Cc-LTP1

Bruchid beetles infest various seeds. The seed coat is the first protective barrier against bruchid infestation. Although non-host seed coats often impair the oviposition, eclosion and survival of the bruchid Callosobruchus maculatus larvae, morphological and biochemical aspects of this phenomenon remain unclear. Here we show that Phaseolus vulgaris (non-host) seed coat reduced C. maculatus female oviposition about 48%, increased 83% the seed penetration time, reduced larval mass and survival about 62 % and 40 % respectively. Interestingly, we found no visible effect on the major events of insect embryogenesis, namely the formation of the cellular blastoderm, germ band extension/retraction, embryo segmentation, appendage formation and dorsal closure. Larvae fed on P. vulgaris seed coat have greater FITC fluorescence signal in the midgut than in the feces, as opposed to what is observed in control larvae fed on Vigna unguiculata. Cysteine protease, α-amylase and α-glucosidase activities were reduced in larvae fed on P. vulgaris natural seed coat. Taken together, our results suggest that although P. vulgaris seed coat does not interfere with C. maculatus embryonic development, food digestion was clearly compromised, impacting larval fitness (e.g. body mass and survivability).

The pulse beetle, Callosobruchus maculatus (F.) is a destructive pest of pulses in both storage and field. It is well known that diatomaceous earth (DE) kill the insects by locally absorbing the epicuticular lipid layers leading to high rate of water loss through the cuticle. However, the effectiveness of DE depends on its ability to kill the adults before copulation and egg-laying. Newly emerged virgin males and females of Callosobruchus maculatus (F.) were exposed to the DEs, Fossil-Shield and Silico-Sec on 30 treated mungbeans (Vigna radita (L)). Fecundity, number of beans used for egg-laying and beans without eggs were evaluated after four days; the number of unhatched eggs was evaluated after ten days. It was determined, that the fecundity of female insects decreased sigmoidely with increasing rate of DE content. Percentages of unhatched eggs and seeds without eggs increased with increasing DE dosages. However, the maximum egg densities (eggs per used secd) occurred at 1200 mg DE/kg for Fossil-Shield and Silico-Sec. The reason for such DE-stimulated behaviour of egg laying expressed as a number of seeds with eggs of C. maculatus is not known, but it may be related to the stress caused by the inert dusts or to the reduction of both chemical and physical (tactile) stimuli. Treatment with DEs altered the surface texture of the beans and caused less cohesion between eggs and the seed surface. Only few larvae managed to penetrate into the grains, possibly due to increased grain roughness and repellent effect of DE. A relatively high number of eggs were laid on the surface of those beans where the amount of dust had been locally reduced by adults' movement and their pick up of DE. Therefore, several larvae tried to penetrate into these treated beans, causing a high larval density per partially cleaned bean. All these reasons lead to a progeny decline.

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae.