Adsorption of Insecticidal Crystal Protein Cry11Aa onto Nano-Mg (OH

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Adsorption of Insecticidal Crystal Protein Cry11Aa onto nanoMg(OH)2: Effects on Bioactivity and Anti-Ultraviolet Ability Xiaohong Pan, Zhangyan Xu, Lan Li, Enshi Shao, Saili Chen, Tengzhou Huang, Zhi Chen, Wenhua Rao, Tianpei Huang, Lingling Zhang, Songqing Wu, and Xiong Guan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03410 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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Journal of Agricultural and Food Chemistry

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Adsorption of Insecticidal Crystal Protein Cry11Aa onto

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nano-Mg(OH)2: Effects on Bioactivity and Anti-Ultraviolet Ability a,b

, Zhangyan Xu a, Lan Li a, Enshi Shao

a,b

, Saili Chen a, Tengzhou Huang a, Zhi

3

Xiaohong Pan

4

Chen a, Wenhua Rao a, Tianpei Huang a,b, Lingling Zhang a,b, Songqing Wu a,b, Xiong Guan a,b,*

5

a

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Biopesticide and Chemical Biology, Ministry of Education & College of Plant Protection &

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College of Resources and Environmental Sciences & College of Life Sciences & Forestry

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College, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P. R. China.

9

b

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State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops & Key Lab of

Fujian-Taiwan Joint Center for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, P. R.

China.

11 12

*

13

591-83789259.

CORRESPONDING

AUTHOR

E-mail:

[email protected];

14 15

ACS Paragon Plus Environment 1

Tel.

&

Fax:

(+086)


16

ABSTRACT

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The traditional Bacillus thuringiensis (Bt) formulation for field applications are not resistant

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to harsh environmental conditions. Hence, the active ingredients of the Bt bioinsecticides could

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degrade quickly and has low anti-ultraviolet ability in the field, which significantly limits its

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practical application. In the present study, we developed an efficient and stable delivery system

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for Bt Cry11Aa toxins. We coated Cry11Aa proteins with Mg(OH)2 nanoparticles (MHNPs), and

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then assessed the effects of MHNPs on bioactivity and anti-ultraviolet ability of the Cry11Aa

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proteins. Our results indicated that MHNPs, like “coating clothes”, could effectively protect the

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Cry protein and enhance the insecticidal bioactivity after UV radiation (the degradation rate was

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decreased from 64.29% to 16.67%). In addtion, MHNPs could improve the proteolysis of

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Cry11Aa in the midgut and aggravate the damage of the Cry protein to the gut epithelial cells,

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leading to increased insecticidal activity against Culex quinquefasciatus. Our results revealed

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that MHNPs, as an excellent nano-carrier, could substantially improve the insecticidal bioactivity

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and anti-ultraviolet ability of Cry11Aa.

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KEY WORDS: Cry11Aa; Nano-Mg(OH)2; Anti-ultraviolet; Bioactivity; Proteolysis

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1. INTRODUCTION

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As one of the most widely used bioinsecticides, Bacillus thuringiensis (Bt) produces

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insecticidal crystal proteins (Cry and Cyt) during the sporulation phase.1-3 Moreover, the Cry

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toxins are effective against lepidopteran, coleopteran, dipteran insect pests and nematodes.4-6

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Mosquitoes are responsible for many severe human diseases, such as malaria, dengue fever,

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yellow fever and so on.7 Bt subsp. israelensis (Bti) is highly active against disease-vector

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mosquitoes, like Aedes, Culex and Anopheles,4 and the efficacy of Bti is due to the presence of

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Cry4A, Cry4B, Cry10A, Cry11A, Cyt1A and Cyt2B proteins.8-10 However, there are also some

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difficulties in using Bti products, such as the degradation of active ingredients, low

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anti-ultraviolet ability and so on.11 Therefore, how to protect the effective component and

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increase the anti-ultraviolet capacity of Bti is of great importance from both economical and

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practical point of view. It has been reported that the Bti products can be adsorbed on clay

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particles, thus protecting their activity (such as UV radiation and microbial degradation) and

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enhancing the persistence of the product.12

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Nanoparticles (NPs) are widely used in industrial, medical, personal and military applications

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due to their small size (one or more dimensions in the order of 100 nm or less), high specific

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surface area, strong adsorption capacity, as well as unique optical and electrical properties.13, 14

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At present, several studies have focused on NPs application in pesticides, and their results have

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indicated that the nano-loading of NPs (such as TiO2, ZnO, SiO2 and CaCO3) can improve the

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utilization rates and other properties.15-20 However, such application in biopesticides also

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requires

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agro-biotechnological applications. As a low cost and nontoxic material, Mg(OH)2 NPs (MHNPs)

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have already been applied in the removal of acid, dye and heavy metals from wastewater, and

relatively

high

cost

and

direct

environmental


exposure,

hindering

the


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previous studies have confirmed their low cell toxicity.21, 22 Therefore, MHNPs are regarded as

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environmentally friendly nanomaterials.23-27 However, till now, only few studies have focused on

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the application of MHNPs in biopesticides.

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In this study, we aimed to develop an efficient and stable delivery system for Cry protein by

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loading MHNPs in biocontrol applications. Cry11Aa toxins was loaded on MHNPs carriers, the

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bioactivity and anti-ultraviolet radiation ability were investigated, the proteolysis of Cry11Aa by

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different treatments was carried out, and the midgut tissues of Culex quinquefasciatus were

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examined. The results indicated that MHNPs could effectively protect the Cry protein after UV

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radiation, enhance the insecticidal bioactivity, improve the proteolysis of Cry11Aa in the midgut

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and aggravate the damage of the Cry protein to the gut epithelial cells. Our findings offered

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valuable information for the practical application of MHNPs in biopesticides.

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2. MATERIALS AND METHODS

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2.1 Preparation and characterization of MHNPs and Cry11Aa toxin

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MHNPs were prepared by co-precipitation of magnesium chloride hexahydrate and sodium

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hydroxide in double distilled water (ddH2O) at room temperature. The resulting suspensions

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were washed with ddH2O for three times and then centrifuged at 10,000 g for 10 min. The pH

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value of the synthesized Mg(OH)2 slurry (1 g/L) was around 10.3. The morphology and size of

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the synthesized nano-Mg(OH)2 samples were characterized on a JSM-6700F scanning electron

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microscope (SEM) (JEOL Ltd., Japan) equipped with an Oxford-INCA energy-dispersive X-ray

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spectroscopy (EDS). Meanwhile, the size distribution of MHNPs was determined by a dynamic

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light scattering (DLS) instrument produced by Malvern Instruments Corporation, MHNPs were

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sonicated before DLS determination, and an average Z-Average value was obtained.


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The Bt strain was grown in 1/2 LB medium until 80% of the crystal was released. After

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centrifugation at 8,000 g for 10 min at 4 ◦C, the mixture of crystals, spores and debris was

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collected and washed with 1 M NaCl, followed by a wash with distilled water. The mixture of

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crystals, spores and debris was directly resuspended in a solubilization buffer (50 mM Na2CO3,

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pH 9.5) and then centrifuged at 13,000 g for 20 min to remove the insoluble debris. The

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supernatant was collected, its pH was adjusted to 4.5 with HAc, and then the supernatant kept at

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4 ◦C for at least 4 h. The pellet was collected by centrifugation at 10,000 g for 15 min, washed

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with distilled water for three times, and dissolved in 50 mM Na2CO3 (pH 10.5). The final

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product was Cry11Aa protein. Cry11Aa protein and the insoluble debris (from centrifugation at

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13,000 g for 20 min) were separately collected and analyzed by SDS-PAGE. All chemicals were

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analytical grade, and the treatment was repeated twice.

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2.2 The stable property analysis of MHNPs

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The pH tolerance experiment: Briefly, 1 mL MES (C6H13NO4S) medium (30 mM, pH=5.6,

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simulated natural environment) was added into MHNPs in 2-mL centrifuge tube at room

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temperature with gentle agitation, and the supernatant was collected every 12 h and replaced

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with fresh MES medium for seven times. The pH value was monitored, and the initial and final

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weights of MHNPs were measured.

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Thermal gravity analysis (TGA): The TGA of MHNPs was recorded by the simultaneous

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thermal analyzer (STA449C, Netzsch Co.) in argon atmosphere within the temperature range of

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27~1000 ◦C and with the heating rate of 10 ◦C/min.

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Ultraviolet radiation resistance capacity: MHNPs were placed at a straight distance of 30

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cm from the front surface of UV lamp and radiated for 18 h (4.5-h UV radiation per time, four

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times). After radiation, the sample was analyzed by X-ray diffraction (XRD) and Fourier



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transforming infrared spectrum (FT-IR) to determine the possible structural change of MHNPs.

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XRD patterns were identified using a PANalytical X’Pert PRO diffractometer with Cu Kα

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radiation (40 kV, 40 mA) in a continuous scanning mode. The 2θ scanning ranged from 5° to 85°

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in steps of 0.017° with a collection time of 20 s per step. The average crystallite size was

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determined from the peak broadening according to the Scherrer equation. FT-IR spectra were

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recorded by the Thermo Nicolet iS50 spectrometer within the range of 400~4,000 cm-1 by the

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KBr pellet method.

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2.3 Cry11Aa protein loading

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MHNPs (3.0 mg) were suspended in 300 µL double distilled water. An aliquot of Cry11Aa in

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Na2CO3 was added to the suspension and ultrasonicated at 4 ◦C for 30 min. The particles were

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collected by centrifugation. After loading, the supernatant and precipitate were extracted by

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centrifugation (10,000 g for 10 min), the supernatant was residual Cry11Aa protein, and the

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precipitate was Cry11Aa-loaded MHNPs (designated as Cry11Aa-MHNPs). The amount of

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Cry11Aa loaded onto MHNPs was calculated by subtracting the amount of residual Cry11Aa

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from the total amount of Cry11Aa added to the sample. The protein concentration was

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determined at a wavelength of 595 nm by Bradford method using bovine serum albumin as the

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standard.

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2.4 The anti-ultraviolet and bioactivity assays

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Briefly, the untreated Cry11Aa protein was set as the control, and both the Cry protein and

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Cry11Aa-MHNPs were placed at a straight distance of 30 cm from the front surface of UV lamp

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and radiated for 4.5 h. After radiation, all samples were analyzed by SDS-PAGE, and the

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concentrations of Cry protein before and after UV radiation were determined as described in

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Section 2.3.


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Meanwhile, the insecticidal activity of the prepared samples was examined against C.

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quinquefasciatus. The C. quinquefasciatus, previously provided by the Jiangsu Center for

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Disease Prevention and Control (Jiangsu Province, China), was reared in an environmentally

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controlled room (T=25±3◦C, RH=80%, L:D=14:10). Mosquitocidal bioassays of different

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samples were assayed against 30 third-instar larvae in 10 mL of dechlorinated water, and the

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samples included Cry11Aa protein, Cry11Aa-MHNPs, Cry11Aa protein-UV 4.5 h and

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Cry11Aa-MHNPs-UV 4.5 h. The initial concentration of Cry11Aa was 0.057 g/L, and the

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volume of protein was set as 50, 100, 150, 200, 250 and 300 µL. Each treatment was replicated

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for three times, and the mortality was recorded at 12 h and 36 h. Moreover, the mean 50% lethal

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concentration (LC50) and 95% confidence limits (CL) was estimated by SPSS analysis (version

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17.0) using statistical parameters.

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2.5 Extraction of C. quinquefasciatus gut proteases and in vitro proteolysis of Cry11Aa

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The extraction of the guts was conducted as previously described.28,

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Briefly, 200 C.

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quinquefasciatus larvae (third-instar) were excised by dissection, placed in 200 µL PBS buffer

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(pH 7.3) and stored at -80 ◦C. Gut tissues in individual tubes were homogenized and then

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centrifuged at 12,000 g for 20 min at 4 ◦C. The supernatants (containing the stomach and midgut

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fractions) were collected, placed into fresh tubes and marked as lumen fractions. In addition, the

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residual pellets were resuspended in 200 µL PBS buffer (pH 7.3), homogenized, labelled as

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membrane fractions and stored at -80 ◦C prior to further analysis.

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The obtained lumen and membrane fractions were incubated with Cry11Aa and

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Cry11Aa-MHNPs, respectively. The Cry11Aa was incubated at 37 ◦C for trypsin activation as

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the positive control. Certain volumes of lumen and membrane fractions were added to 40 µL

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sample (Cry11Aa, Cry11Aa-MHNPs) with a ratio of 10:1 (Cry11Aa: gut samples, w/w). The



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mixtures were incubated at 37 ◦C for different time intervals for the proteolysis of Cry protoxin.

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The reaction was terminated by addition of SDS-PAGE sample buffer (5×loading buffer) and

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immediately heated at 100 ◦C for 5 min. Finally, the proteolysis fragments of Cry11Aa were

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analyzed by SDS-PAGE.

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2.6 Sample preparation for transmission electron microscopy (TEM)

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The third-instar of C. quinquefasciatus larvae were either fed different samples as mentioned

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in Section 2.4 or water only (control). A total of 25 larvae from all replicates were dissected to

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isolate their midguts in 24 h, the peritrophic membranes and malpighian tubules were removed

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under a stereoscopic microscope, and then the isolated midgut tissues were rinsed with PBS for

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three times.30, 31 Subsequently, the midguts were immediately fixed with 2.5% glutaraldehyde in

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phosphate buffer (0.1 M, pH 7.0) for more than 4 h, then post-fixed with 1% OsO4 in phosphate

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buffer (0.1 M, pH 7.0) for 2 h and dehydrated in a graded series of ethanol (30%, 50%, 70%,

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80%, 90%, 95% and 100%) for 5 min at each step. Subsequently, the specimens were embedded

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in 618-resin and sectioned in LEICA EM UC7 ultratome. Then the sections were sequentially

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stained by uranyl acetate and alkaline lead citrate for 5 to 10 min. Transversally sectioned gut

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samples were observed by Hitachi Model H-7650 TEM.

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3. RESULTS AND DISCUSSION

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3.1 Characterization of MHNPs and Cry protein

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The morphology of MHNPs and Cry11Aa-MHNPs was characterized by SEM. We found that

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the synthesized MHNPs contained a lot of small flakes around tens of nanometers (Figure 1a).

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Meanwhile, the size distribution was determined in order to monitor the agglomeration state of

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MHNPs in water. Figure 1d illustrates that the size of MHNPs was well distributed, and the


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average particle size (Z-Average value) was 287.3 nm, indicating that the synthesized MHNPs

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maintained a relatively stable state at nanometer level and had a little agglomeration in water.

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As to Cry11Aa-MHNPs, the MHNPs were densely aggregated but still with structure of

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nano-flakes (Figure 1b), and the EDS analysis revealed that the compound contained C, N and S

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elements (insert picture of Figure 1b), confirming that the Cry11Aa protein was successfully

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loaded onto MHNPs. Additionally, the Cry protein was analyzed by SDS-PAGE, and the

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molecular mass of 68 kDa belonged to Cry11Aa (Figure 1c, Line 2). Moreover, the molecular

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mass of Cry11Aa protein barely changed after loaded onto MHNPs (Figure 1c, Line 1), and the

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protein band at 68 kDa changed shallow, this result also confirming that Cry11Aa was loaded

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onto MHNPs. Furthermore, the adsorption experiments indicated that the adsorption capacity of

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protein by MHNPs could achieve as high as 136 mg/g, suggesting that the MHNPs could be used

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as a good nano-carrier with high adsorption ability of Cry protein.



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Figure 1. Characterization of MHNPs and Cry11Aa loading on MHNPs. a) SEM images of

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MHNPs. b) SEM images of Cry11Aa-MHNPs, the insert picture was EDS analysis (note the C,

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N and S elements). c) SDS-PAGE analysis of Cry11Aa before and after loading with Cry11Aa

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(M: Prestained Markers, which was purchased from Thermo; Line 1: Cry11Aa-MHNPs; Line 2:

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Cry11Aa protein). d) size distribution of MHNPs, the dispersant was water.

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3.2 The stable property analysis of MHNPs in the delivery system

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It has been reported that Mg(OH)2 is an environmentally friendly material owing to its special

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physicochemical properties, such as large availability, high energy storage density, non-toxicity

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and safety.32 In order to evaluate the stable property of synthesized MHNPs in this delivery

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system, we assessed the pH tolerance, thermostability and ultraviolet radiation resistance

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capacity of MHNPs. Our data showed that pH had no obvious changes during the seven cycles


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(Figure 2a), and the weight of MHNPs was slightly decreased (Figure 2b, from 0.01 g to 0.0078

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g). The result indicated that MHNPs could tolerate the pH alteration in natural environment,

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suggesting that MHNPs could maintain the persistent period. Meanwhile, the pH value of

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Cry11Aa-MHNPs in MES medium (Figure 2a) was slight changed during the seven cycles (from

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10.22 to 8.93), and the desorption rate of Cry11Aa in the medium was only 31.5% at the seven

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cycles (data not shown). This result also implied that Cry11Aa could effectively load on MHNPs,

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and the MHNPs would be a stable nano-carrier.

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Figure 2c shows the TGA analysis of MHNPs. The result indicated that the weight loss of

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MHNPs at the temperature of 300 ◦C might be attributed to the H2O loss from the MHNPs.

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Moreover, the weight loss of MHNPs within the temperature range of 300~411 ◦C was about

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27.83%, which might be caused by the decomposition of Mg(OH)2 and subsequent

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transformation into MgO.33 Moreover, we assessed the ultraviolet radiation resistance capacity of

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MHNPs by the XRD and FT-IR. The result of XRD analysis (Figure 2d) indicated that the sizes

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of MHNPs at the (101) direction had slightly changed (from 12.0 ± 0.5 nm to 14.7 ± 0.8 nm)

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after 18 h UV radiation, but all diffraction peaks had no obvious shifted. Meanwhile, FT-IR

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patterns (Figure 2e) of MHNPs had no obvious changes before and after UV radiation. Our data

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suggested that MHNPs possessed super ultraviolet radiation resistance capacity.

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Additionally, previous studies have indicated that the pure Mg(OH)2 material has a relatively

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high heat storage capacity (690 kJ/kg),32 and CO2 can slightly react with MgO and Mg(OH)2

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during heat storage and release processes.34 Their results also suggested that MHNPs exhibit the

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good storage stability. Overall, MHNPs would be a perfect nano-carrier for efficient and stable

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delivery of insecticidal crystal proteins.



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Figure 2. The stable property analysis of MHNPs. a) The pH value of MHNPs treated with

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MES medium and H2O, the pH value of Cry11Aa-MHNPs in MES medium was also monitored;

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b) the weight of MHNPs before and after treatment of MES medium and H2O; c) TGA analysis

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of MHNPs; d) XRD patterns of MHNPs before and after UV radiation, the vertical lines

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represented the reference database for Mg(OH)2 (JCPDF044-1482). e) FT-IR patterns of MHNPs

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before and after UV radiation.

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3.3 The effects on anti-ultraviolet and insecticidal bioactivity

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Ultraviolet exposure caused decreased or lost insecticidal activity of Cry protein. Therefore,

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we evaluated the anti-ultraviolet activity of Cry11Aa before and after loading on MHNPs in

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order to verify the possible protective effects of MHNPs. The toxicities of Cry11Aa against

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third-instar C. quinquefasciatus larvae were compared after different treatments. Figure 3a shows

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that the Cry11Aa protein was partially degraded (Lane 3) under UV radiation compared with the

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control group (Lane 2), while protein bands of Cry11Aa-MHNPs were still clear after UV

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radiation (Lane 5). Meanwhile, we also compared the degree of protein degradation in different


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treatment groups measuring by Bradford method. Figure 3b exhibits that the protein

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concentration of Cry11Aa-MHNPs was decreased after 4.5-h UV radiation (from 0.36 to 0.30

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mg/mL, p

Adsorption of Insecticidal Crystal Protein Cry11Aa onto Nano-Mg (OH

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