Effects of a Reserve Protein on Spodoptera frugiperda Development: A Biochemical and Molecular Approach to the Entomotoxic Mechanism

Abstract

Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda, considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda. The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes.

Keywords: bioinsecticides; enzyme activity; insect gut; lectin properties; multifunctional protein.

Figure 1

Effect of dietary Talisia esculenta reserve protein (talisin) on mass of fifth-instar Spodoptera frugiperda larvae. Inset: size difference in a control larva (left) and a larva fed 0.1, 0.5 and 1% talisin (right). Different letters indicate significant differences (p < 0.05; Tukey’s test). Bar = 1 cm.

Figure 2

Trypsin-like (A), chymotrypsin-like (B) and aminopeptidase (C) activities in control and experimental (0.5% talisin-fed) Spodoptera frugiperda larvae. Different letters indicate significant differences (p < 0.05; Tukey’s test).

Figure 3

In vitro inhibition of proteolytic activity of midgut trypsins of Spodoptera frugiperda larvae fed on control diet (square) and diet containing 0.5% talisin (circle), using the synthetic BApNA substrate.

Figure 4

Peptidase activity of midgut peptidases in zymography gel electrophoresis. Samples fed with control diet or diet containing 0.5% talisin were used for the determination of enzyme profile. The bands were designated SfGP1 through SfGP5. Lane 1 was loaded with midgut juice extract from larvae fed on a control diet devoid of talisin; lane 2, with midgut juice extract from larvae fed on a 0.5% talisin diet; lanes 3 and 5, with midgut juice extract from larvae-fed control diets mixed with TLCK and talisin, respectively; lanes 4 and 6 with midgut juice extract from larvae fed 0.5% talisin diets mixed with TLCK and talisin, respectively.

Figure 5

Resistance of talisin (Talisia esculenta reserve protein) to hydrolysis by Spodoptera frugiperda midgut peptidases. SDS–PAGE of midgut juice extract mixed with talisin at a 1:5 (lectin to midgut homogenates) ratio and incubated at 30 °C for different periods. Digestion of BSA (A) and talisin (C) with midgut juice extract of control-fed larvae and digestion of BSA (B) and talisin (D) with midgut juice extract of talisin-fed larvae. TAL: talisin; BSA: bovine serum albumin; MG: midgut.

Figure 6

Photomicrographs of fifth-instar larvae of Spodoptera frugiperda. (A) Midgut of control larvae, 10×. (B) Midgut of larvae fed on a 0.5% talisin diet, 10× (C) Midgut epithelium of control larvae, 40× (D) Midgut epithelium of larvae fed on a 0.5% talisin diet, 40×. Lu, Lumen; EP, Epithelium; PM, Peritrophic Membrane; (*) Difference between the ectoperitrophic space.

Figure 7

Relative expression of trypsin (A), chymotrypsin (B) and N-aminopeptidase (C) genes of fifth instar of talisin-fed Spodoptera frugiperda larvae. Genes above the dotted line were considered more expressed and genes below the dotted line were considered less expressed. Different letters indicate significant differences (p < 0.05).

Figure 8

Theoretical three-dimensional model for the talisin/Try 6 (A), talisin/Try 12 (B), talisin/Chy 6 (C) and talisin/Chy 12 (D) complexes.