Richard E. Shade

Cysteine digestive proteinases in Coleoptera

Abstract 1. 1. Proteinase activity from midguts of larvae and adult beetles representing several major coleopteran families was optimal at mildly acidic pH. 2. 2. In many beetles, thiol-reducing agents stimulated proteolysis of [ 3 H]methemoglobin, while alkylating agents strongly inhibited it. 3. 3. E-64, a specific inhibitor of cysteine proteinases, was a potent inhibitor in most species. 4. 4. In larvae of red flour beetle ( Tribolium castaneum ), Mexican bean beetle ( Epilachna varivestis ), and cowpea weevil ( Callosobruchus maculatus ), the pH of gut contents was in the range 5–7 and exhibited a negative (reducing) redox potential. 5. 5. These observations suggest that cysteine proteinases are commonly used as digestive enzymes in the Coleoptera.

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor.

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371–379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN‐free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN‐adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN‐induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L‐like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN‐degrading protease activity.

Biological effects of plant lectins on the cowpea weevil.

Abstract Plant lectins have been implicated as antibiosis factors against insects, particularly the cowpea weevil, Callosobruchus maculatus . In the present investigation, 17 plant lectins were screened in an artificial seed system to determine if biological activity toward C. maculatus is widespread among lectins. Five of the 17 plant lectins screened caused a significant delay in C. maculatus developmental time at dietary levels of 0.2 and 1.0% (w/w). These lectins could be classed into one of two groups: lectins with specificity for N -acetylgalactosamine/galactose (GalNAc/Gal) and lectins with specificity for N -acetylglucosamine (GlcNAc). The GalNAc/Gal lectins included osage orange lectin and peanut agglutinin. A dose-response analysis of peanut agglutinin indicated that for every 0.1% increase in dose there was a 0.49 day delay in developmental time. The GlcNAc lectins included potato lectin, jimson weed lectin and wheat germ agglutinin (WGA). The most potent lectin was WGA; the dose-response analysis indicated that for every 0.1% increase in dose there was a 1.47 day delay in developmental time. Additionally, for every 0.1% increase in WGA there was a 2.79% increase in mortality. The results of this investigation indicate that there exists in C. maculatus physiological/biochemical systems vulnerable to selected plant lectins. The genes coding for the effective plant lectins could, in principle, serve as antibiosis factors to use in host plant resistance programs designed to bring resistance into cowpea through recombinant DNA technologies.

An Insecticidal N-Acetylglucosamine-Specific Lectin Gene from Griffonia simplicifolia (Leguminosae)

Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.

Preservation of cowpea grain in sub-Saharan Africa—Bean/Cowpea CRSP contributions

Abstract In sub-Saharan Africa, post-harvest insect pests of cowpeas (Vigna unguiculata (L.) Walp.) degrade the nutritional quality and economic value of the grain and cause producers, in anticipation of losses during storage, to sell at harvest when the price is lowest. Principal pest is the cowpea bruchid, Callosobruchus maculatus (F.), but other bruchids cause losses as well. Beginning in the 1980s, the USAID-funded Bean/Cowpea Collaborative Research Support Program (CRSP) targeted post-harvest insect pests of cowpea as a constraint meriting an investment in research and development. Subsequently, researchers in Senegal, Cameroon, and at Purdue University, created and helped disseminate numerous simple, low cost, and environmentally friendly technologies for managing post-harvest insect pests. Technologies developed and disseminated with the help of NGOs such as World Vision International, the International Institute of Tropical Agriculture’s PRONAF program, and FAO’s Harare, Zimbabwe, office included: (1) a highly effective drum storage technology developed at ISRA, Senegal, and now widely adopted in Senegal; (2) a solar disinfestation technique developed at Purdue and at IRAD, Maroua, Cameroon, now being disseminated in many African countries; (3) an improved ash storage procedure; (4) a bagging technology utilizing triple plastic bags; (5) two cowpea cultivars expressing combined seed and pod wall resistance to cowpea bruchids, released by the Cameroon government in 1999.

Rice and stinging nettle lectins: Insecticidal activity similar to wheat germ agglutinin

Previous studies have shown that lectins with specificity for GlcNAc residues, when fed in the diet of the cowpea weevil,Callosobruchus maculatus, cause delays in its development. We have begun anin vivo structure activity analysis to determine what molecular features lead to maximum lectin toxicity. Although rice lectin has a four-fold greater agglutinating activity toward mammalian erythrocytes than wheat germ agglutinin (WGA), its biological activity when fed toC. maculatus is similar to WGA. Stinging nettle lectin, a poor agglutinin, isca two to four times less effective than WGA.

Efficacy of ash for controlling infestations of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpeas

Abstract Storing cowpeas, Vigna unguiculata (L.) Walpers, with ash to protect them against Callosobruchus maculatus (F.) is a traditional storage method in northern Cameroon. The amount of ash used and the details of the methodology (i.e. mixing vs layering, the source of ash used) varies among farmers. In experiments designed to simulate traditional ash-storage procedures, it was found that a minimum ratio of 3 parts of ash to 4 parts of cowpeas prevented population growth of C. maculatus and that a 3 cm layer of ash on top of stored seeds prevented infestation by adults.

Effect of wheat germ isolectins on development of cowpea weevil

Abstract Wheat germ agglutinin (WGA) isolated from hexaploid wheat is made up of six isolectin dimeric forms. In order to determine if the previously observed biological activity of WGA against the cowpea weevil was due preferentially to particular isolectins, we isolated each of the three isolectin homodimers and bioassayed them in an artificial seed bioassay. We show that each of the isolectins is equally detrimental to the growth and development of the cowpea weevil. Hence, any of the three genes for WGA isolectins could, in principle, be transferred into cowpea to confer bruchid resistance.

The effect of two proteinase inhibitors, E-64 and the Bowman-Birk inhibitor, on the developmental time and mortality of Acanthoscelides obtectus.

An artificial bean seed system was used to evaluate the effects of a cysteine proteinase inhibitor (E‐64) and a serine proteinase inhibitor (Bowman‐Birk inhibitor) on the developmental time and mortality of the common bean weevil, Acanthoscelides obtectus (Say). These inhibitors were incorporated into artificial bean seeds on which the insect fed. To better understand the mode of action of these inhibitors, free amino acids were also added to the seeds, alone and in combination with the inhibitors. E‐64 was found to be highly effective in delaying development and increasing mortality of the insect. Both effects were directly related to the concentration of E‐64. Bowman‐Birk inhibitor had little effect on these parameters. Assays of gut proteolytic activity of insects reared on artificial seeds with various levels of E‐64 demonstrated a direct relationship between E‐64 concentration in the diet and reduction of gut proteolytic activity. Free amino acid supplementation to the diet did not prevent inhibition of gut proteolytic activity by E‐64, but did reverse its effects on developmental time and mortality, strengthening the hypothesis that E‐64 operates by inhibition of essential digestive proteinase activity.

Physical and chemical attributes of cowpea lines resistant and susceptible to Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

Abstract Fifteen lines of cowpeas, Vigna unguiculata (L.) Walpers, were analyzed for physical and chemical characteristics to study their relationship with resistance to Callosobruchus maculatus (F.). No significant differences were found (P