A barley cysteine-protease inhibitor reduces teh performance of two aphid species in artificial diets and transgenic arabidopsis plants

Cystatins from plants have been implicated in plant defense towards insects, based on their role as inhibitors of heterologous cysteine-proteinases. We have previously characterized thirteen genes encoding cystatins (HvCPI-1 to HvCPI-13) from barley (Hordeum vulgare), but only HvCPI-1 C68 → G, a variant generated by direct-mutagenesis, has been tested against insects. The aim of this study was to analyze the effects of the whole gene family members of barley cystatins against two aphids, Myzus persicae and Acyrthosiphon pisum. All the cystatins, except HvCPI-7, HvCPI-10 and HvCPI-12, inhibited in vitro the activity of cathepsin L- and/or B-like proteinases, with HvCPI-6 being the most effective inhibitor for both aphid species. When administered in artificial diets, HvCPI-6 was toxic to A. pisum nymphs (LC50 = 150 μg/ml), whereas no significant mortality was observed on M. persicae nymphs up to 1000 μg/ml. The effects of HvCPI-6 ingestion on A. pisum were correlated with a decrease of cathepsin B- and L-like proteinase activities. In the case of M. persicae, there was an increase of these proteolytic activities, but also of the aminopeptidase-like activity, suggesting that this species is regulating both target and insensitive enzymes to overcome the effects of the cystatin. To further analyze the potential of barley cystatins as insecticidal proteins against aphids, Arabidopsis plants expressing HvCPI-6 were tested against M. persicae. For A. pisum, which does not feed on Arabidopsis, a combined diet-Vicia faba plant bioassay was performed. A significant delay in the development time to reach the adult stage was observed in both species. The present study demonstrates the potential of barley cystatins to interfere with the performance of two aphid specie

Toxicity and mode of action of plant lectins with a ricin-B domain against pest insects

In recent years a lot of attention was given to possible applications of genetic engineering within integrated pest management (IPM). Insect resistant transgenic crops have now been created for many of the world’s economically important crops including maize, cotton, rice and potatoes. Lectins are only one group of proteins that have received a lot of attention because of their insecticidal properties. Although the physiological role of lectins in plants remains enigmatic, it has been clearly shown that the specific carbohydrate-binding activity of lectins plays an important role in plant interactions with other organisms. Entomotoxic effects of different lectins against several orders of plant pest insects including Lepidoptera, Coleoptera, Diptera and Hemiptera have been demonstrated both in vitro and in planta. Since lectins are good candidates to confer insect resistance in transgenic crops, they can be considered of great economic potential for pest management. In this PhD project analyses were performed to investigate the entomotoxic effects of a specific plant lectin family, referred to as the ricin-B related lectins (previously called type 2 ribosome-inactivating proteins (RIPs)). In particular the insecticidal activity of Sambucus nigra agglutinin I (SNA-I) and I’ (SNA-I’) was investigated. SNA-I is a chimeric lectin of 240 kDa composed of an A-domain with N-glycosidase activity and a B-domain with carbohydrate-binding properties for NeuAc(α-2,6)Gal/GalNAc. Furthermore, experiments were carried out to study the mode of action of SNA-I and the hololectin SNA-II. SNA-II is a homodimeric Gal/GalNAc-binding lectin of 60 kDa that consists only of carbohydrate-binding B-domains but lacks enzymatic activity. Chapter 1 gives a detailed overview of the occurrence RIPs in plants and their biological properties. In addition, more information is given on the interaction of plant lectins and insects. In Chapter 2 the effects of the type 2 RIP SNA-I′ delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects, belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae), and Lepidoptera, the beet armyworm (Spodoptera exigua). It was shown that M. nicotianae fed on detached leaves of transgenic lines expressing SNA-I’ reduced adult survival and it was apparent that the type 2 RIP SNA-I’ influenced aphid development by a retardation in the onset of reproduction. In aphids fed on transgenic lines expressing SNA-I’, significant effects were observed on the life parameters, such as intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring. In addition, the experiment with caterpillars showed significant increases in mortality for larvae fed on the transgenic lines expressing SNA-I’ as compared to wild type plants. Further studies revealed a significant reduction in fresh larval mass of caterpillars as well as retardation in development. This study clearly showed the insecticidal activity of SNA-I’ on both insect species. Hence, this information provides further support for RIPs having a role in plant resistance to insect pests. In Chapter 3 the insecticidal activity of the type 2 RIP SNA-I was studied on two hemipteran insect species using both feeding assays with an artificial diet and transgenic lines overexpressing the protein. Feeding of the pea aphid (Acyrthosiphon pisum) on an artificial diet supplemented with different concentrations of the purified SNA-I resulted in reduced survival and fecundity. Similarly, analyses with tobacco aphids (M. nicotianae) on transgenic tobacco plants overexpressing the SNA-I gene revealed a delay in development and reduced adult survival. In addition, the fertility parameters of the surviving aphids were reduced. In the second part of this study a series of experiments was performed using transgenic lines in which a mutant form of SNA-I was expressed. The mutation of one carbohydrate binding site strongly lowered the insecticidal activity of SNA-I, whereas the mutation of both lectin sites in SNA-I abolished its entomotoxic effects on tobacco aphids. From these results it was evident that the carbohydrate-binding activity of SNA-I is essential for its insecticidal activity. At present, the mode of action of RIPs in general and SNA-I in particular towards insects is not known. In Chapter 4 we attempted to get insight into the mode of action of the type 2 RIPs SNA-I. Exposure of insect midgut CF-203 cells to SNA-I showed that this lectin is able to induce cell death with the typical characters of apoptosis such as cell shrinkage, plasma membrane blebbing, nuclear condensation and DNA fragmentation. Exposure of SNA-I to the cells induced caspase-3 like protease activity, suggesting that SNA-I can induce the apoptotic pathway. Interestingly, the hololectin SNA-II revealed a very similar activity towards CF-203 cells. Both SNA-I and SNA-II induced caspase-dependent apoptosis pathway in the low nM range, leading to typical symptoms of cell death. Since SNA-II only has carbohydrate-binding domains, it was concluded that the induction of apoptosis in CF-203 cells by treatment with SNA-I and SNA-II is independent of the activity of the A-chain of SNA-I. In further extend of the study on the mode of action of SNA-I and SNA-II the endocytosis and uptake mechanisms involved in the internalization of SNA-I and SNA-II in CF-203 cells were evaluated in the presence or absence of different inhibitors in Chapter 5. Therefore cytotoxicity of SNA-I and SNA-II on cells was evaluated and the effect of different inhibitors was scored. Furthermore, the internalization of FITC-labeled SNA-I and SNA-II was quantified. Microscopic studies revealed that the uptake of both SNA-I and SNA-II was dose dependent. In addition, it was shown that both SNA-I and SNA-II were taken up in the CF-203 cells by a receptor-mediated endocytosis mechanism that is clathrin- and caveolae-dependent. Since similar results were obtained for both SNA-I and SNA-II these results confirmed that the A-chain of the type 2 RIP is not involved in the internalization process. Taking into account the results of previous chapters which clearly showed the entomotoxic properties of SNA-I and its effect on insect cells experiments were set up in Chapter 6 to investigate the effect of SNA-I at insect level. Therefore, A. pisum and S. exigua were fed a diet containing SNA-I. Feeding bioassays with artificial diet supplemented with purified SNA-I or SNA-II on aphids showed insecticidal activities. Also caterpillars fed on diet mixed with SNA-I showed reduction in larval mass. In this study it was shown that SNA-I also induced the apoptotic characteristics at the level of the organism (insect). Caspase-3 like activity and DNA fragmentation were observed in the gut tissues of beet armyworms and pea aphids. In conclusion, the data of this PhD project demonstrated that the elderberry lectins SNA-I and SNA-II show insecticidal activity, which makes them potential tools for crop protection against pest insects

Expression of Sambucus nigra agglutinin (SNA-I′) from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species

Tobacco plants (Nicotiana tabacum cv Samsun NN) have been transformed with the gene encoding the type-2 ribosome-inactivating protein (RIP) SNA-I' from elderberry (Sambucus nigra) under the control of the Cauliflower Mosaic Virus 35S promoter. Previous research confirmed that these plants synthesize, correctly process and assemble a fully active RIP. Variability in protein expression was observed within the transgenic lines. The effects of the type-2 RIP SNA-I' delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae) and Lepidoptera, the beet armyworm (Spodoptera exigua). In the experiment with aphids, significant effects were observed on the life parameters, such as survival, intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring, whereas with caterpillars significant reduction in fresh weight as well as retardation in development were observed. In addition, significant increases in mortality were noted for insects fed on the transgenic lines as compared to wild type plants. This information provides further support for RIPs having a role in plant resistance to insect pest species

Insecticidal activity of the essential oils from yarrow (Achillea wilhelmsii L.) and sweet asafetida (Ferula assa-foetida L.) against Aphis gossypii Glover. (Hemiptera: Aphididae) under controlled laboratory conditions

The essential oils of two medicinal plants from yarrow (Achillea wilhelmsii L.) and sweet asafetida (Ferula assa-foetida L.) were studied for fumigant toxicity, contact toxicity and repellent activity against two-day old nymphs of Aphis gossypii Glover. The mortality increased with increase of concentration and exposure time from 12 to 24 h in fumigant toxicity and from 24 to 48 h in contact toxicity. After 12 h, the LC50 values in fumigant toxicity of A. wilhelmsii and F. assa-foetida essential oils were estimated to be 23.4 and 16.8 mu l l(- 1) air respectively, and the toxicity was increased after 24 h to LC50 of 16.16 and 15.10 mu l l(- 1) air. LC50 values in contact toxicity of yarrow and sweet asafetida essential oils were estimated 6620and 2040 mu l l(- 1) after 24 h and 2478 and 882 mu l l(- 1) after 48 h, respectively. Furthermore, repellent activity was observed for all essential oils at the highest concentration (10 mu l ml(- 1)). The structure of the essential oil was analysed by (GC/MS). The highest components were (E)-1-propenyl sec-butyl disulfide (43.16%) and (Z)-1-propenyl sec-butyl disulfide (27.45) in sweet asafetida; camphore (29.03%) and 1,8-Cineole (12.86%) in Yarrow, which are compounds that possess insecticidal activity against various insect species. Results from this research could be a basic and essential step for application of present plant materials as a potential substitution of chemical insecticides for control of A. gossypii

Lethal and sublethal effects of spirotetramat and abamectin on predatory beetles (Menochilus sexmaculatus) via prey (Agonoscena pistaciae) exposure, important for integrated pest management in pistachio orchards

Menochilus sexmaculatus Fabricius (Coleoptera: Coccinellidae) is an important biological control agent in pistachio orchards, especially against Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), which is the most damaging pest of pistachio. In this project we exposed M. sexmaculatus adults to two important commonly-used insecticides through feeding on treated prey (A. pistaciae) to evaluate the side-effects on this predator. We tested spirotetramat, which belongs to the keto-enol group inhibiting lipid biosynthesis in insects, at 2/1, 1/1 and 1/2 of the maximum field recommended concentration (MFRC), and abamectin, which is a mixture of avermectins and a natural fermentation product of the bacterium Streptomyces avermitilis, at 1/1, 1/2, 1/4, 1/8 and 1/16 of its MFRC. Spirotetramat did not affect adult survival of M. sexmaculatus at all three concentrations when ingested via treated prey, while in marked contrast abamectin caused 100% adult mortality of M. sexmaculatus when ingested via treated prey at 1/1, 1/2, 1/4 and 1/8 of the MFRC. At sublethal levels, spirotetramat reduced total and daily fecundity of M. sexmaculatus at all three concentrations tested, but did not affect egg hatching at 1/1 and 1/2 of the MFRC. Moreover, prey consumption was decreased when beetles were exposed to the prey treated with spirotetramat at 1/1 and 2/1 of the MFRC concentrations. With abamectin, even at 1/16 of the MFRC, total fecundity, daily fecundity and prey consumption of M. sexmaculatus adults were significantly affected. In conclusion, no acute toxicity was observed on M. sexmaculatus by ingestion of prey treated with spirotetramat, although reproduction parameters and prey consumption were affected at MFRC and lower concentrations. In marked contrast, abamectin was notably very harmful at its MFRC and also at lower concentrations. This research highlighted the importance of toxicity risk assessments, including lethal and sublethal effects, to obtain a more accurate estimation of the compatibility of insecticides in current integrated pest management (IPM) programs