Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens

Abstract

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens. Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

Biochemical characterization of digestive membrane‐associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis

“…The effects of several Cry toxins have been previously demonstrated in larvae of A. gemmatalis (Bel et al, ; Mushtaq et al, , ; Fiuza et al, ). The mode of action and toxicity of some different Cry toxins were recently described by Mushtaq et al () and indicated that the domain III of Cry1Ac, which has a lectin fold, is not important for toxicity.…”

“…It is known that the binding of Cry toxin to target proteins results in the formation of pores in the cell membrane leading to cell death and tissue degradation (Gomez et al, ; de Maagd, Bravo, & Crickmore, ). In Brazil, transgenic soybean expressing Cry1Ac (one type of Bt toxin) has been commercialized since 2013 (Bel, Sheets, Tan, Narvac, & Escriche, ). The effect of Cry toxins against A. gemmatalis has been previously described, but its receptor still remains unidentified (Fiuza, Knaak, Fernando, Antônio, & Henriques, ).…”

“…The effect of Cry toxins against A. gemmatalis has been previously described, but its receptor still remains unidentified (Fiuza, Knaak, Fernando, Antônio, & Henriques, ). Some recent works demonstrated the susceptibility of A. gemmatalis larvae to several Cry toxins, and Cry1Ac was pointed as the most successful (Bel et al, ; Mushtaq et al, ; Mushtaq, Shakoori, & Jurat‐Fuentes, ). The authors observed the binding of Cry toxins to brush border membrane vesicles, suggesting the participation of receptors in the midgut.…”

Domain III of Cry1Ac Is Critical to Binding and Toxicity against Soybean Looper (Chrysodeixis includens) but Not to Velvetbean Caterpillar (Anticarsia gemmatalis)

“…Analysis of Cry2Ac7 domain III determined that out of the N506, Q509, and Y513 amino acid residues in Cry1Ac involved in binding to GalNAc [ 14 ], only Q509 is conserved. Consequently, we designed a toxin hybrid (H1.2Ac) combining domains I and II from Cry1Ac as the most active Bt toxin against A. gemmatalis and C. includens , and domain III from Cry2Ac7 as a novel toxin with toxicity against both species but predicted not to share binding sites with Cry1Ac [ 18 ].…”

“…However, the fact that H1.2Ac and Cry1Ac are equally active against A. gemmatalis larvae supports the finding that interactions with the binding sites recognized by domain III of Cry1Ac may not be relevant to toxicity against A. gemmatalis . Dependence on domain III for recognition of this second population of Cry1Ac binding sites in A. gemmatalis BBMV would also help explain why GalNAc significantly inhibited Cry1Ac binding to BBMV from C. includens, while the effect was more modest on Cry1Ac binding to A. gemmatalis BBMV [ 18 ]. Interestingly, previous reports suggest the existence of two populations of Cry1Ac binding sites in C. includens , but not in A. gemmatalis [ 18 ].…”