plants that were treated with regurgitant from caterpillars of the moth Spodoptera frugiperda (Noctuidae) or were directly attacked by the caterpillars. The results show that the generalist egg parasitoid Tr. pretiosum is innately attracted by volatiles from freshly-damaged plants 0–1 and 2–3 h after regurgitant treatment. During this interval, the volatile blend consisted of green leaf volatiles (GLVs) and a blend of aromatic compounds, mono- and homoterpenes, respectively. Behavioral assays with synthetic GLVs confirmed their attractiveness to Tr. pretiosum. The generalist learned the more complex volatile blends released 6–7 h after induction, which consisted mainly of sesquiterpenes. The specialist T. remus on the other hand was attracted only to volatiles emitted from fresh and old damage after associating these volatiles with oviposition. Taken together, these results strengthen the emerging pattern that egg and larval parasitoids behave in a similar way in that generalists can respond innately to HIPVs, while specialists seems to rely more on associative learning.

Proceedings of the 23rd Annual Biochemical Engineering Symposium

The 23rd Annual Biochemical Engineering Symposium was held at the University of Oklahoma on April 17, 1993. The objectives of the symposium were to provide 1) a forum for informal discussion of biochemical engineering research being carried at the participating universities and 2) an opportunity for students to present and publish their work. Thirteen papers presented at the symposium are included in the proceedings. Because final publication usually takes place in refereed journals, the articles included here are typically brief and often cover work in progress. The program of the symposium and a list of participants are included in the proceedings. ContentsA Low-Cost Bioreactor Strategy for RNA Synthesis, H. Anthony Marble, Eleni Chrisikos, and Robert H. Davis Development of a CELSS Bioreactor: Oxygen Transfer and Micromixing in Parabolic Flight, P.E. Villeneuve, K.S. Wenger, B.G. Thompson, T. Kedar, and E.H. Dunlop Scale-up of Dexter Murine Bone Marrow Cultures Utilizing a Three-Dimensional Fiberglass Support Matrix, John G. Highfill, Paul Todd, Steve Haley, and Dhinaker Kompala Modeling and Estimation of States of Recombinant Fermentations Using Nonlinear Input/Output Models, Vicotr M. Saucedo and M. Nazmul Karim Deadent Microfiltration of Bovine Serum Albumin Suspension Through Yeast Cake Layers and Assymetric Polymeric Membranes, Naveen Arora and Robert H. Davis Monitoring the Fate of Toluene and Phenol in the Rhizosphere, N. Muralidharan, Lawrence C. Davis, and Larry E. Erickson Hydrodynamic Motions Associated with Bubble Coalescence and Breakup, T.Y. Yiin, L.A. Glasgow, and L.E. Erickson Expression and Purification of a-Human Atrial Natriuretic Peptide in Escherichia coli by Fusion with L-Asparaginase, Nien-Tung Ma and Roger G. Harrison High Pressure Crystallization of Proteins, Mungara V. Saikumar, Charles E. Glatz, and Maurice A. Larson Structure/Function Relationships in the Catalytic and Starch Binding Domains of Glucoamylase, Pedro M. Coutinho, Clark Ford, Peter J. Reilly Cellular Responses of Insect Cell Spodoptera frugiperda to Environmental Stresses, Paul Yeh, Grace Y. Sun, Gary A. Weisman, Rakesh Bajpai A Novel Approach to Understanding the Antimicrobial Activity of Peptides, Naveen Pathak, Marie-Helene Janna, Gael Ruche, David McCarthy, and Roger Harrison Mass Transfer in the Bioremediation of Soils Contaminated with Trapped Non-Aqueous Phase Liquids, Xiaoqing Yang, Larry E. Jacobson, and L.T. Fan

Evolutionary conservation of apoptosis mechanisms: Lepidopteran and baculoviral inhibitor of apoptosis proteins are inhibitors of mammalian caspase-9

We cloned a new inhibitor of apoptosis protein (IAP) homolog, SfIAP, from Spodoptera frugiperda Sf-21 cells, a host of insect baculoviruses. SfIAP contains two baculovirus IAP repeat domains followed by a RING domain. SfIAP has striking amino acid sequence similarity with baculoviral IAPs, CpIAP and OpIAP, suggesting that baculoviral IAPs may be host-derived genes. SfIAP and baculoviral CpIAP inhibit Bax but not Fas-induced apoptosis in human cells. Their apoptosis-suppressing activity in mammalian cells requires both baculovirus IAP repeat and RING domains. Further biochemical data suggest that SfIAP and CpIAP are specific inhibitors of mammalian caspase-9, the pinnacle caspase in the mitochondria/cytochrome c pathway for apoptosis, but are not inhibitors of downstream caspase-3 and caspase-7. Thus the mechanisms by which insect and baculoviral IAPs suppress apoptosis may involve inhibition of an insect caspase-9 homologue. Peptides representing the IAP-binding domain of the Drosophila cell death protein Grim abrogated human caspase suppression by SfIAP and CpIAP, implying evolutionary conservation of the functions of IAPs and their inhibitors.

Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1

We have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1.

Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system

In the “selective” cholesteryl ester (CE) uptake process, surface-associated lipoproteins [high density lipoprotein (HDL) and low density lipoprotein] are trapped in the space formed between closely apposed surface microvilli (microvillar channels) in hormone-stimulated steroidogenic cells. This is the same location where an HDL receptor (SR-BI) is found. In the current study, we sought to understand the relationship between SR-BI and selective CE uptake in a heterologous insect cell system. Sf9 (Spodoptera frugiperda) cells overexpressing recombinant SR-BI were examined for (i) SR-BI protein by Western blot analysis and light or electron immunomicroscopy, and (ii) selective lipoprotein CE uptake by the use of radiolabeled or fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control Sf9 cells do not express SR-BI, show microvillar channels, or internalize CEs. An unexpected finding was the induction of a complex channel system in Sf9 cells expressing SR-BI. SR-BI-expressing cells showed many cell surface double-membraned channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the channels, and high levels of selective HDL-CE uptake. Thus, double-membraned channels can be induced by expression of recombinant SR-BI in a heterologous system, and these specialized structures facilitate both the binding of HDL and selective HDL-CE uptake.

Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells

A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K+in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated K+in currents half-maximally with an inhibitor concentration (IC50) of 105 μm. An almost identical high Cs+ sensitivity (IC50 = 90 μm) was found for the potato guard-cell K+in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.

Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.

A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from Bacillus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in approximately 15% of Bacillus strains analyzed. The sequence of the vip3A(b) gene, a homologue of vip3A(a) isolated from B. thuringiensis strain AB424 is also reported. Vip3A(a) and (b) proteins confer upon Escherichia coli insecticidal activity against the lepidopteran insect larvae mentioned above. The sequence of the gene predicts a 791-amino acid (88.5 kDa) protein that contains no homology with known proteins. Vip3A insecticidal proteins are secreted without N-terminal processing. Unlike the B. thuringiensis 5-endotoxins, whose expression is restricted to sporulation, Vip3A insecticidal proteins are expressed in the vegetative stage of growth starting at mid-log phase as well as during sporulation. Vip3A represents a novel class of proteins insecticidal to lepidopteran insect larvae.

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.