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Bowman-Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy-associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI-fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a Blast search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

One function of plant lectins such as wheat germ agglutinin is to serve as defences against herbivorous insects. The midgut is one critical site affected by dietary lectins. We observed marked cellular, structural and gene expression changes in the midguts of Drosophila melanogaster third instar larvae that were fed wheat germ agglutinin. Some of these changes were similar to those observed in the midguts of starved D. melanogaster. Dietary wheat germ agglutinin caused shortening, branching, swelling, distortion and in some cases disintegration of the midgut microvilli. Starvation was accompanied primarily by shortening of the microvilli. Microarray analyses revealed that dietary wheat germ agglutinin evoked differential expression of 61 transcripts; seven of these were also differentially expressed in starved D. melanogaster. The differentially transcribed gene clusters in wheat germ agglutinin-fed larvae were associated with (1) cytoskeleton organization; (2) digestive enzymes; (3) detoxification reactions; and (4) energy metabolism. Four possible transcription factor binding motifs were associated with the differentially expressed genes. One of these exhibited substantial similarity to MyoD, a transcription factor binding motif associated with cellular structures in mammals. These results are consistent with the hypothesis that wheat germ agglutinin caused a starvation-like effect and structural changes of midgut cells of D. melanogaster third-instar larvae.

Oligoarray analysis was used to determine the number and nature of genes expressed in third instar Drosophila melanogaster larval midguts. The majority of transcripts were associated with protein synthesis and metabolism. Serine proteases were the main proteolytic enzymes detected. Some 40% of the cytochrome P450 genes and 74% of the glutathione S transferases (GSTs) in the genome of D. melanogaster were observed to be expressed in the midgut by oligoarray analysis. We also identified potential transcription factor binding motifs (TFBMs) of P450s, GSTs and carboxylesterases. Many of the midgut-expressed GST genes contained candidate TFBMs homologous to TFBMs in mammals that have been associated with responses to oxidative stress. We also investigated the response of GSTs in the midgut to dietary H 2 O 2 , which showed a dosagebased differential response.

The cowpea bruchid, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae: Bruchini), is aworldwide pest of stored cowpea grain [ Vigna unguiculata (L.) Walp. (Leguminosae)], causing tensof millions of dollars’ worth of damage annually. One method of managing this pest involves plantingcultivars whose seeds are resistant to bruchids. Despite extensive research, we still do not know themechanism by which TVu 2027, the original resistant cowpea line, resists C. maculatus . Using micro-CAT (computerized axial tomography) scan imaging, we visualized the feeding patterns of bruchidsliving and growing within cowpea seeds. We present evidence that an interior zone in TVu 2027 seedsis responsible for the high mortality and developmental de lays experienced by avirulent larval bruchids.We observed that both virulent as well as avirulent bruchid strains have different feeding patterns inTVu 2027 seeds compared to susceptible seeds. It appears that the resistance factor is most concen- trated in a zone in each cotyledon adjacent to the air space separating the two seed halves.

Consumer preference information is essential to targeting research. This paper reports an effort of a multi-disciplinary team to measure the market value of cowpea characteristics. Five samples were purchased once per month in seven markets in Ghana and Cameroon starting in September 1996. In the market, price and vendor characteristics were noted. In the laboratory, size of grains, testa color, testa texture, eye color and damage levels were recorded. A hedonic pricing regression model was used. Results indicate that grain size is the most important characteristic. Consumers seem more sensitive to bruchid (Callosobruchus maculates) damage than hypothesized. Cowpeas with white testa command a clear premium only in one of the Ghanaian markets. In Ghana, black eyes sell at a premium, but in Cameroon black eyes are discounted. In general, this study indicates that quality characteristics are very important in West African food markets. Even low income consumers are willing to pay a premium for products that match their preferences, and they are vigilant in identifying products that do not meet their standards. Purchasing samples on a regular basis and hedonic pricing offers a practical way for biological scientists and economists to work together to measure these consumer preferences.

We describe here the first systematic work to discover insect genes involved in food breakdown using a cDNA library enriched for gut-expressed transcripts from Callosobruchus maculatus. A total of 1056 clones were screened for cDNA insert-containing plasmids, and 503 nonredundant open reading frames were discovered. Twenty-three inferred genes potentially involved in digestive processes in cowpea weevil were identified, including proteinases and amylases. The predicted catalytic sites were identified in the inferred cysteine and aspartic acid proteinases, and in alpha-amylases. Transcriptome analysis of the cowpea bruchid will potentially permit gene discovery in other beetles, an insect order of major economic and ecological importance that is poorly represented in genomic databases.

*Summary Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 10 10 ‐10 11 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower K i (2.1 pM) than parental scN, due to its increased association rate constant (2.6 6 0.09 3 10 7 M ‐1 sec ‐1 ). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

Griffonia simplicifolia leaf lectin II (GS-II) is a N-acetylglucosamine (GlcNAc) binding protein, consisting of large (rGS-II) and small (sGS-II) subunits, that is presumed to function in plant defense because both the holoprotein and the rGS-II inhibited insect growth and development in feeding bioassays. A cDNA encoding sGS-II has been isolated and the deduced peptide has sequence similarity to class III chitinases. However, neither the GS-II holoprotein nor bacterially expressed recombinant sGS-II proteins had detectable chitinase or lysozyme activities. Both rGS-II and sGS-II mRNAs accumulated in response to methyl jasmonate (Me-JA) treatment but not after wounding in local leaves unless injury was followed by treatment with the ethylene action inhibitor norbornadiene (NBD). Salicylic acid (SA) suppressed wounding/NBD induction of GS-II transcript accumulation and ethylene inhibited Me-JA-induced expression. Apparently, defensive gene expression induced by signal transduction through the octadecanoid pathway in local leaves is suppressed by stress induced ethylene that is produced as a consequence of wounding. However, in leaves systemic to the wound site, rGS-II mRNA levels increased substantially indicating that physiological levels of ethylene are insufficient to down regulate defensive gene expression away from the site of injury. It seems that G. simplicifolia has evolved a rather specialized response to herbivore attack whereby local activation of defensive gene expression is attenuated in order to mount a more substantial defense distal to the site of invasion.

Rice weevils, Sitophilus oryzae (L.), complete their development from egg to early adulthood inside grains of wheat, rice or maize, but little is known about their feeding behavior within the seeds. An ultrasonic insect feeding monitor was used to characterize the feeding patterns of individual rice weevils as they developed in maize grains. Weevils reared in grains held at ca. 40% r.h. took longer to develop from egg to pupation than those reared in grains kept at ca. 70% r.h. Feeding patterns revealed that larvae developing in grains held at ca. 70% r.h. had only four instars, whereas supernumerary molts (five instars) occurred in certain individuals reared in grain held at ca. 40% r.h. The supernumerary molts may represent a response of the insect to stresses associated with low moisture levels in the seeds.

An artificial maize seed bioassay was developed to evaluate potential resistance factors against the rice weevil, Sitophilus oryzae. Weevils reared in artificial seeds compared to those reared in whole maize seeds: (i) developed faster, (ii) had similar within-seed developmental mortalities, (iii) were lighter in weight upon emergence and (iv) oviposited the same number of eggs. Using this bioassay we found that E-64, a cysteine protease inhibitor, decreased the number of emerged adults per seed and delayed within-seed developmental time, suggesting that the rice weevil utilizes a cysteine protease to digest its dietary protein. Weevils fed inhibitors of trypsin and chymotrypsin, Bowman-Birk and Kunitz inhibitors respectively, developed normally. Para-amino-l-phenylalanine (PAPA), a non-protein amino acid implicated as an insect resistance factor in Vigna vexillata, was lethal at dietary levels of 0.2% (w/w) and higher. An extract from Amaranthus caudatus seeds delayed the developmental time of the rice weevil at dietary levels of 0.2% (w/w) and increased mortality at dietary levels of 1.0% (w/w). Several proteins tested, including Griffonia simplicifolia agglutinin II, phytohemagglutinin extract containing common bean α-amylase inhibitor, pokeweed agglutinin, Bacillus thuringiensis CRY1A/CRY1B endotoxin, and an α-amylase inhibitor from wheat, had no effect on the rice weevil. The artificial maize seed bioassay was adapted by pelleting the seed for use with an ultrasonic insect feeding monitor to determine the finding activity of rice weevils as they developed from egg hatch to pupation.

Thirty cowpea, Vigna unguiculata (L.) Walpers, cultivars were evaluated as intact pods to determine if any possessed resistance to the cowpea weevil, Callosobruchus maculatus (Fabr.). Pod resistance was measured as pre-establishment larval mortality (PreM); those larvae dying after egg hatch but before penetrating into the seeds, and as post-establishment within-seed mortality (PostM); those larvae dying after penetrating into the seeds. Among the 30 varieties examined, PreM ranged from 57.9% to 99.4% and PostM ranged from 6.7%, to 82.6%. Ten varieties exhibited total intact pod mortality (mortality from egg hatch to adult emergence from the seed) greater than 95%. Physical measurements were made of several pod and seed characteristics to ascertain whether the observed pod resistance was due to seed factors, pod-wall factors, or to interactions between the pod and seeds. Although resistance to breakage (from handling plus pod dehiscence) is a necessary characteristic for overall pod protection against C. maculatus; other factors are also important. Among the other pod and seed characteristics measured to identify major resistance factors, seed coat thickness was the one most highly correlated with pod resistance. Our results suggest that interactions between pod-wall and seed coat characteristics play a large role in pod resistance of cowpeas to C. maculatus.

The effects of plant age and growing conditions on the wound-induced responses of tomatoes (Lycopersicon esculentum Mill) was studied. The impact of these variables on the suitability of tomato foliage as food was assessed both by growth rates of neonate Manduca sexta Johannson larvae and by radiometric assay for proteinase inhibitor concentration. Plant quality (as assessed by M. sexta growth rates) was decreased by wounding regardless of plant age or environmental conditions at the time of wounding. The decrease in quality was not necessarily associated with increased concentrations of proteinase inhibitor. When tested directly, high concentrations of proteinase inhibitor did not affect larval growth rates. Induction of foliar proteinase inhibitors in response to wounding was found to peak while the plants were young and decrease as the plants matured. Resume Croissance de Manduca sexta sur pieds de tomates blesses: influence des inhibiteurs de proteinases induits Le feuillage de pieds de tomates (Lycospersicon esculentum Mill) a ete endommage a differents âges et dans differentes conditions ecologiques. Les modifications de sa valeur alimentaire ont ete testees avec des chenilles neonates de Manduca sexta Johannson et par des dosages radiometriques de sa concentration en inhibiteurs de proteinase. Des plantes endommagees a 2 semaines provoquent une croissance larvaire plus lente que des plantes endommagees a 4 semaines. Des plantes soumises a une forte intensite lumineuse et a des temperatures elevees lors de leur alteration provoquent une croissance larvaire plus lente que des plantes maintenues en serre pendant cette periode. Les changements de la qualite des plantes mis en evidence par les taux de croissance larvaire n'etaient pas lies a l'importance de l'induction des inhibiteurs de proteinase dans le feuillage. Quand les plantes sont endommagees a 3 jours d'intervalle, 10 a 30 jours apres la germination, l'induction des inhibiteurs de proteinase est maximale le 16c jour et diminue ensuite. M. sexta se developpe aussi bien sur les feuilles mures de tomate, qu'elles contiennent ou non des concentrations elevees en inhibiteurs de proteinase. Ces resultats montrent que la blessure a un impact negatif sur la valeur comme aliment pour M. sexta du feuillage de tomate, qui ne depend pas de la quantite d'inhibiteurs de proteinase induite dans les feuilles.

Evidence is presented that the Colorado potato beetle (CPB) (Leptinotarsa decemlineata (Say)) depends, at least partially, on cysteine proteinases for protein digestion. Midgut homogenates of CPB larvae have a mildly acidic pH and exhibit major proteolytic activity in the mildly acidic pH range. This proteolytic activity is activated by reducing agents, is inhibited by E-64 (a specific cysteine proteinase inhibitor), and is not inhibited by serine proteinase inhibitors. In addition, consumption of E-64 treated potato leaves by CPB larvae at rates as low as 0.8 μg/cm2 of leaf tissue has a deleterious effect on larval growth and development. Resume Suppression par des inhibiteurs de proteinases intestinales, de la croissance et du developpement des larves de doryphores Les proteinases intestinales des larves de Leptinotarsa decemlineata sont en partie caracterisees par les pH limitant leur activite, et par l'effet d'inhibiteurs de proteinases de specificites connues. Les proteinases ont ete testees avec la methemoglobine tritiee comme substrat et nous avons determine les taux relatifs de peptides radioactives TCA solubles, liberees, dans des conditions precises, pour des pH compris entre 2,0 et 12,0. Le taux le plus eleve d'activite proteolytique a ete observe pour des pH 5,0 a 6,0, bien qu'il y ait eu une activite appreciable aux pH 4,0 et 8,0. Parmi les inhibiteurs examines, le E-64, inhibiteur proteinase cysteine tres selectif, a ete le plus efficace sur l'activite proteinase. La pepstatine, inhibiteur proteinase aspartique, a ete actif, mais seulement dans une gamme plus moin de pH. Les inhibiteurs proteinase serine, ont ete pratiquement inactifs sur l'activite proteinase. Chez des larves de doryphores nourries de feuilles de pommes de terre traitees avec differentes concentrations de E-64 ou de pepstatine, la consommation de E-64 a retarde fortement la croissance larvaire et le developpement, la pepstatine a provoque un retard du developpement plus limitee. Nos resultats suggerent que le doryphore depend, tout au moins partiellement, des proteinases cysteine pour l'assimilation des proteines.