Open Access
How to translate text using browser tools
13 January 2021 Effect of Natural and Artificial Diets on Protease Activity in the Midgut of Spodoptera cosmioides and Spodoptera eridania (Lepidoptera: Noctuidae) Larvae
Francelina Aparecida Duarte Rocha, Yaremis Beatriz Meriño-Cabrera, Eliseu José Guedes Pereira, José Cola Zanuncio, Wellington Garcia Campos, José Eduardo Serrão, Maria Goreti Almeida Oliveira
Author Affiliations +

Spodoptera cosmioides Walker and Spodoptera eridania Stoll (both Lepidoptera: Noctuidae) are herbivorous insects affecting crop yield. Understanding midgut digestive enzyme properties in these caterpillars when feeding on different resources is important for control strategies of these agricultural pests. This study evaluated the activity of midgut digestive enzyme total proteases, trypsin, cysteine proteases, and chymotrypsin in S. cosmioides and S. eridania feeding on natural soybean and cotton leaves and artificial diets. The proteolytic activities of midgut digestive enzymes in S. cosmioides and S. eridania vary according to diet, suggesting adaptation of these caterpillars to different host plants in order to avoid the inhibitory effects of secondary metabolites through the overexpression of proteases. High activities occur for trypsin and total proteases in both insects indicating that these enzymes are potential targets for inhibition in pest control programs.

Spodoptera cosmioides Walker and Spodoptera eridania Stoll (both Lepidoptera: Noctuidae) are phytophagous insects that damage soybean and cotton crops (Souza et al. 2013). These caterpillars are controlled with synthetic insecticides, which have low efficacy due to induced tolerance by excessive use of these compounds (Diez-Rodríguez & Omoto 2001; Carvalho et al. 2013). Thus, chemical control may result in financial losses, unbalance the food web, and select for resistant strains, which may overlap with alternative pest control methods (Mills & Kean 2010; Vianna et al. 2011).

Production of defense proteins, such as protease inhibitors, is a strategy of some plants to avoid herbivory (Wasternack & Hause 2013). The protease inhibitors affect food digestion, reducing or inhibiting the protein synthesis required for insect growth, development, and reproduction (Meriño-Cabrera et al. 2018)

The Kunitz (Onesti et al. 1991) and Bowman-Birk inhibitors (Song & Suh 1998) are found in large amounts in soybeans, and gossypol is found in cotton (Meisner 1978; Souza et al. 2006), all inhibiting digestive trypsin and chymotrypsin (Liener 1994; Gariani & Leatherbarrow 1997).

Phytophagous insects have physiological adaptations to reduce the negative effects of the ingestion of protease inhibitors produced by plants (Moon et al. 2004; Patarroyo-Vargas et al. 2017; Meriño-Cabrera et al. 2018). These adaptations include increased levels of digestive proteases (Pilon et al. 2006, 2009; Scott et al. 2010; Meriño-Cabrera et al. 2018), and isoform synthesis, which do not bind to protease inhibitors, or bind and degrade protease inhibitors (Srinivasan et al. 2006; Zhang et al. 2010; Jamal et al. 2012).

Midgut proteases have been studied for use in insect pest control due to their role in peptide bond hydrolysis and releasing of amino acids for insect growth, reproduction and survival (Mahdavi et al. 2013; Shi et al. 2013).

Inhibition of serine proteases (E.C. 3.4.21), the main digestive enzymes in Lepidoptera, decrease the total digestive activity in these insects by up to 95% (Pilon et al. 2006). In this sense, protease inhibitors in natural and artificial diets are potential compounds to control these pests (Gatehouse 1999; Kidd 2000; Pilon et al. 2006; Rosell et al. 2008; Moreira et al. 2011). The total spectrum of midgut proteases and adaptation of insects to modify the activity of their proteases in different diets should be studied to allow the use of these compounds in pest management (Jongsma & Bolter 1997; Visôtto et al. 2009a, b).

Given that the production and activities of digestive enzymes in S. eridania and S. cosmioides change according to diet (Kotkar et al. 2012; Sarate et al. 2012), this study evaluated the digestive enzyme activity in these species fed on a natural diet rich in protease inhibitors (soybean and cotton leaves) and an artificial diet free of inhibitors.

Materials and Methods


Spodoptera cosmioides and S. eridania caterpillars were obtained from mass rearing in the Insect-Plant Interaction Laboratory of the Department of Entomology of the Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil.

Adults were kept in cages fed with honey (10.5 g), beer (350 mL), sucrose (60 g), ascorbic acid (1.05 g), nipagine (1.05 g), and water (1,000 mL). Spodoptera cosmioides and S. eridania oviposition began after 3 d. Old females and eggs were collected every 2 d and maintained at 27.5 °C, 75% relative humidity, and a 14:10 h (L:D) photoperiod.

Hatched caterpillars were transferred to containers (500 mL) and fed on artificial diet (Greene et al. 1976), soybean leaves (TMG 1264 RR, V3 stage) or cotton leaves (FM 910, V4 stage) until the fifth instar, when the midguts were dissected for enzyme assays.


The midguts of 10 S. cosmioides and 10 S. eridania caterpillars were macerated with 1 mL 1 mM HCl at 4 °C and centrifuged at 10,000 g for 10 min at 4 °C (Paixão et al. 2013).

Total protein concentration in triplicate of the midgut extracts was determined according to Bradford methods (1976) using bovine serum albumin as a standard.


The amidasic trypsin activity was determined using the chromogenic substrate N-benzoyl-L-arginyl-p-nitroanilide (L-BApNA 2 mM) in 0.1 M Tris-HCl buffer, pH 8.2 containing 20 mM CaCl2 (25 °C) (Erlanger et al. 1961). The control had enzyme substrate buffer without midgut extract. The product formation was calculated at 410 nm for 2.5 min, using molar extinction coefficient of 8800 (M-1 cm-1).

The esterase trypsin activity was determined using 0.1 mM N-α-p-tosyl-L-arginine methyl ester (L-TAME) substrate in 0.1M Tris-HCl pH 8.2 buffer, containing 20 mM CaCl2 (25 °C) (Hummel 1959). The product formation was calculated at 247 nm for 2.5 min using 540 M-1 cm-1 molar extinction coefficient. All analyses were performed in technical triplicate.


The amidasic chymotrypsin activity was determined with 1.2 mM N-Benzoyl-L-tyrosine-p-nitroanilide (L-BTpNA) substrate in 0.1M Tris-HCl buffer pH 8.2 with 20 mM CaCl2 (25 °C). For the control, the midgut extract was omitted from the reaction. The product formation was calculated at 410 nm for 2.5 min using 8 800 (M-1 cm-1) molar extinction coefficient.

The esterase chymotrypsin activity was determined with 0.1 mM N-Acetyl-L-tyrosine ethyl ester monohydrate (ATEE) substrate in 0.1M Tris-HCl pH 8.2 buffer containing 20 mM CaCl2. The control contained substrate and buffer without midgut extract. The product formation was calculated at 247 nm for 2.5 min. All analyses were performed in technical triplicate.


The activity of cysteine proteases was determined according to Mendonça et al. (2011). Briefly, the reaction mixture had 500 µL 0.5 mM L-BapNA substrate (25 °C), 500 µL of 0.1 M Tris-HCl buffer pH 8.2, containing 20 mM CaCl2 and 5 mM Dithiothreitol (DTT), 100 µL of 1 mM benzamidineand 10 µL of the midgut extract. For control, the midgut extract was omitted from the mixture. The product formation was calculated at 410 nm for 2.5 min using 8800 M-1 cm-1 molar extinction coefficient. The analyses were performed in technical triplicate.


The activity of total proteases was determined with 2% azocasein substrate in 0.1 M Tris-HCl buffer pH 8.2 containing 20 mM CaCl2 (37 °C) (w/v) (Tomarelli et al. 1949). The reaction mixture had 50 µL of substrate and 60 µL of midgut extract and was incubated at 37 °C for 30 min. The reaction was blocked with 240 µL of 10% trichloroacetic acid (w/v).

Samples were then homogenized, maintained in ice for 15 min, and centrifuged at 8,000× g for 5 min at 25 °C. The supernatant (240 µL) was transferred to 280 µL of 1M NaOH and analyzed at 440 nm in a spectrophotometer.


The experiment was conducted in a completely randomized design with 3 technical replicates from a pool of 10 caterpillars. The presuppositions of normality and homoscedasticity were verified with SAS residue analysis (PROC MIXED followed by PROC UNIVARIATE and PROC GPLOT) (SAS 2013). The means obtained for each enzyme analysis were compared using t test at 5% level of significance, protected by ANOVA. Data were processed with SAS software version 9.1 (SAS 2013).


The analysis of the effect of the different diets on proteolytic activity in the S. cosmioides midgut showed that trypsin activity changed according larval diet (Fig. 1A, B), with those fed on cotton showing esterase and amidasic trypsin activity of 2,098.2 ± 654.4 and 86.4 ± 40.5 µmol s-1 per µg of protein, respectively, higher than those fed on soybean leaves and artificial diet.

For chymotrypsin enzyme, larvae fed on soybean leaves presented activity of 39.0 ± 13.0 µmol s-1 per µg of protein for esterase chymotrypsin (Fig. 1C), higher than for larvae fed on cotton and artificial diet (P ≤ 0.05). Amidasic chymotrypsin activity was higher in larvae fed on cotton leaves (13.4 ± 6.4 µmol s-1 per µg of protein) than with soybean leaves and artificial diet (Fig. 1D).

Fig. 1.

Proteolytic activity in the midgut of Spodoptera cosmioides and Spodoptera eridania (Lepidoptera: Noctuidae) larvae fed on cotton, soybean, and artificial diets. (A) Amydasic trypsin. (B) Esterase trypsin. (C) Esterase chymotrypsin. (D) Amydasic chymotrypsin. (E) Cysteine protease. (F) Total protease. Gray bars: larvae fed with cotton; white bars: larvae fed with artificial diet; black bars: larvae fed with soybean. Bars with asterisks differ (P > 0.05) by the Fisher minimum difference test protected by analysis of variance.


In relation to cysteine protease activity, the higher activity was found in caterpillars fed on artificial diet (P ≤ 0.05), with 11.5 ± 3.4 µmol s-1 per µg of protein (Fig. 1E).

The higher content of total proteases was found in the midgut of larvae fed on cotton leaves (784.2 ± 0.7 µg per µL) compared with the other treatments (P ≤ 0.05) (Fig. 1F).

Spodoptera eridania showed higher trypsin activity in larvae fed on an artificial diet, (P ≤ 0.05), with esterase and amidasic trypsin activities of 2,040.7 ± 42.9 and 233.3 ±33.8 µmol s-1 per µg of protein, respectively, followed by soybean and cotton leaves (Fig. 1A, B).

In the analyses of the chymotrypsin enzymes, the larvae fed on the artificial diet showed activity of 214.2 ± 7.2 µmol s-1 per µg of protein for esterase chymotrypsin and 27.0 ± 2.5 µmol s-1 per µg of protein for amidasic, followed by cotton and soybean leaves (Fig. 1C, D).

For cysteine protease, higher activity was found in caterpillars fed on artificial diet in comparison with cotton and soybean diets, with 30.0 ± 1.0 µmol s-1 per µg of protein (Fig. 1E).

The higher content of total proteases was found in the midgut of larvae fed on cotton leaves (654.7 ± 2.7 µg per µL) compared to the other diets (P ≤ 0.05) (Fig. 1F).

The activity of serine proteases (trypsin and chymotrypsin), cysteine proteases, and total proteases varied according to insect species (P < 0.05) (Table 1). In addition, these enzymes, except for amidasic chymotrypsin, varied according to diet type (Table 1).


The high serine protease activity in S. cosmioides larvae fed on cotton leaves suggests adaptation of this species to this plant. Cotton plants produce gossypol, which plays a role as protease inhibitor (Lara 1991; Calhoun 1994) and insects may bypass inhibitory effects over-expressing proteases (Jongsma et al. 1994; Mosolov & Valueva 2008; Moreira et al. 2011). Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) larvae fed on plants expressing trypsin inhibitors, present hyperproduction of this enzyme as an adaptation to protease inhibitors (De Leo et al. 1998).

The higher serine protease activity in S. eridania larvae fed on artificial diet may be due to this diet containing high protein content and some starch without compounds that decrease the activity of this enzyme (Mendonça et al. 2009). The esterase trypsin activity in S. eridania fed on soybean leaves is 3 times higher than S. cosmioides fed on the same diet, suggesting an increase in the expression of serine protease genes, favoring their polyphagous feeding habits. Spodoptera frugiperda is non-sensitive to soybean Kunitz trypsin inhibitor (SKTI) and soybean Bowman-Birk inhibitor (SBBI) due to an evolutionary mechanism favoring its highly polyphagous nature (de Oliveira et al. 2013).

Our findings show higher esterase activity of serine proteases (trypsin) compared to amidasic activity in both species studied, suggesting high affinity for L-TAME substrate (Oliveira et al. 2005). Proteases have acylation with slow acyl-enzyme formation and deacylation with rapid product formation during amidase activity, the latter being slow during esterase activity. The acylation with formation of acyl-enzyme is the main step in the hydrolysis reaction of amide substrates by trypsin enzymes, whereas deacylation is the main step of the ester substrate hydrolysis with product formation (Inagami 1972; Fastrez & Fersht 1973; Xavier et al. 2005).

The esterase chymotrypsin activity increases in S. cosmioides fed on soybean, suggesting higher soybean consumption compared with S. eridania (Bortolotto et al. 2015) and serine protease trypsin sensitivity to inhibitors in soybean plants. This compensatory feeding is a consequence of the absence of amino acids available for protein synthesis, increasing other serine proteases such as chymotrypsin (Scriber & Slansky 1981; Simpson & Simpson 1990) for insect growth (Broadway & Duffey 1986; Ryan 1990).

Table 1.

Source of variation, degrees of freedom, and analysis of variance (ANOVA) for specific activities of proteolytic enzymes of Spodoptera cosmioides and Spodoptera eridania (Lepidoptera: Noctuidae).


The higher cysteine protease activity for both species fed on artificial diet may be due to this diet not containing substances that affect the activity of this enzyme. However, survival of Helicoverpa armigera Hübner (Lepidoptera: Noctuidade) is low with artificial diet plus a combination of trypsin and cysteine inhibitors, proving the importance of this enzyme for digestion in Lepidoptera even with low activity (Senthilkumar et al. 2010).

Cysteine proteases have low activity in the midgut of the larvae studied here, confirming that serine proteases are the most active proteolytic enzymes in Lepidoptera (Kipgen & Aggarwal 2014; Meriño-Cabrera et al. 2018).

The high total protease content in the midgut of S. cosmioides and S. eridania may be explained by the efficient use of plant proteins and artificial diet by both species. This indicates that the level of free amino acids and low molecular weight soluble proteins are sufficient for the development of S. cosmioides and S. eridania larvae, as reported for Cameraria ohridella (Deschka & Dimic) (Lepidoptera: Gracillariidae) (Stygar et al. 2010).

It has been claimed that the profile of midgut digestive enzymes is specific according to insect order (Terra & Ferreira 1994; Fialho et al. 2012; 2013). Our findings suggest that the variation in protease activity in the 2 species studied may be associated with their feeding habits. Nevertheless, whether changes in the amino acid composition or mechanism of action of these enzymes occurs due to evolutionary divergence or selective pressure exerted by different feeding habits remains uncertain.

Our findings show that diet-biased protease activity (serine and cysteine proteases and total proteases) presents high plasticity, suggesting that diet affects the amount of specific digestive proteases available for digestion, which may be related to de novo synthesis or post-translational activation of proteinases (Bolter & Jongsma 1995). In addition, this diet-biased variability may be explained by the rapid change in digestive proteolytic metabolism in these insects following ingestion of proteinase inhibitors from plants (Bolter & Jongsma 1995; Overney et al. 1997). Digestive proteases in animals are affected by both the amount and type of the proteins ingested (Lhoste et al. 1993; Noriega et al. 1994).

Overall, this study shows that the variability of proteolytic activity according to insect species may reflect different adaptations of these pests to the nutritional composition of plants with protease inhibitors.


This research was supported by Brazilian research agencies National Council for Scientific and Technological Development (CNPq), Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES), and Minas Gerais State Research Agency (FAPEMIG).

References Cited


Bolter CJ, Jongsma MA. 1995. Colorado potato beetles (Leptinotarsa decemlineata) adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. Journal of Insect Physiology 41: 1071–1078. Google Scholar


Bortolotto OC, Pomari-Fernandes A, Bueno RDF, Bueno ADF, Queiroz AP, Sanzovo A, Ferreira RB. 2015. The use of soybean integrated pest management in Brazil: a review. Agronomy Science and Biotechnology 1: 25–32. Google Scholar


Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248–254. Google Scholar


Broadway RM, Duffey SS. 1986. Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exiqua. Journal of Insect Physiology 32: 827–833. Google Scholar


Calhoun DS, Jones JE, Caldwell WD, Burris E, Leonard BR, Moore SH, Aguillard W. 1994. Registration of La. 850082 FN and La. 850075 FHG, two cotton germplasm lines resistant to multiple insect pests. Crop Science 34: 316–317. Google Scholar


Carvalho RA, Omoto C, Field LM, Williamson MS, Bass C. 2013. Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda. PLoS ONE 8: e62268. Google Scholar


De Leo F, Bonadé-Bottino MA, Ceci LR, Gallerani R, Jouanin L. 1998. Opposite effects on Spodoptera littoralis larvae of a low and high expression level of a trypsin proteinase inhibitor in transgenic plants. Plant Physiology 119: 997–1004. Google Scholar


de Oliveira CFR, de Paula Souza T, Parra JRP, Marangoni S, Silva-Filho MdC, Macedo MLR. 2013. Insensitive trypsins are differentially transcribed during Spodoptera frugiperda adaptation against plant protease inhibitors. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 165: 19–25. Google Scholar


Diez-Rodríguez GI, Omoto C. 2001. Herança da resistência de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) a lambda-cialotrina. Neotropi-cal Entomology 30: 311–316. Google Scholar


Erlanger BF, Kokowsky N, Cohen W. 1961. The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics 95: 271–278. Google Scholar


Fastrez J, Fersht AR. 1973. Demonstration of the acyl-enzyme mechanism for the hydrolysis of peptides and anilides by chymotrypsin. Biochemistry 12: 2025–2034. Google Scholar


Fialho MCQ, Moreira NR, Zanuncio JC, Ribeiro AF, Terra WR, Serrão JE. 2012. Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae). Journal of Insect Physiology 58: 850–856. Google Scholar


Fialho MCQ, Terra WR, Moreira NR, Zanuncio JC, Serrão JE. 2013. Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae). Arthropod, Structure and Development 42: 277–285. Google Scholar


Gariani T, Leatherbarrow RJ. 1997. Stability of protease inhibitors based on the Bowman-Birk reactive site loop to hydrolysis by proteases. The Journal of Peptide Research 49: 467–475. Google Scholar


Gatehouse AM, Norton E, Davison GM, Babbé SM, Newell CA, Gatehouse JA. 1999. Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo. Journal of Insect Physiology 45: 545–558. Google Scholar


Greene GL, Leppla NC, Dickerson WA. 1976. Velvetbean caterpillar: a rearing procedure and artificial medium. Journal of Economic Entomology 69: 487–488. Google Scholar


Hummel BCW. 1959. A modified spectrophotometric determination of chymotrypsin, trypsin and trombin. Canadian Journal of Biochemistry and Physiology 37: 1393–1399. Google Scholar


Inagami T 1972. Trypsin, pp. 1–83 In Funatsu M, Hiromi K, Imahori K, Murachi T, Narita K [eds.], Proteins: Structure and Function. Kodanska, Tokyo, Japan. Google Scholar


Jamal F, Pandey PK, Singh D, Khan MY. 2012. Serine protease inhibitors in plants: nature's arsenal crafted for insect predators. Phytochemistry Reviews 12: 1–34. Google Scholar


Jongsma MA, Bakker PL, Visser B, Stiekema WJ. 1994. Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack, wounding and virus infection. Planta 195: 29–35. Google Scholar


Jongsma MA, Bolter C. 1997. The adaptations of insects to plant proteinase inhibitors. Journal of Insect Physiology 43: 885–895. Google Scholar


Kidd H. 2000. Human exposure to pesticide residues, natural toxins and GMOs and real and perceived risks. Pesticide Outlook 11: 215–216. Google Scholar


Kipgen L, Aggarwal KK. 2014 Gut protease profiles of different instars of Helicoverpa armigera (Lepidoptera: Noctuidae). International Journal of Tropical Insect Science 34: 172–178. Google Scholar


Kotkar HM, Bhide JA, Gupta SV, Giri PA. 2012. Amylase gene expression patterns in Helicoverpa armigera upon feeding on a range of host plants. Gene 501: 1–7. Google Scholar


Lara FM. 1991. Princípios de resistência das plantas a insetos. Ícone, Rio de Janeiro, Brazil. Google Scholar


Lhoste EF, Fiszlewicz M, Gueugneau AM, Wicker-Planquart C, Puigserver A, Corring T. 1993. Effects of dietary proteins on some pancreatic mRNAs encoding digestive enzymes in the pig. The Journal of Nutritional Biochemistry 4: 143–152. Google Scholar


Liener IE. 1994. Implications of antinutritional components in soybean foods. Critical Reviews in Food Science & Nutrition 34: 31–67. Google Scholar


Mahdavi A, Ghadamyari M, Sajedi RH, Sharifi M, Kouchaki B. 2013. Identification and partial characterization of midgut proteases in the lesser mulberry pyralid, Glyphodes pyloalis. Journal of Insect Science 13: 81. Google Scholar


Meisner J, Ishaaya I, Ascher KRS, Zur M. 1978. Gossypol inhibits protease and amylase activity of Spodoptera littoralis larvae. Annals of the Entomological Society of America 71: 5–8. Google Scholar


Mendonça EG, de Almeida OMG, Evangelista VL, Guedes RNC, Rainha RF, de Oliveira JA. 2009. Determinação da atividade enzimática e do número de bactérias associadas ao intestino médio da lagarta da soja, Anticarsia gemmatalis, criada em diferentes dietas. Revista Ceres 56: 18–24. Google Scholar


Mendonça EG, Visôtto LE, Costa NCS, Ribeiro FR, DE Oliveira JA, Oliveira MGdA. 2011. Enzymatic characterization of cysteine protease isoforms of Anticarsia gemmatalis (Hübner, 1818). Ciência e Agrotecnologia 35: 446–454. Google Scholar


Meriño-Cabrera Y, Zanuncio JC, Silva RS, Solis-Vargas M, Cordeiro G, Ribeiro FR, Campos WG, Picanço MC, Oliveira MGA. 2018. Biochemical response between insects and plants: an investigation of enzyme activity in the digestive system of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and leaves of Coffea arabica (Rubiaceae) after herbivory. Annals of Applied Biology 172: 236–243. Google Scholar


Mills NJ, Kean JM. 2010. Behavioral studies, molecular approaches and modeling: methodological contributions to biological control success. Biology Control 52: 255–262. Google Scholar


Moon J, Salzman RA, Ahn JE, Koiwa H, Zhu-Salzman K. 2004. Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defence protease inhibitor. Insect Molecular Biology 13: 283–291. Google Scholar


Moreira LF, Campos WG, Ribeiro FR, Guedes RNC, Oliveira MGA. 2011. Survival and developmental impairment induced by the trypsin inhibitor bis-benzamidine in the velvetbean caterpillar (Anticarsia gemmatalis). Crop Protection 30: 1285–1290. Google Scholar


Mosolov VV, Valueva TA. 2008. Proteinase inhibitors in plant biotechnology: a review. Applied Biochemistry and Microbiology 44: 233–240. Google Scholar


Noriega FG, Barillas-Mury C, Wells MA. 1994. Dietary control of late trypsin gene transcription in Aedes aegypti. Insect Biochemistry and Molecular Biology 24: 627–631. Google Scholar


Oliveira MGA, Simone S, Xavier L, Guedes RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comparative Biochemistry and Physiology 140B: 369–380. Google Scholar


Onesti S, Brick P, Blow DM. 1991. Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina coffra seeds. Journal of Molecular Biology 217: 153–176. Google Scholar


Overney S, Fawe A, Yelle S, Michaud D. 1997 Diet-related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say). Archives of Insect Biochemistry and Physiology 36: 241–250. Google Scholar


Paixao GP, Lourenção AL, Silva CR, Eduardo EG, Silva PL, Oliveira JA, Zanuncio JC, Oliveira MGA. 2013. Biochemical responses of Anticarsia gemmatalis (Lepidoptera: Noctuidae) in soybean cultivars sprayed with the protease inhibitor berenil. Journal of Agricultural and Food Chemistry 61: 8034–8038. Google Scholar


Patarroyo-Vargas AM, Meriño-Cabrera Y, Zanuncio JC, Rocha FAD, Garcia WC, Oliveira MGA. 2017. Kinetic characterization of Anticarsia gemmatalis digestive serine-proteases and the inhibitory effect of synthetic peptides. Protein Peptide Letters 24: 1040–1047. Google Scholar


Pilon AM, Oliveira MGA, Guedes RNC. 2006. Protein digestibility, protease activity and post-embryonic development of the velvetbean caterpillar (Anticarsia gemmatalis) exposed to the trypsin-inhibitor benzamidine. Pesticide Biochemistry and Physiology 86: 23–29. Google Scholar


Pilon AM, Oliveira MGA, Pilon FM, Guedes RNC, Oliveira JA, Fazollo A. 2009. Adaptação da lagarta da soja Anticarsia gemmatalis Hübner (Lepdoptera: Noctuidae) ao inibidor de protease benzamidina. Revista Ceres 56: 744–748. Google Scholar


Rosell G, Quero C, Guerrero A. 2008. Biorational insecticides in pest management. Journal Pesticide Science 33: 103–121. Google Scholar


Ryan CA. 1990. Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28: 425–449. Google Scholar


Sarate PJ, Tamhane VA, Kotkar HM, Ratnakaran N, Susan N, Gupta VS, Girig AP. 2012. Developmental and digestive flexibilities in the midgut of a polyphagous pest, the cotton bollworm, Helicoverpa armigera. Journal of Insect Science 12: 42. Google Scholar


SAS. 2013. SAS user's manual, vers. 9.1. SAS Institute, Cary, North Carolina, USA. Google Scholar


Scott IM, Thaler JS, Scott JG. 2010. Response of a generalist herbivore Trichoplusia ni to jasmonate-mediated induced defense in tomato. Journal of Chemical Ecology 36: 490–499. Google Scholar


Scriber JM, Slansky F. 1981. The nutritional ecology of immature insects. Annual Review of Entomology 26: 183–211. Google Scholar


Senthilkumar R, Cheng CP, Yeh KW. 2010. Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco. Plant Biotechnology Journal 8: 65–75. Google Scholar


Shi M, Zhu N, Yi Y, Chen X. 2013. Four serine protease cDNAs from the midgut of Plutella xylostella and their proteinase activity are influenced by the endoparasitoid, Cotesia vestalis. Archives of Insect Biochemistry and Physiology 83: 101–114. Google Scholar


Simpson SJ, Simpson CL. 1990. The mechanisms of nutritional compensation by phytophagous insects. Insect Plant Interactions 2: 111–160. Google Scholar


Song HK, Suh SW. 1998. Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator. Journal of Molecular Biology 275: 347–363. Google Scholar


Souza BHS, Bottega DB, Silva AG, Boiça Junior AL. 2013. Feeding non-preference by Spodoptera frugiperda and Spodoptera eridania on tomato genotypes. Ceres 60: 21–29. Google Scholar


Souza DMM, Amorim TML, Sales MP, Vidal MS. 2006. Identificação de genótipos de algodão (Gossypium spp.) quanto à presença de inibidores de protease. Estudos Biológicos 28: 97–103. Google Scholar


Srinivasan A, Giri AP, Gupta VS. 2006. Structural and functional diversities in lepidoptera serine proteases. Cellular & Molecular Biology Letters 11: 132–154. Google Scholar


Stygar D, Dolezych B, Nakonieczny M, Migula P, Michalczyk K, Zaak M. 2010. Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae). Comptes Rendus Biologies 333: 725–735. Google Scholar


Terra WR, Ferreira C. 1994. Insect digestive enzymes: properties, compartmentalization and function. Comparative Biochemistry and Physiology 109B: 1–62. Google Scholar


Tomarelli RM, Charney J, Harding ML. 1949. The use of azoalbumin as a substrate in the colorimetric determination of peptic and tryptic activity. Journal of Laboratory and Clinical Medicine 34: 428–433. Google Scholar


Vianna UR, Pratissoli D, Zanuncio JC, Alencar JRCC, De Zinger FD. 2011. Espécies e/ou linhagens de Trichogramma spp. (Hymenoptera: Trichogrammatidae) para o controle de Anticarsia gemmatalis (Lepidoptera: Noctuidae). Arquivos do Instituto Biológico 78: 81–87. Google Scholar


Visôtto LE, Oliveira MGA, Guedes RNC, Ribon AOB. 2009. Contribution of gut bacteria to digestion and development of the velvetbean caterpillar: Anticarsia gemmatalis. Journal of Insect Physiology 55: 185–191. Google Scholar


Visôtto LE, Oliveira MGA, Ribon AOB, Mares-Guia TR, Guedes RNC. 2009. Characterization and identification of proteolytic bacteria from the gut of the velvetbean caterpillar (Lepidoptera: Noctuidae). Environmental Entomology 38: 1078–1085. Google Scholar


Xavier LP, Oliveira MGA, Guedes RNC, Santos AV, De Simone SG. 2005. Trypsin-like activity of membrane-bound midgut proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae). European Journal of Entomology 102: 147–153. Google Scholar


Wasternack C, Hause B. 2013. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. Annals of Botany 111: 1021–1058. Google Scholar


Zhang C, Zhou D, Zheng S, Lin L, Tao S, Yang L, Hu S, Feng Q. 2010. A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: cloning, characterization, developmental and induced expression patterns, and localization. Journal of Insect Physiology 56: 788–799. Google Scholar
Francelina Aparecida Duarte Rocha, Yaremis Beatriz Meriño-Cabrera, Eliseu José Guedes Pereira, José Cola Zanuncio, Wellington Garcia Campos, José Eduardo Serrão, and Maria Goreti Almeida Oliveira "Effect of Natural and Artificial Diets on Protease Activity in the Midgut of Spodoptera cosmioides and Spodoptera eridania (Lepidoptera: Noctuidae) Larvae," Florida Entomologist 103(4), 452-457, (13 January 2021).
Published: 13 January 2021
Back to Top