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Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 85 (2004) 182–187 www.elsevier.com/locate/yjipa
Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies Carmen Sara Hern andez, Ana Rodrigo, and Juan Ferre* Departament de Genetica, Universitat de Valencia, 46100 Burjassot (Valencia), Spain Received 1 March 2004; accepted 8 March 2004
Abstract Binding assays with brush border membrane vesicles (BBMV) from insect midguts are commonly used in the study of the interactions between Bacillus thuringiensis Cry toxins and their receptors. Collaboration between laboratories often require that frozen insect samples are sent in dry ice. Because of customs restrictions and delays, sample thawing is always a risk and often the biological material becomes ruined during shipping. We have tested lyophilization as an alternative method for preserving insect midguts for binding studies with B. thuringiensis Cry toxins. For this purpose, BBMV were prepared from both frozen and lyophilized midguts from three lepidopteran species: Spodoptera exigua, Manduca sexta, and Helicoverpa armigera. Higher membrane protein recovery was always obtained from lyophilized midguts compared to frozen midguts, and similar membrane marker enzyme activities were found in BBMV from either treatment. Comparable equilibrium dissociation constants and binding site concentrations, calculated from binding experiments with labeled 125 I-Cry1Ab toxin, were found using BBMV from either method. In the light of these results, lyophilization is a good preserving method of lepidopteran midguts to study binding of B. thuringiensis Cry toxins. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Tissue preservation; Cry toxins; BBMV; Membrane receptors
1. Introduction Bacillus thuringiensis (Bt) is a bacterium widely used for the biological control of insect pests of the order Lepidoptera, Diptera, and Coleoptera. Its activity is due to parasporal crystalline inclusions containing one or more insecticidal proteins (d-endotoxins or Cry proteins) that are toxic by ingestion. These proteins are solubilized in the larval midgut and processed by proteases that activate them to their toxic forms. The activated toxins bind to specific receptors in the brush border membrane of epithelial cells in the insect midgut. The insertion of these toxins into the membrane gives rise to the formation of pores which cause an osmotic imbalance, leading to cell lysis and the insect death (Rajamohan et al., 1998; Schnepf et al., 1998).
* Corresponding author. Fax: +34-96-354-3029. E-mail address: [email protected] (J. Ferre´).
0022-2011/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.03.002
Even if each step in the mode of action (solubilization, proteolytic processing, receptor binding, and membrane insertion) may be decisive for toxicity, the specificity of Cry toxins is often determined by their interaction with the high-affinity receptors present in the insect gut (Van Rie et al., 1989, 1990). Different Cry toxins can share the same receptor or they can bind to different receptors in the brush border membrane of the insect. In lepidopterans, aminopeptidases and cadherinlike proteins have been proposed as Cry toxin receptors (Hara et al., 2003; Whalon and Wingerd, 2003). Alteration of binding to specific receptors in the midgut is the most common and best characterized mechanism of resistance to Cry toxins. Insect strains with resistance to more than one Cry toxin have been described to have altered a common receptor for such toxins (Ferre and Van Rie, 2002). Therefore, knowledge of specific toxin–receptor interactions is a key element to design specific strategies to prevent cross-resistance. The usual procedure for the study of toxin–receptor interactions requires the preparation of brush border
C.S. Hernandez et al. / Journal of Invertebrate Pathology 85 (2004) 182–187
membrane vesicles (BBMV) from either fresh or frozen larvae midguts (Wolfersberger et al., 1987). Sending live samples of insect pests among laboratories around the world involves a serious environmental risk and, for binding studies, this type of biological material is typically shipped frozen. In a previous study, brush border membrane fractions, maintaining functional activity of Bacillus sphaericus binary toxin receptors, were prepared from whole mosquito larvae preserved by drying (Poopathi et al., 2002). In the present study we have tested lyophilization as a way to preserve midguts for the preparation of BBMV maintaining the B. thuringiensis Cry toxin receptors unaffected. Dissected midguts from larvae of three lepidopteran species (Spodoptera exigua, Manduca sexta, and Helicoverpa armigera) were either lyophilized or stored at )80 °C. BBMV prepared from both types of samples were compared in terms of protein yield, membrane marker enzymatic activities, and binding assays with labeled Cry1Ab to check whether lyophilized midguts can be used as reliable material for binding studies with B. thuringiensis Cry toxins.
2. Materials and methods 2.1. Tissue handling and BBMV preparation Approximately 3–5 g of midguts were dissected from last instar larvae of S. exigua, M. sexta, and H. armigera, washed in ice-cold MET buffer (250 mM mannitol, 17 mM Tris–HCl, and 5 mM EGTA; pH 7.5), and frozen in liquid nitrogen. For each insect species, dissected midguts were divided in two equal portions. One portion was stored at )80 °C as regularly, whereas the other portion was lyophilized overnight at 6 )40 °C and 6 1 mbar of pressure, and stored at 4 °C for at least a week to simulate an habitual refrigerated shipment. BBMV were prepared by the differential magnesium precipitation method described by Wolfersberger et al. (1987) from midguts preserved by the two treatments, and then frozen in liquid nitrogen and kept at )80 °C until used. The protein concentration in the BBMV preparations was determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as standard. 2.2. Enzymatic assays Alkaline phosphatase and leucine aminopeptidase were used as membrane enzymatic markers for the BBMV preparations. Alkaline phosphatase assay was done according to the manufacturerÕs protocol (Biosystems, Barcelona, Spain) using 1 lg of BBMV protein and 4-nitrophenilphosphate as substrate. Activity was calculated in time intervals during which product for-
183
mation increased linearly. Leucin aminopeptidase activity was measured in 200 ng of BBMV protein using L leucil-b-naphthylamide as substrate, following a Sigma procedure (No. 251, Sigma Diagnostics, St. Louis, MO) with slight modifications: reaction volumes were 30% reduced, incubation time was adjusted to 10 min, and color development was determined after 10 min. 2.3. Cry1Ab toxin labeling Cry1Ab toxin was prepared from the recombinant B. thuringiensis strain EG7077 (Ecogen), activated and chromatographically purified as previously described (Sayyed et al., 2000). Toxin (20 lg) was labeled with 0.3 mCi of Na125 I (Amersham Little Chalfont, UK) using the chloramine T method (Van Rie et al., 1989). 2.4. Binding assays Previously to use, BBMV were centrifuged for 10 min at 16,000g and resuspended in binding buffer (8 mM Na2 HPO4 , 2 mM KH2 PO4 , 150 mM NaCl; pH 7.4; and 0.1% bovine serum albumin). For qualitative binding experiments, increasing amounts of BBMV were incubated with 1 nM 125 I-Cry1Ab in a final volume of 0.1 ml of binding buffer for 1 h at room temperature. An excess of unlabeled toxin was used to determine nonspecific binding. After incubation, samples were centrifuged at 16,000g for 10 min, and the pellets were washed twice with 0.5 ml of cold binding buffer. Radioactivity retained in the pellet was measured in a model 1282 Compugamma CS gamma counter (LKB Pharmacia). Homologous competition experiments were done by incubating 4 lg of BBMV with 1.3 nM 125 I-Cry1Ab for 1 h at room temperature. Increasing amounts of unlabeled Cry1Ab toxin were used to compete binding. Reactions were stopped by centrifugation at 16,000g for 10 min. Pellets were washed and radioactivity measured as described above. Dissociation constants and concentration of receptors were calculated using the LIGAND program (Munson and Rodbard, 1980).
3. Results 3.1. Yield and quality of BBMV preparations Starting with approximately the same amount of midgut tissue, protein yield was higher in BBMV preparations from lyophilized midguts than from frozen midguts for the three lepidopteran species (Table 1). The BBMV protein yield from S. exigua, M. sexta, and H. armigera was 25, 95, and 37% greater, respectively, than the yield obtained from frozen midguts. To assess the quality of the BBMV prepared from lyophilized midguts, the specific activity of two brush
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C.S. Hernandez et al. / Journal of Invertebrate Pathology 85 (2004) 182–187
Table 1 Protein yield and enzymatic activities from BBMV preparations obtained from frozen and lyophilized midguts Species
S. exigua M. sexta H. armigera
Yielda
Enzymatic activitiesb Alkaline phosphatase
Leucine aminopeptidase
Freezing
Lyophilization
Freezing
Lyophilization
Freezing
Lyophilization
0.83 1.24 0.81
1.04 2.42 1.11
1.0 0.2 3.7 0.6 4.5 1.2
1.0 0.2 3.7 0.5 5.0 0.8
91 32 5 32 6
19 3 24 3 28 1
a
Yield expressed as mg BBMV protein/g midgut. Means SD. Specific activity is expressed as lmol/min per mg protein. For each insect species, specific activities were calculated from at least two replicates. b
border membrane marker enzymes was compared with that of BBMV prepared from frozen midguts (Table 1). In the three insect species, no significant differences (P
values >0.05) were obtained with alkaline phosphatase specific activities between BBMV prepared from frozen and lyophilized midguts. Similarly, leucine
Fig. 1. Binding of 125 I-Cry1Ab as a function of BBMV concentration from S. exigua (A and B), M. sexta (C and D), and H. armigera (E and F). Each data point is a mean from duplicate samples. Data are from BBMV prepared from frozen midguts (A, C, and E) and lyophilized midguts (B, D, and F). Symbols: (j), total binding; (), nonspecific binding.
C.S. Hernandez et al. / Journal of Invertebrate Pathology 85 (2004) 182–187
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aminopeptidase activity was not significantly different (P values >0.05) in assays with BBMV from both treatments for M. sexta and H. armigera. In S. exigua, this activity was significantly higher (P value <0.05) in BBMV from lyophilized midguts than in BBMV from frozen midguts. 3.2. Binding of labeled Cry1Ab to BBMV Qualitative binding experiments were performed to determine if 125 I-Cry1Ab bound specifically to BBMV from lyophilized midguts and to estimate a BBMV concentration suitable for competition binding experiments. Increasing concentrations of BBMV from S. exigua, M. sexta, and H. armigera were incubated with a predetermined quantity of labeled toxin. Specific binding of Cry1Ab to BBMV from the two treatments was found in all species (Fig. 1). According to the binding curves, protein BBMV concentration of 40 lg/ml was chosen for subsequent competition assays for all lepidopteran species. Homologous competition binding assays were done using increasing concentrations of unlabeled Cry1Ab as competitor. Toxin competition was observed in experiments with BBMV from lyophilized midguts of S. exigua, M. sexta, and H. armigera (Fig. 2). From these assays, quantitative binding parameters were obtained and compared with those obtained with BBMV from frozen midguts. No significant differences were found in the equilibrium dissociation constant (Kd ) and the binding site concentration (Rt ) (P values >0.05) for each insect (Table 2).
4. Discussion Binding assays are important to establish models that relate Cry toxins with one or more membrane receptors. The knowledge of the toxin–receptor interactions in a given insect pest is crucial for the design of transgenic plants expressing more than one insecticidal protein genes. Resistance development to a given toxin may also cause cross-resistance to other toxins if they recognize the same receptor, thus the recommended strategy to prevent cross-resistance entails to avoid the use of combinations of toxins that share a common receptor (Ferre and Van Rie, 2002). The information on toxin– Table 2 Binding parameters from competition experiments with Species
S. exigua M. sexta H. armigera a
125
Fig. 2. Binding of 125 I-Cry1Ab to BBMV from S. exigua (A), M. sexta (B), and H. armigera (C) at different concentrations of unlabeled Cry1Ab competitor. Each data point represent the mean value of at least two independient experiments. Data obtained from BBMV prepared from frozen midguts (, broken line) and lyophilized midguts (j, solid line).
receptor interactions is also useful when searching for new B. thuringiensis strains to replace former ones for which insects have already started to evolve resistance. Insect pests to be analyzed for toxin–receptor interactions are not always maintained at the laboratories where binding assays are carried out. Likewise, insect
I-Cry1Ab and BBMV from frozen and lyophilized midguts
Kd (nM)a
Rt (pmol/mg)a
Freezing
Lyophilization
Freezing
Lyophilization
2.7 0.6 1.9 0.3 1.3 0.3
2.2 0.5 1.8 0.4 1.5 0.4
9.8 1.7 11.8 1.9 12.2 2.0
7.6 1.4 10.3 1.6 14.3 2.2
Means SE. For each insect species, Kd and Rt were calculated from at least two replicates.
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C.S. Hernandez et al. / Journal of Invertebrate Pathology 85 (2004) 182–187
resistant colonies are frequently far away from laboratories that would analyze their resistance mechanism by toxin–receptor assays. Shipping insect pests between laboratories is common practice even though it is not free of numerous difficulties. To avoid the environmental hazard that shipping live insect pests may pose, larvae or dissected guts are shipped frozen in dry ice. This practice increases costs and many courier companies refuse handling this type of samples. Moreover, quite often some packets become stuck at customs waiting for approval before being cleared, which causes a waste of time and material, sometimes without possibility of replacement. To avoid the inconveniences of the above method, a preserving method other than deep freezing is desirable to facilitate the exchange of insect samples between laboratories. We have tested lyophilization of insect midguts as a preserving alternative to ultrafreezing for the shipping of samples intended for preparation of BBMV with full binding capacity to Cry toxins. In the three insect species tested, we have found that the protein yield of BBMV preparation was higher from lyophilized midguts than from frozen midguts. Lyophilization, besides drying the midguts, reduces them to a fine powder which resuspends easily into a homogeneous mixture after rehydration. We have observed that rehydrated and homogenized powder suspensions had a more uniform appearance than suspensions from homogenized thawed midguts. Most likely, the homogenization degree is crucial in the final protein yield of BBMV preparations, as deduced by comparing two studies dealing with BBMV preparation from whole larvae, in which inclusion of steps in the protocol to eliminate hard debris before homogenization seemed to render a higher yield of BBMV protein (Escriche et al., 1995; MacIntosh et al., 1994). Two brush border membrane marker enzymes were used to monitor the quality of BBMV. Both alkaline phosphatase and leucine aminopeptidase showed similar or even higher enzymatic activities in BBMV preparations from lyophilized midguts with regard to frozen midguts. This result indicates that these membrane proteins remain functional, keeping their activity after lyophilization. To test if lyophilization affected Cry receptors, binding assays with BBMV from the three lepidopteran species were performed. B. thuringiensis Cry1Ab toxin was selected as ligand for these experiments. This toxin is commonly found in many insecticidal formulations of B. thuringiensis and Cry1Ab binding characteristics have been determined in the selected insect species (Chen et al., 1995; Escriche et al., 1997; Estela et al., 2004). Assays with BBMV from lyophilized midguts showed specific binding of Cry1Ab in all species tested. To verify whether the binding capacity of Cry1Ab receptors in BBMV from lyophilized midguts was comparable to
that from frozen midguts, equilibrium binding parameters were calculated. Comparison by t test indicated no significant differences in Kd and Rt values between samples from the two treatments, corroborating that lyophilization preserved the binding capacity of Cry receptors as efficiently as freezing. Our results with B. thuringiensis Cry1Ab and those from a previous study with the B. sphaericus Bin2 toxin and dried whole mosquito larvae (Poopathi et al., 2002) show that different insect midgut membrane receptors preserve their ability to bind to their respective toxins after lyophilization. Furthermore, enzyme markers from BBMV were also preserved with this treatment in both studies. It is likely that other midgut membrane receptors and enzymes may also remain unaltered after midgut drying. Since frozen midguts have been shown to be as good as fresh ones for the preparation of BBMV (Wolfersberger et al., 1987), now it seems reasonable to conclude that BBMV from lyophilized midguts are as reliable as those obtained from fresh midguts. With lyophilization of midguts as a preserving method, many shipping difficulties could be avoided and a greater number of samples could arrive unspoiled to their destinations.
Acknowledgments We thank M.S. Ibiza for toxin labeling. This work was supported by the Spanish Ministry of Science and Technology (Project No. AGL2003-09282-C03-01).
References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chen, X.J., Curtiss, A., Alcantara, E., Dean, D.H., 1995. Mutations in domain I of Bacillus thuringiensis d-endotoxin CryIAb reduce the irreversible binding of toxin to Manduca sexta brush border membrane vesicles. J. Biol. Chem. 270, 6412–6419. Escriche, B., Ferre, J., Silva, F.J., 1997. Occurrence of a common binding site in Mamestra brassicae, Phthorimaea operculella, and Spodoptera exigua for the insecticidal crystal proteins CryIA from Bacillus thuringiensis. Insect Biochem. Mol. Biol. 27, 651–656. Escriche, B., Silva, F.J., Ferre, J., 1995. Testing suitability of brush border membrane vesicles prepared from whole larvae from small insects for binding studies with Bacillus thuringiensis CryIA(b) crystal protein. J. Invertebr. Pathol. 65, 318–320. Estela, A., Escriche, B., Ferre, J., 2004. Interaction of Bacillus thuringiensis with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae). Appl. Environ. Microbiol. 70, 1378–1384. Ferre, J., Van Rie, J., 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 47, 501–533. Hara, H., Atsumi, S., Yaoi, K., Nakanishi, K., Higurashi, S., Miura, N., Tabunoki, H., Sato, R., 2003. A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae. FEBS Lett. 538, 29–34.
C.S. Hernandez et al. / Journal of Invertebrate Pathology 85 (2004) 182–187 MacIntosh, S.C., Lidster, B.D., Kirkham, C.L., 1994. Isolation of brush border membrane vesicles from whole diamondback moth (Lepidoptera: Plutellidae) larvae. J. Invert. Pathol. 63, 97–98. Munson, P., Rodbard, D., 1980. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107, 220–239. Poopathi, S., Charles, J.F., Nielsen-LeRoux, C., 2002. Alternative method for preservation of mosquito larvae to study binding mechanisms of Bacillus sphaericus binary toxin. J. Invert. Pathol. 79, 132–134. Rajamohan, F., Lee, M.K., Dean, D.H., 1998. Bacillus thuringiensis insecticidal proteins: molecular mode of action. Prog. Nucleic Acid Res. Mol. Biol. 60, 1–27. Sayyed, A.H., Haward, R., Herrero, S., Ferre, J., Wright, D.J., 2000. Genetic and biochemical approach for characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in a field population of the diamondback moth, Plutella xylostella. Appl. Environ. Microbiol. 66, 1509–1516.
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Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Van Rie, J., Jansens, S., Hofte, H., Degheele, D., Van Mellaert, H., 1989. Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur. J. Biochem. 186, 239–247. Van Rie, J., Jansens, S., Hofte, H., Degheele, D., Van Mellaert, H., 1990. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins. Appl. Environ. Microbiol. 56, 1378–1385. Whalon, M.E., Wingerd, B.A., 2003. Bt: mode of action and use. Arch. Insect Biochem. Physiol 54, 200–211. Wolfersberger, M.G., Luethy, P., Maurer, P., Parenti, P., Sacchi, V.F., Giordana, B., Hanozet, G.M., 1987. Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol. 86A, 301–308.
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 85 (2004) 182–187 www.elsevier.com/locate/yjipa
Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies Carmen Sara Hern andez, Ana Rodrigo, and Juan Ferre* Departament de Genetica, Universitat de Valencia, 46100 Burjassot (Valencia), Spain Received 1 March 2004; accepted 8 March 2004
Abstract Binding assays with brush border membrane vesicles (BBMV) from insect midguts are commonly used in the study of the interactions between Bacillus thuringiensis Cry toxins and their receptors. Collaboration between laboratories often require that frozen insect samples are sent in dry ice. Because of customs restrictions and delays, sample thawing is always a risk and often the biological material becomes ruined during shipping. We have tested lyophilization as an alternative method for preserving insect midguts for binding studies with B. thuringiensis Cry toxins. For this purpose, BBMV were prepared from both frozen and lyophilized midguts from three lepidopteran species: Spodoptera exigua, Manduca sexta, and Helicoverpa armigera. Higher membrane protein recovery was always obtained from lyophilized midguts compared to frozen midguts, and similar membrane marker enzyme activities were found in BBMV from either treatment. Comparable equilibrium dissociation constants and binding site concentrations, calculated from binding experiments with labeled 125 I-Cry1Ab toxin, were found using BBMV from either method. In the light of these results, lyophilization is a good preserving method of lepidopteran midguts to study binding of B. thuringiensis Cry toxins. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Tissue preservation; Cry toxins; BBMV; Membrane receptors
1. Introduction Bacillus thuringiensis (Bt) is a bacterium widely used for the biological control of insect pests of the order Lepidoptera, Diptera, and Coleoptera. Its activity is due to parasporal crystalline inclusions containing one or more insecticidal proteins (d-endotoxins or Cry proteins) that are toxic by ingestion. These proteins are solubilized in the larval midgut and processed by proteases that activate them to their toxic forms. The activated toxins bind to specific receptors in the brush border membrane of epithelial cells in the insect midgut. The insertion of these toxins into the membrane gives rise to the formation of pores which cause an osmotic imbalance, leading to cell lysis and the insect death (Rajamohan et al., 1998; Schnepf et al., 1998).
* Corresponding author. Fax: +34-96-354-3029. E-mail address: [email protected] (J. Ferre´).
0022-2011/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.03.002
Even if each step in the mode of action (solubilization, proteolytic processing, receptor binding, and membrane insertion) may be decisive for toxicity, the specificity of Cry toxins is often determined by their interaction with the high-affinity receptors present in the insect gut (Van Rie et al., 1989, 1990). Different Cry toxins can share the same receptor or they can bind to different receptors in the brush border membrane of the insect. In lepidopterans, aminopeptidases and cadherinlike proteins have been proposed as Cry toxin receptors (Hara et al., 2003; Whalon and Wingerd, 2003). Alteration of binding to specific receptors in the midgut is the most common and best characterized mechanism of resistance to Cry toxins. Insect strains with resistance to more than one Cry toxin have been described to have altered a common receptor for such toxins (Ferre and Van Rie, 2002). Therefore, knowledge of specific toxin–receptor interactions is a key element to design specific strategies to prevent cross-resistance. The usual procedure for the study of toxin–receptor interactions requires the preparation of brush border
C.S. Hernandez et al. / Journal of Invertebrate Pathology 85 (2004) 182–187
membrane vesicles (BBMV) from either fresh or frozen larvae midguts (Wolfersberger et al., 1987). Sending live samples of insect pests among laboratories around the world involves a serious environmental risk and, for binding studies, this type of biological material is typically shipped frozen. In a previous study, brush border membrane fractions, maintaining functional activity of Bacillus sphaericus binary toxin receptors, were prepared from whole mosquito larvae preserved by drying (Poopathi et al., 2002). In the present study we have tested lyophilization as a way to preserve midguts for the preparation of BBMV maintaining the B. thuringiensis Cry toxin receptors unaffected. Dissected midguts from larvae of three lepidopteran species (Spodoptera exigua, Manduca sexta, and Helicoverpa armigera) were either lyophilized or stored at )80 °C. BBMV prepared from both types of samples were compared in terms of protein yield, membrane marker enzymatic activities, and binding assays with labeled Cry1Ab to check whether lyophilized midguts can be used as reliable material for binding studies with B. thuringiensis Cry toxins.
2. Materials and methods 2.1. Tissue handling and BBMV preparation Approximately 3–5 g of midguts were dissected from last instar larvae of S. exigua, M. sexta, and H. armigera, washed in ice-cold MET buffer (250 mM mannitol, 17 mM Tris–HCl, and 5 mM EGTA; pH 7.5), and frozen in liquid nitrogen. For each insect species, dissected midguts were divided in two equal portions. One portion was stored at )80 °C as regularly, whereas the other portion was lyophilized overnight at 6 )40 °C and 6 1 mbar of pressure, and stored at 4 °C for at least a week to simulate an habitual refrigerated shipment. BBMV were prepared by the differential magnesium precipitation method described by Wolfersberger et al. (1987) from midguts preserved by the two treatments, and then frozen in liquid nitrogen and kept at )80 °C until used. The protein concentration in the BBMV preparations was determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as standard. 2.2. Enzymatic assays Alkaline phosphatase and leucine aminopeptidase were used as membrane enzymatic markers for the BBMV preparations. Alkaline phosphatase assay was done according to the manufacturerÕs protocol (Biosystems, Barcelona, Spain) using 1 lg of BBMV protein and 4-nitrophenilphosphate as substrate. Activity was calculated in time intervals during which product for-
183
mation increased linearly. Leucin aminopeptidase activity was measured in 200 ng of BBMV protein using L leucil-b-naphthylamide as substrate, following a Sigma procedure (No. 251, Sigma Diagnostics, St. Louis, MO) with slight modifications: reaction volumes were 30% reduced, incubation time was adjusted to 10 min, and color development was determined after 10 min. 2.3. Cry1Ab toxin labeling Cry1Ab toxin was prepared from the recombinant B. thuringiensis strain EG7077 (Ecogen), activated and chromatographically purified as previously described (Sayyed et al., 2000). Toxin (20 lg) was labeled with 0.3 mCi of Na125 I (Amersham Little Chalfont, UK) using the chloramine T method (Van Rie et al., 1989). 2.4. Binding assays Previously to use, BBMV were centrifuged for 10 min at 16,000g and resuspended in binding buffer (8 mM Na2 HPO4 , 2 mM KH2 PO4 , 150 mM NaCl; pH 7.4; and 0.1% bovine serum albumin). For qualitative binding experiments, increasing amounts of BBMV were incubated with 1 nM 125 I-Cry1Ab in a final volume of 0.1 ml of binding buffer for 1 h at room temperature. An excess of unlabeled toxin was used to determine nonspecific binding. After incubation, samples were centrifuged at 16,000g for 10 min, and the pellets were washed twice with 0.5 ml of cold binding buffer. Radioactivity retained in the pellet was measured in a model 1282 Compugamma CS gamma counter (LKB Pharmacia). Homologous competition experiments were done by incubating 4 lg of BBMV with 1.3 nM 125 I-Cry1Ab for 1 h at room temperature. Increasing amounts of unlabeled Cry1Ab toxin were used to compete binding. Reactions were stopped by centrifugation at 16,000g for 10 min. Pellets were washed and radioactivity measured as described above. Dissociation constants and concentration of receptors were calculated using the LIGAND program (Munson and Rodbard, 1980).
3. Results 3.1. Yield and quality of BBMV preparations Starting with approximately the same amount of midgut tissue, protein yield was higher in BBMV preparations from lyophilized midguts than from frozen midguts for the three lepidopteran species (Table 1). The BBMV protein yield from S. exigua, M. sexta, and H. armigera was 25, 95, and 37% greater, respectively, than the yield obtained from frozen midguts. To assess the quality of the BBMV prepared from lyophilized midguts, the specific activity of two brush
184
C.S. Hernandez et al. / Journal of Invertebrate Pathology 85 (2004) 182–187
Table 1 Protein yield and enzymatic activities from BBMV preparations obtained from frozen and lyophilized midguts Species
S. exigua M. sexta H. armigera
Yielda
Enzymatic activitiesb Alkaline phosphatase
Leucine aminopeptidase
Freezing
Lyophilization
Freezing
Lyophilization
Freezing
Lyophilization
0.83 1.24 0.81
1.04 2.42 1.11
1.0 0.2 3.7 0.6 4.5 1.2
1.0 0.2 3.7 0.5 5.0 0.8
91 32 5 32 6
19 3 24 3 28 1
a
Yield expressed as mg BBMV protein/g midgut. Means SD. Specific activity is expressed as lmol/min per mg protein. For each insect species, specific activities were calculated from at least two replicates. b
border membrane marker enzymes was compared with that of BBMV prepared from frozen midguts (Table 1). In the three insect species, no significant differences (P
values >0.05) were obtained with alkaline phosphatase specific activities between BBMV prepared from frozen and lyophilized midguts. Similarly, leucine
Fig. 1. Binding of 125 I-Cry1Ab as a function of BBMV concentration from S. exigua (A and B), M. sexta (C and D), and H. armigera (E and F). Each data point is a mean from duplicate samples. Data are from BBMV prepared from frozen midguts (A, C, and E) and lyophilized midguts (B, D, and F). Symbols: (j), total binding; (), nonspecific binding.
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aminopeptidase activity was not significantly different (P values >0.05) in assays with BBMV from both treatments for M. sexta and H. armigera. In S. exigua, this activity was significantly higher (P value <0.05) in BBMV from lyophilized midguts than in BBMV from frozen midguts. 3.2. Binding of labeled Cry1Ab to BBMV Qualitative binding experiments were performed to determine if 125 I-Cry1Ab bound specifically to BBMV from lyophilized midguts and to estimate a BBMV concentration suitable for competition binding experiments. Increasing concentrations of BBMV from S. exigua, M. sexta, and H. armigera were incubated with a predetermined quantity of labeled toxin. Specific binding of Cry1Ab to BBMV from the two treatments was found in all species (Fig. 1). According to the binding curves, protein BBMV concentration of 40 lg/ml was chosen for subsequent competition assays for all lepidopteran species. Homologous competition binding assays were done using increasing concentrations of unlabeled Cry1Ab as competitor. Toxin competition was observed in experiments with BBMV from lyophilized midguts of S. exigua, M. sexta, and H. armigera (Fig. 2). From these assays, quantitative binding parameters were obtained and compared with those obtained with BBMV from frozen midguts. No significant differences were found in the equilibrium dissociation constant (Kd ) and the binding site concentration (Rt ) (P values >0.05) for each insect (Table 2).
4. Discussion Binding assays are important to establish models that relate Cry toxins with one or more membrane receptors. The knowledge of the toxin–receptor interactions in a given insect pest is crucial for the design of transgenic plants expressing more than one insecticidal protein genes. Resistance development to a given toxin may also cause cross-resistance to other toxins if they recognize the same receptor, thus the recommended strategy to prevent cross-resistance entails to avoid the use of combinations of toxins that share a common receptor (Ferre and Van Rie, 2002). The information on toxin– Table 2 Binding parameters from competition experiments with Species
S. exigua M. sexta H. armigera a
125
Fig. 2. Binding of 125 I-Cry1Ab to BBMV from S. exigua (A), M. sexta (B), and H. armigera (C) at different concentrations of unlabeled Cry1Ab competitor. Each data point represent the mean value of at least two independient experiments. Data obtained from BBMV prepared from frozen midguts (, broken line) and lyophilized midguts (j, solid line).
receptor interactions is also useful when searching for new B. thuringiensis strains to replace former ones for which insects have already started to evolve resistance. Insect pests to be analyzed for toxin–receptor interactions are not always maintained at the laboratories where binding assays are carried out. Likewise, insect
I-Cry1Ab and BBMV from frozen and lyophilized midguts
Kd (nM)a
Rt (pmol/mg)a
Freezing
Lyophilization
Freezing
Lyophilization
2.7 0.6 1.9 0.3 1.3 0.3
2.2 0.5 1.8 0.4 1.5 0.4
9.8 1.7 11.8 1.9 12.2 2.0
7.6 1.4 10.3 1.6 14.3 2.2
Means SE. For each insect species, Kd and Rt were calculated from at least two replicates.
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resistant colonies are frequently far away from laboratories that would analyze their resistance mechanism by toxin–receptor assays. Shipping insect pests between laboratories is common practice even though it is not free of numerous difficulties. To avoid the environmental hazard that shipping live insect pests may pose, larvae or dissected guts are shipped frozen in dry ice. This practice increases costs and many courier companies refuse handling this type of samples. Moreover, quite often some packets become stuck at customs waiting for approval before being cleared, which causes a waste of time and material, sometimes without possibility of replacement. To avoid the inconveniences of the above method, a preserving method other than deep freezing is desirable to facilitate the exchange of insect samples between laboratories. We have tested lyophilization of insect midguts as a preserving alternative to ultrafreezing for the shipping of samples intended for preparation of BBMV with full binding capacity to Cry toxins. In the three insect species tested, we have found that the protein yield of BBMV preparation was higher from lyophilized midguts than from frozen midguts. Lyophilization, besides drying the midguts, reduces them to a fine powder which resuspends easily into a homogeneous mixture after rehydration. We have observed that rehydrated and homogenized powder suspensions had a more uniform appearance than suspensions from homogenized thawed midguts. Most likely, the homogenization degree is crucial in the final protein yield of BBMV preparations, as deduced by comparing two studies dealing with BBMV preparation from whole larvae, in which inclusion of steps in the protocol to eliminate hard debris before homogenization seemed to render a higher yield of BBMV protein (Escriche et al., 1995; MacIntosh et al., 1994). Two brush border membrane marker enzymes were used to monitor the quality of BBMV. Both alkaline phosphatase and leucine aminopeptidase showed similar or even higher enzymatic activities in BBMV preparations from lyophilized midguts with regard to frozen midguts. This result indicates that these membrane proteins remain functional, keeping their activity after lyophilization. To test if lyophilization affected Cry receptors, binding assays with BBMV from the three lepidopteran species were performed. B. thuringiensis Cry1Ab toxin was selected as ligand for these experiments. This toxin is commonly found in many insecticidal formulations of B. thuringiensis and Cry1Ab binding characteristics have been determined in the selected insect species (Chen et al., 1995; Escriche et al., 1997; Estela et al., 2004). Assays with BBMV from lyophilized midguts showed specific binding of Cry1Ab in all species tested. To verify whether the binding capacity of Cry1Ab receptors in BBMV from lyophilized midguts was comparable to
that from frozen midguts, equilibrium binding parameters were calculated. Comparison by t test indicated no significant differences in Kd and Rt values between samples from the two treatments, corroborating that lyophilization preserved the binding capacity of Cry receptors as efficiently as freezing. Our results with B. thuringiensis Cry1Ab and those from a previous study with the B. sphaericus Bin2 toxin and dried whole mosquito larvae (Poopathi et al., 2002) show that different insect midgut membrane receptors preserve their ability to bind to their respective toxins after lyophilization. Furthermore, enzyme markers from BBMV were also preserved with this treatment in both studies. It is likely that other midgut membrane receptors and enzymes may also remain unaltered after midgut drying. Since frozen midguts have been shown to be as good as fresh ones for the preparation of BBMV (Wolfersberger et al., 1987), now it seems reasonable to conclude that BBMV from lyophilized midguts are as reliable as those obtained from fresh midguts. With lyophilization of midguts as a preserving method, many shipping difficulties could be avoided and a greater number of samples could arrive unspoiled to their destinations.
Acknowledgments We thank M.S. Ibiza for toxin labeling. This work was supported by the Spanish Ministry of Science and Technology (Project No. AGL2003-09282-C03-01).
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