Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, <i>Tenebrio molitor</i> L.

Prabhakar, Sheila; Chen, M.-S.; Elpidina, Elena N.; Vinokurov, Konstantin S.; Smith, C. Michael; Marshall, Jeremy L.; Oppert, Brenda

doi:10.1111/j.1365-2583.2007.00740.x

Cited by 74 publications

(80 citation statements)

References 78 publications

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Section: Cysteine and Serine Proteases In The Banana Weevil Midgutsupporting

confidence: 71%

“…3A) but merely detectable at pH 6.5 (not shown), which points again to the presence of serine proteinases in the midgut extracts, as also reported for other Coleoptera predominantly relying on cysteine proteases for dietary protein digestion (e.g. Novillo et al, 1997; Vinokurov et al, 2006a,b;Prabhakar et al, 2007). …”

supporting

confidence: 76%

“…3A), suggesting the presence of cathepsin L-like, cathepsin B-like and aminopeptidase (cathepsin H-like) activities in larval extracts, as observed earlier for a number of coleopteran insects (e.g. Thie and Houseman, 1990;Michaud et al, 1993;Bown et al, 2004;Montesdeoca et al, 2005; Vinokurov et al, 2006a,b;Prabhakar et al, 2007).Trypsin-and chymotrypsin-like serine proteases hydrolyzing, respectively, Z-R-MCA and succinyl-AAPF-MCA were detected at pH 8.5 (Fig. 3A) but merely detectable at pH 6.5 (not shown), which points again to the presence of serine proteinases in the midgut extracts, as also reported for other Coleoptera predominantly relying on cysteine proteases for dietary protein digestion (e.g.…”

supporting

confidence: 71%

“…These insects have developed over time effective strategies to elude the inhibitory effects of plant protease inhibitors, involving the use of complex digestive protease systems with proteases from different mechanistic classes acting in a complementary, coordinated manner (Brunelle et al, 1999;Hernandez et al, 2003;Gruden et al, 2003;Vinokurov et al, 2006a,b;Prabhakar et al, 2007); the over-expression of target proteases following cystatin ingestion to outnumber the inhibitory proteins (Cloutier et al, 2000;Ahn et al, 2004); the constitutive or diet-induced expression of cysteine cathepsins weakly sensitive to the ingested cystatin, the so-called 'cystatin-insensitive proteases' (Michaud et al, 1993(Michaud et al, , 1995aGirard et al, 1998a;Cloutier et al, 1999Cloutier et al, , 2000ZhuSalzman et al, 2003;Brunelle et al, 2004;Gruden et al, 2004;Liu et al, 2004;Koo et al, 2008); the over-expression of proteases from alternative mechanistic classes following cystatin ingestion (Zhu-Salzman et al, 2003;Brunelle et al, 2004;Rivard et al, 2004;Oppert et al, 2005); and the degradation of defensive protease inhibitors using non-target, insensitive proteases (Michaud et al, 1995b;Girard et al, 1998b;Giri et al, 1998;Gruden et al, 2003;Zhu-Salzman et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

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Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

Kiggundu

Muchwezi

Vyver

et al. 2009

Arch Insect Biochem Physiol

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The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of active cysteine proteases following inhibitor ingestion. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were first conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet bioassay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1)and papaya cystatin (CpCYS1), on the overall growth rate of young weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg- MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the diagnostic cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with these broad inhibitory effects of cystatins, OsCYS1 andCpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatininfiltrated banana stem disks. These promising results, which illustrate the natural susceptibility of C. sordidus to plant cystatins, are discussed in the light of current genomic data on coleopteran cysteine cathepsins and recent hypotheses suggesting a key role for digestive cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.Keywords: Banana weevil (Cosmopolites sordidus); banana (Musa spp.); cathepsin B-like proteases; cysteine cathepsins; plant cystatins; oryzacystatin I Plant cystatins against the banana weevil Page 3 INTRODUCTIONThe banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is an insect pest of considerable importance in Africa, associated with the rapid decline of banana and plantain plantations (Gold, 1999a(Gold, , 2000Swennen and Vuylsteke, 2001;Gold et al., 2005). At the adult stage, banana weevil females deposit their eggs inside host plant tissues, at the base of the pseudostem or on an exposed corm. On hatching, the larvae tunnel through the corm to feed and develop, where they damage tissues, compromise water and mineral uptake, and weaken the colonized organs. Banana weevil infestations cause important losses in the field by a direct negative impact on harvestable bunch weight and a weakening effect on infested organs causing plant toppling during windstorms (Sengooba, 1986;Rukazambuga et al., 1998).Banana weevil control in Africa relies essentially on cultural and sanitation practices such as the use of clean planting material, the systematic trapping of adult weevils to prevent population build-up, an...

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Section: Cysteine and Serine Proteases In The Banana Weevil Midgutsupporting

confidence: 71%

supporting

confidence: 76%

supporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

Kiggundu

Muchwezi

Vyver

et al. 2009

Arch Insect Biochem Physiol

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“…Cloning of TmCad1 cDNA-A partial cDNA encoding a T. molitor cadherin was identified among randomly selected clones from a T. molitor larval midgut cDNA library (34). Genespecific primers were designed based on this sequence, and the complete coding sequence (TmCad1) was obtained from larval midgut mRNA by 5Ј-and 3Ј-rapid amplification of cDNA ends using the GeneRacer TM kit from Invitrogen and SuperTaq…”

Section: Methodsmentioning

confidence: 99%

A Novel Tenebrio molitor Cadherin Is a Functional Receptor for Bacillus thuringiensis Cry3Aa Toxin

Fabrick

Oppert

Lorenzen

et al. 2009

Journal of Biological Chemistry

106

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Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera.The mode of action of Bacillus thuringiensis insecticidal Cry toxins has been extensively studied in lepidopteran larvae (1). Our current understanding is that the major factors that contribute to Cry toxicity in insects include solubilization and activation of the crystalline toxin as well as interactions between toxin and midgut receptors. In lepidopterans, several insect midgut proteins have been proposed as Cry toxin receptors (2).Cry1A receptor functionality has been demonstrated for cadherin proteins from Bombyx mori (3, 4), Manduca sexta (5, 6), Ostrinia nubilalis (7), and Heliothis virescens (8). The specific toxin-binding region in lepidopteran cadherins has been localized proximal to the cell membrane insertion site (9 -11). Mutations in toxin binding motifs of lepidopteran cadherin genes are genetically linked to Cry1Ac resistance in H. virescens (12, 13), Helicoverpa armigera (14, 15), and Pectinophora gossypiella (9, 16).Interactions between Cry toxins and cadherin receptors and the implications for toxicity have been studied in Lepidoptera more extensively than in any other insect order (2). According to the model proposed by Bravo et al. (17), Cry toxin binding to cadherin is followed by toxin oligomerization. Toxin oligomers reportedly are intermediates required for effective pore formation and ultimately toxicity (18). A fragment of the BtR1 cadherin from M. sexta, corresponding to repeat 12 and containing a critical toxin-binding region, enhanced the activity of Cry1A toxins in Lepidoptera (19) by promoting toxin oligomerization (20). However, other studies in Lepidoptera have found that cadherin fragments can reduce Cry1A toxicity (11,21). An alternative model suggests that Cry toxin binding to cadherin receptors activates intracellular pathways leading to cell death (22). Notably, in both models, cadherin is a critical contact point for Cry toxins that is pivotal for intoxication.In contrast to the lepidopteran model, r...

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Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum)

Vinokurov

Elpidina

Zhuzhikov

et al. 2009

Arch Insect Biochem Physiol

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The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6-6.0 in the anterior to 7.0-7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p-nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin-, chymotrypsin-, and elastase-like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases.

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Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L.

Cited by 74 publications

References 78 publications

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

A Novel Tenebrio molitor Cadherin Is a Functional Receptor for Bacillus thuringiensis Cry3Aa Toxin

Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum)

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