Structural and mechanical properties of silk from different instars of

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Structural and mechanical properties of silk from different instars of Bombyx mori Zhangchuan Peng, Xi Yang, Chun Liu, Zhaoming Dong, Feng Wang, Xin Wang, Wenbo Hu, Xia Zhang, Ping Zhao, and Qingyou Xia Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01576 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Biomacromolecules

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Structural and mechanical properties of silk from

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different instars of Bombyx mori

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Zhangchuan Peng,†,§ Xi Yang,†,§ Chun Liu,†,‡,∫ Zhaoming Dong,†,‡,∫ Feng Wang,†,‡,∫ Xin Wang,†,‡,∫

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Wenbo Hu,†,‡,∫ Xia Zhang,† Ping Zhao,†,‡,∫ Qingyou Xia*,†,‡,∫

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†

Biological Science Research Center Southwest University, Chongqing 400716, China

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‡

Chongqing Key Laboratory of Sericultural Science Chongqing 400716, China

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∫

Chongqing Engineering and Technology Research Center for Novel Silk Materials, Chongqing

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400716, China

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ABSTRACT. Silkworm silk has excellent mechanical properties, biocompatibility, and promising

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applications in biomedical sector. Silkworms spin silk at the beginning and end of each of their

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five instar stages, as well as spinning mature silk after the fifth instar. We evaluated the

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mechanical properties and structure of 10 kinds of silk fibers from different stages. A tensile test

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showed that instar beginning silk, instar end silk, and mature silk possess distinct properties.

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Attenuated total reflectance Fourier-transform infrared spectroscopy and X-ray diffraction results

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showed that the excellent mechanical properties of instar end silk are attributed to higher β-sheet

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content and suitable crystallinity. Liquid chromatography−tandem mass spectrometry showed that

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P25 protein content in IV-E silk is 2.9 times higher than that of cocoon silk. This study can offer

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guidelines for further biomimetic investigations into the design and manufacture of artificial silk

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protein fibers with novel function.

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Keywords: silkworm silk, mechanical properties, secondary structure, crystallinity, multilevel

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structure

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INTRODUCTION

ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Silkworm (Bombyx mori) silk has been used in textiles for several thousand years and is

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regarded as a model high-performance polymeric material because of its excellent balance of

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strength and extensibility. Its applications include composite materials, regenerative medicine,

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military products, and beauty products1-6. Previous studies have attempted to develop silkworm

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silk with mechanical properties that exceed those of spider silk by adding special substances7-10,

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creating high-performance regenerated silk fibroin fiber11-14, and using transgenic technology to

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transform the mechanical properties of silk15-21. However, a strategy ignored by many people is

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that we can get a lot of inspiration to develop better silkworm silk by researching some kinds of

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silk which has a unique structure and mechanical properties.

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Silkworms spin different silks for specific purposes at different growth stages. Silkworm larvae

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go through five instars and molt four times before cocooning. Molting is the transition stage from

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one instar to another. Silkworms spin silk at the beginning and end of each instar (except the

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beginning stage of the fifth instar), but they spin very little silk in between these times. There are

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10 kinds of silk in the whole life cycle of silkworm: first instar beginning silk (I-B silk), first instar

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end silk (I-E silk), second instar beginning silk (II-B silk), second instar end silk (II-E silk), third

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instar beginning silk (III-B silk), third instar end silk (III-E silk), fourth instar beginning silk (IV-B

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silk), fourth instar end silk (IV-E silk), scaffold silk, and cocoon silk (Fig. 1). Instar beginning silk

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and instar end silk may also be called larval silk, while scaffold silk and cocoon silk are called

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mature silk, because they are spun by mature silkworms. Instar beginning silk is used to hold the

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silkworms’ bodies to the substrate to prevent them from falling during feeding and moving; instar

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end silk is used not only to hold their bodies, but also to fix themselves to the substrate while the

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silkworms cast off their old larval skins. In addition, scaffold silk is used to attach the cocoon to

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the substrate. Cocoon silk is a protective covering for the pupa and has been extensively studied22-

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25.


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Biomacromolecules

B. mori cocoon silk is composed of fibroin (75%) and sericin (25%). Fibroin is the useful

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portion from the point of view of industry and is the main source of the silk’s mechanical

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properties. Fibroin is oriented fiber protein complexes that consist of 350 kDa heavy chains (Fib

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H), 25 kDa light chains (Fib L), and a 30 or 27 kDa fibrohexamerin/P25, depending on the degree

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of glycosylation26-28. Quantitative ELISA assay revealed that the molar ratios of the Fib H, Fib L,

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and P25 were about 6:6:1 in the cocoon silk28. The Fib H and Fib L are linked by a single disulfide

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bond, and P25 associates with disulfide-linked Fib H and Fib L by noncovalent interactions.

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Current thinking suggests that 1 P25 and 6 heterodimers of disulfide-linked Fib H and Fib L form

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the basic unit of silk fibroin28-31. The composition of silk protein is the basis of structure and

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mechanical properties of silk.

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According to previous work, there are great differences in the protein composition of silk at

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different instars, including protein type and protein content25. This suggests that there may be

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significant differences in the mechanical properties of silk at different instars. In this study, we

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collected 10 kinds of silk from different developmental instars, then measured and compared their

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mechanical properties and structures, and also discussed the relationship between multilevel

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structures and properties. A solid understanding of the silk from different instars will provide

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important lessons for the design of new composite materials with an interesting balance of

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ductility, strength, and toughness.



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Fig. 1. Silkworm life cycle, including the egg stage, five instars, the pupal stage, and the moth

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stage. Silkworms spin silk at the beginning and end of each instar during the larval period, but not

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during the fifth instar beginning stage. I: first instar, II: second instar, III: third instar, IV: fourth

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instar, V: fifth instar. I-B: first instar beginning stage. I-E: first instar end stage, II-B: second instar

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beginning stage, II-E: second instar end stage, III-B: third instar beginning stage, III-E: third instar

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end stage, IV-B: fourth instar beginning stage, IV-E: fourth instar end stage, Scaffold: fifth instar

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seventh day, Cocooning: cocooning stage.

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EXPERIMENTAL SECTION

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Study organisms and silk collection

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B. mori (872, Dazao, C108, and Yun strains) were provided by the State Key Laboratory of

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Silkworm Genome Biology at Southwest University. The silkworms were reared on mulberry


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Biomacromolecules

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leaves at a stable temperature of 25 °C. All 10 kinds of silk were collected: I-B silk, I-E silk, II-B

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silk, II-E silk, III-B silk, III-E silk, IV-B silk, IV-E silk, scaffold silk, and cocoon silk. Silk tangle

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was used for attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) and

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X-ray diffraction (XRD). The silk was spun naturally by silkworms in a 9-mm culture dish and

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was stored in a 1.5-mL centrifuge tube at 24°C until required (Fig. 2A). Due to the difficulty of

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separating a single silk fiber (here“a single silk fiber” means a single bave fiber, has two brins.

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The meaning of the phrase is the same in the following.) from a silk tangle, we used an alternate

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technique, gently lifting a single silk fiber from around the spinneret with a toothpick and placing

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it on a paper mold for the tensile test (Fig. 3A)32.

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Fig. 2. (A) Collecting silk to characterize structure, (B) Silk tangle for each instar silk, (C) Body

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weight of each stage.



89 90

Fig. 3. (A) Collecting silk for diameter measurement and tensile test, (B) Single silk fiber from

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each instar silk, (C) Average long axis diameter of each type of silk.

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Observation of silk and calculation of average long axis diameter

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The overall appearance of silk in a silk tangle from each instar was viewed using a Zeiss Stemi

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2000C stereomicroscope, and the micromorphology of silk tangles was viewed using a Hitachi

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SU3500 scanning electron microscope (SEM) with an accelerating voltage of 5 kV and a

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magnification of 500–2000×. The morphology of a single silk strand from each instar was

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observed using a Zeiss EVO 18 SEM with an accelerating voltage of 10 kV and a magnification of

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1000–10000×. The magnification of silk from each instar was 10,000, 5,000, 5,000, 5,000, 5,000,

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5,000, 5,000, 5,000, 1,000, and 1,000 respectively (I-B silk→cocoon silk). When we calculate the


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Biomacromolecules

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cross sectional area of silk, we regarded the cross section of each instar silk as an ellipse and

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thought the ratio of the long axis to the short axis of the ellipse is 2:1.15, 32 So we only need to get

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the long axis diameter can calculate the cross sectional area of silk. To measure the average long

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axis diameter of silk from each instar, 15 photos of single silk strands from 15 silkworms of

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similar size were used, and 5 different locations in each photo were measured.

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And then we calculated the equivalent diameter according to cross sectional area. Because

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tensile test instrument needs equivalent diameter rather than cross sectional area. The equivalent

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diameter of each instar silk could obtain according to the following formula:

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The equivalent diameter = 2 *

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Tensile test

2

the cross sectional area of silk π

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The tensile force of a single silk fiber during the first five stages, from I-B to III-B, was not

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detected by the instrument, because the fibers were too thin. Instead, multiple silk fibers were

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included in each tensile sample: 100 strands of I-B silk, 100 of I-E silk, 50 of II-B silk, 20 of II-E

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silk, and 10 of III-B silk. The tensile tests were repeated 8, 8, 4, 6, and 16 times, respectively, so

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the amount of silk measured for the first five stages was 800, 800, 200, 120, and 160 strands

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(Table. 2). A single silk fiber was utilized in each tensile sample for the remaining stages (III-E

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silk, IV-B silk, IV-E silk, scaffold silk, and cocoon silk). For each stage, 25 ± 5 silk fibers were

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measured (Table. 2). The tensile tests were performed on a dynamic mechanical analyzer (DMA

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Q800; TA Universal Analysis, USA and the mininum detection force of the load cell is 0.00001

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N) under the following test conditions: gauge length, 10 mm; stretching speed, 1 mm/min; ambient

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temperature, 24°C; and relative humidity, 60%32.

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The diameter of each silk fiber (each of which contained two filaments called brins) was measured using an SEM according to the previous literature15, 32. In both experiments, specimens



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were equilibrated under the indicated conditions for 24 h prior to tensile testing. Experimental data

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were analyzed using TA Universal Analysis software to export the raw data on stress-strain

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curves. Subsequently, the raw data were used to calculate mechanical performance parameters

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using ORIGIN 8.0. (OriginLab, Northampton, MA), including elongation, maximum strength,

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elastic modulus, and toughness32, 33.

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ATR-FTIR analysis

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Infrared spectroscopy in attenuated total internal reflection mode was performed using a

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Thermo Scientific Nicolet iN10 with a Slide-On ATR objective lens. The spectra of the samples

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were recorded in the 650–4000 cm-1 range at a resolution of 8 cm-1 with 256 scans for each

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measurement34. The applied ATR current pressure was 75. OMNIC 9 software (Thermo Scientific)

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was used to collect and process the spectral data. We collected the background before collecting

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the ATR spectra of the samples. Spectral data analysis, including baseline correction,

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deconvolution of amide I bands, and peak fitting, was performed using OMNIC 9 software

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(Thermo Scientific) and PeakFit software (Seasolve, version 4.12) according to the literature35-39.

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XRD analysis

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Direct identification of the crystalline phases present in micro-samples was performed using

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XRD. X-ray diffraction was performed by a X'Pert³ Powder X-ray diffractometer (PANalytical

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Netherlands) with Cu-Kɑ radiation from a source operated at 40 kV and 40 mA.

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Before scanning, silk tangle from different instars were cut into tiny pieces. Cocoon silk, scaffold

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silk and IV-E silk were filled up the big sample pool (20 mm *20 mm *0.5 mm). Limited by the

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quantity, silk tangle samples from other periods were filled up the small sample pool (5 mm *5

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mm *0.5 mm). Then, all sample pools were mounted on aluminum frames and scanned from 5° to

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50° (2θ) at a speed of 2.0°/min. The relative crystallinity of samples was calculated by using MDI

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JADE 6.5 software. During the deconvolution process, the numbers and positions of the peaks


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Biomacromolecules

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were fixed by using the data reported in the literature40-43. The crystallinity of samples was

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evaluated according to the following formula: crystallinity = (X/Y) × 100%, where X is the net

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area of diffracted peaks, and Y is the net area of diffracted peaks + background area37, 39, 44.

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Liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis

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To understand the foundation of structure and mechanical properties of the silkworm silks at the

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protein molecular level. We used shotgun LC−MS/MS45 to analyze two silk proteomes, including

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the IV-E silk and the cocoon silk. A 20-mg sample of these two silks was weighed and then

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dissolved in 2 mL of 9 M LiBr. The detailed sample pretreatment process, mass spectrometry

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detection process, and data processing were reported in previous work25.

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Statistical analysis

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All data results were expressed as mean ± standard deviations (except for crystallinity). One-

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way analysis of variance followed by unpaired two-tailed Student’s t-test was performed. The

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levels of statistically significant difference were set at *p-value < 0.05, **p-value < 0.01, and

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***p-value < 0.001.

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RESULTS AND DISCUSSION

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Morphological characteristics of silk from each instar

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Photographs of the silk tangles and single silk fibers under SEM of silk from each instar are

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shown in Figures 2B and 3B. Figure 2B shows that the earlier the period, the more easily the silk

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sticks together and the tighter the silk tangle is. There were differences in color and luster of the

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silk tangle associated with the other differences between each instar silks. As shown in Figure 3B,

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all silk fibers consist of two fibroin brins surrounded by sericin. The fiber morphology seemed to

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be unaffected by developmental stages, but the diameter obviously increased with increase in the

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age and weight of the silkworm (Fig. 2C, Fig. 3C and Table 1).

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Table 1. Silk diameter and body weight of each instar silkworm larva



Page 10 of 34

Silk from each instar

Average equivalent diameter (μm)

Instar or larval stage

Weight of each larva (mg)

I-B silk

0.5 ± 0.07

I-B

0.5 ± 0.09

I-E silk

1.1 ± 0.2

I-E

5.9 ± 0.9

II-B silk

1.6 ± 0.4

II-B

6.0 ± 0.5

II-E silk

1.6 ± 0.6

II-E

43.2 ± 0.7

III-B silk

2.7 ± 0.4

III-B

41.3 ± 0.6

III-E silk

2.9 ± 0.5

III-E

219.4 ± 8.3

IV-B silk

2.9 ± 0.6

IV-B

193.2 ± 4.3

IV-E silk

5.3 ± 1.4

IV-E

892.6 ± 38.8

Scaffold silk

15.4 ± 2.6

Scaffold

4,762.4 ± 67.8

Cocoon silk

21.1 ± 1.9

Cocooning

1,384.0 ± 31.7

171 172

Mechanical properties of each instar silk

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The silk fibers of each instar were carefully tested under quasi-static tensile mode (Fig. 3A)32.

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Figure 4 and Table 2 present the mechanical properties of the silk from each instar. As shown in

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Figure 4A, the stress–strain curves of all instar silk in the present study are similar to those in

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previously reported data32, 33, 37. All the instar end silk curves are noticeably higher than those of

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the instar beginning silk and mature silk, and all the instar beginning silk curves are slightly higher

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than those of mature silk. Figure 4B shows that the elongation of silk before the fourth instar end

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stage was significantly lower than that of mature silk. Interestingly, the elongation of silk

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gradually increased as the larvae aged. We hypothesized that the structure of each instar’s silk

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likely changes by a gradual process as well. Figure 4C shows that instar end silk has the highest

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elastic modulus, followed by instar beginning silk, and then mature silk. The maximum strength

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(Fig. 4D) follows a similar progression: instar end silk is noticeably stronger than instar beginning

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silk, and instar beginning silk is slightly stronger than mature silk. The toughness (Fig. 4E)

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followed a slightly different order; instar end silk was the toughest, followed by mature silk and


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Biomacromolecules

then instar beginning silk.

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The results indicate that instar beginning silk (I-B silk, II-B silk, III-B silk, and IV-B silk),

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instar end silk (I-E silk, II-E silk, III-E silk, and IV-E silk), and mature silk (scaffold silk and

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cocoon silk) represent three different types of silk with different mechanical properties. Instar end

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silk displays the highest elastic modulus, with maximum strength and toughness values that are far

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higher than those of instar beginning silk and mature silk (Fig. 4B–E). Smaller diameter maybe is

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one of the reasons why instar end silk has a higher maximum strength than mature silk46, 47.

193

Diameter of instar end silk and instar beginning silk are all smaller than mature silk, but

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mechanical properties of instar end silk are much higher than that of instar beginning silk and

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mature silk. And there is no phenomenon that the smaller the diameter, the better the mechanical

196

properties. Therefore, there are some other reasons why instar end silk has very good mechanical

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properties and protein composition and secondary structure are more likely the main cause,

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however, further proof is needed. Instar end silk may be regarded as a novel fiber material that has

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excellent mechanical properties, nearly at the level of spider silk48, 49, which has not to date been

200

reported in B. mori. Secondly, although instar beginning silk’s maximum strength is similar to that

201

of mature silk (Fig. 4D), its elongation and toughness values are almost all significantly lower than

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those of mature silk (Fig. 4B and E) and its elastic modulus values are all significantly higher than

203

those of mature silk (Fig. 4C). Thus, instar beginning silk shows different mechanical properties

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from instar end silk and mature silk, indicating that it likely has a different structure.

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In fact, the mechanical properties of silk at different stages are consistent with the biological

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functions at each stage. At each instar end stage (before molting), silkworm larva spin small

207

amounts of silk to facilitate molting by anchoring their eight pairs of ventral feet and the old

208

cuticle to the substratum22, 25, 50. We know that Bombyx mori evolved from wild silkworm. When

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the wild silkworm larvae molt, their bodies are immobilized by powerful silk fibers that keep them



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from falling out of the tree, even when the wind blows or the tree shakes. If instar end silk is not

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strong enough, the silkworm larvae will die from falling or failing to molt to the next instar. We

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speculate that in the course of evolution, the characteristics of instar end silk did not degenerate.

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Therefore, spinning silk with superb mechanical properties before molting is critical to the growth

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and development of silkworms, especially wild silkworms. In contrast, instar beginning silk and

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mature silk do not need such robust mechanical properties. Instar beginning silk, which is spun

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after molting, does not need to be as strong because the larvae can grasp the branches with their

217

feet rather than relying solely on silk fibers. In the case of mature silk (cocoon silk and scaffold

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silk), many silk fibers are clustered together, making the cocoon strong enough to protect the

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developing pupa, although a single silk strand is not very strong51-53.


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Biomacromolecules

220 221

Fig. 4. Mechanical properties of each instar silk. (A) Stress-strain curve, (B) elongation, (C) elastic

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modulus, (D) maximum strength, (E) toughness.

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Table 2. Mechanical properties and structure of silk from each instar



Tensile

Silk

Silk from

Maximum

Elastic

Elongation test

number

each instar

Page 14 of 34

Toughness strength

modulus

β-sheet

Relative

content

crystalli-

（%）

nity (%)

(MJ/m3)

(%) (MPa)

(GPa)

8.8 ± 3.0

376.3 ± 68.3

11.5 ± 2.1

22.6 ± 8.0

24.5 ± 1.8

67.3

100

10.8 ± 2.2

1227.3 ± 51.9

31.8 ± 3.3

93.2 ± 23.5

27.1 ± 2.4

68.0

4

50

16.0 ± 1.0

457.2 ± 76.1

11.8 ± 2.4

51.0 ± 4.2

19.3 ± 2.5

-

II-E silk

6

20

17.8 ± 2.6

984.1 ± 169.5

26.7 ± 3.9

141.3 ± 39.7

25.2 ± 1.8

66.2

III-B silk

16

10

14.0 ± 1.9

405.1 ± 53.6

13.0 ± 2.9

40.1 ± 8.0

20.9 ± 1.8

-

III-E silk

20

1

16.0 ± 4.5

1049.3 ± 208.9

27.1 ± 6.4

127.6 ± 57.3

26.1 ± 1.9

60.3

IV-B silk

21

1

10.2 ± 2.8

388.3 ± 181.6

13.9 ± 6.5

26.4 ± 11.5

17.3 ± 2.68

-

IV-E silk

33

1

22.9 ± 5.8

1179.4 ± 274.3

25.9 ± 6.4

205.6 ± 90.4

28.0 ± 2.5

58.1

25

1

18.0 ± 3.8

367.4 ± 105.1

8.3 ± 2.8

47.1 ± 14.4

21.5 ± 2.4

50.8

28

1

22.3 ± 2.6

348.2 ± 29.2

5.7 ± 1.0

56.2 ± 10.5

21.4 ± 2.0

50.5

repeats

in a test

I-B silk

8

100

I-E silk

8

II-B silk

Scaffold silk Cocoon silk

224 225 226

Secondary structural characteristics of silk fibers In order to obtain a better insight into the molecular basis of mechanical properties, the

227

secondary structure content of each instar silk was investigated by ATR-FTIR. FTIR is commonly

228

used to study the protein secondary structures of silk-based biopolymers. However, the

229

conventional FTIR technique is seldom used to test silkworm larvae single silk fibers because the

230

spot size of the conventional globar light source (which is usually a minimum of 10 μm × 10 μm)

231

is too large compared with the diameter of a single silk filament (usually less than 5 μm, Fig. 2C

232

and Table 1). Therefore, only a small part of the infrared beam can illuminate a larvae single silk

233

filament, resulting in a very poor-quality image and an almost useless spectrum. Thus, ATR-FTIR

234

was used to characterize the secondary structures, because it is not limited by sample diameter and

235

size34, 54-57.


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236

Biomacromolecules

The quality of ATR-FTIR spectra of cocoon silk fibroin (Fig. S3A) is the same as that of

237

ordinary FTIR spectra36-39. ATR-FTIR spectra of silk fibroin fall principally in the amide I, amide

238

II, and amide III absorption bands (Fig. S3A, Fig. 5A)58, 59. Interestingly, there was a very obvious

239

special absorption peak at about 1318 cm-1 in II-B, III-B, and IV-B silk (Fig. 5A), which is an

240

absorption peak of calcium oxalate60-62. we also observed a large number of calcium oxalate

241

crystal particles in the silk tangles of these three instar stages, as well as a small amount in the II-

242

E, III-E, and IV-E stages (Fig. S2). We hypothesize that calcium oxalate granules originated in the

243

silkworm epidermis and were scraped off because of friction when a large number of silkworms

244

were crowded together, but this explanation requires further research to confirm.

245 246

Fig. 5. Secondary structural characteristics of silk from each instar. (A) ATR-FTIR spectrum from

247

1000 cm-1 to 1800 cm-1 of silk from each instar, (B) Deconvolution of the corresponding amide III

248

band, (C) β-sheet content of silk from each instar. ACS Paragon Plus Environment


249

The amide I10, 11, 46, 57, 63 and amide III35-39, 64 bands are commonly used to analyze protein

250

secondary structure and the composition of secondary structures in silk materials. To understand

251

the compositions of different secondary structures, including β-sheet and α-helix/random coil,

252

peak deconvolution analysis was carried out on the amide III band, at 1200–1300 cm−1 (Fig. 5B,

253

Fig. S3 B–K). The amide III band was chosen because calcium oxalate has another main

254

absorption peak at about 1619 cm-1, which interferes with the peak deconvolution analysis of the

255

amide I band (about 1600–1700 cm-1). In previous studies, the quantitative analysis of the β-sheet

256

structure was carried out by investigating the spectral region of ATR spectra: the peaks at about

257

1266 cm−1 represent the β-sheet structure10, 46, 56, 63, 65, and the peaks at about 1233 cm−1 represent

258

the α-helix/random coil structure (as the helical and random coil conformations in the B. mori silk

259

fibroin are difficult to distinguish) (Fig. 5B, Fig. S3 B–K)10, 37, 56, 66.

260

The deconvolution result of FTIR spectra in the amide III band (Fig. S3 B–K) revealed the

261

quantitative content of the secondary structure of the silk from each instar (Fig. 5D, Table 2). The

262

β-sheet content was higher in all the instar end silks than in mature silk, but that in instar

263

beginning silk (except the I-B silk) was lower than in mature silk. These results provide additional

264

confirmation that instar beginning silk, instar end silk, and mature silk are three distinct kinds of

265

silk.

266

In general, all silkworm silks may be considered semi-crystalline biopolymers with highly

267

organized antiparallel β-sheet nanocrystals embedded in an amorphous matrix67. The β-sheet

268

structure is regarded as a physical crosslinking point in silks and the dominant factor that is

269

responsible for the physical properties of silks37, 68, 69. For silkworm silk, therefore, silk with a

270

higher β-sheet content will have stronger mechanical properties. The β-sheet content of instar end

271

silk was obviously higher than that of mature silk (Fig. 5D, Table 2). This result was consistent

272

with the fact that the elastic modulus, maximum strength, and toughness values of instar end silk


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Biomacromolecules

273

were all higher than those of mature silk, proving that the β-sheet plays an important role in

274

determining the properties of silks37, 70, 71.

275

The β-sheet structure content of instar beginning silk (except I-B silk) was slightly lower than

276

that of cocoon silk, but the maximum strength was slightly better than that of cocoon silk. This

277

finding indicated that these silks may have different structural foundations. The mechanical

278

properties of the I-B silk were similar to those of other instar beginning silks (Fig. 4), but its β-

279

sheet structure content was obviously higher than that of other instar beginning silks and mature

280

silk (Fig. 5D). Thus, I-B silk is also a special silk with a unique structure and mechanical

281

properties, which should be confirmed through further research.

282

Crystal morphological characteristics of each instar silk fibers

283

One of the most widely used methods of characterizing the crystal morphological characteristics

284

of silk is XRD. In previous studies, the XRD patterns in protein fiber material were determined as

285

follows: 11.95°, 21.4°, and 24.02° for α-helix crystal structure, and 9.1° (9.2°), 16.71°, 20.34°

286

(20.6°), 24.49° (24.6°), 30.90°, 34.59°, 40.97°, and 44.12° for β-sheet crystal structure40-43. In the

287

present study, crystal diffraction peaks of silk from each instar were detected at about 9.0°, 20.5°,

288

and 24.5° (Fig. 6A–B). At the same time, the diffraction peaks of calcium oxalate (at about 14.93°,

289

23.56°, 24.40°, 29.72°, and 30.11°) were also detected in larvae silk60-62. Especially in instar

290

beginning silk, the diffraction peaks of calcium oxalate were very obvious (Fig. 6, B right). These

291

findings supported the ATR-FTIR result (Fig. 5A). In addition, the peak at about 21.4° in larva

292

silk should belong to an unknown crystal diffraction peak, because the XRD spectrum for pure silk

293

generally does not contain sharp peaks (Fig. S4 A–E)10, 72, 73. By comparison, the relative intensity

294

of silk diffraction peaks of II-B, III-B, and IV-B silk was too low to be used to calculate the

295

crystallinity. However, it could still be used to estimate the crystallinity of the remaining kinds of

296

silk (Fig. S4 A–G), because the relative intensity of diffraction peaks was sufficient and



297

background diffraction of calcium oxalate crystals was minimal.

298 299

Fig. 6. (A) The X-ray diffraction diffractograms of silk from each instar. (B) Deconvolution of the

300

cocoon silk diffractogram, with intensity as a function of scattering angle 2θ. The peaks are fitted

301

as the sum of six Gaussians: three crystalline peaks (light red) and three amorphous halo (pale

302

green). Three crystalline peaks are indexed as 9.0°, 20.5°, and 24.5°. (C) Relative crystallinity of

303

silk from each instar.


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Biomacromolecules

304

Crystallinity analysis showed that crystallinity of the silk from each instar gradually decreased

305

from I-B silk to cocoon silk, even though there was no result from II-B silk, III-B silk, or IV-B silk

306

but we still believe in such trends. (Fig. 6C, Table 2). The high crystallinity of larvae silk may be

307

explained by differences in protein components, differences in the silk secretion process, small

308

diameter of filaments, and so on. The β-sheet content differed from the crystallinity results, as it

309

was highest in instar end silk, followed by mature silk and then instar beginning silk (Fig. 6C,

310

Table 2). These two results are not contradictory, because the crystal structure contains not only

311

the β-sheet crystal structure but also the α-helix crystal structure40-43. The higher the crystallinity,

312

the lower the amorphous portion that affects the elongation of silk. Thus, the elongation of larvae

313

silk is lower than that of mature silk, and in general, the smaller the instar, the smaller the

314

elongation of silk (Fig. 4B, Fig. 6C)37, 68, 69.

315

In general, the increase in crystallinity is associated with an increase in the rigid connections

316

between molecules in silk fiber and with increases in the strength, elastic modulus, and toughness

317

of silk fiber, because crystal structure has high density37, 68, 69. Nevertheless, why do the elastic

318

modulus and strength of II-B silk, III-B silk and IV-B silk increase only a little, while the

319

toughness decreases (compared with mature silk)? The reason may be that the silk gland just

320

completed its renewal event and the silk protein synthesis and assemblies are not very mature at

321

the beginning stage of each instar22. Thus, the silk has an incomplete secondary molecular

322

structure and poor mechanical properties. In addition, the calcium oxalate on the surface of these

323

beginning silks maybe decrease the mechanical properties21. As for I-B silk, its main diffraction

324

peak appeared at about 22.5°. However, the main diffraction peak of other samples appeared at

325

about 20.5° (β-sheet crystal structure) (Fig. 6 A, Fig. S4 H). A diffraction peak near 22° should

326

belong to the α-helix crystal structure74. This indicates that the β-sheet structure did not exist in I-

327

B silk. In addition, the peak at about 16.32 is also an α-helix crystal structure. Therefore, the α-



328

helix crystal structure is the main crystal structure in I-B silk. The I-B silk may be formed through

329

unique protein synthesis and assembly at its beginning stage. However, many details of I-B silk

330

filament formation are not clear.

331

There are some commonalities between instar end silk and mature silk, both of which are

332

formed at the end of each period. The elastic modulus, strength, and toughness of instar end silk,

333

which has higher crystallinity, are much greater than those of mature silk (Fig. 4, Fig. 6, Table 2).

334

Of the end silks, IV-E silk had the best comprehensive mechanical properties, but its crystallinity

335

values were not the highest. Thus, increases in crystallinity are not always correlated to increases

336

in mechanical properties. The best mechanical properties of silk are achieved only when the β-

337

sheet crystal structure, α-helix crystal structure, α-helix amorphous structure, and random coil are

338

balanced.

339

The structure of cocoon silk is most stable, but its mechanical properties are also not the best. In

340

order to improve the mechanical properties of silkworm silk, the silk in the larvae stage (especially

341

instar end silk) should be studied further. IV-E silk is a good choice because it has the best

342

comprehensive mechanical properties. We also compared the mechanical properties of IV-E silk

343

and cocoon silk of Dazao, C108, and Yun strains. The results showed that the mechanical

344

properties of these kinds of silk were all better than that of cocoon silk (Fig. S5, Fig. S6 and Fig.

345

S7). It indicated that IV-E silk is a ubiquitous high-performance fiber material and has the value of

346

being studied deeply.

347

Protein component difference between IV-E silk and the cocoon silk

348

Structural foundations determine performance. Previous studies in our laboratory proved that

349

there are significant differences in protein composition and content among the instar silks25.

350

Presumably, the difference in protein composition and content is the most fundamental reason for

351

the difference in the secondary structure and mechanical properties of each instar silks. In the


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Biomacromolecules

352

present study, LC–MS/MS was used to identify the proteome of IV-E silk and the cocoon silk. The

353

method of detailed analysis is described in previous studies25, 45, 75, 76.

354

Using a combined analysis of triplicate samples, 222 proteins were identified (Table S1). A total

355

of 184 proteins were identified in IV-E silk and 115 proteins were identified in cocoon silk (Table

356

S1). Differential protein analysis was performed to explain why IV-E silk has the best

357

comprehensive mechanical properties. The result showed that the percentage content of 53

358

proteins was significantly higher in IV-E silk than in cocoon silk, and the percentage content of 9

359

proteins was significantly lower in IV-E silk than in cocoon silk (Fig. 7A, Table S1). We divided

360

the identified proteins into eight categories (Table S1). The top 10 proteins that were up- and

361

downregulated are presented in Figures 7B and 7C, respectively. Comparative analysis shows that

362

among many differentially regulated proteins, fibroin and sericin are the most abundant. These are

363

the main ingredients of silk, and other proteins are low-level auxiliary components related to silk25,

364

77.

365

and sericin 2 protein are significantly higher in IV-E silk than in cocoon silk, sericin 1 and sericin

366

3 proteins are significantly lower in IV-E silk than in cocoon silk, and Fib H and Fib L proteins are

367

not changed. This result indicates that the core structure of IV-E silk is similar to that of cocoon

368

silk, but there may be differences in molecular assembly. Because the mechanical properties of

369

silk mainly come from silk fibroin, protein changes in silk fibroin may be the main reason for the

370

change in mechanical properties. Thus, the change in sericin may be linked to the secretion of silk

371

and the viscosity of silk. Fibroin P25 is the only silk fibroin that changed (Table 3 and Table S1).

372

Therefore, the content change of P25 protein appears to be the main cause for the change in

373

mechanical properties.

we analyzed the changes to the main silk proteins (Table 3) and determined that P25 protein



374 375

Fig. 7. (A) Protein content differential statistics (IV-E silk vs. cocoon silk). (B) Top 10 proteins

376

that were significantly upregulated in IV-E silk. (C) Top nine proteins that were significant

377

downregulated in IV-E silk. (D) The ratio of three fibroin proteins in IV-E silk and cocoon silk

378

from 872 strain and Dazao strain25.

379 380 381


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Biomacromolecules

382 383 384

Table 3. Changes in major silk proteins Av iBAQ Protein

Fold

Protein ID IV-E silk

Cocoon silk

p-valueb

thresholdc

changea

Fibroin heavy chain

BGIBMGA005111-PA

10998666667

8957900000

1.2

0.01

NoChange

Fibroin light chain

BGIBMGA009393-PA

7698200000

6113266667

1.3

0.3

NoChange

Fibroin P25

BGIBMGA001347-PA

3526200000

1234400000

2.9

0.007

Up

Sericin 1

BGIBMGA001793-PA

322516667

6829166667

0.05

0.003

Down

Sericin 2

BGIBMGA011901-PA

7032266667

305337

>1000

0.0005

Up

Sericin 3

BGIBMGA012002-PA

2040433

164653333

0.01

0.005

Down

Osiris 9

BGIBMGA000013-PA

0

3600266667

0.01 (Table S1).

390

Table 3 shows that P25 protein content increased by 2.857 times and the molar ratio of three

391

fibroin proteins changed. For example, the molar ratios of the heavy chain, light chain, and P25

392

were close to 6:6:1 in the cocoon silk, which was consistent with the literature28. The ratio in IV-E

393

silk was close to 3:3:1 (Fig. 7D), a significant enough change to affect the structural and

394

mechanical properties. This phenomenon occurred in both the 872 strain and the Dazao strain. A

395

previous study provided similar results25. In the Dazao strain, the ratio of Fib H:Fib L:P25 in

396

cocoon silk was close to 6:6:1, and that in IV-E silk was close to 2:2:1 (Fig. 7D). In addition, the

397

mechanical properties of IV-E silk were stronger than those of cocoon silk in the Dazao strain

398

(Fig. S5). Therefore, the multiplied increase of fibroin P25 protein content is likely the main



399

reason for the enhancement of mechanical properties in IV-E silk.

400

Relationship between the structural and mechanical properties of IV-E silk

401

Previous studies reported that the mature P25 protein contains 203 amino acid residues, and

402

there are 8 Cys residues and 3 N- glycosylation sites in the peptide chain78, 79. The P25 molecule is

403

rather compact due to intramolecular disulfide bonds, and three N-linked oligosaccharides chains

404

(high mannose type) are on the periphery of the protein core. P25 is in the center of the elementary

405

unit of fibroin model and plays a role in connecting the FibH-FibL complex28, 31. P25 is not only

406

responsible for the efficient secretion and intracellular transport of fibroin, but also is involved in

407

inducing the proper folding of the H-chain by hydrophobic interactions. The N-linked

408

oligosaccharides in P25 may also be involved in the folding of H-chain and facilitate the formation

409

of a stable complex, as the N-linked high mannose type oligosaccharide chains of P25 exhibit

410

molecular chaperone-like behavior26, 28. Therefore, P25 probably plays an important role in

411

maintaining the structure and mechanical properties of silk. Thus, doubling of the P25 protein

412

content will contribute to form more β-sheet structures and increase crystalline properties,

413

strengthening the connections between macromolecules.

414

On the basis of the above discussion, we propose a model that describes the molecular assembly

415

and hierarchical structure in cocoon silk and IV-E silk to explain the enhancement of IV-E silk’s

416

mechanical properties (Fig. 8). Both the cocoon silk and IV-E silk are composed of numerous

417

interlocking nano-fibrils3, 66, 67, 80. Inside the nano-fibrils, there are a certain number of molecular

418

beams (estimated to be fewer than 581, 82) that are made up of fibroin elementary units in a regular

419

arrangement (Fig. 8B and D)15, 26, 28, 31, 83. Inside the fibroin elementary unit, Fib H, Fib L, and P25

420

are present in a ratio of 6:6:1 and the crystalline region is connected by the amorphous region to

421

form a three-dimensional network structure (Fig. 8C)66-68, 81, 82, 84, 85. The crystalline region is

422

mainly composed of stacked β-sheets with peptide chains connected by hydrogen bonds67, 81, 82, 84,


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Biomacromolecules

423

85.

424

form hydrogen bonds with hydroxyls of the side chain functional groups, Ser and Tyr (Fig. 8C).

425

So as for in IV-E silk, we speculated that the extra P25 are easily embedded where two fibroin

426

elementary units meet head to tail, between two fibroin elementary units and inside the fibroin

427

elementary unit through noncovalent interaction (Fig. 8D). In addition, the hydroxyls of P25 easily

428

form hydrogen bonds with H2O. These interactions result in the presence of more moisture in IV-E

429

silk. The excess moisture, like a slip agent, contributes to the formation of the β-sheet86. Therefore,

430

we hypothesize that P25 plays an important role in promoting Fib H protein folding, promoting the

431

formation of additional β-sheet structures and crystal structures, linking macromolecules, and

432

stabilizing the structure of nano-fibril to enhance the mechanical properties of IV-E silk.

433

Contrasting IV-E silk and cocoon silk, like the case of a brick wall, the addition of different kinds

434

of adhesives or adhesives of different quality greatly influences the mechanical properties of the

435

wall, known as brick wall effect. Maybe we can use this to transform the mechanical properties of

436

silk.

Three oligosaccharide molecules of P25 contain a large number of hydroxyls, which readily

437



438

Fig. 8. Model describing the structural hierarchy and molecular assembly in cocoon silk and IV-E

439

silk. (A) Down: The silk consisted of two approximately triangular fibroin fibers and external

440

sericin layer. Fibroin is composed of numerous fibrils. Up: Fibril is composed of numerous nano-

441

fibrils. (B) Nano-fibril is composed of numerous interlocking fibroin elementary units. (C) Down

442

left: Fibroin elementary unit is composed of Fib H, Fib L, and P25 which are present in a ratio of

443

6:6:1. Down right: Fib H contains N terminal, C terminal, 11 amorphous regions and 12 crystalline

444

regions. The crystalline region is connected by the amorphous region. Up: P25 is composed of a

445

protein core and 3 mannose type oligosaccharides. There are four active hydroxyls in a mannose

446

molecule. (D) Left: Nano-fibril model of IV- E silk. Up right: the extra P25 are embedded between

447

two fibroin elementary units meet head to tail. Middle right: the extra P25 are embedded between

448

two fibroin elementary units. Down right: the extra P25 are embedded inside the fibroin

449

elementary unit.

450

CONCLUSION

451

In the present study, we comprehensively and systematically compared mechanical properties

452

and secondary structure of silkworm silk from each instar. The elastic modulus, maximum

453

strength, and toughness of instar end silk are all much higher than those of instar beginning silk

454

and mature silk. In instar beginning silk, only the elastic modulus is significantly greater than in

455

mature silk. Its maximum strength is almost the same as that of mature silk, and its toughness is

456

slightly lower than that of mature silk. Therefore, we hypothesized that instar beginning silk, instar

457

end silk, and mature silk represent three different kinds of silk fibers with different mechanical

458

properties and structural characteristics. Results of secondary structural characterization showed

459

that the instar end silk has the highest β-sheet content, followed by mature silk and then instar

460

beginning silk. It again suggested that the β-sheet structure is an important contributor to the

461

excellent mechanical properties of silk. XRD results showed that the crystallinity of silk gradually ACS Paragon Plus Environment

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Biomacromolecules

462

decreases as the instar stage increases. we can conclude that higher β-sheet structure content and

463

suitable crystallinity contribute to improving the mechanical properties of silk. An analysis of IV-

464

E silk and cocoon silk by LC−MS/MS revealed that P25 protein is the main reason for the

465

enhancement of mechanical properties of IV-E silk. The P25 content of IV-E silk in both the 872

466

strain and the Dazao strain were two to three times higher, and the mechanical properties also

467

greatly exceeded those of cocoon silk. Thus, we hypothesize that the mechanical properties of silk

468

will be greatly improved by increasing the content of a suitable adhesive component in silk fibers

469

(a small protein or other chemical molecule with viscosity), assuming the content of Fib H protein

470

remains unchanged. These molecules may be able to promote the formation of more β-sheet and

471

crystalline structure. The brick wall effect is also very relevant to the effort to improve the

472

mechanical properties of silkworm silk or other fiber materials.

473

ASSOCIATED CONTENT

474

Supporting Information

475

This material includes: Fig. S1. Stress-strain curves of each instar silk. Fig. S2. Surface

476

morphology SEM photograph of each instar silk tangles. Fig. S3. Deconvolution of ATR-FTIR of

477

each instar silks. Fig. S4. Deconvolution of diffractogram of XRD of each instar silks. Fig. S5,

478

Fig. S6 and Fig. S7. Mechanical properties of IV-E silk and cocoon silk of other silkworm strains.

479

(PDF) Table S1. Identified proteins from IV-E silk and cocoon silk. (.xlsx)

480

AUTHOR INFORMATION

481

Corresponding Author

482

*Address: Qingyou Xia State Key Laboratory of Silkworm Genome Biology Southwest University

483

216 Tiansheng Road Chongqing 400716, P. R. China. Tel: 86-23-68250099; Fax: 86-23-



484

68251128; E-mail: [email protected].

485

Author Contributions

486

§These authors contributed equally to this work. The manuscript was written through

487

contributions of all authors. All authors have given approval to the final version of the manuscript.

488

Notes

489

The authors declare no competing financial interest.

490

ACKNOWLEDGMENTS

491

This work was supported by grants from the key program of the National Natural Science

492

Foundation of China (31530071), the National Natural Science Foundation of China (31772532).

493

REFERENCES

494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514

1. Gellynck, K.; Verdonk, P.; Almqvist, F.; Van Nimmen, E.; De Bakker, D.; Van Langenhove, L.; Mertens, J.; Verbruggen, G.; Kiekens, P., A spider silk supportive matrix used for cartilage regeneration. In Medical Textiles and Biomaterials for Healthcare, Elsevier: 2006; pp 350-354. 2. Porter, D.; Vollrath, F., Silk as a biomimetic ideal for structural polymers. Adv Mater 2009, 21, (4), 487-492. 3. Hakimi, O.; Knight, D. P.; Vollrath, F.; Vadgama, P., Spider and mulberry silkworm silks as compatible biomaterials. Compos Part B-Eng 2007, 38, (3), 324-337. 4. Murphy, A. R.; Kaplan, D. L., Biomedical applications of chemically-modified silk fibroin. J Mater Chem 2009, 19, (36), 6443-6450. 5. Vepari, C.; Kaplan, D. L., Silk as a biomaterial. Prog Polym Sci 2007, 32, (8-9), 991-1007. 6. Xiong, R.; Grant, A. M.; Ma, R.; Zhang, S.; Tsukruk, V. V., Naturally-derived biopolymer nanocomposites: Interfacial design, properties and emerging applications. Mat Sci Eng R 2018, 125, 1-41. 7. Wang, J. T.; Li, L. L.; Feng, L.; Li, J. F.; Jiang, L. H.; Shen, Q., Directly obtaining pristine magnetic silk fibers from silkworm. Int J Biol Macromol 2014, 63, 205-209. 8. Wang, J. T.; Li, L. L.; Zhang, M. Y.; Liu, S. L.; Jiang, L. H.; Shen, Q., Directly obtaining high strength silk fiber from silkworm by feeding carbon nanotubes. Mat Sci Eng C-Mater 2014, 34, (1), 417-421. 9. Wang, Q.; Wang, C.; Zhang, M.; Jian, M.; Zhang, Y., Feeding Single-Walled Carbon Nanotubes or Graphene to Silkworms for Reinforced Silk Fibers. Nano Lett 2016, 16, (10), 6695-6700. 10. Guo, Z.; Xie, W.; Gao, Q.; Wang, D.; Gao, F.; Li, S.; Zhao, L., In situ biomineralization by


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Structural and mechanical properties of silk from different instars of

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