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Proteases inhibitors-insensitive cysteine proteases allow Nezara viridula to feed on growing seeds of field-grown soybean

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Abstract

The southern green stink bug, Nezara viridula is one of the primary soybean pests and causes significant economic losses around the world. In spite of the high proteases inhibitor (PI) levels, N. viridula can feed on developing seeds of field-grown soybean and reduce crop yields. Although the PI-induced responses have been extensively investigated in many pest insects, there is lack of knowledge about the mechanisms that stink bugs employ to withstand cysteine PIs of soybean seeds. This study demonstrated that feeding on developing seeds of field-grown soybean inhibited total proteases activity of N. viridula, as result of inhibition of cathepsin B-like activity in the gut. In addition, from the 30 digestive cathepsins recognized in this study, 6 were identified as cathepsin B-like. Stink bugs that fed on growing seeds of field-grown soybean had similar gut pH to those reared in the laboratory, and both cathepsin B- and L-like had an optima pH of 6.5. Therefore, using specific proteases inhibitors we found that the main proteolytic activity in the gut is from cysteine proteases when N. viridula feeds on soybean crops. Since cathepsin L-like activity was not inhibited by soybean PIs, our results suggested that N. viridula relays on cathepsin L-like to feed on soybean. To our knowledge no study before has shown the impact of seed PIs of field-grown soybean on digestive proteases (cathepsin B- and L-like) of N. viridula. This study suggests that the activity of PI-insensitive cathepsins L-like in the gut would be part of an adaptive strategy to feed on developing soybean seeds. In agreement, the expansions of cathepsin L-like complement observed in pentatomids could confer to the insects a higher versatility to counteract the effects of different PIs.

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... Cysteine protease activity was determined on the supernatant as described in previous studies, using the specific substrate p-Glu-Phe-Leu-pNA (Sigma, St Louis, MO, USA; as explained in S7). 42 Membrane lipid peroxidation was analyzed through thiobarbituric acid reactive substances (TBARS) by high-performance liquid chromatography (HPLC) 43 (as explained in the Methods S8). TBARS are formed as a byproduct of lipid peroxidation. ...
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... 13,16,34,35 However, stink bugs are able to avoid plant defenses by inducing digestive proteases inhibitors-insensitive cysteine proteases or by non-transit gut microbiota that deactivates protease inhibitors. 36,37 Currently, in Argentina an infestation rate of four D. furcatus adults per m 2 of soybean at seed development stage (R5) 38 decreases crop yield by around 10%, showing the growing economic burden of this pest. 12 The Principles of Conservation Agriculture have been broadly adopted by Argentine and Brazilian agricultural producers since the 90s. ...
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Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.
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Cysteine peptidases (CP) play a role as digestive enzymes in hemipterans similar to serine peptidases in most other insects. There are two major CPs: cathepsin L (CAL), which is an endopeptidase and cathepsin B (CAB) that is both an exopeptidase and a minor endopeptidase. There are thirteen putative CALs in Dysdercus peruvianus, which in some cases were confirmed by cloning their encoding genes. RNA-seq data showed that DpCAL5 is mainly expressed in the anterior midgut (AM), DpCAL10 in carcass (whole body less midgut), suggesting it is a lysosomal enzyme, and the other DpCALs are expressed in middle (MM) and posterior (PM) midgut. The expression data were confirmed by qPCR and enzyme secretion to midgut lumen by a proteomic approach. Two CAL activities were isolated by chromatography from midgut samples with similar kinetic properties toward small substrates. Docking analysis of a long peptide with several DpCALs modeled with digestive Tenebrio molitor CAL (TmCAL3) as template showed that on adapting to luminal digestion DpCALs (chiefly DpCAL5) changed in relation to their ancestral lysosomal enzyme (DpCAL10) mainly at its S2 subsite. A similar conclusion arrived from structure alignment-based clustering of DpCALs based on structural similarity of the modeled structures. Changes mostly on S2 subsite could mean the enzymes turn out less peptide-bond selective, as described in TmCALs. R. prolixus CALs changed on adapting to luminal digestion, although less than DpCALs. Both D. peruvianus and R. prolixus have two digestive CABs which are expressed in the same extension as CALs, in the first digestive section of the midgut, but less than in the other midgut sections. Mahanarva fimbriolata does not seem to have digestive CALs and their digestive CABs are mainly expressed in the first digestive section of the midgut and do not diverge much from their lysosomal counterparts. The data suggest that CABs are necessary at the initial stage of digestion in CP-dependent Hemipterans, which action is completed by CALs with low peptide-bond selectivity in Heteroptera species. In M. fimbriolata protein digestion is supposed to be associated with the inactivation of sap noxious proteins, making CAB sufficient as digestive CP. Hemipteran genomes and tran-scriptome data showed that CALs have been recruited as digestive enzymes only in heteropterans, whereas digestive CABs occur in all hemipterans.
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The southern green stink bug, Nezara viridula is a polyphagous pest of commercially important crops during both nymph and adult stages. This insect has recently transitioned from a secondary agricultural pest to one of primary concern. Novel management solutions are needed due to the limited effectiveness of current control strategies. We performed biochemical and transcriptomic analyses to characterize digestive enzymes in the salivary glands and along midgut tissues of N. viridula nymphs and adults fed on sweet corn. The digestive profiles were more distinct between midgut regions (M1 to M3) than between life stages. Aminopeptidase and chymotrypsin activities declined from the M1 (anterior) toward the M3 midgut region. Cysteine protease activity was higher in the M2 and M3 regions than in M1. Differences in sensitivity to chymotrypsin inhibitors between midgut regions suggest that distinct genes or isoforms are expressed in different regions of the gut. In nymphs, DNA and RNA degradation was higher in M1 than in M3. Adult nuclease activity was low across all midgut regions, but high in salivary glands. The differences in protease activities are reflected by transcriptomic data and functional enrichment of GO terms. Together, our results show that different regions of the digestive tract of N. viridula have specific and distinct digestive properties, and increase our understanding of the physiology of this organism.
Article
The phytophagous stink bug, Nezara viridula (L.) infests multiple plant species and impacts agricultural production worldwide. We analyzed the transcriptomes of N. viridula accessory salivary gland (ASG), principal salivary gland (PSG) and gut, with a focus on putative digestive proteases and nucleases that present a primary obstacle for the stability of protein- or nucleic acid-based stink bug control approaches. We performed high throughput Illumina sequencing followed by de novo transcriptome assemblies. We identified the sequences of 141 unique proteases and 134 nucleases from the N. viridula transcriptomes. Analysis of relative transcript abundance in conjunction with previously reported proteome data (Lomate and Bonning, 2016) supports high levels of serine protease expression in the salivary glands and high cysteine protease expression in the gut. Specifically, trypsin and chymotrypsin transcripts were abundant in the PSG, and cathepsin L-like cysteine protease transcripts were abundant in the gut. Nuclease transcript levels were generally lower than those of the proteases, the exception being abundant transcripts of ribonuclease-C20 in the PSG. The abundance of chymotrypsin, trypsin, and some carboxypeptidase transcripts suggests a significant role for the PSG in production of digestive enzymes. This result is at odds with the premise that the ASG produces watery saliva, which is high in enzymatic activity, while the PSG produces only sheath saliva. We have generated a comprehensive transcriptome sequence dataset from the digestive organs of N. viridula, identified major protease and nuclease genes and confirmed expression of the most abundant enzymes thereby providing greater insight into the digestive physiology of N. viridula.
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Solar UV-B radiation has been reported to enhance constitutive and inducible plant defenses against herbivore insects in many species. However, the induction of plant defenses depends on the phytohormone profile induced by the specific herbivore feeding guild. No study has shown the impact of soybean leaf chemical defenses induced by thrips herbivory in combination with solar UV-B radiation on thrips performance. To uncover plant responses to herbivory in crop conditions, we proposed the hypothesis that solar UV-B radiation will increase constitutive and inducible defenses and phytohormones related with defenses in field-grown soybean, therefore affecting thrips performance. In this study two soybean cultivars (cv.) were grown in field conditions under attenuated or solar UV-B radiation and damaged by 6 days of herbivory of Caliothrips phaseoli. Our field experiments showed similar survivorship levels of thrips that fed on foliage grown under either attenuated or solar UV-B radiation, while survivorship of thrips that fed on cv. Williams was lower than those that fed on cv Charata. Cv Williams produced different flavonols and higher trypsin protease inhibitor (TPI) activity levels and more genistin than cv Charata. The increment of jasmonic acid (JA)-regulated defenses against insects in foliage of cv Williams was explained by the induction of JA and JA-Ile after herbivory and solar UV-B exposure. Independently of the UV-B environment herbivory induced salicylic acid (SA) and 12-oxo-phytodienoic acid (cis-OPDA) in both cvs. To our knowledge no study before has showed a complete profile of defensive hormones and defensive compounds induced by thrips feeding and solar UV-B.
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Hemipteran ancestors probably lost their digestive serine peptidases on adapting to a plant sap diet. On returning to protein ingestion, these insects start using cathepsin (lysosomal) peptidases as digestive enzymes, from which the less known is cathepsin D. Nine of the ten cathepsin D transcribing genes found in Disdercus peruvianus midgut are expressed exclusively in this tissue and only DpCatD10 is also expressed in other tissues. The main action of cathepsins D is in the first (V1) (from three, V1-3) midgut regions, where 40% of the total proteolytic activity was assigned to aspartic peptidases with an optimum pH of 3.5. The most expressed cathepsins D were identified in the midgut luminal contents by proteomics. The data indicate that D. peruvianus have kept a lysosomal gene expressed in all tissues and evolved another set of genes with a digestive function restricted to midgut. Digestive cathepsins D apparently complement the action of digestive cathepsin L and they are arguably responsible for the hydrolysis of cysteine peptidase inhibitors known to be present in the cotton seeds eaten by the insect, before they meet cathepsin L.
Article
Background: Southern green stink bugs (Nezara viridula) invade field-grown soybean crops, where they feed on developing seeds and inject phytotoxic saliva that causes yield reduction. Although leaf responses to herbivory are well studied, no information is available about the regulation of defenses in seeds. Results: This study demonstrated that mitogen-activated protein kinases (MPK) 3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean, and regulates a defensive response after stink bug damage. Although 10-20 min after stink bug feeding of seeds induced expression of MPK3, MPK6 and MPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also early induced and with increasing levels up to 72 h after damage. Damaged seeds up-regulated defensive genes typically modulated by JA/ET or SA, which in turn decreased the activity of digestive enzymes in the gut of stink bugs. Induced seeds were less preferred by stink bugs. Conclusion: This study shows that stink bug damage induces seed defenses, which is perceived early by MPKs that may activate defense metabolic pathways in developing seeds of field grown-soybean.
Chapter
The aim of this chapter is to review the recent and spectacular progress in the study of insect digestive biochemistry. It tries to establish uniform parameters for studying insect digestive enzymes, providing an overview of the biochemistry of insect digestion, and discusses factors affecting digestive enzymes in vivo. It reviews digestive enzymes and microvillar proteins, with the emphasis on molecular aspects. Further it describes the details of the digestive biochemical process alongside insect evolution and discusses data on digestive enzyme secretion mechanisms. Digestion is the process by which food molecules are broken down into smaller molecules that are absorbed by cells in the gut tissue. This process is controlled by digestive enzymes and is dependent on their localization in the insect gut. Enzyme kinetic parameters are meaningless unless assays are performed in conditions in which enzymes are stable. If researchers adopt uniform parameters and methods, comparisons among similar and different insect species will be more meaningful.
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The 5000 arthropod genomes initiative (i5k) has tasked itself with coordinating the sequencing of 5000 insect or related arthropod genomes. The resulting influx of data, mostly from small research groups or communities with little bioinformatics experience, will require visualization, dissemination and curation, preferably from a centralized platform. The National Agricultural Library (NAL) has implemented the i5k Workspace@NAL (http://i5k.nal.usda.gov/) to help meet the i5k initiative's genome hosting needs. Any i5k member is encouraged to contact the i5k Workspace with their genome project details. Once submitted, new content will be accessible via organism pages, genome browsers and BLAST search engines, which are implemented via the open-source Tripal framework, a web interface for the underlying Chado database schema. We also implement the Web Apollo software for groups that choose to curate gene models. New content will add to the existing body of 35 arthropod species, which include species relevant for many aspects of arthropod genomic research, including agriculture, invasion biology, systematics, ecology and evolution, and developmental research.
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Clustal Omega is a completely rewritten and revised version of the widely used Clustal series of programs for multiple sequence alignment. It can deal with very large numbers (many tens of thousands) of DNA/RNA or protein sequences due to its use of the mBED algorithm for calculating guide trees. This algorithm allows very large alignment problems to be tackled very quickly, even on personal computers. The accuracy of the program has been considerably improved over earlier Clustal programs, through the use of the HHalign method for aligning profile hidden Markov models. The program currently is used from the command line or can be run on line.
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Stink bugs, primarily southern green stink bug, Nezara viridula (Hemiptera: Pentatomidae), are a major pest complex of soybeans (Glycine max) throughout the southern United States. Densities sometimes peak during late R6 and R7 soybean growth stages when soybeans are approaching physiology maturity and the rate of injury from stink bugs is reduced. Field cage trials were conducted from 2005 to 2008 to examine the type and extent of soybean damage caused by southern green stink bugs during the R7 growth stage. The yield response was variable, but overall was not significant. The impact of southern green stink bugs on quality was more consistent. Test weight decreased, and heat damage and total damage increased as stink bug density increased. Based on these data, three economic injury models were developed using different assumptions. The model that assumes no yield loss, does not predict economic injury within the range of stink bug densities tested. However, if the statistically non-significant yield losses are accepted as real, then the models suggest that the southern green stink bug economic injury level and action threshold for soybeans during R7 stage is generally between nine and 15 stink bugs per row m.
Article
Caged soybean plants were infested artificially with Nezara viridula (L.) at various population densities to determine effects of feeding damage on yield and quality of soybeans. Significant reductions in seed yield and significant increases in seed damage were recorded from population densities of 1, 3, and 5 bugs/row-ft. Seeds harvested from cages containing 40 pods infested on 4 dates with a single bug exhibited 63.9-78.5% damage. Germination, emergence, and seedling survival were reduced significantly by all degrees of southern green stink bug damage. Total oil content decreased and total protein content increased as percentage and degree of southern green stink bug damage increased.
Article
Changes in the trypsin inhibitor activity and in the phytic acid, tannin, and catechin content of lentils (Lens culinaris var. Vulgaris) were investigated after soaking in distilled water, citric acid, and sodium bicarbonate solutions. The effect of cooking, after the seeds mere presoaked in the above-mentioned solutions and both the soaking and cooking solutions were discarded, was also studied. Finally, two varieties of lentils (L. culinaris var. Vulgaris and Variabilis) were germinated for 6 days, and the effect on the trypsin inhibitor activity and the phytic acid, tannin, and catechin contents was also measured. Soaking did not modify the trypsin inhibitor activity, decreased the phytic acid content, and increased the tannin and catechin contents. Cooking the presoaked seed brought about the total removal of trypsin inhibitor activity, a reduction of the phytic acid level, and an increase of the content of tannins and catechins. The trypsin inhibitor activity and the phytic acid content showed a large decrease after 6 days of germination, while amounts of tannins and catechins in the two lentil varieties studied increased. Cooking and germination seem to be good procedures to improve the quality of lentil flour from the nutritional point of view, despite the fact that a large variation on the effects of processing, related to the different legume varieties, has been observed.
Article
1. Phenotypic diversity is the fuel that powers evolution. 2. Asexual organisms rely on mutation whereas sexual organisms combine mutation with recombination. 3. Few organisms provide examples of species that are both sexual and asexual, but aphids do. 4. To examine evolution on perceptible timescales requires strong evolutionary forces and, as Darwin noted, agricultural practices provide strong selection. In the case of aphids, insecticides provide a considerable force in the elimination of genotypes. 5. Insecticide resistance in Myzus persicae (Sulzer) has arisen independently through point mutation and gene amplification on a number of occasions and at different times. Resistance to organophosphates, pyrethroids, and pirimicarb (a dimethyl carbamate) is now widespread. 6. In this paper, we examine these three elements: sexual recombination, clonal expansion, and insecticide selection in the peach–potato aphid M. persicae in relation to the evolution of insecticide resistance and survival of the fittest clone.
Article
Ingestion of soybean Kunitz trypsin inhibitor (SKTI) by larvae of the phytophagous insect pest Helicoverpa armigera induced production of inhibitor-insensitive protease activity. The induced activity was not due to proteolytic enzymes of different mechanistic classes, but rather to variants of the existing enzymes. Characterization of cDNAs showed that sequences encoding proteins of the serine protease family were abundant in gut tissue of both control and SKTI-fed insects. The majority of serine protease family cDNAs encode enzymes closely homologous to trypsin and chymotrypsin; comparison of these sequences shows variation in amino acid residues within the region which would be in contact with a protein protease inhibitor. More diverged sequences which may not encode active proteases are also present. All the cDNAs examined were found to derive from multigene families; at least 28 different genes are present in the serine protease family. Chronic ingestion of SKTI results in some serine protease-encoding mRNA species increasing in level, whereas others decrease. Chymotrypsin-encoding mRNAs tend to increase in level as a result of SKTI ingestion, but no clear trend is shown by trypsin-encoding mRNAs. It is suggested that multiple, varying protease-encoding genes are an adaptive mechanism for reducing the deleterious effects of plant protease inhibitors.
Article
Potato plants were treated with gaseous methyl jasmonate (MJ) to obtain leaves with high induced levels of cysteine and aspartic proteinase inhibitors. Induced papain inhibitor activity was estimated at 4% of total protein. Other conditions produced leaves with low and moderate levels of this inhibitor. Development of Colorado potato beetle larvae was similar when they were reared on leaves containing low, moderate and high levels of papain inhibitor. Nevertheless, general proteinase activity was significantly reduced (42%) in insects reared on the high inhibitor diet, while proteinase activity that was insensitive to induced inhibitors in juice from MJ-treated leaves had increased two-fold. Activities towards the specific cysteine proteinase substrate p-Glu-Phe-Leu-pNA were the same in guts from insects reared on the three leaf types. However, juice from MJ-treated leaves inhibited as much as 67% of this activity in guts of insects reared on the low inhibitor diet compared to only 27% of the activity in gut extracts from insects reared on MJ-treated leaves, indicating a 2.5-fold induction of cysteine proteinase activity insensitive to potato proteinase inhibitors. None of the activities towards another specific cysteine proteinase substrate l-Arg-pNA were sensitive to inhibitors from MJ-treated leaves, but guts of insects fed these leaves had a 3.5-fold induction of this proteinase activity compared to those reared on plants containing low papain inhibitor levels. These data suggest that Colorado potato beetle larvae compensated for inhibited gut proteolytic activity during chronic intake of papain inhibitors by synthesizing insensitive proteinase(s).
Article
We compared soybean trypsin inhibitor and potato proteinase inhibitor II in relation to their effects on the growth and digestive physiology of larval H. zea (Boddie) and S. exiqua (Hubner). When incorporated into an artificial diet, both proteinase inhibitors significantly reduced the growth and development of the larvae. However, when the artificial diet containing the proteinase inhibitor was supplemented with methionine, the reduction of growth did not occur. The proteinase inhibitors had no effect on the in vivo digestion of protein, indicating that they do not reduce the level of digested protein. However, when the larvae chronically ingest the proteinase inhibitors, there was significant elevation of the level of tryptic activity (the primary alimentary protease for both species of insect). Thus, we conclude that the mode of action of proteinase inhibitors is to cause the pernicious hyperproduction of trypsin. This, coupled with insufficient dietary availability of sulphur-containing amino acids (i.e. methionine) needed for enzyme synthesis, results in inhibition of growth. Thus, protein quantity/quality is very important in dictating the anti-nutritional effects of plant proteinase inhibitors on herbivorous insects.
Article
The resistance of a transgenic line of oilseed rape expressing constitutively the cysteine proteinase inhibitor oryzacystatin I (OCI) was assessed against Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). The levels of OCI expression in the transformed line averaged 0.2% and 0.05% of total soluble protein in leaves and petioles respectively. In vitro analyses showed that P. chrysocephala larvae use both cysteine and serine proteinases for protein digestion, and that all the cysteine proteolytic activity is OCI-sensitive. However, bioassays showed that adults fed identically on leaf discs from control or transformed plants. When larvae were reared on transgenic plants expressing OCI, they showed an increase in weight gain compared to those reared on control plants. Furthermore, those larvae from transgenic plants exhibited a 2-fold increase in both cysteine and serine proteolytic activity as a reponse to the presence of OCI. The plasticity of insect digestive physiology and feeding behaviour are discussed, as well as the relevance of engineering a genotype expressing both types of proteinase inhibitors.
Article
Euschistus heros (F.) (Heteroptera: Pentatomidae) is the most prevalent stink bug pest in Brazil, and populations can be difficult to manage using organophosphates or endosulfan. Because E. heros is difficult to rear in the laboratory, no baseline insecticide susceptibility data have been published. Therefore, we conducted dose-mortality studies using field-collected insects, and, using the results from the dose-mortality studies, we surveyed susceptibility to acephate, methamidophos, and endosulfan in populations from southern and central Brazil. In addition, esterase activity was evaluated among populations as these enzymes have been related to organophosphate resistance. Finally, newer chemistries were evaluated for E. heros population management in small-plot field studies. In the dose-mortality bioassays, variation in susceptibility to methamidophos and endosulfan among populations was significant, but the variation in susceptibility to acephate was not. The population from Londrina (at the Embrapa Soybean Research Station, northern Parana state) was among the most susceptible, whereas the population from Pedrinhas Paulista (southwestern Sao Paulo state) was among the least susceptible. Significant variation among populations was observed in esterase activity with the population from Pedrinhas Paulista having the highest esterase levels and the population from Londrina having among the lowest levels. To survey populations, we used the dose-mortality data for the Londrina population (because it was, consistently, one of the most susceptible populations) to estimate the insecticide concentration that killed 99% of the population (LC99). The corresponding doses were 5.9 microg (AI) of acephate adult(-1), 0.7 microg (AI) methamidophos adult(-1), and 1,600 microg (AI) endosulfan adult(-1). The survey data confirmed the data from the dose-mortality bioassays in that the population from Pedrinhas Paulista was identified as one of the least susceptible and the population from Londrina was one of the most susceptible. The field tests confirmed that older chemistries provided minimal control (< 50% control), whereas the products containing mixtures of pyrethroids and neonicotinoids provided better control (> 60%).
Article
When fed on a diet containing a proteinaceous cysteine protease inhibitor from soybean (scN), cowpea bruchid larvae enhance their overall digestive capacity to counter the inhibitory effect. Elevated proteolytic activity is attributed not only to the major digestive cysteine proteases (CmCPs), but also to aspartic proteases, a minor midgut protease component. In this study, we isolated a CmCatD cDNA from cowpea bruchid midgut that shares substantial sequence similarity with cathepsin D-like aspartic proteases of other organisms. Its transcript profile was developmentally regulated and subject to alteration by dietary scN. CmCatD transcripts were more abundant in scN-fed 3rd and 4th instar midguts than in control. The bacterially expressed recombinant CmCatD proprotein was capable of autoprocessing under acidic conditions, and mature CmCatD also exhibited pH-dependent proteolytic activity which was inhibited specifically by pepstatin A, indicative of its aspartic protease nature. CmCatD trans-activated CmCPs and vice versa, suggesting a cooperation between the minor midgut CmCatD and major digestive CmCPs. Further, CmCatD was able to degrade scN after extensive incubation. This activity partially restored CmCP proteolytic activity otherwise inhibited by scN. Thus CmCatD could facilitate insects' coping with the challenge of dietary scN by exerting its scN-insensitive and scN-degrading activity, freeing cysteine proteases for food degradation. Taken together, cowpea bruchids coordinate the functionality of the two classes of digestive proteases to fend off the negative effect of scN, and fulfill their nutrient requirements.
Article
The peritrophic membrane (PM) is an anatomical structure surrounding the food bolus in most insects. Rejecting the idea that PM has evolved from coating mucus to play the same protective role as it, novel functions were proposed and experimentally tested. The theoretical principles underlying the digestive enzyme recycling mechanism were described and used to develop an algorithm to calculate enzyme distributions along the midgut and to infer secretory and absorptive sites. The activity of a Spodoptera frugiperda microvillar aminopeptidase decreases by 50% if placed in the presence of midgut contents. S. frugiperda trypsin preparations placed into dialysis bags in stirred and unstirred media have activities of 210 and 160%, respectively, over the activities of samples in a test tube. The ectoperitrophic fluid (EF) present in the midgut caeca of Rhynchosciara americana may be collected. If the enzymes restricted to this fluid are assayed in the presence of PM contents (PMC) their activities decrease by at least 58%. The lack of PM caused by calcofluor feeding impairs growth due to an increase in the metabolic cost associated with the conversion of food into body mass. This probably results from an increase in digestive enzyme excretion and useless homeostatic attempt to reestablish destroyed midgut gradients. The experimental models support the view that PM enhances digestive efficiency by: (a) prevention of non-specific binding of undigested material onto cell surface; (b) prevention of excretion by allowing enzyme recycling powered by an ectoperitrophic counterflux of fluid; (c) removal from inside PM of the oligomeric molecules that may inhibit the enzymes involved in initial digestion; (d) restriction of oligomer hydrolases to ectoperitrophic space (ECS) to avoid probable partial inhibition by non-dispersed undigested food. Finally, PM functions are discussed regarding insects feeding on any diet.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
The biochemical interactions between digestive proteinases of the Coleoptera pest black vine weevil (Otiorynchus sulcatus) and two plant cysteine proteinase inhibitors, oryzacystatin I (OCI) and oryzacystatin II (OCII), were assessed using gelatin-polyacrylamide gel electrophoresis, OCI-affinity chromatography, and recombinant forms of the two plant inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of them being totally or partially inactivated by OCI and OCII. The maximal inhibitory effect of both OCs at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of OC-insensitive proteases, indicating the stability of the OCI and OCII effects. After removing OC-sensitive proteinases from the insect crude extract by OCI-affinity chromatography, the effects of the insect cystatin-insensitive proteases on the structural integrity of the free OCs were analyzed. While OCI remained stable, OCII was subjected to limited proteolysis leading to its gradual transformation into a approximately 10.5-kDa unstable intermediate, OCIIi. As shown by the degradation pattern of a glutathione S-transferase (GST)/OCII fusion protein, the appearance of OCIIi resulted from the C-terminal truncation of OCII. Either free or linked to GST, OCIIi was as active against papain and human cathepsin H as OCII, and the initial specificities of the inhibitor for these two cysteine proteinases were conserved after cleavage. Although these observations indicate the high conformational stability of OCII near its active (inhibitory) site, they also suggest a general conformational destabilization of this inhibitor following its initial cleavage, subsequently leading to its complete hydrolysis. This apparent susceptibility of OCII to proteolytic cleavage by the insect proteinases could have major implications when planning the use of this plant cystatin for insect pest control.
Article
Incorporation of genes encoding proteinase inhibitors into oilseed rape genome could confer resistance to Coleoptera, which are the major pests on rape in Europe. A detailed study of the digestive proteinase of a model cruciferous-feeding Coleoptera, Phaedon cochleariae, showed that this insect relies on a complex proteolytic system including serine, cysteine, aspartyl proteinases, and leucine aminopeptidases. The inhibition of general and specific activities by a range of proteinase inhibitors in vitro suggested that oryzacystatin I (OCI) and Bowman-Birk inhibitor (BBI) would have adverse effects when ingested by the larvae. However, the growth and the feeding of larvae reared on oilseed rape leaf discs treated with a high dose of OCI and/or BBI were not affected. Moreover, the levels and patterns of proteolytic activities were not modified in these larvae. The study of the interactions between P. cochleariae larval proteinases and OCI and BBI revealed that both inhibitors were rapidly cleaved by serine proteinases in association with leucine aminopeptidases, and consequently lost their inhibitory capacity. This mechanism of resistance is very efficient, and may be widespread among Coleoptera. The major implications for insect control using proteinase inhibitor-based strategies are discussed.
Article
Protease inhibitors have been proposed as potential control molecules that could be engineered into potato plants for developing crops resistant to the Colorado potato beetle, Leptinotarsa decemlineata, a major pest of potato and other Solanaceae. In this study, we examined the effects of feeding young female beetles with foliage from a cultivar of the "Kennebec" potato line (K52) transformed with a gene encoding oryzacystatin I (OCI), a specific cysteine proteinase inhibitor with proven activity against cathepsin H-like enzymes of larvae and adults of the potato beetle. To evaluate the insect's performance, we collected data over a 16-d postemergence period on survival, diapause incidence, foliage consumption, weight gain, and oviposition of females. Tested individuals were fed untransformed (control) and OCI-transformed foliage at two stages of potato leaf differentiation, corresponding to "low" and "high" levels of OCI expression in leaves of K52. The OCI-expressing foliage did not affect female survival (close to 100%), incidence of diapause (15-30%), relative growth rate (RGR) during postemergence growth (5-9% d(-1)) or maximum weight reached (140-160 mg). Neither did it affect female reproductive fitness as measured by preoviposition time (8-9 d), 16-d fecundity (220-290 eggs), or egg eclosion incidence (86-91%). However, nutritional stress to females feeding on OCI foliage was evident, as reflected in their lower efficiency of conversion of ingested foliage (ECI) during postemergence growth, increased foliage consumed per egg laid (up to 119% more), and adaptation of their digestive proteolytic system to the inhibitory effect of OCI. Interestingly, beetles fed foliage expressing the highest level of OCI reacted rapidly to the presence of OCI by producing OCI-insensitive proteases, and exhibiting strong hypertrophic behavior by ingestion of 2.4-2.5 times more OCI rich foliage apparently as a compensatory response for nutritional stress due to the protease inhibitor in their diet.
Article
Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.