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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.

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.