Larry L. Murdock

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.

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.

Arabidopsis vegetative storage protein is an anti-insect acid phosphatase

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the proteins secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.

Diversity in digestive proteinase activity among insects.

AbstractpH-optimum curves for proteolytic activity from midgut homogenates of 23 species of insects were determined using a radiometric proteinase assay. Optima for digestion of radiolabeled methemoglobin in these insects ranged from pH 3.0 to pH 12.0. Since pH optima vary among the different mechanistic classes of proteinases, these data suggest that there is high diversity in midgut digestive proteinases among various insects.

Formamidine Pesticides: Octopamine-Like Actions in a Firefly

The formamidine pesticide chlordimeform and its N-demethylated metabolites cause the light organ of the firefly Photinus pyralis L. to glow brightly. Monodemethyl chlordimeform is active at doses as low as 5 nanograms per insect when applied topically. This action is postsynaptic and probably involves membrane-bound receptors since cyproheptadine blocks the glows induced by both monodemethyl chlordimeform and octopamine, the putative neurotransmitter in the light organ. The pesticidal and pestistatic properties of the formamidines may result from actions on octopaminergic systems.

Suppression of larval Colorado potato beetle growth and development by digestive proteinase inhibitors

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.

Development of cowpea cultivars and germplasm by the Bean/Cowpea CRSP

This paper reviews accomplishments in cowpea cultivar and germplasm development by the Bean/Cowpea Collaborative Research Support Program (CRSP) which was funded by the United States Agency for International Development for a period of about 20 years. Drought-adapted, pest and disease resistant cultivars ‘Mouride’, ‘Melakh’ and ‘Ein El Gazal’ were developed for rainfed production in the tropical Sahelian zone of Africa. Cultivars ‘CRSP Niebe’ and ‘Lori Niebe’ which have seed and pod resistance to cowpea weevil and some disease resistance were developed for rainfed production in the tropical Savanna zone of West Africa. Cultivar ‘California Blackeye No. 27’ was developed for irrigated production in subtropical California, USA and is a semidwarf with heat tolerance and broad-based resistance to root-knot nematodes and Fusarium wilt. Various cultivars with persistent-green seed color including ‘Bettergreen’ and ‘Charleston Greenpack’ were bred for use in the food freezing industry in the USA. Germplasms were developed with unique traits including: snap-type pods, green manure/cover crop capabilities, heat tolerance during reproductive development, chilling tolerance during emergence, delayed leaf senescence as a mechanism of adaptation to mid-season drought and high grain yields, differences in stable carbon isotope discrimination, harvest index, rooting and plant water- and nutrient-relations traits, broad-based resistance to root-knot nematodes and Fusarium wilt, and resistance to flower thrips, cowpea aphid, lygus bug and cowpea weevil, and various quality traits including all-white and sweet grain. These germplasms provide a valuable resource for breeding additional cowpea cultivars for Africa and the USA.

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.

FARMER ACCEPTABILITY CRITERIA IN BREEDING COWPEA

Cowpea (Vigna unguiculata) farmers from diverse geographical areas in northern Cameroon selected breeding lines from on-station trials for their own fields and explained their reasons (criteria) for making the selections. The average selection intensity employed by farmers varied from 6 to 17% and was similar to that employed by plant breeders. Their selection criteria fell into three broad categories relating to yield, preference and quality, and labour, which were very consistent over years, locations and gender. Selection criteria were strongly influenced by market preferences, reflecting the increasing role of cowpea as a cash crop. Implications ofthese findings for breeding strategies and genetic resource conservation are discussed.