Soybean protease inhibitors increase Bacillus thuringiensis subs. israelensis toxicity against Hypothenemus hampei




biological control, bacterial toxins, soybean meal, protease inhibitors, synergism


Introduction. The coffee berry borer (Hypothenemus hampei Ferrari, CBB) is one of the most devastating pests on coffee plantations around the world. Although CBB is susceptible to the effect of δ–endotoxins of Bacillus thuringiensis subs. israelensis (Bti) at laboratory level, the efficacy of this control method is poor in the field, presumably due to the inactivation by digestive proteases different to those required for protoxin activation. Objective. To study whether the addition of a soybean flour extract enriched with protease inhibitors (PI), mixed with Bti crystals and spores (Bti-sc) in an artificial diet, could improve the toxicity of Bti against CBB. Materials and methods. This study was performed in San José, Costa Rica, between 2012 and 2013. A set of adult female CBB insects was exposed to a mixture containing different concentrations of a partially purified soybean meal extract with active PI and lyophilized Bti-sc, and were tested through a bioassay in artificial diet to estimate the sub-lethal concentration (LC50). The mortality results were validated by observing the dissected midgut, whose ultrastructure was analyzed by transmission electron microscopy. Results. The soybean extracts partially degraded the Bti-sc complex, it reduced its LC50 by almost four times (from 1.135 to 0.315 µg µl-1) and enhanced CBB mortality in a concentration-dependent manner. Histological analyses of the midgut confirmed this synergistic effect, since severe epithelial damage to the intestinal epithelium of CBB exposed to Bti-sc + PI was visualized compared to Bti-sc alone. Conclusions. The combination of a soybean extract enriched in PI and Bti-sc enhanced the mortality effect over CBB, which was confirmed by the midgut collapse. Soybean flour is a cost-effective supplement that could increase Bti effectiveness against CBB and delay the appearance of biological resistance.


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Acuña-González, P., y W. Betanco Velásquez. 2007. Evaluación de la incidencia natural de Beauveria bassiana (Bals) Vuill, sobre Hypothenemus hampei (Ferrari) y Leucoptera coffeella (Guérin-Méneville) en el cultivo de café en dos zonas cafetaleras de Nicaragua. Tesis Diploma, Universidad Nacional Agraria, Managua, NIC.

Anderson, R.L., and W.J. Wolf. 1995. Compositional changes in trypsin inhibitors, phytic acid, saponins and isoflavones related to soybean processing. J. Nutr. 125:581S-588S. doi:10.1093/jn/125.3_Suppl.581S

Aristizábal, L.F., M. Jiménez, A.E. Bustillo, H.I. Trujillo, and S.P. Arthurs. 2015. Monitoring coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae), populations with alcohol-baited funnel traps in coffee farms in Colombia. FL Entomol. 98:381-383. doi:10.1653/024.098.0165

Aristizábal, L.F., S. Shriner, R. Hollingsworth, and S. Arthurs. 2017. Flight activity and field infestation relationships for coffee berry borer in commercial coffee plantations in Kona and Kau districts, Hawaii. J. Econ. Entomol. 110:2421–2427. doi:10.1093/jee/tox215

Belayneh-Mulaw, T., C.P. Kubicek, and I.S. Druzhinina. 2010. The rhizosphere of Coffea arabica in its native highland forests of Ethiopia provides a niche for a distinguished diversity of Trichoderma. Diversity 2:527-549. doi:10.3390/d2040527

Cantor, F., V.L.R.M. Benassi, e C.J. Fanton. 2001. Broca-do-café, Hypothenemus hampei (Coleoptera: Scolytidae). Em: E. Ferrera et al., editores, Histórico e impacto das pragas introduzidas no Brasil. Holos, São Paulo, BRA. p. 99-105.

Cárdenas, S.I. 2007. Caracterización morfológica y agronómica de la colección núcleo de café (Coffea arabica L.) del CATIE. Tesis MSc., CATIE, Turrialba, CRI.

Constantino, L.M., L. Navarro, A. Berrio, F.E. Acevedo, D. Rubio, y P. Benavides. 2011. Aspectos biológicos, morfológicos y genéticos de Hypothenemus obscurus e Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae). Rev. Colomb. Entomol. 37:173-182.

Cotabarren, J., D. Lufrano, M.G. Parisi, and W.D. Obregón. 2020. Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review. Plant Sci. 292:110398. doi:10.1016/j.plantsci.2019.110398

De-la-Rosa, W., M. Figueroa, and J. Ibarra. 2005. Selection of Bacillus thuringiensis strains native to Mexico active against the coffee berry borer Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae). Vedalia 12:3-9.

de-Oliveira, C.F.R., I.M. Vasconcelos, R. Aparicio, M.D.G.M. Freire, P.A. Baldasso, S. Marangoni, and M.L.R. Macedo. 2012. Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds. Proc. Biochem. 47:929-935. doi:10.1016/j.procbio.2012.02.022

El-latif, A.O.A. 2015. Protease purification and characterization of a serine protease inhibitor from Egyptian varieties of soybean seeds and its efficacy against Spodoptera littoralis. J. Plant Prot. Res. 55:16-25. doi:10.1515/jppr-2015-0003

Erlanger, B.F., N. Kokowsky, and W. Cohen. 1961. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95:271-278. doi:10.1016/0003-9861(61)90145-X

Fabrick, J., C. Behnke, T. Czapla, K. Bala, A.G. Rao, K.J. Kramer, and G.R. Reeck. 2002. Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae). Insect Biochem. Mol. Biol. 32:405-415. doi:10.1016/S0965-1748(01)00117-5

Finney, D.J., and W.L. Stevens. 1948. A table for the calculation of working probits and weights in probit analysis. Biometrika 35:191-201. doi:10.1093/biomet/35.1-2.191

Franco, O.L., S.C. Dias, C.P. Magalhães, A.C.S. Monteiro, C. Bloch, F.R. Melo, O.B. Oliveira-Neto, R.G. Monnerat, and M.F. Grossi-de-Sá. 2004. Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis). Phytochemistry 65:81-89. doi:10.1016/j.phytochem.2003.09.010

García-Carreño, F.L., and N.F. Haard. 1993. Characterization of proteinase classes in langostilla (Pleuroncodes planipes) and crayfish (Pacifastacus astacus) extracts. J. Food Biochem. 17:97-113. doi:10.1111/j.1745-4514.1993.tb00864.x

Ghodke, A.B., S.G. Chavan, B.V. Sonawane, and A.A. Bharose. 2013. Isolation and in vitro identification of proteinase inhibitors from soybean seeds inhibiting Helicoverpa gut proteases. J. Plant Interact. 8:170-178. doi:10.1080/17429145.2012.668952

Gillman, J.D., W.S. Kim, and H.B. Krishnan. 2015. Identification of a new soybean Kunitz trypsin inhibitor mutation and its effect on Bowman-Birk protease inhibitor content in soybean seed. J. Agric. Food Chem. 63:1352-1359. doi:10.1021/jf505220p

González, V. 2019. Costa Rica: Coffee annual: Coffee production and trade. Annual report. USDA, USA. (accessed Sep. 29, 2019).

Gu, C., X. Song, L. Zhao, S. Pan, and G. Qin. 2014. Purification and characterization of Bowman-Birk trypsin inhibitor from soybean. J. Food Nutr. Res. 2:546-550. doi:10.12691/jfnr-2-9-3

Guedidi, S., Y. Yurekli, A. Deratani, P. Déjardin, C. Innocent, S.A. Altinkaya, S. Roudesli, and A. Yemenicioglu. 2010. Effect of enzyme location on activity and stability of trypsin and urease immobilized on porous membranes by using layer-by-layer self-assembly of polyelectrolyte. J. Membr. Sci. 365:59-67. doi:10.1016/j.memsci.2010.08.042

Gujar, T., V. Kalia, A. Kumari, and T.V. Prasad. 2004. Potentiation of insecticidal activity of Bacillus thuringiensis subsp. kurstaki HD-1 by proteinase inhibitors in the American bollworm, Helicoverpa armigera (Hübner). Indian J. Exp. Biol. 42(2):157-163.

Hernández-Soto, A., M.C. Del Rincón-Castro, A.M. Espinoza, and J.E. Ibarra. 2009. Parasporal body formation via overexpression of the Cry10Aa toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa synergism. Appl. Environ. Microbiol. 75:4661-4667. doi:10.1128/AEM.00409-09

Jiménez, A.J. 2009. As enzimas presentes no trato digestivo dos insetos: um alvo susceptível de inhibição. Tese Dou., Universidade de Brasília, Brasilia, BRA.

Jouzani, G.S., E. Valijanian, and R. Sharafi. 2017. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Appl. Microbiol. Biotechnol. 101:2691-2711. doi:10.1007/s00253-017-8175-y

Kakade, M.L., J.J. Rackis, J.E. McGhee, G. Puski, and R.S. Usda. 1974. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem. 51:376-381.

Kirouac, M., V. Vachon, D. Quievy, J.L. Schwartz, and R. Laprade. 2006. Protease inhibitors fail to prevent pore formation by the activated Bacillus thuringiensis toxin Cry1Aa in insect brush border membrane vesicles. Appl. Environ. Microbiol. 72:506-515. doi:10.1128/AEM.72.1.506-515.2006

Kuhar, K., R. Kansal, B. Subrahmanyam, K.R. Koundal, K. Miglani, and V.K. Gupta. 2013. A Bowman–Birk protease inhibitor with antifeedant and antifungal activity from Dolichos biflorus. Acta Physiol. Plant. 35:1887-1903. doi:10.1007/s11738-013-1227-8

Lecadet, M.M., M.O. Blondel, and J. Ribier. 1980. Generalized transduction in Bacillus thuringiensis var. berliner 1715 using bacteriophage CP-54Ber. Microbiol. 121:203-212. doi:10.1099/00221287-121-1-203

Li, J., and H.A. Chase. 2010. Applications of membrane techniques for purification of natural products. Biotechnol. Lett. 32:601-608. doi:10.1007/s10529-009-0199-7

Lomate, P.R., and V.K. Hivrale. 2013. Effect of Bacillus thuringiensis (Bt) Cry1Ac toxin and protease inhibitor on growth and development of Helicoverpa armigera (Hübner). Pest. Biochem. Physiol. 105(2):77-83. doi:10.1016/j.pestbp.2013.01.002

López-Pazos, S.A., J.E. Cortázar-Gómez, and J.A. Cerón-Salamanca. 2009. Cry1B and Cry3A are active against Hypothenemus hampei Ferrari (Coleoptera: Scolytidae). J. Invertebr. Pathol. 101:242-245. doi:10.1016/j.jip.2009.05.011

Macedo, M.L.R, Md. Freire, O.L. Franco, L. Migliolo, and C.F. de-Oliveira. 2011. Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the control of Homalinotus coriaceus. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 158:164-172. doi:10.1016/j.cbpb.2010.11.005

MacIntosh, S.C., G.M. Kishore, F.J. Perlak, P.G. Marrone, T.B. Stone, S.R. Sims, and R.L. Fuchs. 1990. Potentiation of Bacillus thuringiensis insecticidal activity by serine protease inhibitors. J. Agric. Food Chem. 38:1145-1152. doi:10.1021/jf00094a051

Mansour, S.A., M.S. Foda, and A.R. Aly. 2012. Mosquitocidal activity of two Bacillus bacterial endotoxins combined with plant oils and conventional insecticides. Ind. Crop Prod. 35:44-52. doi:10.1016/j.indcrop.2011.06.001

McDonald, K. 2007. Cryopreparation methods for electron microscopy of selected model systems. In: J.R. McIntosh, editor, Cellular electron microscopy. Academic Press, Elsiever, San Diego, CA, USA. p. 23-56. doi:10.1016/S0091-679X(06)79002-1

Méndez-López, I., R. Basurto-Ríos, and J.E. Ibarra. 2003. Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae). FEMS Microbiol. Lett. 226:73-77. doi:10.1016/S0378-1097(03)00557-3

Molina, D., A. Blanco-Labra, y H. Zamora. 2011. Inhibidores de proteasas de plantas efectivos contra las aspártico proteasas de Hypothenemus hampei. Rev. Colomb. Entomol. 37:183-191.

Oliveira, C.M., A.M. Auad, S.M. Mendes, and M.R. Frizzas. 2013. Economic impact of exotic insect pests in Brazilian agriculture. J. Appl. Entomol. 137:1-15. doi:10.1111/jen.12018

Oppert, B., T.D. Morgan, and K.J. Kramer. 2011. Efficacy of Bacillus thuringiensis Cry3Aa protoxin and protease inhibitors against coleopteran storage pests. Pest Manag. Sci. 67:568-573. doi:10.1002/ps.2099

Pan, D., A.P. Hill, A. Kashou, K.A. Wilson, and A. Tan-Wilson. 2011. Electrophoretic transfer protein zymography. Anal. Biochem. 411:277-283. doi:10.1016/j.ab.2011.01.015

Panchal, B.M., and M. Kachole. 2012. Identification of potent inhibitors of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) gut proteinase from plant gum PIs. Int. J. Sci. Technol. 1:662-670.

Pardey, A.E.B. 2006. Una revisión sobre la broca del café, Hypothenemus hampei (coleoptera: Curculionidae: Scolytinae), en Colombia. Rev. Colomb. Entomol. 32:101-116.

Perić, V., M. Srebrić, L. Jankuloski, M. Jankulovska, S. Žilić, V. Kandić, and S. Mladenović-Drinić. 2009. The effects of nitrogen on protein, oil and trypsin inhibitor content of soybean. Genetika 41:137-144. doi:10.2298/GENSR0902137P

Portilla, R.M., y D. Streett. 2006. Nuevas técnicas de producción masiva automatizada de Hypothenemus hampei sobre la dieta artificial Cenibroca modificada. Cenicafé 57(1):37-50.

Rojas, M. 2012. Manejo sostenible de la broca del café (Hypothenemus hampei) mediante poda sistemática del cafeto en Costa Rica. Agron. Costarricense 36(2):71-79.

Roosta, H.R., T. Javadi, and F. Nazari. 2011. Isolation and characterization of trypsin inhibitors (Kunitz soybean trypsin inhibitor, Bowman-Birk inhibitor) in soybean. Adv. Environ. Biol. 5:145-153.

Rosenheim, J.A., and M.A. Hoy. 1989. Confidence intervals for the Abbott’s formula correction of bioassay data for control response. J. Econ. Entomol. 82:331-335. doi:10.1093/jee/82.2.331

Ruan, J., J. Yan, S. Hou, H. Chen, Q. Wu, and X. Han. 2015. Expression and purification of the trypsin inhibitor from tartary buckwheat in Pichia pastoris and its novel toxic effect on Mamestra brassicae larvae. Mol. Biol. Rep. 42:209-216. doi:10.1007/s11033-014-3760-y

Schünemann, R., N. Knaak, and L. Fiuza. 2014. Mode of action and specificity of Bacillus thuringiensis toxins in the control of caterpillars and stink bugs in soybean culture. ISRN Microbiol. 2014:135675. doi:10.1155/2014/135675

Shamim, M., N.A. Khan, and K.N. Singh. 2011. Inhibition of midgut protease of yellow stem borer (Scirpophaga incertulas) by cysteine protease-like inhibitor from mature jackfruit (Artocarpus heterophyllus) seed. Acta Physiol. Plant. 33:2249-2257. doi:10.1007/s11738-011-0764-2

Shamsi, T.N., R. Parveen, A. Ahmad, R.R. Samal, S. Kumar, and S. Fatima. 2018. Inhibition of gut proteases and development of dengue vector, Aedes aegypti by Allium sativum protease inhibitor. Acta Ecol. Sin. 38:325-328. doi:10.1016/j.chnaes.2018.01.002

Shamsi, T.N., R. Parveen, M. Amir, M.A. Baig, M.I. Qureshi, S. Ali, and S. Fatima. 2016. Allium sativum protease inhibitor: a novel Kunitz trypsin inhibitor from garlic is a new comrade of the serpin family (EA Permyakov, Ed.). PLoS One 11(11):e0165572. doi:10.1371/journal.pone.0165572

Singh, S., A. Singh, S. Kumar, P. Mittal, and I.K. Singh. 2018. Protease inhibitors: recent advancement in its usage as a potential biocontrol agent for insect pest management. Insect Sci. 27:186-201. doi:10.1111/1744-7917.12641

Sriket, C., S. Benjakul, W. Visessanguan, and K. Hara. 2011. Effect of legume seed extracts on the inhibition of proteolytic activity and muscle degradation of fresh water prawn (Macrobrachium rosenbergii). Food Chem. 129:1093-1099. doi:10.1016/j.foodchem.2011.05.080

Suckling, D.M., L.D. Stringer, A.E. Stephens, B. Woods, D.G. Williams, G. Baker, and A.M. El-Sayed. 2014. From integrated pest management to integrated pest eradication: technologies and future needs. Pest Manag. Sci. 70:179-189. doi:10.1002/ps.3670.

Valencia, A., and J. Arboleda. 2005. Digestion of the inhibition αAI by Hypothenemus hampei aspartic proteinases. Rev. Colomb. Entomol. 31:117-121.

Valerio-Oviedo, A. 2006. Evaluación de la incorporación de diferentes fungicidas y dosis en dietas artificiales para la reproducción de la broca de café con miras a la multiplicación de sus parasitoides bajo condiciones controladas. Tesis Bach., Instituto Tecnológico de Costa Rica, Cartago, CRI.

Vasudev, A., and S. K. Sohal. 2016. Partially purified Glycine max proteinase inhibitors: potential bioactive compounds against tobacco cutworm, Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae). Turk. J. Zool. 40:379-387. doi:10.3906/zoo-1508-20

Vega, F., J. Jaramillo, A. Castillo, and F. Infante. 2009. The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae): a short review, with recent findings and future research directions. Terr. Arthropod. Rev. 2:129-147. doi:10.1163/187498209X12525675906031

Vidal-Quist, J.C., P. Castañera, and J. González-Cabrera. 2010. Cyt1Aa protein from Bacillus thuringiensis (Berliner) serovar israelensis is active against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Pest Manag. Sci. 66:949-955. doi:10.1002/ps.1965

Vorlová, L. 2011. Important minor soybens proteins: soybean allergens and enzymes inhibitors. In: H. El-Shemy, editor, Soybean and health. InTech, Brno, CZE. p. 425-432. doi:10.5772/1007

Wei-Salas, S., y A. Durán-Quirós. 2015. Caracterización del uso del suelo en las principales áreas agrícolas de la gran área metropolitana (GAM) de Costa Rica. Agron. Costarricense 39(1):149-160.

Zahiri, N.S., and M.S. Mulla, 2005. Non-larvicidal effects of Bacillus thuringiensis israelensis and Bacillus sphaericus on oviposition and adult mortality of Culex quinquefasciatus Say (Diptera: Culicidae). J. Vector Ecol. 30:155-162.

Zhao, A., Y. Li, C. Leng, P. Wang, and Y. Li. 2019. Inhibitory effect of protease inhibitors on larval midgut protease activities and the performance of Plutella xylostella (Lepidoptera: Plutellidae). Front Physiol. 9:1963. doi:10.3389/fphys.2018.01963

Zhu-Salzman, K., and R. Zeng. 2015. Insect response to plant defensive protease inhibitors. Ann. Rev. Entomol. 60:233-252. doi:10.1146/annurev-ento-010814-020816

Zibaee, I., A.R. Bandani, J.J. Sendi, R. Talaei-Hassanloei, and B. Kouchaki. 2010. Effects of Bacillus thuringiensis var. kurstaki and medicinal plants on Hyphantria cunea drury (Lepidoptera: Arctiidae). Invertebr. Surviv. J. 7:251-261.

Zorzetti, J., A.P.S. Ricietto, F.A.P. Fazion, A. M. Meneghin, P.M.O.J. Neves, L.A. Vilas-Boas, and G.T. Vilas-Bôas. 2018. Isolation, morphological and molecular characterization of Bacillus thuringiensis strains against Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae). Rev. Bras. Entomol. 62:198-204. doi:10.1016/j.rbe.2018.07.002



How to Cite

Mesén-Porras, E. A., Dahdouh-Cabia, S., Jiménez-Quirós, C., Mora-Castro, R., Rodríguez, C., & Pinto-Tomás, A. A. (2020). Soybean protease inhibitors increase Bacillus thuringiensis subs. israelensis toxicity against Hypothenemus hampei. Agronomía Mesoamericana, 31(2), 461–478.