Adult Colorado potato beetles,Leptinotarsa decemlineata compensate for nutritional stress on oryzacystatin I-transgenic potato plants by hypertrophic behavior and over-production of insensitive proteases

Abstract

Protease inhibitors have been proposed as potential control molecules that could be engineered into potato plants for developing crops resistant to the Colorado potato beetle, Leptinotarsa decemlineata, a major pest of potato and other Solanaceae. In this study, we examined the effects of feeding young female beetles with foliage from a cultivar of the "Kennebec" potato line (K52) transformed with a gene encoding oryzacystatin I (OCI), a specific cysteine proteinase inhibitor with proven activity against cathepsin H-like enzymes of larvae and adults of the potato beetle. To evaluate the insect's performance, we collected data over a 16-d postemergence period on survival, diapause incidence, foliage consumption, weight gain, and oviposition of females. Tested individuals were fed untransformed (control) and OCI-transformed foliage at two stages of potato leaf differentiation, corresponding to "low" and "high" levels of OCI expression in leaves of K52. The OCI-expressing foliage did not affect female survival (close to 100%), incidence of diapause (15-30%), relative growth rate (RGR) during postemergence growth (5-9% d(-1)) or maximum weight reached (140-160 mg). Neither did it affect female reproductive fitness as measured by preoviposition time (8-9 d), 16-d fecundity (220-290 eggs), or egg eclosion incidence (86-91%). However, nutritional stress to females feeding on OCI foliage was evident, as reflected in their lower efficiency of conversion of ingested foliage (ECI) during postemergence growth, increased foliage consumed per egg laid (up to 119% more), and adaptation of their digestive proteolytic system to the inhibitory effect of OCI. Interestingly, beetles fed foliage expressing the highest level of OCI reacted rapidly to the presence of OCI by producing OCI-insensitive proteases, and exhibiting strong hypertrophic behavior by ingestion of 2.4-2.5 times more OCI rich foliage apparently as a compensatory response for nutritional stress due to the protease inhibitor in their diet.

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

“…Our bioassays with OsCYS1 and CpCYS1 were conducted with excess concentrations of inhibitor, estimated at ~0.6 mg/g.fresh weight of infiltrated stem disk after re-extraction (not shown). Such concentrations would represent accumulation rates reaching ~2% of leaf soluble proteins in tissues of transgenic banana lines, compared to lower rates of 0.1-1.0% typically observed in cystatinexpressing transgenic plants (Cloutier et al, 2000;Van der Vyver et al, 2003). In practice, a strong inhibitory potency of the protease inhibitor selected is critical for an effective inhibition effect, as it directly determines the minimal amount of recombinant inhibitor required in the modified host plant to provide sufficient pesticidal effects (De Leo et al, 1998;Goulet et al, 2008).…”

“…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).…”

“…The negative effects of E-64 on growth and fecundity of several coleopteran species, including cystatin-resistant insects such as the Colorado potato beetle Leptinotarsa decemlineata (Bolter and Latoszek-Green, 1997) and the cowpea weevil Callosobruchus maculatus (Murdock et al, 1988), were systematically associated with a broadspectrum inhibition of cysteine proteases in midgut extracts (Michaud et al, 1993;Fabrick et al, 2002;Kim and Mullin, 2003). By contrast, the reported inefficiency of plant cystatins against a number of Coleoptera was typically associated with the presence of cystatin-insensitive cysteine proteases, thought to help the target insects compensating for the loss of sensitive protease activities following cystatin intake (Cloutier et al, , 2000Zhu-Salzman et al, 2003;Gruden et al, 2003Gruden et al, , 2004. In line with previous studies reporting detrimental effects for cystatins or other plant cysteine-type inhibitors against some coleopteran species (Zhao et al, 1996;Koiwa et al, 2000;Fabrick et al, 2002), the negative effects of OsCYS1 and CpCYS1 against the banana Plant cystatins against the banana weevil Page 16 weevil were correlated with a broad inhibitory spectrum of the two cystatins against Z-FR-MCA-(cathepsin L-like) and Z-RR-MCA-hydrolyzing (cathepsin B-like) cysteine proteases in midgut extracts, comparable to the overall inhibitory effect of E-64 against the same enzymes.…”

“…On the other hand, the occurrence of cathepsin B variants sensitive to cystatins in the midgut of the banana weevil (Fig. 3B) [and other cystatin-susceptible insects (Bown et al, 2004)] is somewhat intriguing since these enzymes were identified as possible key players in the resistance of Coleoptera to dietary cystatins (Michaud et al, 1993;Cloutier et al, 2000;Goulet et al, 2008;Koo et al, 2008). Unlike other proteases of the papain family, cathepsin B and cathepsin B-like homologues bear an extra structural element, the 'occluding loop', that physically hinders the substrate binding cleft and prevents free access to the inhibitory loops of large competitive inhibitors such as cystatins (Musil et al, 1991;Illy et al, 1997).…”

“…Several papers have been published assessing the effectiveness of plant cystatins against herbivorous Coleoptera, with conclusive results in some cases (Leplé et al, 1995;Kuroda et al, 1996;Lecardonnel et al, 1999;Koiwa et al, 2000;Alvarez-Alfageme et al 2007;Ninkovic et al, 2007) but disappointing results in other cases owing to the ability of the target insects to readily elude or even counteract the antidigestive effects of natural or recombinant inhibitors (e.g. Girard et al, 1998a,b;Cloutier et al, 1999Cloutier et al, , 2000Zhu-Salzman et al, 2003;Gruden et al, 2003Gruden et al, , 2004Ahn et al, 2004Ahn et al, , 2007Oppert et al, 2005;Koo et al, 2008). Here we document the broad inhibitory effects of plant cystatins against midgut cysteine cathepsins of the banana weevil, and the detrimental effects of these proteins on larval growth using a novel bioassay set-up with cystatin-infiltrated banana tissues.…”