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Callosobruchus maculatus is the main pest cowpea (Vigna unguiculata). Given its relevance as an insect pest, studies have focused in finding toxic compounds which could prevent its predatory action towards the seeds. Clitoria fairchildiana is a native Amazon species, whose seeds are refractory to insect predation. This characteristic was the basis of our interest in evaluating the toxicity of its seed proteins to C. maculatus larvae. Seed proteins were fractioned, according to their solubility, to albumins (F1), globulins (F2), kaphyrins (F3), glutelins (F4), linked kaphyrins (F5) and cross-linked glutelins (F6). The fractionated proteins were quantified, analysed by tricine-SDS-PAGE and inserted into the diet of this insect pest in order to evaluate their insecticidal potential. The most toxic fraction to C. maculatus, the propanol soluble F3, was submitted to molecular exclusion chromatography and all of the peaks obtained, F3P1, F3P2, F3P3, caused a reduction of larval mass, especially F3P1, seen as a major ~12 kDa electrophoretic band. This protein was identified as a vicilin-like protein by mass spectrometry and BLAST analysis. The alignment of the Cfvic (C. fairchildiana vicilin) peptides with a V. unguiculata vicilin sequence, revealed that Cfvic has at least five peptides (ALLTLVNPDGR, AILTLVNPDGR, NFLAGGKDNV, ISDINSAMDR, NFLAGEK) which lined up with two chitin binding sites (ChBS). This finding was corroborated by chitin affinity chromatography and molecular docking of chitin-binding domains for N-Acetyl-D-glucosamine and by the reduction of Cfvic chitin affinity after chemical modification of its Lys residues. In conclusion, Cfvic is a 12 kDa vicilin-like protein, highly toxic to C. maculatus, acting as an insect toxin through its ability to bind to chitin structures present in the insect midgut.

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Schinus terebinthifolius, Raddi, has been extensively studied due to its anti-inflammatory and antibiotic properties. S. terebinthifolius was also toxic to some insects, however little has been explored about the nature of its insecticide compounds or the toxicity of this plant to insect species. In this work, we investigate the toxicity of S. terebinthifolius seed flour against the insect C. maculatus. S. terebinthifolius seed flour interfered with the post hatch development of the C. maculatus larvae, decreasing larval survival, mass and length. Using DEAE-cellulose chromatography, five protein fractions were isolated, a non-retained fraction (NRF) and four retained fractions, eluted with 0.25, 0.5, 0.7 and 1.0 M NaCl. Proteins with varying molecular masses were observed in all fractions. The majority protein bands were identified by mass spectrometry analysis and among the main identified proteins are 11S globulins (such glycinin), lipoxygenase, chitinases, 7S globulins (vicilins, canavalin and ß conglycinin), annexin, catalase and sucrose binding protein. All DEAE-protein fractions were toxic to the insect, interfering with the post hatch larval development and survival. Decreases greater than 90% were observed in the larval mass and length at 20 days after oviposition (DAO) for larvae raised on diet containing 0.5% of some fractions. Alterations in the level of proteins, glucose and in the activity of the enzymes lipases and cysteine proteases were also detected in these larvae. Our results show that seeds of S. terebinthifolius have an arsenal of toxic proteins with potential for the control of the insect C. maculatus.

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BACKGROUND: Stored grain insects are controlled with fumigant insecticides which can select resistant insect populations and cause environmental and applicator contamination. Thus, resistant cultivars and chemical constituents of essential oils are an alternative to the almost exclusive use of these insecticides. The effects of the combination of cowpea cultivars Vigna unguiculata (L.) Walp. with chemical constituents of essential oils against Callosobruchus maculatus were determined. Four cowpea cultivars: BRS Tracuateua, BR 17 Gurgueia, Epace 10 and Sempre Verde (insect rearing) untreated were used in the experiments and combined with chemical constituents of essential oil: eugenol, geraniol and trans-anethole. The biological parameters observed were: total egg number and eggs per grain, egg viability (%), insects emerged and insects per grain, immature stage viability (%), instantaneous rate of growth (ri), insect dry weight (mg), grain weight loss (%) and egg-adult period. RESULTS: When comparing all biological parameters, the cultivars BRS Tracuateua and BR 17 Gurgueia were harmful to C. maculatus. In the toxicity tests, the results showed that LC30 and LC50 of the chemical constituents ranged from 54.77 to 103.48 ppm and 60.99 to 125.18 ppm, respectively. In most of the biological parameters, LC50 had adverse effects significantly higher than LC30 and BR 17 Gurgueia treated were harmful to C. maculatus. CONCLUSIONS: Overall, the findings showed that BR 17 Gurgueia combined with eugenol and geraniol more significantly affected the biological parameters of C. maculatus than when associated with trans-anethole, reducing egg number, insects emerged and egg viability. © 2020 Society of Chemical Industry.

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The objective of this study was to evaluate the enzymatic activity of homogenates of insects fed on grain of cowpea, Vigna unguiculata (L.), cultivars grown with different nitrogen sources. For the experiment we used aliquots of the homogenate of 100 unsexed adult insects, emerged from 10 g of grain obtained from four cowpea cultivars: 'BRS Acauã', 'BRS Carijó', 'BRS Pujante', and 'BRS Tapaihum' grown under different regimes of nitrogen sources: mineral fertilizer, inoculation with strains of diazotrophs (BR 3267, BR 3262, BR 3299; INPA 03-11B, 03-84 UFLA, as well as the control (with soil nitrogen). The parameters evaluated were enzymatic activities of insect protease, amylase and lipase and the starch content of the grains. There were differences in the enzymatic activity of amylase, lipase and protease of insect homogenate according to the food source. A lower activity of the enzyme amylase from C. maculatus homogenate was observed when insects were fed grain of the cultivar BRS Carijó. A lower activity of lipase enzyme from C. maculatus homogenate was observed when the insects fed on grain from the interaction of the cultivar Tapaihum inoculated with BR 3262 diazotrophs. The lowest proteolytic activity was observed in homogenate of insects fed on interaction of 'BRS Carijó' inoculated with BR 3262 diazotrophs. Starch content correlated positively with the amylase activity of C. maculatus homogenate. The cultivar BRS Carijó had a different behavior from the other cultivars, according to the cluster analysis.

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Compostos secundários obtidos de plantas podem ser utilizados no controle de Callosobruchus maculatus, como uma tática alternativa potencial aos inseticidas sintéticos. Foram testados óleos essenciais (Cymbopogon martini Roxb., Piper aduncum L., Piper hispidinervum C.DC., Melaleuca sp., Lippia gracillis Shau) e fixos (Helianthus annus L., Sesamum indicum L., Gossypium hirsutum L., Glycine max L. e Caryocar brasiliense Camb.), na concentração de 50µl/20g, de acordo com estudos anteriores. Grãos de caupi, cv. Sempre Verde, foram impregnados com os óleos, em recipientes de vidro e submetidos à agitação manual por dois minutos. Cada parcela de 20g foi infestada com oito fêmeas de C. maculatus com 0 a 48h de idade, durante quatro dias. Os óleos foram avaliados logo após a impregnação e aos 30, 60, 90 e 120 dias de armazenamento. Na primeira avaliação, todos os óleos essenciais provocaram 100 por cento de mortalidade e para os óleos fixos, a mortalidade variou entre 35 por cento (G. hirsutum) e 67,5 por cento (G. max). Com o prolongamento do período de armazenamento, houve um aumento do número de ovos viáveis e de insetos emergidos, exceto para P. aduncum. Em relação aos óleos fixos, S. indicum, G. max, G. hirsutum e C. brasiliense foram os mais eficientes até os 30 dias de armazenamento. Os resultados indicam que os óleos testados na concentração de 50µl/20g apresentam baixo efeito residual, com exceção de P. aduncum, que foi efetivo durante todo o período de armazenamento.

The secondary compounds extracted from plants are considered potential alternative to synthetic insecticides in the control of agricultural pests. Essential oils (Cymbopogon martini Roxb., Piper aduncum L., P. hispidinervum C.DC., Melaleuca sp. and Lippia gracillis Shau) and fixed oils (Helianthus annus L., Sesamum indicum L., Gossypium hirsutum L., Glycine max L. and Caryocar brasiliense Camb.) at the concentration of 50µl/20g were tested according to previous studies. Samples of cowpea cv. Sempre Verde were impregnated with these oils in glass recipients and submitted to manual agitation for two minutes. Each plot of 20g was infested with eight females of C. maculatus up to 48 hours old, during four days. The oil efficiency was evaluated right after the impregnation and after 30, 60, 90 and 120 days of storage. In the first evaluation, all essential oils caused 100 percent of mortality and the fixed oils caused low mortality, varying from 35 percent (G. hirsutum) to 67,5 percent (G. max). With longer storage period, there was an increase in the number of viable eggs and emerged insects, except for P. aduncum. Among the fixed oils, S. indicum, G. max, G. hirsutum and C. brasiliense were the most efficient up to 30 days of storage. The results showed the low residual effect of the tested oils for the control of C. maculatus, excep P. aduncum, which was efficient throughout the 120 days of storage.

RESUMO

O caruncho, Callosobruchus maculatus (Fabr.), é considerado a praga mais importante do caupi, Vigna unguiculata (L.) Walp., armazenado em regiões tropicais e subtropicais. Visando minimizar os efeitos indesejáveis dos inseticidas químicos sintéticos, o controle dessa praga com óleos de origem vegetal vem se constituindo numa alternativa promissora, de baixo custo e segura para os aplicadores e consumidores. Foram testados os óleos essenciais [(Cymbopogon martini (Roxb.) J.F. Watson], Piper aduncum L., Piper hispidinervum C.DC., Melaleuca sp., Lippia gracillis Shauer) e fixos (Helianthus annuus L, Sesamum indicum L, Gossypium hirsutum L., Glycine max (L.) Merr. e Caryocar brasiliense Camb.), em grãos de caupi, cv. Sempre Verde. Os óleos foram utilizados nas concentrações 10, 20, 30, 40 e 50mL/20g, correspondendo a 0,5, 1,0, 1,5, 2,0 e 2,5 L/t e impregnados aos grãos no interior de recipientes de plástico, com auxílio de pipetador automático e agitados manualmente durante dois minutos. Parcelas de 20g de caupi foram infestadas com oito fêmeas de C. maculatus, com 0 a 48 h de idade. Cada óleo foi testado, separadamente, em delineamento inteiramente casualisado com seis repetições. Os óleos essenciais de C. martini, P. aduncum e L. gracillis causaram 100 por cento de mortalidade em todas as concentrações, P. hispidinervum a partir de 1,5 L/t e Melaleuca sp. nas concentrações de 2,0 e 2,5 L/t. A redução do número de ovos viáveis e de insetos emergidos foi de 100 por cento para todos os óleos essenciais, exceto Melaleuca sp. Por outro lado, os óleos fixos, apesar de apresentarem baixa mortalidade em todas as concentrações testadas, reduziram em praticamente 100 por cento o número de ovos viáveis e de insetos emergidos.

The weevil Callosobruchus maculatus (Fabr.) is considered the most important pest of cowpea, Vigna unguiculata (L.) Walp., stored in tropical and subtropical regions. Focusing to minimize the undesired effects of synthetic chemical insecticides, vegetal origin oils are a promissory alternative to control it because of its low cost and safety to the applicator and consumers. Essential oils [(Cymbopogon martini (Roxb.) J.F. Watson], Piper aduncum L., Piper hispidinervum C.DC., Melaleuca sp. and Lippia gracillis Shauer) and fixed oils (Helianthus annuus L., Sesamum indicum L., Gossypium hirsutum L., Glycine max (L.) Merr. and Caryocar brasiliense Camb.) were tested in cowpea grains (cv. "Sempre Verde"). These oils were used in concentrations of 10, 20, 30, 40 and 50mL/20g, corresponding to 0.5, 1.0, 1.5, 2.0 and 2.5 L/t and impregnated to the grains into the plastic recipients using an automatic pipette and handly agitated for two minutes. Plots of 20g from cowpea were infested with eight females of C. maculatus from 0 to 48 hours old. Each oil was tested separately in a randomly arrangement with six replicates. The essential oils of C. martini, P. aduncum and L. gracillis provoked 100 percent of mortality in all concentrations, P. hispedinervum from 1.5 L/t and Melaleuca sp. in the concentrations of 2.0 and 2.5 L/t. The reduction of the viable eggs and emerged insects was about 100 percent, except for Melaleuca sp. On the other hand, the fixed oils, in spite of its low mortality in all tested concentrations, reduced to almost 100 percent the number of the viable eggs and emerged insects.