Research articleBioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds
Graphical abstract
This work reports in vitro and in vivo biochemical information about a novel insecticidal Kunitz trypsin inhibitor purified from the Catanduva tree (Piptadenia moniliformis) seeds. Furthermore, data here reported focus on biotechnological potential of inhibitor for insect pest control.
Introduction
Proteinaceous inhibitors are proteins widely distributed in nature possessing the ability to interact with enzymes of different origins, suppressing partial or totally the catalytic activity of these enzymes. In plants, they have been found in storage tissues, such as leaves, fruits, tubers and seeds. A variety of physiological functions have been attributed for these proteins, especially its role in the plant-defense mechanisms against insect pest attack [1], [2]. Serine protease inhibitors of the Kunitz superfamily have gained particular attention due to the potent activity against insect larval midgut serine proteases. The use of proteinaceous inhibitors as candidates for control strategies of insects is hopeful, since insect digestive proteases are promising targets in the control of various insects. Kunitz inhibitors were capable of inhibiting the proteolytic activity of serine proteases from lepidopterans, such as the black cutworm (Agrotis ipsilon Hufnagel), the corn earworm (Heliothis zea), the tobacco budworm (Heliothis virenscens Fab.), the western spruce budworm (Choristeneura occidentalis) [3], and coleopterans such as the cotton boll weevil (Anthonomus grandis) [4], the cowpea weevil (Callosobruchus maculatus) [5], [6], [7], [8] and the bean weevil (Zabrotes subfasciatus) [8], [9] and dipterans such as Ceratitis capitata [6], [8], [9], [10]. Increased mortality, several deformations, decreased weight gain, and reduction of the fecundity and oviposition were common effects observed when Kunitz inhibitors were added to the artificial diets of insects [5], [6], [7], [11].
The fruit fly (Diptera: Tephritidae) is an insect pest that attacks a great variety of fruit-tree species that are economically important in Brazil such as mango, caja, caja-mirim, siriguela, guava, guabiroba, jabuticaba, rose apple, cherry, carambola, orange, tangerine, abiu and sapodilla trees. The species C. capitata (Wiedemann), commonly known as the Mediterranean fruit fly, is considered the main pest of fruticulture in the world, attacking an extensive host range including more than 350 plant species. Millions of dollars are annually lost and many programs were created to control and to eradicate this pest [12]. Great quantities of insecticides are often used, causing damage to the environment and consumer health. Solutions for this situation are based on the development of novel, target-specific compounds reducing the impact on the environment and increasing effectiveness in the combat against this insect [13], [14]. Compounds such as lectins, vicilins, proteases and amylases inhibitors can be considered possible alternatives to the use of pesticides to control insect pests [5], [6], [15].
In this view, the tropical Fabaceae tree of the Mimosoideae subfamily, Piptadenia moniliformis (Benth.), known popularly as Catanduva and angico-de-bezerro, typically found in the caatinga northeastern part of Brazil, had a Kunitz-type trypsin inhibitor purified and biochemically characterized. The inhibitory activity was tested: in vitro assays of insect pests from different orders and in vivo bioassays models during the larval development of C. capitata (fruit fly).
Section snippets
Purification of P. moniliformis trypsin inhibitor
The PmTKI purity degree was observed by SDS-PAGE and MALDI-ToF mass spectrometry analyses (Fig. 1). Both methodologies showed a major molecular mass of 19.296 Da. This average molecular mass also showed, by MS analysis, a double charge (M + H+2) of 9.648 Da, indicating the absence of contaminants and higher purity degree Moreover, this mass was unmodified in the presence or absence of reducing agent as observed by SDS-PAGE gel. Moreover, purification procedures resulted in a purification of a
Material
Enzymes: Papain, Bromelain, Bovine chymotrypsin, Bovine Trypsin and Porcine Elastase; Substrates: BApNA, BTpNA and Azocasein were purchased from Sigma Chemical Co. (St. Louis, MO). The following abbreviations are used: BApNA, N-benzoyl-DL-arginyl-p-nitroanilide; BTpNA, N-benzoyl-l-tyrosyl-p-nitroanilide; and SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. All were purchased from Sigma (St. Louis, MO, USA).
Purification of P. moniliformis trypsin inhibitor
Catanduva (P. moniliformis) seeds were obtained from ICMBio/MMA
Acknowledgments
The authors are thankful for the grants and support from CAPES, CNPq, FAPDF, FAPERN and RENORBIO-FINEP. Our sincere acknowledgements to Mauricio Pereira de Sales, a great researcher and professor (in memoriam).
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