Local changes in individual spider silk fibers from tubuliform and major-ampullate glands under varying humidity and tensile strain

Local changes in individual spider silk fibers from tubuliform and major-ampullate glands under varying humidity and tensile strain | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Local changes in individual spider silk fibers from tubuliform and major-ampullate glands under varying humidity and tensile strain Helga Lichtenegger, Karolina Peter, Michael Sztucki, Sebastian Kalbfleisch, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8335511/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Spider silk’s exceptional mechanics and environmental adaptability stem from a hierarchical structure of crystalline β-sheet nanodomains embedded in an amorphous protein matrix. While major ampullate (MA) silk’s structural responses to strain and environment are well studied, tubuliform (TU) silk—critical for egg sac construction and promising for biomedical uses—remains poorly understood. We investigate TU and MA silk under varying relative humidity (RH) and mechanical strain using spatially resolved, high‑resolution nanobeam X‑ray diffraction. Increasing RH markedly reduces Young’s modulus, yield strain, and yield stress in both silks, with TU showing greater humidity sensitivity. Ultrastructural analysis reveals key contrasts: TU exhibits pronounced changes in nanocrystal dimensions, molecular alignment, and lattice spacing with strain and humidity, whereas MA maintains more stable crystalline organization, reflecting optimization for tensile strength. By mapping structural parameters across single-fiber diameters (edge, transition, bulk), we provide spatially resolved insights into TU’s ultrastructure and hierarchical mechanisms. Notably, structural parameters are predominantly homogeneous across the fiber diameter for both silks at the studied length scales. These distinct structural responses offer design templates for biomimetic materials. Future work should examine additional environmental variables (e.g., temperature, solvents) and finer length scales to further unlock spider silk’s potential as a model biomaterial. Biological sciences/Structural biology/X-ray crystallography/Nanocrystallography Physical sciences/Materials science/Biomaterials/Biomaterials – proteins Biological sciences/Structural biology/X-ray crystallography/Nanocrystallography Physical sciences/Materials science/Biomaterials/Biomaterials – proteins Synchrotron Radiation In-situ Tensile Testing X-ray Scattering Ultrastructure Humidity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Spider dragline silk has long fascinated scientists due to its exceptional combination of high tensile strength and extensibility, resulting in a toughness amongst the highest values of most natural and synthetic fibers (Braxton et al., 2025 ; Gosline et al., 1999 ; Nova et al., 2010 ). These extraordinary mechanical properties are attributed to its hierarchical structure, which features highly ordered crystalline β-sheet nanostructures embedded within an amorphous protein matrix (Termonia, 1994 ). The β-sheet nanostructures, with a nearly orthorhombic unit cell (Marsh et al., 1955 ; Warwicker, 1960 ), provide strength, while the amorphous regions contribute to extensibility and energy dissipation (Du et al., 2006 ; Giesa et al., 2011 ; Lefevre & Auger, 2016 ). This unique architecture allows spider silk to exhibit remarkable adaptability under varying environmental conditions, making it a model biomaterial for both fundamental research and practical applications. Applications include recombinant spider silk bio-inks (Lechner et al., 2022 ), adhesives (Ye et al., 2023 ), and spider silk inspired fibers for sensing, high strength textiles, and water collection (Li et al., 2022 ). Many possible applications are found in the field of medicine (Altman et al., 2003 ; Rising & Johansson, 2015 ). In particular, the treatment of peripheral nerve injuries (Bergmann et al., 2022 ) is an area where silk scaffolds may be impactful. These injuries often result in significant loss of motor and sensory function, and current treatments, such as autografts, are limited by donor site morbidity and availability (Kornfeld et al., 2021 ; Puranik et al., 2025 ; Radtke et al., 2011 ). Spider silk offers a biocompatible, mechanically robust alternative, with the ability to support cellular adhesion, migration, and proliferation (Naghilou et al., 2023 ; Stadlmayr et al., 2025 ). In a previous study, we compared the ability of tubuliform (TU) silk and major ampullate (MA) silk present in the egg sac of spiders to guide Schwann cells. MA silk is produced by the major-ampullate gland whereas TU silk is produced by the tubuliform gland of the female spider. We found that both silk types are part of the egg sac, which is a protective cover for the spider’s eggs. Interestingly, TU silk exhibited a superior ability to promote Schwann cell adhesion, migration, and alignment (Peter et al., 2025 ). We found that a combination of morphology and other structural features likely make TU silk particularly interesting for Schwann cells and therefore nerve regeneration. Nevertheless, our knowledge about TU silk is still limited, especially when it comes to its response to environmental and mechanical stimuli. Clarifying these structural adaptations is critical for optimizing TU silk’s use in biomimetic applications, particularly in nerve repair and regenerative medicine. Among the environmental factors influencing spider silk, humidity plays a particularly critical role. One of the most striking effects of high humidity is supercontraction, where some types of silk, including MA silk, shrink by up to 50% of its original length (Work & Morosoff, 1982 ). This phenomenon is driven by the diffusion of water molecules into the silk, causing a transition from a glassy to a rubbery state, the dissociation of hydrogen bonds, and swelling of the fiber (Cohen, 2023 ; Cohen et al., 2021 ). These changes lead to a loss of molecular orientation and an increase in entropy, fundamentally altering the silk’s mechanical behavior(Schäfer et al., 2008 ; Vehoff et al., 2007 ). For TU silk it has been reported, that supercontraction is absent (Ruiz et al., 2019 ). Relative humidity (RH) significantly alters the silk’s mechanical properties, such as stiffness, strength, and extensibility (Plaza et al., 2006 ; Vehoff et al., 2007 ; Yazawa et al., 2020 ). For example, Yazawa et al. ( 2020 ) showed that toughness of MA silk is enhanced under moderate humidity (43% RH) and high strain rates while Young’s modulus decreases with increasing RH (Yazawa et al., 2020 ). Vehoff et al. ( 2007 ) confirmed humidity's substantial effect on mechanical properties through large-scale testing (Vehoff et al., 2007 ). For silkworm silk, Seydel et al. ( 2011 ) further demonstrated that tensile strain increases molecular mobility of water and amorphous polymer chains in the amorphous regions of humid silk, contributing to the observed diffusion processes (Seydel et al., 2011 ). While these studies have advanced our understanding of dragline spider silk’s adaptations to changes in humidity, they focus primarily on MA silk and silkworm silk. No such studies have been conducted on TU silk. Furthermore, they have predominantly focused on bulk properties or average structural changes, leaving critical aspects of spatially resolved structural differences within an individual fiber unexplored. So far, one study has combined wide-angle X-ray scattering and tensile testing on bundles of 400–600 dragline silk fibers to achieve sufficient scattering intensity (Yazawa et al., 2020 ). Another publication focused on tensile testing under various environmental conditions but excludes structural analysis (Vehoff et al., 2007 ). To the best of our knowledge, only one study has combined X-ray diffraction and tensile testing on single spider silk fibers (Glišović et al., 2008 ). This study, however could not resolve local structural differences in individual fibers due to the relatively large spot size of 7 µm, which covers the diameter of an average silk fiber (Glišović et al., 2008 ). Local differences, however, have been described in the literature in dry state, e.g. by applying X-ray nanodiffraction a 1 µm thick skin layer composed of nanofibrils was identified for Argiope bruennichi (Riekel et al., 2017 ). It is therefore essential to study them to gain a full understanding of deformation mechanisms and local influence of humidity. The aim of our study was to elucidate differences in humidity-driven structural and mechanical changes between the two silk types (TU and MA silk) in a highly position-resolved way, in order to gain, for the first time, information on local ultrastructural changes within individual fibers. Furthermore, this study is the first to investigate the poorly studied TU silk using an in-situ-tensile synchrotron experiment. By exploring the spatially resolved structural differences across the diameter of individual fibers and across fiber types, this work provides a deeper understanding of the hierarchical organization and mechanical behavior of spider silk. These results can serve as a template to correlate protein structure to humidity response and mechanics, enabling improved design of biomimetic materials. Results and Discussion We address these questions by employing high-resolution nanobeam X-ray diffraction at synchrotron facilities to map the spatially resolved inner structure of single spider silk fibers during in-situ mechanical stretching under controlled environmental conditions (Figure S1). In brief we applied a stretching cell and a humidity cell to stretch a single silk fiber up to a certain amount at a specific constant level of RH. For details on the experimental setup see section Materials and Methods and Figure S1. Our approach enables discrimination of distinct zones within the fiber (Bulk, Transition, and Edge), providing insights into the ultrastructure of spider silk fibers and possible core-shell arrangements. Based on SAXS intensity maps we defined the three zones as shown in Figure 1. To obtain position-resolved structural information, the SAXS intensity map was used to identify the position and orientation of the fiber within the scan. Coordinates corresponding to lines parallel to the fiber’s long axis were defined, and the scattering intensity along each line was averaged. The averaged lines were then divided into three distinct zones: Edge zone (the outermost 1 µm from each edge of the fiber), Transition zone (the next 1 µm inward from the edge zone), Bulk zone (the remaining central region of the fiber). The zone dimensions were based on a previous research investigating the skin-core morphology of Argiope bruennichi MA silk, which found a 1 µm thick proteinaceous skin layer serving as an elastic sheath (Riekel et al., 2017) . In the following, the results are presented and discussed. i. Mechanical parameters upon RH increase The mechanical properties of TU and MA silk fibers, as determined by tensile testing of individual fibers as described above, were strongly influenced by RH, with both silk types showing a notable reduction in stiffness as RH increased (Figure 2a). Young’s modulus, fitted between 0.2% and 1% strain, decreased at higher RH levels, with the most pronounced drop observed at 65% RH compared to 30% RH. Between 30% and 45% RH, the decrease was more pronounced for TU silk than for MA silk, while both exhibited very pronounced reductions between 45% and 65% RH. Similarly, yield strain, determined using the 0.2% offset method, was higher at 30% RH than at 65% RH for both silk types, with TU silk showing a more pronounced decrease (Figure 2b). Moderate reductions in yield strain were also observed between 45% and 65% RH for both silk types. Yield stress followed the same trend, with significant decreases as RH increased, particularly between 30% and 65% RH (Figure 2c). Maximum stress and strain at break were not evaluated, as the stretching was manually interrupted at predefined extension values. These changes align with previous studies that have shown softening of MA silk under wet conditions (Glišović et al., 2008; Vehoff et al., 2007). The reduction in stiffness can be attributed to the disruption of hydrogen bonds in the amorphous regions of the silk, which increases molecular mobility (Cohen et al., 2021). Water molecules act as a plasticizer, enhancing chain mobility in the amorphous regions and lowering the energy required for deformation. The absence of supercontraction means that the silk remained below its glass transition temperature and below a certain critical humidity (23 °C and up to 65% RH)(Plaza et al., 2006). Consistent with this, we did not observe any supercontraction in our experiments with both types of silk fibers. The distinct responses of TU and MA silk to RH reflect differences in their structural composition and biological roles (Peter et al., 2025). TU silk exhibited greater sensitivity to humidity, with more pronounced reductions in mechanical properties compared to MA silk. We infer, that at least part of this effect can be attributed to the crystal structure and the size and arrangement of nanocrystals. Variations in the amorphous matrix may also play a role, potentially allowing for greater water infiltration in TU silk. Biologically, TU silk serves in the egg sac of spiders as a protective layer against humidity and temperature fluctuations (Ewunkem & Agee, 2022; Peter et al., 2025). In contrast MA silk ­­­- optimized for high tensile strength and serving as a lifeline for the spider (Blackledge, 2012) - showed a more stable response to changes in RH, likely due to its more robust and compact crystal structure. i Ultrastructural parameters upon extension Ultrastructural parameters were derived by analysing the 2D scattering patterns from the experiments (Figure 3). High-resolution nanobeam X-ray diffraction revealed distinct trends in structural parameters for TU and MA silk under extension, with notable differences between the two silk types and across RH levels. These trends provide insights into the hierarchical structure and strain-induced changes of the silks. The unit cell of spider silk can be described as a nearly orthorhombic structure. In a fiber, the crystallites’ c axis aligns with the direction of the fiber axis while the a- and b-axes are randomly oriented around the fiber axis (Marsh et al., 1955; Warwicker, 1956). The unit cell parameters of the two different silk types were examined as a function of extension, RH and zone (Edge, Transition and Bulk) (Figure 4). Among these, the b parameter, which reflects the intersheet spacing within β-sheet nanocrystals, showed particularly notable differences between TU and MA silk when exposed to strain under varying RH conditions, and it was higher for TU silk (Figure 4b). We also observed that the b parameter of TU silk was remarkably sensitive to strain, showing distinct changes with extension: at 30% RH, the b parameter decreased for all zones, whereas for higher RH (45% and 65%) the same parameter increased under extension in the Edge zone. In contrast, the b parameter of MA silk remained relatively stable. This difference suggests that TU silk’s nanocrystals are more susceptible to water infiltration, which may disrupt hydrogen bonds between the β-sheets of the nanocrystals. The sensitivity of the b parameter to strain was more pronounced in TU silk, particularly at higher RH levels. This demonstrates that TU silk undergoes greater molecular alignment and structural changes even in the crystalline domains under strain, consistent with its role in egg sac construction, where flexibility and extensibility are critical. As reported in our previous work (Peter et al., 2025), the b parameter reflecting the intersheet dimensions of the nanocrystallites is larger in TU silk than in MA silk, which can be attributed to larger side-chain amino-acids in TU silk’s nanocrystals (Tian & Lewis, 2006). This structural difference likely contributes to the greater extensibility of TU silk. We also performed statistical analysis (Figure S2, S3). We analyzed the behavior of the parameters upon extension using a linear weighted least-squares (WLS) regression fit (Figure S2). For the unit cell parameter b (Figure 4 b), the difference between TU and MA silk at 30% RH included significant decreases for all zones for TU silk and non-significant increases in case of MA silk. In TU silk, there is a trend of higher b values in the Edge zone, predominantly at high RH. The unit cell parameter a (Figure 4 a) remained fairly constant with only small absolute changes. However, statistical analysis showed that the parameter decreased slightly with increasing extension for both TU and MA silk, but the trend was less significant at higher RH levels (Figure S2). This suggests that strain-induced structural changes in the interchain direction upon extension are less pronounced under humid conditions (Figure 4 a). The unit cell parameter c (Figure 4 c), which corresponds to the fiber axis, increased with extension for both silk types, with limited statistical significance due to the weak (002) reflection and corresponding Fit uncertainty (Figure S4). This is consistent with findings from Glišović et al. (2008), who observed a nearly linear correlation between the c value and strain in dragline silk (Glišović et al., 2008). The Herman’s orientation parameter is a measure of the degree of molecular alignment within a fiber (De Volder et al., 2014), with values ranging from 0 (completely random orientation) to 1 (perfect alignment along the fiber axis). This parameter provides insights into how the crystalline and amorphous regions of the silk align under mechanical strain. For TU silk, the Herman’s orientation parameter increased with extension, with significant increases observed at 30% RH in the Transition and Edge zones. This indicates that molecular alignment is enhanced under low-humidity conditions. In contrast, MA silk exhibited mixed trends: a non-significant increase at 30% RH, a non-significant decrease at 45% RH, and variable trends at 65% RH. These findings align with previous studies on MA silk, such as Glišović et al. (2008), who reported little to no dependency of crystalline orientation on strain for MA silk at moderate RH levels (Glišović et al., 2008). The significant increases in molecular alignment for TU silk highlight its dynamic structural response to strain, particularly in the Transition and Edge zones. The L 020 particle size did not exhibit significant trends for either silk type, consistent with previous findings for MA silk at 50% RH (Glišović et al., 2008). However, L 210 showed decreasing trends, with significant decreases for TU silk at 30% and 45% RH and for MA silk in some zones at the same RH levels. This may reflect unfolding or splitting of β-sheet crystallites along the hydrogen bond direction under strain (Glišović et al., 2008) or might be the effect of transverse contraction under applied strain. The lattice spacing d 210 generally decreased with extension for both silk types, indicating transverse contraction of the crystalline structure under strain. For TU silk, this trend was highly significant at 30% RH, less significant at 45% RH, and non-significant at 65% RH. For MA silk, d 210 showed a highly significant decrease at 30% RH, significant decreases in the Edge zone at 45% RH, and no significant trends elsewhere. Unlike previous studies that reported no change in peak positions under strain (Glišović et al., 2008; Yazawa et al., 2020), our findings suggest densification of the silks’ crystalline structure under mechanical strain, which is less pronounced at higher humidity levels, i.e. mitigated by humidity. For TU silk this densification is accompanied by increased reorientation of crystallites. ii. Ultrastructural parameters upon RH increase When analyzing the trends of structural parameters with increasing RH, distinct differences between TU and MA silk emerged, providing additional insights into their structural changes under varying environmental conditions (Figure S3). For TU silk, the unit cell parameter b is highly dependent on changes in RH. The intersheet spacing (b) increases with increasing RH across all zones. In contrast, MA silk shows no clear trend (Figure S3). One possible explanation could be the distinct amino-acid profile of TU silk. β-sheets are formed based on structural domains like AAQAASAA, AAQAA, and AASQAA (Tian & Lewis, 2006). This leads to larger spacing between the β-sheets compared to the poly-A n /(GA) n model of MA silk and therefore to a more hydrophilic character of TU silk (Hu et al., 2005). This fact may allow water molecules to enter and increase the distance between the β-sheets even more. For MA silk, the crystallinity parameter (Figure S5) showed a general decreasing trend with increasing RH, with some significant decreases observed (Figure S3). This suggests that the crystalline regions of MA silk become less ordered under humid conditions, potentially due to water infiltration disrupting hydrogen bonds. In contrast, no clear trend was observed for TU silk, indicating that its crystallinity is less affected by RH. These findings differ from previous studies, which reported constant crystallinity for MA silk under varying RH conditions (Yazawa et al., 2020). The orientation parameter (Figure S6) for MA silk exhibited a trend of increasing values with increasing RH, with significant increases in the Bulk and Edge zones (Figure S3). These contrasts previous studies reporting a decrease in orientation with increasing RH for MA silk (Lefèvre et al., 2009; Yazawa et al., 2020). For TU silk, the trends in orientation were mixed and non-significant, suggesting, that its molecular alignment is less sensitive to changes in RH. The increase in orientation parameter for MA silk with RH may reflect a structural adaptation to maintain alignment of nanocrystallites under humid conditions, which could contribute to its mechanical stability. The L 020 particle size (Figure S7) for MA silk exhibited an increasing trend with RH, with a significant increase in the Bulk zone at 2% extension (Figure S3). This suggests that the crystalline regions of MA silk expand slightly under humid conditions, possibly due to water-induced swelling. For TU silk, L 020 showed a decreasing trend in the Bulk zone, with significant decreases at 2% and 8% extension. This is noteworthy, since the b parameter significantly increases in TU-silk at higher RH, which would suggest a greater particle size in the intersheet direction. However, as the Scherrer width does not directly provide the particle size but the length of coherently scattering domains in a certain crystal direction, the decrease in L 210 points towards a loss of order upon increasing RH. This corresponds well with the observation of greater swelling in b direction in TU silk than in MA silk, which could cause disorder on the molecular scale. The L 210 particle size (Figure S8) for TU silk showed an overall increasing trend with RH, with significant increases at 10% extension in the Edge zone and at 0% extension in the Transition and Edge zones (Figure S3). This suggests that the β-sheet crystallites in TU silk may reorganize under humid conditions. Previous studies reported no dependency of L 210 particle size on RH for MA silk (Glišović et al., 2008), which aligns with the mixed trends observed in this study. The lattice spacing d 210 (Figure S9) for TU silk generally increased with RH, with significant increases at 0% extension in the Bulk zone and at 0%, 6%, and 10% extension in the Transition zone (Figure S3). This trend suggests that the crystalline structure of TU silk becomes less dense under humid conditions, likely due to water infiltration. This is also in line with a partly significant increase of unit cell parameter b in all zones with increasing RH for TU silk. For MA silk, d 210 exhibited mixed trends, with some significant decreases at 0% extension, these findings support previous reports of limited dependency of d-spacings on RH for MA silk (Glišović et al., 2008). iii. Trends between different zones The analysis of structural parameters differing across the Bulk, Transition and Edge zones revealed that, for the majority of parameters and conditions, there were no statistically significant differences between the zones. Across all tested combinations of RH and extension steps, the p-values for most parameters exceeded the significance threshold of 0.05. One exception to this overall trend was observed for TU silk and the parameter d 210 at 30% RH and 0% extension, where a significant difference was found between the Edge and Bulk zones. The mean difference between these zones was 0.4758, with a p-value of 0.0205. This localized variation suggests that spatially dependent adaptations may occur under specific environmental conditions. The Edge zone, being more exposed to environmental factors, may experience greater water loss or structural rearrangement, leading to differences in lattice spacing compared to the Bulk zone. However, no significant differences were observed between the Bulk and Transition zones or between the Transition and Edge zones for d 210 or any other parameter. Furthermore, it seems that the crystallinity parameter is decreased in the edge zone compared to the rest for both silk types (Figure S5). Another edge effect we observe in TU silk is an increase in b parameter at higher RH in the edge zone compared to the other zones (Figure 4 b). Despite these exceptions, the overall homogeneity of the studied parameters of the silk structure across the zones was consistent, even under varying RH and extension conditions for both silk types. These findings suggest a relatively homogeneous distribution of the studied structural features within the silk fibers at the measured length scales, aligning with the studies that emphasize the uniform hierarchical structure of silk (Lin et al., 2017; Qiu et al., 2019). This rather homogeneous structural organization contrasts with earlier studies using experimental removal (Sponner et al., 2007; Yazawa et al., 2018), X-ray nanodiffraction (Riekel et al., 2017), imaging (Du et al., 2006), spectroscopic methods (Brown et al., 2011), and computational modeling (Giesa et al., 2011; Keten & Buehler, 2010), which suggest that dragline silk contains a lipid/glycoprotein coat, a skin, and at least one core region (often subdivided into outer and inner components) (Stadlmayr et al., 2025). These layers serve distinct functions: the core (including crystalline β-sheet domains) usually provides strength, stiffness, and energy dissipation, whereas the skin acts as a protective or elastic barrier (Yazawa et al., 2019). In detail, Sponner et al. reported based on biochemical analysis that MA silk consists of 5 different layers, from which the three layers described together as the shell were described to be no larger than 250 nm together (Sponner et al., 2007). An outer spidroin protein core with mainly MaSp1 and a thickness of up to 400 nm was compared to an inner core with a mixture of MaSp1 and MaSp2 (Sponner et al., 2007). These differences in protein composition can be only hardly detected by X-ray nanodiffraction and glycoprotein and lipid layers are too small in dimension to be detected. However, by applying X-ray nanodiffraction Riekel et al. (2017) discovered a 1 µm thick skin layer composed of nanofibrils. Our results indicate that such differentiation may not be as pronounced as previously suggested, at least for the structural parameters and length scales analyzed in this study. This suggests that at least for the silk type investigated in this study - the hierarchical structure is more uniformly distributed than generally assumed. Nevertheless, a clear understanding of the hierarchical structure of silk is still lacking, since the observed features and length scales depend very much on the sample preparation and experimental techniques (Wang & Schniepp, 2019). b. Comparative Insights Between TU and MA Silk The differences observed between TU and MA silk in their response to extension and RH underscore the importance of silk type and environmental conditions in determining mechanical and structural behavior. The differences reflect the unique hierarchical organization and biological roles of the two silk types. 5 gives an overview of the major effects of strain and changes in RH on both silk types. The significant decrease in the b parameter with extension for TU silk at 30% RH contrasts with the non-significant positive trend for MA silk. This suggests that TU silk nanocrystals which are characterized by larger side-chain amino acids (Peter et al., 2025; Tian & Lewis, 2006), undergo compression and deformation under strain, leading to a reduction in b. In contrast, at higher RH (45% and 65%) we observe an increase in the b parameter upon extension for TU silk, but only in the Edge zone. This is remarkable, since we would normally expect transverse contraction upon longitudinal elongation. The transverse expansion suggests decreased interaction between the β-sheets upon hydration to the point where other deformation mechanisms may occur, such as inter-sheet sliding. We hypothesize that the occurrence of this effect at higher humidity and in the edge zone only might be due to increased exchange of water molecules with the environment in these regions. In contrast, MA silk’s b parameter remains more stable, reflecting its more compact and robust crystalline structure. This stability aligns with MA silk’s role as dragline silk, where high tensile strength and stiffness are prioritized over extensibility. Similarly, the increasing Herman’s orientation parameter with extension for TU silk especially at 30% RH in the Transition and Edge zones indicates enhanced molecular alignment under low-humidity conditions, which may contribute to its superior extensibility. In contrast, MA silk exhibited mixed trends in molecular alignment dependent on extension. Furthermore, we observe generally larger values for the crystallinity parameter in MA silk than for TU silk at higher RH and therefore a relative reduction in the crystallinity parameter at higher RH for TU silk (Figure S5). This might be one reason for the generally higher tensile strength of MA silk compared to TU silk (Peter et al., 2025). Additional differences were observed in particle size and lattice spacing. For example, L 020 showed a decreasing trend for TU silk in the Bulk zone, while MA silk exhibited an increasing trend with RH increase. Given the increase of the b parameter in TU silk, the decrease in L 020 points to a greater disorder in intersheet direction for this type of silk with increasing RH. The lattice spacing d 210 for TU silk generally increased with RH increase, reflecting a loosening of the crystalline structure, whereas MA silk showed mixed trends, consistent with its more stable crystalline organization. These comparative insights provide a deeper understanding of the hierarchical mechanisms underlying the mechanical behavior of spider silk and offer valuable guidance for the design of biomimetic materials. For instance, TU silk’s flexibility and structural changes could inspire the development of dynamic scaffolds for tissue engineering, while MA silk’s stability could inform the design of high-strength fibers for load-bearing applications. c. Limitations and Future Directions While this study provides significant insights into the adaptive mechanisms of spider silk, it is limited by the length scales studied (100–250 nm) and the range of environmental conditions tested. The focus on this specific length scale and the need to average data within pre-defined zones may have constrained our ability to capture finer structural details, such as molecular-level interactions within the crystalline and amorphous regions. Computational molecular modeling could help address this limitation by providing a more detailed understanding of these interactions. However, challenges remain, as the full composition of the amorphous matrix and the protein sequences may not yet be fully characterized, complicating the modeling process. Additionally, the environmental conditions tested in this study were limited to variations in RH at a constant temperature. Other factors known to influence spider silk’s properties, such as temperature fluctuations, UV exposure, and cyclic environmental stress, were not explored (Wharton et al., 2025). Investigating these effects could provide a more complete picture of spider silk’s adaptability and durability. Additionally, studying other silk types, such as flagelliform silk or aciniform silk, and their responses to dynamic environmental changes could further expand our understanding of the diverse mechanical and structural properties of spider silk. These limitations may have influenced the generalizability of the findings to other silk types or real-world conditions. These limitations may have influenced the generalizability of the findings to other silk types and species or actual environmental conditions. Future research should address these limitations by exploring finer length scales to capture additional structural details. Combining nanobeam X-ray diffraction with complementary techniques, such as neutron scattering or Raman spectroscopy, could provide a more comprehensive understanding of silk’s hierarchical structure and its dynamic responses to environmental stimuli. For instance, neutron scattering could offer insights into hydrogen bonding and water interactions, while Raman spectroscopy could reveal molecular-level changes in silk’s chemical structure under strain or varying humidity. Expanding the range of environmental conditions tested is another critical direction for future research. Investigating the effects of temperature, UV exposure, and cyclic environmental changes could provide a more complete picture of spider silk’s adaptability and durability. Additionally, studying other silk types, such as flagelliform, aciniform silk, or attaching silk, and their responses to dynamic environmental changes could further expand our understanding of the diverse mechanical and structural properties of spider silk. Addressing the limitations of our study and pursuing these future directions will deepen our understanding of spider silk’s remarkable adaptability to environmental changes and enhance its potential for biomimetic applications. Conclusion This study provides new insights into the hierarchical structure and environmental adaptability of spider silk, with a focus on the distinct responses of tubuliform (TU) and major ampullate (MA) silk from Trichonephila ( T.) inaurata’s egg sac to mechanical strain and relative humidity (RH) (Fig. 5 ). Using high-resolution nanobeam X-ray diffraction at synchrotron facilities, we revealed that increasing RH significantly reduces Young’s modulus, yield strain, and yield stress for both silk types, with TU silk exhibiting a slightly higher sensitivity to humidity. Structural analysis highlighted key differences, such as the pronounced adaptability of TU silk’s nanocrystals and molecular alignment under strain, compared to the more stable crystalline organization of MA silk. Another example is the intersheet spacing being highly sensitive to strain and RH for TU silk. These differences reflect the unique biological roles of TU silk in egg sac construction and MA silk as dragline silk, emphasizing their specialized mechanical and structural properties. Our findings expand the understanding of spider silk’s hierarchical mechanisms, particularly for the underexplored TU silk, and challenge some previously reported trends for MA silk. Notably, we observe predominantly homogeneous ultrastructural parameters across the diameter of the silk fiber. Therefore, no clear evidence of core-shell differences was observed at the studied length scales and for the studied parameters, suggesting that T. inaurata’s MA and TU silk’s hierarchical structure might be more homogeneous than previously hypothesized. These findings challenge earlier models proposing distinct functional roles for the core and shell regions of spider silk fibers. Future studies should explore finer structural details and additional environmental factors, such as temperature and UV exposure, to further unravel the remarkable adaptability of spider silk. Materials and Methods a. Preparation of Samples The samples used in this study were prepared from egg sac silk of T. inaurata spiders. The housing of the spiders, as well as the harvesting of the silk, were conducted following previous protocols (Peter et al., 2025 ; Stadlmayr et al., 2024 ). Two types of silk fibers were extracted from the egg sacs: MA silk and TU silk. The extraction process was performed under a digital microscope (Keyence VHX-5000) to ensure precision and minimize damage to the fibers as previously described (Peter et al., 2025 ). Individual silk fibers were carefully glued onto 3D-printed plastic support frames using superglue. To avoid pre-stressing the fibers, minimal force was applied during the manipulation and mounting process. The length and diameter of each fiber were measured using a digital microscope (Keyence VHX-5000). The diameter was determined by averaging fifteen measurements taken at different positions along the length of the fiber to ensure accuracy and account for any variability in thickness. The gauge length was approx. 4 mm for each sample. After preparation, the samples were stored in small plastic containers at ambient temperature and humidity until further use. b. Experimental Setup and Measurement procedure To investigate spatially-resolved ultrastructural changes in single threads of spider silk during stretching, two synchrotron experiments were conducted using X-ray nanodiffraction in transmission mode. The general experimental setup, including the incoming X-ray beam, the in-situ stretching setup, a flight tube, and detector, is shown in Figure S1 . The first experiment was performed at the NanoMAX beamline (MAX IV) (Carbone et al., 2022 ; Johansson et al., 2021 ) under ambient conditions (conditions were measured to be 22°C, 30% RH), while the second was conducted at the nanofocus beamline ID13 (ESRF) using a custom-designed humidity cell to test samples at varying RH levels (30%, 45%, and 65% RH). The customised humidity controller uses a PID algorithm to dynamically mix dry and humidified nitrogen at room temperature, based on feedback from a Sensirion humidity sensor installed few millimeters below the silk fibres.In both experiments, high-speed mesh scans were employed, scanning the fibers line by line perpendicular to their long axis with continuously moving motors (fly scan) and a line spacing of 1 µm to minimize radiation damage while maintaining high spatial resolution. For this, we evaluated if the ultrastructural parameters changed upon increasing scanning time. There was no major change of parameters when comparing them from one scan line to the next one. Therefore, we assume that we scanned fast enough and chose a far enough distance between the scan lines (1 µm) so we could avoid major structural changes due to radiation damage. At NanoMAX, the fibers were mounted perpendicular to the monochromatic X-ray beam (λ = 0.08856 nm, 14 keV, 5.57×10 10 photons/s). The beam had a full width at half maximum (FWHM) of 100 nm, and nano-diffraction measurements were performed with exposure times of 10–20 ms and step sizes of 100–250 nm during continuous movement (fly scan). The sample-to-detector distance (0.1496 m) was calibrated using an LaB 6 (NIST 660C) standard and processed with the pyFAI software package. The detector used was a single photon counting pixel detector (Pilatus3X 1M, Dectris, Switzerland). The specific in-situ stretching setup, including a schematic sketch of the nanoindenter device (Alemnis AG, Thun, Switzerland)) used for stretching the fiber and the clamping mechanism for the 3D-printed sample support, is shown in Fig. 1 b. The sides of the plastic supports were cut using a hot thread cutter (TZ1300 Thread Zap II, The Beadsmith), allowing the load to be exclusively carried by the fiber. Once the load was exclusively carried by the fiber, alignment corrections were applied using the nanoindenter motors. Stretching was performed incrementally, starting with small 0.01 mm adjustments to detect a slight force increase. Afterwards the fibers were stretched in 2% extension steps, where time for stretching was calculated based on the initial length of each fiber. The stretching speed was set to 50 µm/s, and a mesh scan was initiated immediately after each extension. Stress relaxation was observed during scanning. New fibers were used for each extension step. Extensions were performed up to a maximum of 18%, and force-displacement data was recorded to calculate mechanical parameters such as Young’s modulus. Higher extensions were not possible since a large amount of fibers ruptured at higher extensions. Stress-strain curves of TU and MA silk are shown in Figure S10. At ID13, the fibers were mounted similarly, perpendicular to the monochromatic X-ray beam (λ = 0.08157 nm, 15.2 keV, 1.01×10 11 photons/s). Nano-diffraction measurements were performed with a beam FWHM of 100 nm, exposure times of 7–20 ms, and step sizes of 100–250 nm perpendicular to the fiber’s long axis and 1µm step size between lines. The sample-to-detector distance (0.1381 m) was calibrated using an Al 2 O 3 standard and processed with pyFAI. The detector used was a single photon counting pixel detector (EigerX 4M, Dectris, Switzerland). The setup including a custom-made stretching cell with a humidity-controlled chamber, as shown in Figure S1 c, was used to test fibers at 30%, 45%, and 65% RH. The same 3D-printed sample supports and hot thread cutter were used as in the NanoMAX experiment. Stretching followed the same procedure as at NanoMAX, with incremental 2% extensions and mesh scans performed after each step. Force-displacement data was recorded to analyze mechanical behavior under varying humidity conditions. c. Data processing and analysis The mechanical properties of the silk fibers, including Young’s modulus, yield stress and yield strain, were determined from force-displacement data obtained during tensile testing. The force-displacement data was converted into stress-strain data by incorporating the diameter and length of the fiber. The original cross-sectional area of the fiber was calculated using the measured diameter, assuming a circular cross-section. In our previous research (Peter et al., 2025 ), we found, that TU silk shows an irregular shaped fiber surface with longitudinal grooves on the surface. For the analysis of the mechanical parameters, we assumed a circular cross section also for TU silk. True stress and true strain were also calculated, with true stress accounting for the reduction in cross-sectional area during deformation and true strain calculated as the natural logarithm of the engineering strain increment. The stress-strain data was filtered to isolate the linear elastic region, defined as the strain range between 0.2% and 1%. This range was chosen to ensure accurate determination of Young’s modulus while avoiding non-linear effects. Linear regression was applied to the filtered data to calculate the slope of the stress-strain curve, representing Young’s modulus. The standard error of the slope was calculated to estimate the uncertainty in Young’s modulus. The 0.2% offset method was used to determine the yield stress and yield strain. A parallel line to the linear fit, offset by 0.2% strain, was calculated, and the intersection of this offset line with the stress-strain curve was identified as the yield point. For statistical analysis a 1-way ANOVA (Origin 2025) and a mean value comparison by a Tukey test were performed. Scattering data was generated as HDF5 files, and integration was performed using the python libraries pyFAI (Kieffer et al., 2025 ) for ESRF data and azint (Jensen et al., 2022 ) for MAX IV data. The number of data points was set to 1000 in the q-range and 360 in the azimuthal range. The scattering patterns from each zone were averaged, and background intensity was calculated from areas without fiber. The background was subtracted, and the intensity was normalized against the incoming beam intensity. The resulting Intensity versus q plots were fitted using the lmfit library in Python. For the analysis of the (020), (210), (002) and (x)-peaks, the azimuthal range was selected based on the caked scattering intensity map. We named the (x)-peak according to our previous research on TU and MA silk (Peter et al., 2025 ). The peaks were fitted using Gaussian functions, where some are referred to as Short-Range-Order (SRO) peaks, and the rest are referred to as Bragg peaks based on previous fits on experimental data of spider silk (Riekel et al., 2019 ; Sampath et al., 2012 ) (Fig. 3 ). The lattice spacing d was calculated using Eq. 1 , where q is the center position of the respective peak. Particle size was determined using the Scherrer formula with k = 0.9, and a crystallinity parameter was estimated by fitting the overall azimuth-averaged Intensity versus q curve (Figure S11). $$\:d=2\pi\:/q$$ 1 Herman’s orientation parameter f was derived by fitting the scattering Intensity versus the azimuth for specific q ranges corresponding to the (020) and (210) peaks (Figure S12). Information on the crystallite orientation about the fibers' long axis was obtained from the peaks’ FWHM (full width at half maximum). Herman’s orientation function f (Eq. 2 ) ranges from 0 (no preferred orientation) to 1 (perfect alignment) for crystals along the measurement direction (Du et al., 2006 ). The angle between the c-axis and the fiber axis is referred to as Φ (Blamires et al., 2018 ). $$\:f=(3⟨{cos}^{2}\varphi\:⟩-1)/2\:$$ 2 Due to the orientation of the (020) and (210) reflections in the equatorial plane Grubb et al. defined the following relationship (3) with A = 0.8 and B = 1.2 since (210) is 65 ° from (020) (Grubb & Jelinski, 1997 ). Since the reflections are measured perpendicular to the fiber axis, φ can be referred to as 90° - Φ. Therefore, 〈cos 2 ϕ〉 = 1 - 〈cos 2 φ〉. Since the fitted functions are Gaussians, 〈cos 2 φ 1 〉 can be approximated by {cos(0.4FWHM 210 )}x 2 and 〈cos 2 φ 1 〉 by {cos(0.4FWHM 020 )} 2 (Du et al., 2006 ). $$\:⟨{cos}^{2}\varphi\:⟩=1-A⟨{cos}^{2}{\varphi\:}_{1}⟩-B⟨{cos}^{2}{\varphi\:}_{2}⟩$$ 3 Exemplary scattering data with fitted functions for all other parameters that are not shown in Fig. 3 can be found in the supplementary information. d. Statistical analysis The statistical analysis was performed using Python (version 3.9). The analysis aimed to identify trends in key parameters as a function of extension, RH, and measurement zones (Bulk, Transition, and Edge). Parameters of interest included Herman’s orientation factor, lattice spacings, crystallinity parameter, and the unit cell parameters. For each parameter, error values originating from fitting were incorporated into the analysis. To analyze trends as a function of extension, weighted least-squares (WLS) regression was applied for each silk type, RH, and measurement zone. WLS regression was chosen because it accounts for heteroscedasticity in the data by weighting each data point according to the inverse square of its associated error. This ensures that data points with smaller errors contribute more to the regression model, improving the reliability of the results. The weights were based on the inverse square of the parameter’s error. For each regression, the slope, p-value, and direction of trend (increasing or decreasing) were extracted. Statistical significance was determined based on the p-value, with thresholds of p < 0.05 (significant), p < 0.01 (moderately significant), and p < 0.001 (highly significant). Similarly, trends as a function of RH were analyzed for each silk type, extension, and measurement zone and WLS regression was applied. To assess potential differences between the zones for various parameters, a statistical analysis was conducted. The dataset included measurements for multiple parameters across different combinations of RH and extension steps. For each parameter, the data was grouped by zone, and statistical tests were performed to determine whether significant differences existed between the zones. The data was grouped by RH and extension step, and statistical tests were applied to each unique combination. A one-way ANOVA was used to test for differences in means between the zones when the data met the assumptions of normality and homogeneity of variance. For cases where these assumptions were not met, or where data was limited, a non-parametric Kruskal-Wallis test was used as an alternative. A significance level of 0.05 was applied, and p-values below this threshold were considered indicative of statistically significant differences between zones. For cases where significant differences were detected in the ANOVA, a post-hoc Tukey’s HSD test was performed to identify which specific pairs of zones differed. Abbreviations MA Major-ampullate RH Relative humidity SAXS Small-angle X-ray scattering SRO Short-range order T. Trichonephila TU Tubuliform WAXS Wide-angle X-ray scattering WLS Weighted least-squares Declarations Acknowledgements The authors would like to thank Maja Vasiljevic, Arno Frank, and Charbel Sakr for their assistance during the experiments conducted at the synchrotron facilities. Their support and expertise were instrumental in the successful completion of this work. Part of the experiments were performed on beamline ID13 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France We acknowledge the ESRF for the provision of synchrotron radiation, and we would like to thank the beamline staff for assistance and support in using the beamline. We acknowledge the MAX IV Laboratory for beamtime on the NanoMAX beamline under proposal 20230683. Research conducted at MAX IV, a Swedish national user facility, is supported by Vetenskapsrådet (Swedish Research Council, VR) under contract 2018-07152, Vinnova (Swedish Governmental Agency for Innovation Systems) under contract 2018-04969 and Formas under contract 2019-02496. Funding Declaration This research was funded in whole by the Austrian Science Fund (FWF) [10.55776/P33613]. Data Availability Statement Data is provided online under DOI: Competing Interest Declaration The authors declare no competing interests. References Altman, G. H., Diaz, F., Jakuba, C., Calabro, T., Horan, R. L., Chen, J., Lu, H., Richmond, J., & Kaplan, D. L. (2003). Silk-based biomaterials. Biomaterials , 24 (3), 401-416. https://doi.org/10.1016/s0142-9612(02)00353-8 Bergmann, F., Stadlmayr, S., Millesi, F., Zeitlinger, M., Naghilou, A., & Radtke, C. (2022). The properties of native Trichonephila dragline silk and its biomedical applications. Biomaterials Advances , 140 , 213089. https://doi.org/https://doi.org/10.1016/j.bioadv.2022.213089 Blackledge, T. A. (2012). Spider silk: a brief review and prospectus on research linking biomechanics and ecology in draglines and orb webs. The Journal of Arachnology , 40 (1), 1-12, 12. https://doi.org/10.1636/M11-67.1 Blamires, S. J., Nobbs, M., Martens, P. J., Tso, I. M., Chuang, W.-T., Chang, C.-K., & Sheu, H.-S. (2018). Multiscale mechanisms of nutritionally induced property variation in spider silks. PLoS One , 13 (2), e0192005. https://doi.org/10.1371/journal.pone.0192005 Braxton, T., Holland, C., & Greco, G. (2025). The circular argument behind spider and silkworm silk mechanical properties. Materials & Design , 260 , 115224. https://doi.org/https://doi.org/10.1016/j.matdes.2025.115224 Brown, C. P., MacLeod, J., Amenitsch, H., Cacho-Nerin, F., Gill, H. S., Price, A. J., Traversa, E., Licoccia, S., & Rosei, F. (2011). The critical role of water in spider silk and its consequence for protein mechanics [10.1039/C1NR10502G]. Nanoscale , 3 (9), 3805-3811. https://doi.org/10.1039/C1NR10502G Carbone, D., Kalbfleisch, S., Johansson, U., Bjorling, A., Kahnt, M., Sala, S., Stankevic, T., Rodriguez-Fernandez, A., Bring, B., Matej, Z., Bell, P., Erb, D., Hardion, V., Weninger, C., Al-Sallami, H., Lidon-Simon, J., Carlson, S., Jerrebo, A., Norsk Jensen, B., . . . Roslund, L. (2022). Design and performance of a dedicated coherent X-ray scanning diffraction instrument at beamline NanoMAX of MAX IV. Journal of Synchrotron Radiation , 29 (3), 876-887. https://doi.org/doi:10.1107/S1600577522001333 Cohen, N. (2023). The underlying mechanisms behind the hydration-induced and mechanical response of spider silk. Journal of the Mechanics and Physics of Solids , 172 , 105141. https://doi.org/10.1016/j.jmps.2022.105141 Cohen, N., Levin, M., & Eisenbach, C. D. (2021). On the Origin of Supercontraction in Spider Silk. Biomacromolecules , 22 (2), 993-1000. https://doi.org/10.1021/acs.biomac.0c01747 De Volder, M., Park, S., Tawfick, S., & Hart, A. J. (2014). Strain-engineered manufacturing of freeform carbon nanotube microstructures. Nature Communications , 5 (1), 4512. https://doi.org/10.1038/ncomms5512 Du, N., Liu, X. Y., Narayanan, J., Li, L., Lim, M. L., & Li, D. (2006). Design of superior spider silk: from nanostructure to mechanical properties. Biophys J , 91 (12), 4528-4535. https://doi.org/10.1529/biophysj.106.089144 Ewunkem, A. J., & Agee, K. (2022). Spider Parental Care and Awe-Inspiring Egg Sac (Cocoon). International Journal of Zoology , 2022 , 6763306. https://doi.org/10.1155/2022/6763306 Giesa, T., Arslan, M., Pugno, N. M., & Buehler, M. J. (2011). Nanoconfinement of Spider Silk Fibrils Begets Superior Strength, Extensibility, and Toughness. Nano Letters , 11 (11), 5038-5046. https://doi.org/10.1021/nl203108t Glišović, A., Vehoff, T., Davies, R. J., & Salditt, T. (2008). Strain Dependent Structural Changes of Spider Dragline Silk. Macromolecules , 41 (2), 390-398. https://doi.org/10.1021/ma070528p Gosline, J. M., Guerette, P. A., Ortlepp, C. S., & Savage, K. N. (1999). The mechanical design of spider silks: from fibroin sequence to mechanical function. J Exp Biol , 202 (Pt 23), 3295-3303. https://doi.org/10.1242/jeb.202.23.3295 Grubb, D. T., & Jelinski, L. W. (1997). Fiber Morphology of Spider Silk: The Effects of Tensile Deformation. Macromolecules , 30 (10), 2860-2867. https://doi.org/10.1021/ma961293c Hu, X., Lawrence, B., Kohler, K., Falick, A. M., Moore, A. M. F., McMullen, E., Jones, P. R., & Vierra, C. (2005). Araneoid Egg Case Silk: A Fibroin with Novel Ensemble Repeat Units from the Black Widow Spider, Latrodectus hesperus. Biochemistry , 44 (30), 10020-10027. https://doi.org/10.1021/bi050494i Jensen, A. B., Christensen, T. E. K., Weninger, C., & Birkedal, H. (2022). Very large-scale diffraction investigations enabled by a matrix-multiplication facilitated radial and azimuthal integration algorithm: MatFRAIA. Journal of Synchrotron Radiation , 29 (6), 1420-1428. https://doi.org/doi:10.1107/S1600577522008232 Johansson, U., Carbone, D., Kalbfleisch, S., Bjorling, A., Kahnt, M., Sala, S., Stankevic, T., Liebi, M., Rodriguez Fernandez, A., Bring, B., Paterson, D., Thanell, K., Bell, P., Erb, D., Weninger, C., Matej, Z., Roslund, L., Ahnberg, K., Norsk Jensen, B., . . . Vogt, U. (2021). NanoMAX: the hard X-ray nanoprobe beamline at the MAX IV Laboratory. Journal of Synchrotron Radiation , 28 (6), 1935-1947. https://doi.org/doi:10.1107/S1600577521008213 Keten, S., & Buehler, M. J. (2010). Nanostructure and molecular mechanics of spider dragline silk protein assemblies. Journal of the Royal Society Interface , 7 (53), 1709-1721. https://doi.org/10.1098/rsif.2010.0149 Kieffer, J., Orlans, J., Coquelle, N., Debionne, S., Basu, S., Homs, A., Santoni, G., & De Sanctis, D. (2025). Application of signal separation to diffraction image compression and serial crystallography. Journal of Applied Crystallography , 58 (1), 138-153. https://doi.org/doi:10.1107/S1600576724011038 Kornfeld, T., Nessler, J., Helmer, C., Hannemann, R., Waldmann, K. H., Peck, C. T., Hoffmann, P., Brandes, G., Vogt, P. M., & Radtke, C. (2021). Spider silk nerve graft promotes axonal regeneration on long distance nerve defect in a sheep model. Biomaterials , 271 , 120692. https://doi.org/10.1016/j.biomaterials.2021.120692 Lechner, A., Trossmann, V. T., & Scheibel, T. (2022). Impact of Cell Loading of Recombinant Spider Silk Based Bioinks on Gelation and Printability. Macromolecular Bioscience , 22 (3), 2100390. https://doi.org/https://doi.org/10.1002/mabi.202100390 Lefevre, T., & Auger, M. (2016). Spider silk as a blueprint for greener materials: a review. International Materials Reviews , 61 (2), 127-153. https://doi.org/10.1080/09506608.2016.1148894 Lefèvre, T., Pézolet, M., Cooper, G., Cruz, D. H., West, M. M., Obst, M., Hitchcock, A. P., Karunakaran, C., & Kaznatcheev, K. (2009). Mapping molecular orientation in dry and wetNephila clavipesdragline spider silk. Journal of Physics: Conference Series , 186 , 012089. https://doi.org/10.1088/1742-6596/186/1/012089 Li, J., Li, S., Huang, J., Khan, A. Q., An, B., Zhou, X., Liu, Z., & Zhu, M. (2022). Spider Silk-Inspired Artificial Fibers. Advanced Science , 9 (5), 2103965. https://doi.org/https://doi.org/10.1002/advs.202103965 Lin, T.-Y., Masunaga, H., Sato, R., Malay, A. D., Toyooka, K., Hikima, T., & Numata, K. (2017). Liquid Crystalline Granules Align in a Hierarchical Structure To Produce Spider Dragline Microfibrils. Biomacromolecules , 18 (4), 1350-1355. https://doi.org/10.1021/acs.biomac.7b00086 Marsh, R. E., Corey, R. B., & Pauling, L. (1955). The structure of tussah silk fibroin (with a note on the structure of [beta]-poly-l-alanine). Acta Crystallographica , 8 (11), 710-715. https://doi.org/doi:10.1107/S0365110X5500217X Naghilou, A., Peter, K., Millesi, F., Stadlmayr, S., Wolf, S., Rad, A., Semmler, L., Supper, P., Ploszczanski, L., Liu, J., Burghammer, M., Riekel, C., Bismarck, A., Backus, E. H. G., Lichtenegger, H., & Radtke, C. (2023). Insights into the material properties of dragline spider silk affecting Schwann cell migration. International Journal of Biological Macromolecules , 244 , 125398. https://doi.org/https://doi.org/10.1016/j.ijbiomac.2023.125398 Nova, A., Keten, S., Pugno, N. M., Redaelli, A., & Buehler, M. J. (2010). Molecular and Nanostructural Mechanisms of Deformation, Strength and Toughness of Spider Silk Fibrils. Nano Letters , 10 (7), 2626-2634. https://doi.org/10.1021/nl101341w Peter, K., Stadlmayr, S., Naghilou, A., Ploszczanski, L., Hofmann, M., Riekel, C., Liu, J., Burghammer, M., Gusenbauer, C., Konnerth, J., Schniepp, H. C., Rennhofer, H., Sinn, G., Radtke, C., & Lichtenegger, H. C. (2025). Exploring the Unique Properties and Superior Schwann Cell Guiding Abilities of Spider Egg Sac Silk. ACS Applied Bio Materials , 8 (2), 1307-1319. https://doi.org/10.1021/acsabm.4c01587 Plaza, G. R., Guinea, G. V., Pérez‐Rigueiro, J., & Elices, M. (2006). Thermo‐hygro‐mechanical behavior of spider dragline silk: Glassy and rubbery states. Journal of Polymer Science Part B: Polymer Physics , 44 (6), 994-999. https://doi.org/10.1002/polb.20751 Puranik, N., Tiwari, S., Kumari, M., Yadav, S. K., Dhakal, T., & Song, M. (2025). Advanced Bioactive Polymers and Materials for Nerve Repair: Strategies and Mechanistic Insights. Journal of Functional Biomaterials , 16 (7), 255. https://www.mdpi.com/2079-4983/16/7/255 Qiu, W., Patil, A., Hu, F., & Liu, X. Y. (2019). Hierarchical Structure of Silk Materials Versus Mechanical Performance and Mesoscopic Engineering Principles. Small , 15 (51), 1903948. https://doi.org/https://doi.org/10.1002/smll.201903948 Radtke, C., Allmeling, C., Waldmann, K. H., Reimers, K., Thies, K., Schenk, H. C., Hillmer, A., Guggenheim, M., Brandes, G., & Vogt, P. M. (2011). Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep. PLoS One , 6 (2), e16990. https://doi.org/10.1371/journal.pone.0016990 Riekel, C., Burghammer, M., Dane, T. G., Ferrero, C., & Rosenthal, M. (2017). Nanoscale Structural Features in Major Ampullate Spider Silk. Biomacromolecules , 18 (1), 231-241. https://doi.org/10.1021/acs.biomac.6b01537 Riekel, C., Burghammer, M., & Rosenthal, M. (2019). Nanoscale X-Ray Diffraction of Silk Fibers. Frontiers in Materials , 6 . https://doi.org/10.3389/fmats.2019.00315 Rising, A., & Johansson, J. (2015). Toward spinning artificial spider silk. Nat Chem Biol , 11 (5), 309-315. https://doi.org/10.1038/nchembio.1789 Ruiz, V., Jiang, P., Müller, C., Jorge, I., Vázquez, J., Ridruejo, Á., Aznar-Cervantes, S. D., Cenis, J. L., Messeguer-Olmo, L., Elices, M., Guinea, G. V., & Pérez-Rigueiro, J. (2019). Preparation and characterization of Nephila clavipes tubuliform silk gut [10.1039/C9SM00212J]. Soft Matter , 15 (14), 2960-2970. https://doi.org/10.1039/C9SM00212J Sampath, S., Isdebski, T., Jenkins, J. E., Ayon, J. V., Henning, R. W., Orgel, J. P. R. O., Antipoa, O., & Yarger, J. L. (2012). X-ray diffraction study of nanocrystalline and amorphous structure within major and minor ampullate dragline spider silks. Soft Matter , 8 (25), 6713. https://doi.org/10.1039/c2sm25373a Schäfer, A., Vehoff, T., Glišović, A., & Salditt, T. (2008). Spider silk softening by water uptake: an AFM study. European Biophysics Journal , 37 (2), 197-204. https://doi.org/10.1007/s00249-007-0216-5 Seydel, T., Knoll, W., Greving, I., Dicko, C., Koza, M. M., Krasnov, I., & Müller, M. (2011). Increased molecular mobility in humid silk fibers under tensile stress. Physical Review E , 83 (1), 016104. https://doi.org/10.1103/PhysRevE.83.016104 Sponner, A., Vater, W., Monajembashi, S., Unger, E., Grosse, F., & Weisshart, K. (2007). Composition and hierarchical organisation of a spider silk. PLoS One , 2 (10), e998. https://doi.org/10.1371/journal.pone.0000998 Stadlmayr, S., Mautner, A., Bacher, M., Peter, K., Mentler, A., Schulz, S., Lichtenegger, H., Brecker, L., Bismarck, A., Naghilou, A., & Radtke, C. (2025). Holistic Analysis of Material Properties in Phylogenetically Diverse Spider Silks and Their Influence on Cell Adhesion. Advanced Functional Materials , 35 (15), 2415945. https://doi.org/https://doi.org/10.1002/adfm.202415945 Stadlmayr, S., Peter, K., Millesi, F., Rad, A., Wolf, S., Mero, S., Zehl, M., Mentler, A., Gusenbauer, C., Konnerth, J., Schniepp, H. C., Lichtenegger, H., Naghilou, A., & Radtke, C. (2024). Comparative Analysis of Various Spider Silks in Regard to Nerve Regeneration: Material Properties and Schwann Cell Response. Advanced healthcare materials , 13 (8), 2302968. https://doi.org/https://doi.org/10.1002/adhm.202302968 Termonia, Y. (1994). Molecular Modeling of Spider Silk Elasticity. Macromolecules , 27 (25), 7378-7381. https://doi.org/10.1021/ma00103a018 Tian, M., & Lewis, R. V. (2006). Tubuliform silk protein: A protein with unique molecular characteristics and mechanical properties in the spider silk fibroin family. Applied Physics A , 82 (2), 265-273. https://doi.org/10.1007/s00339-005-3433-8 Vehoff, T., Glišović, A., Schollmeyer, H., Zippelius, A., & Salditt, T. (2007). Mechanical Properties of Spider Dragline Silk: Humidity, Hysteresis, and Relaxation. Biophysical Journal , 93 (12), 4425-4432. https://doi.org/https://doi.org/10.1529/biophysj.106.099309 Wang, Q. J., & Schniepp, H. C. (2019). Nanofibrils as Building Blocks of Silk Fibers: Critical Review of the Experimental Evidence. Jom , 71 (4), 1248-1263. https://doi.org/10.1007/s11837-019-03340-y Warwicker, J. O. (1956). The crystal structure of silk fibroins [10.1039/TF9565200554]. Transactions of the Faraday Society , 52 (0), 554-557. https://doi.org/10.1039/TF9565200554 Warwicker, J. O. (1960). Comparative studies of fibroins. II. The crystal structures of various fibroins. J Mol Biol , 2 , 350-362. https://doi.org/10.1016/s0022-2836(60)80046-0 Wharton, D., Weston, N., & Goodacre, S. (2025). Come rain or shine: effects of external conditions on the properties of linyphiid silk [Original Research]. Frontiers in Arachnid Science , Volume 4 - 2025 . https://doi.org/10.3389/frchs.2025.1580992 Work, R. W., & Morosoff, N. (1982). A Physico-Chemical Study of the Supercontraction of Spider Major Ampullate Silk Fibers. Textile Research Journal , 52 (5), 349-356. https://doi.org/10.1177/004051758205200508 Yazawa, K., Malay, A. D., Masunaga, H., Norma-Rashid, Y., & Numata, K. (2020). Simultaneous effect of strain rate and humidity on the structure and mechanical behavior of spider silk. Communications Materials , 1 (1), 10. https://doi.org/10.1038/s43246-020-0011-8 Yazawa, K., Malay, A. D., Masunaga, H., & Numata, K. (2018). Role of Skin Layers on Mechanical Properties and Supercontraction of Spider Dragline Silk Fiber. Macromolecular Bioscience , 19 (3). https://doi.org/10.1002/mabi.201800220 Yazawa, K., Malay, A. D., Masunaga, H., & Numata, K. (2019). Role of Skin Layers on Mechanical Properties and Supercontraction of Spider Dragline Silk Fiber. Macromolecular Bioscience , 19 (3), 1800220. https://doi.org/https://doi.org/10.1002/mabi.201800220 Ye, L., Liu, X., Li, K., Li, X., Zhu, J., Yang, S., Xu, L., Yang, M., Yan, Y., & Yan, J. (2023). A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials. International Journal of Biological Macromolecules , 253 , 127125. https://doi.org/https://doi.org/10.1016/j.ijbiomac.2023.127125 Additional Declarations There is NO Competing Interest. Supplementary Files SIInsitusilksubmissioncommmat.docx Supplemental Material GraphicalAbstract.png Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8335511","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":571314909,"identity":"8108256a-cfa0-493e-bc1a-2dd482bb4ee2","order_by":0,"name":"Helga Lichtenegger","email":"data:image/png;base64,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","orcid":"","institution":"BOKU University","correspondingAuthor":true,"prefix":"","firstName":"Helga","middleName":"","lastName":"Lichtenegger","suffix":""},{"id":571314910,"identity":"29bda0cd-01c2-4b0d-8edb-cb3b10e18395","order_by":1,"name":"Karolina Peter","email":"","orcid":"https://orcid.org/0000-0001-6288-7979","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Karolina","middleName":"","lastName":"Peter","suffix":""},{"id":571314924,"identity":"4f2e84da-05de-4eec-8f3b-541293af9af9","order_by":2,"name":"Michael Sztucki","email":"","orcid":"https://orcid.org/0000-0002-5314-9402","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Sztucki","suffix":""},{"id":571314925,"identity":"7f52d955-ad22-4d35-af32-59b2b597dbe1","order_by":3,"name":"Sebastian Kalbfleisch","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Kalbfleisch","suffix":""},{"id":571314913,"identity":"d8dcba10-f8ed-4d16-bfb7-68fc233b8e07","order_by":4,"name":"Manfred Burghammer","email":"","orcid":"https://orcid.org/0000-0002-2262-3431","institution":"European Synchrotron Radiation Facility","correspondingAuthor":false,"prefix":"","firstName":"Manfred","middleName":"","lastName":"Burghammer","suffix":""},{"id":571314914,"identity":"25ba3dbb-b2a3-49ed-aebd-0fcf791cb607","order_by":5,"name":"Christian Riekel","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Riekel","suffix":""},{"id":571314915,"identity":"b4683965-042f-47b7-b074-c87d32f06da4","order_by":6,"name":"Jiliang Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiliang","middleName":"","lastName":"Liu","suffix":""},{"id":571314916,"identity":"33a37e14-e780-447d-b106-7200faa6139e","order_by":7,"name":"Sarah Stadlmayr","email":"","orcid":"https://orcid.org/0000-0002-1461-6274","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Stadlmayr","suffix":""},{"id":571314917,"identity":"1e753686-8b00-471d-affd-ef037915364f","order_by":8,"name":"Aida Naghilou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Aida","middleName":"","lastName":"Naghilou","suffix":""},{"id":571314918,"identity":"14b3fc8d-aa49-4022-bbbf-c32f1bd325ab","order_by":9,"name":"Leon Ploszczanski","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Leon","middleName":"","lastName":"Ploszczanski","suffix":""},{"id":571314919,"identity":"d6794011-5d2c-430a-8d7d-65547c2efb9a","order_by":10,"name":"Gerhard Sinn","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gerhard","middleName":"","lastName":"Sinn","suffix":""},{"id":571314933,"identity":"e3ca5af6-d219-4851-9f91-e09682961917","order_by":11,"name":"Harald Rennhofer","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Harald","middleName":"","lastName":"Rennhofer","suffix":""},{"id":571314921,"identity":"625db0d6-54ed-4c3b-9148-c554a12f839f","order_by":12,"name":"Christine Radtke","email":"","orcid":"","institution":"Medical University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"","lastName":"Radtke","suffix":""}],"badges":[],"createdAt":"2025-12-11 10:30:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8335511/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8335511/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100342370,"identity":"ea63f189-2bef-4abe-82ea-0aa1e7da1164","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2771236,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptInsitusilksubmissioncommmat.docx","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/f112f9672dce5864eca49caa.docx"},{"id":100342366,"identity":"25deedf4-4b25-4905-ac15-700a2918c58d","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12472,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSMAT251252.json","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/66b9236f240b0b95dcca9e28.json"},{"id":100373142,"identity":"d8b091e8-1ea8-41a3-9dea-c9cd4e0d7256","added_by":"auto","created_at":"2026-01-16 08:13:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3613892,"visible":true,"origin":"","legend":"","description":"","filename":"SIInsitusilksubmissioncommmat.docx","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/039594f948635b7c82289a22.docx"},{"id":100342373,"identity":"36509af9-748f-4f42-848e-c0938397def3","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177273,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSMAT2512520enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/0692a1f2f2168ce33c8cab29.xml"},{"id":100373646,"identity":"8f645f4d-e434-487c-bbe2-232d1dbd689a","added_by":"auto","created_at":"2026-01-16 08:15:55","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":179604,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/626de0477dbe9e73348a8a26.png"},{"id":100373901,"identity":"3032b6c5-275d-49c2-a936-c9a69748fa3c","added_by":"auto","created_at":"2026-01-16 08:21:03","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":975205,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/843fd8a50dcea230b10caa27.png"},{"id":100342378,"identity":"441c5657-f45e-4014-a38a-4c591306a9ec","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88316,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/bc2d42418493df86e001c546.png"},{"id":100342388,"identity":"dd762a7e-3b8a-4f24-b57a-d3b2c6216441","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":851950,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/c12132d44ab3fde7a4cb85cd.png"},{"id":100342382,"identity":"a4759373-bb97-4d65-9dc5-221d951b0888","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":308496,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/74bd718be1c04d14352ed681.png"},{"id":100342380,"identity":"8356332c-4de3-409e-b1a7-12c826d2438e","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":230549,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/9e57094b83a9fb55bd78cfd9.png"},{"id":100342385,"identity":"6a28c13b-0382-481c-931f-4e127898f70d","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41731,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/b2c3ec4c9ebf684fdc11bbbe.png"},{"id":100377852,"identity":"c2c36b9c-84aa-453d-87f0-a611dac62798","added_by":"auto","created_at":"2026-01-16 08:48:36","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100666,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/fed69af8bafc7dadf792257f.png"},{"id":100342387,"identity":"a8140ac0-aafc-439f-b014-72b945d8feb4","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24519,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/fff78d29876a1f15831ff29f.png"},{"id":100342386,"identity":"17a06c03-e2b4-4532-a8cd-144e87b04b4e","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111343,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/8c40749d39b97dd7ddfe0e57.png"},{"id":100372913,"identity":"83605b7e-5948-4d38-8e44-4eab8b3ed189","added_by":"auto","created_at":"2026-01-16 08:13:24","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":73012,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/9d23c060647f2ecd3e844807.png"},{"id":100373552,"identity":"0dd469d6-6a86-4713-af38-0d0e7eac93fa","added_by":"auto","created_at":"2026-01-16 08:14:52","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51289,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/f379d49160f82ae48154d3b2.png"},{"id":100342390,"identity":"3e011284-a262-4e92-8462-263d7113777b","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174310,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSMAT2512520structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/dc3ebaaed0a72b550259d2dc.xml"},{"id":100342389,"identity":"e6638eea-535c-4480-bf88-cbe9b29797a0","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186693,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/db20101f1351d8349f1e023c.html"},{"id":100342367,"identity":"3a410c98-1e23-45a8-abe5-9d4cb88f312a","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":534044,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Sample Silk fibers as digital microscope images of a) TU silk and c) MA silk. Corresponding X-ray nano-diffraction mesh scans (ID13) of b) TU silk and d) MA silk with line-wise scanning direction indicated by white arrows. Colormap derived by mapping SAXS intensity. Edge-, Transition and Bulk-zone are separated by black dashed lines.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/86494c3f449d60107100654a.png"},{"id":100373630,"identity":"7f6465f6-51d9-4a55-bc69-625cf1b31f8c","added_by":"auto","created_at":"2026-01-16 08:15:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62930,"visible":true,"origin":"","legend":"\u003cp\u003eMechanical parameters calculated from true stress-strain curves for TU and MA silk at different relative humidity levels (30, 45 and 65% RH). 30% RH data includes ESRF and MAX IV data. (a) Young’s modulus, (b) yield strain determined with the 0.2% offset method and (c) yield stress. Empty dots are outliers. All values are mean values ± SD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/163933c602faa6c75285009c.png"},{"id":100373129,"identity":"1a71558c-8677-4e11-a660-36188b482b04","added_by":"auto","created_at":"2026-01-16 08:13:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":400307,"visible":true,"origin":"","legend":"\u003cp\u003eExemplary scattering data at 2% extension and 30% RH averaged from the Bulk from a-d) ID13 (ESRF) beamline and e-h) NanoMAX (MAX IV) beamline. Directions in the polyalanine nanocrystals are shown in the middle at the top of the figure. 001 or c direction corresponds to the fiber long axis, (020) or b corresponds to the intersheet dimension and 100 or a to the interchain dimension. a-b) and e-f) are showing data from MA silk, where a) and e) are the 1d scattering curves with Intensity versus scattering vector q and b) and f) are the corresponding 2d scattering patterns. In c-d) and g-h) data from TU silk is displayed. C) and g) are the 1d scattering curves and d) and h) the 2d scattering patterns. In the 1d scatting curves Bragg and short-range-order (SRO) peaks are displayed by Gaussians. The corresponding area in the 2d scattering pattern is indicated by white dashed lines and the corresponding (020) and (210) peaks as labels. The double-sided white arrow points in the long axis of the silk fiber.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/81d1d9e5033be62b2632bdd5.png"},{"id":100342372,"identity":"8ff6a3d2-3de3-4e7a-be7d-2bfa3255368a","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":172675,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of Unit cell parameters dependent on extension and at different levels of relative humidity (RH). The average values for each zone are separately shown indicated by a color code. In a) unit cell parameter a is displayed at a RH of 30%, 45% and 65% RH, in b) unit cell parameter b is shown at the same levels of RH, and in c) unit cell parameter c is displayed. All values are mean values ± SD.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/2ea79356bb9d3d947fd5d2f4.png"},{"id":100377809,"identity":"f48e0bfb-e087-4e0f-8e5e-bd468180d36b","added_by":"auto","created_at":"2026-01-16 08:48:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276288,"visible":true,"origin":"","legend":"\u003cp\u003eOverview on major effects observed in this study displayed by schematic silk fibers and the crystalline domains (nanocrystallites) embedded in an amorphous matrix (grey). In each panel on the left side the original state of the fiber and on the right side the altered state. a +b) Effects of stretching the fiber on crystallites dimensions and their orientation of a) TU silk and b) MA silk. Major effects of increase in relative humidity (RH) in c) TU silk and d) MA silk.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/2640b8394ad0cb15b4a49e81.png"},{"id":100421511,"identity":"665cc576-faab-4557-a105-9d31b1122e51","added_by":"auto","created_at":"2026-01-16 13:33:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2332774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/f1b14baf-7afa-4602-9ea1-734d4b93529b.pdf"},{"id":100342376,"identity":"4e1e18cb-7506-4a06-927f-c0229c6395e1","added_by":"auto","created_at":"2026-01-16 00:06:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3613892,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"SIInsitusilksubmissioncommmat.docx","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/68ba351c075365ceb2cc1e6f.docx"},{"id":100377789,"identity":"23d6657e-959b-4c9f-a3d6-2d0a2e29b01c","added_by":"auto","created_at":"2026-01-16 08:48:24","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":124375,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8335511/v1/0c12ced686164913e11a5f4c.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Local changes in individual spider silk fibers from tubuliform and major-ampullate glands under varying humidity and tensile strain","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpider dragline silk has long fascinated scientists due to its exceptional combination of high tensile strength and extensibility, resulting in a toughness amongst the highest values of most natural and synthetic fibers (Braxton et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Gosline et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Nova et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These extraordinary mechanical properties are attributed to its hierarchical structure, which features highly ordered crystalline β-sheet nanostructures embedded within an amorphous protein matrix (Termonia, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The β-sheet nanostructures, with a nearly orthorhombic unit cell (Marsh et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1955\u003c/span\u003e; Warwicker, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1960\u003c/span\u003e), provide strength, while the amorphous regions contribute to extensibility and energy dissipation (Du et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Giesa et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lefevre \u0026amp; Auger, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This unique architecture allows spider silk to exhibit remarkable adaptability under varying environmental conditions, making it a model biomaterial for both fundamental research and practical applications. Applications include recombinant spider silk bio-inks (Lechner et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), adhesives (Ye et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and spider silk inspired fibers for sensing, high strength textiles, and water collection (Li et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Many possible applications are found in the field of medicine (Altman et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Rising \u0026amp; Johansson, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In particular, the treatment of peripheral nerve injuries (Bergmann et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) is an area where silk scaffolds may be impactful. These injuries often result in significant loss of motor and sensory function, and current treatments, such as autografts, are limited by donor site morbidity and availability (Kornfeld et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Puranik et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Radtke et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Spider silk offers a biocompatible, mechanically robust alternative, with the ability to support cellular adhesion, migration, and proliferation (Naghilou et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Stadlmayr et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In a previous study, we compared the ability of tubuliform (TU) silk and major ampullate (MA) silk present in the egg sac of spiders to guide Schwann cells. MA silk is produced by the major-ampullate gland whereas TU silk is produced by the tubuliform gland of the female spider. We found that both silk types are part of the egg sac, which is a protective cover for the spider\u0026rsquo;s eggs. Interestingly, TU silk exhibited a superior ability to promote Schwann cell adhesion, migration, and alignment (Peter et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). We found that a combination of morphology and other structural features likely make TU silk particularly interesting for Schwann cells and therefore nerve regeneration. Nevertheless, our knowledge about TU silk is still limited, especially when it comes to its response to environmental and mechanical stimuli. Clarifying these structural adaptations is critical for optimizing TU silk\u0026rsquo;s use in biomimetic applications, particularly in nerve repair and regenerative medicine.\u003c/p\u003e \u003cp\u003eAmong the environmental factors influencing spider silk, humidity plays a particularly critical role. One of the most striking effects of high humidity is supercontraction, where some types of silk, including MA silk, shrink by up to 50% of its original length (Work \u0026amp; Morosoff, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). This phenomenon is driven by the diffusion of water molecules into the silk, causing a transition from a glassy to a rubbery state, the dissociation of hydrogen bonds, and swelling of the fiber (Cohen, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Cohen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These changes lead to a loss of molecular orientation and an increase in entropy, fundamentally altering the silk\u0026rsquo;s mechanical behavior(Sch\u0026auml;fer et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Vehoff et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). For TU silk it has been reported, that supercontraction is absent (Ruiz et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRelative humidity (RH) significantly alters the silk\u0026rsquo;s mechanical properties, such as stiffness, strength, and extensibility (Plaza et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Vehoff et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yazawa et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, Yazawa et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) showed that toughness of MA silk is enhanced under moderate humidity (43% RH) and high strain rates while Young\u0026rsquo;s modulus decreases with increasing RH (Yazawa et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Vehoff et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) confirmed humidity's substantial effect on mechanical properties through large-scale testing (Vehoff et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). For silkworm silk, Seydel et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) further demonstrated that tensile strain increases molecular mobility of water and amorphous polymer chains in the amorphous regions of humid silk, contributing to the observed diffusion processes (Seydel et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile these studies have advanced our understanding of dragline spider silk\u0026rsquo;s adaptations to changes in humidity, they focus primarily on MA silk and silkworm silk. No such studies have been conducted on TU silk. Furthermore, they have predominantly focused on bulk properties or average structural changes, leaving critical aspects of spatially resolved structural differences within an individual fiber unexplored. So far, one study has combined wide-angle X-ray scattering and tensile testing on bundles of 400\u0026ndash;600 dragline silk fibers to achieve sufficient scattering intensity (Yazawa et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another publication focused on tensile testing under various environmental conditions but excludes structural analysis (Vehoff et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). To the best of our knowledge, only one study has combined X-ray diffraction and tensile testing on single spider silk fibers (Glišović et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This study, however could not resolve local structural differences in individual fibers due to the relatively large spot size of 7 \u0026micro;m, which covers the diameter of an average silk fiber (Glišović et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Local differences, however, have been described in the literature in dry state, e.g. by applying X-ray nanodiffraction a 1 \u0026micro;m thick skin layer composed of nanofibrils was identified for \u003cem\u003eArgiope bruennichi\u003c/em\u003e (Riekel et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is therefore essential to study them to gain a full understanding of deformation mechanisms and local influence of humidity.\u003c/p\u003e \u003cp\u003eThe aim of our study was to elucidate differences in humidity-driven structural and mechanical changes between the two silk types (TU and MA silk) in a highly position-resolved way, in order to gain, for the first time, information on local ultrastructural changes within individual fibers.\u003c/p\u003e \u003cp\u003eFurthermore, this study is the first to investigate the poorly studied TU silk using an in-situ-tensile synchrotron experiment. By exploring the spatially resolved structural differences across the diameter of individual fibers and across fiber types, this work provides a deeper understanding of the hierarchical organization and mechanical behavior of spider silk. These results can serve as a template to correlate protein structure to humidity response and mechanics, enabling improved design of biomimetic materials.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eWe address these questions by employing high-resolution nanobeam X-ray diffraction at synchrotron facilities to map the spatially resolved inner structure of single spider silk fibers during in-situ mechanical stretching under controlled environmental conditions (Figure S1). In brief we applied a stretching cell and a humidity cell to stretch a single silk fiber up to a certain amount at a specific constant level of RH. For details on the experimental setup see section Materials and Methods and Figure S1. Our approach enables discrimination of distinct zones within the fiber (Bulk, Transition, and Edge), providing insights into the ultrastructure of spider silk fibers and possible core-shell arrangements. Based on SAXS intensity maps we defined the three zones as shown in Figure 1. To obtain position-resolved structural information, the SAXS intensity map was used to identify the position and orientation of the fiber within the scan. Coordinates corresponding to lines parallel to the fiber\u0026rsquo;s long axis were defined, and the scattering intensity along each line was averaged. The averaged lines were then divided into three distinct zones: \u0026nbsp;Edge zone (the outermost 1 \u0026micro;m from each edge of the fiber), Transition zone (the next 1 \u0026micro;m inward from the edge zone), Bulk zone (the remaining central region of the fiber). The zone dimensions were based on a previous research investigating the skin-core morphology of \u003cem\u003eArgiope bruennichi\u0026nbsp;\u003c/em\u003eMA silk, which found a 1 \u0026micro;m thick proteinaceous skin layer serving as an elastic sheath (Riekel et al., 2017)\u003cem\u003e.\u003c/em\u003e In the following, the results are presented and discussed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMechanical parameters upon RH increase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mechanical properties of TU and MA silk fibers, as determined by tensile testing of individual fibers as described above, were strongly influenced by RH, with both silk types showing a notable reduction in stiffness as RH increased (Figure 2a). Young\u0026rsquo;s modulus, fitted between 0.2% and 1% strain, decreased at higher RH levels, with the most pronounced drop observed at 65% RH compared to 30% RH. Between 30% and 45% RH, the decrease was more pronounced for TU silk than for MA silk, while both exhibited very pronounced reductions between 45% and 65% RH. \u0026nbsp;Similarly, yield strain, determined using the 0.2% offset method, was higher at 30% RH than at 65% RH for both silk types, with TU silk showing a more pronounced decrease (Figure 2b). Moderate reductions in yield strain were also observed between 45% and 65% RH for both silk types. Yield stress followed the same trend, with significant decreases as RH increased, particularly between 30% and 65% RH (Figure 2c). Maximum stress and strain at break were not evaluated, as the stretching was manually interrupted at predefined extension values.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese changes align with previous studies that have shown softening of MA silk under wet conditions (Gli\u0026scaron;ović et al., 2008; Vehoff et al., 2007). The reduction in stiffness can be attributed to the disruption of hydrogen bonds in the amorphous regions of the silk, which increases molecular mobility (Cohen et al., 2021). Water molecules act as a plasticizer, enhancing chain mobility in the amorphous regions and lowering the energy required for deformation. The absence of supercontraction means that the silk remained below its glass transition temperature and below a certain critical humidity (23 \u0026deg;C and up to 65% RH)(Plaza et al., 2006). Consistent with this, we did not observe any supercontraction in our experiments with both types of silk fibers.\u003c/p\u003e\n\u003cp\u003eThe distinct responses of TU and MA silk to RH reflect differences in their structural composition and biological roles (Peter et al., 2025). TU silk exhibited greater sensitivity to humidity, with more pronounced reductions in mechanical properties compared to MA silk. We infer, that at least part of this effect can be attributed to the crystal structure and the size and arrangement of nanocrystals. Variations in the amorphous matrix may also play a role, potentially allowing for greater water infiltration in TU silk. Biologically, TU silk serves in the egg sac of spiders as a protective layer against humidity and temperature fluctuations (Ewunkem \u0026amp; Agee, 2022; Peter et al., 2025). In contrast MA silk \u0026shy;\u0026shy;\u0026shy;- optimized for high tensile strength and serving as a lifeline for the spider (Blackledge, 2012) - showed a more stable response to changes in RH, likely due to its more robust and compact crystal structure.\u003c/p\u003e\n\u003ch2\u003ei Ultrastructural parameters upon extension\u003c/h2\u003e\n\u003cp\u003eUltrastructural parameters were derived by analysing the 2D scattering patterns from the experiments (Figure 3). High-resolution nanobeam X-ray diffraction revealed distinct trends in structural parameters for TU and MA silk under extension, with notable differences between the two silk types and across RH levels. These trends provide insights into the hierarchical structure and strain-induced changes of the silks.\u003c/p\u003e\n\u003cp\u003eThe unit cell of spider silk can be described as a nearly orthorhombic structure. In a fiber, the crystallites\u0026rsquo; c axis aligns with the direction of the fiber axis while the a- and b-axes are randomly oriented around the fiber axis (Marsh et al., 1955; Warwicker, 1956).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe unit cell parameters of the two different silk types were examined as a function of extension, RH and zone (Edge, Transition and Bulk) (Figure 4). Among these, the b parameter, which reflects the intersheet spacing within\u0026nbsp;\u0026beta;-sheet nanocrystals, showed particularly notable differences between TU and MA silk when exposed to strain under varying RH conditions, and it was higher for TU silk (Figure 4b). We also observed that the b parameter of TU silk was remarkably sensitive to strain, showing distinct changes with extension: at 30% RH, the b parameter decreased for all zones, whereas for higher RH (45% and 65%) the same parameter increased under extension in the Edge zone. In contrast, the b parameter of MA silk remained relatively stable. This difference suggests that TU silk\u0026rsquo;s nanocrystals are more susceptible to water infiltration, which may disrupt hydrogen bonds between the\u0026nbsp;\u0026beta;-sheets of the nanocrystals. The sensitivity of the b parameter to strain was more pronounced in TU silk, particularly at higher RH levels. This demonstrates that TU silk undergoes greater molecular alignment and structural changes even in the crystalline domains under strain, consistent with its role in egg sac construction, where flexibility and extensibility are critical. As reported in our previous work\u0026nbsp;(Peter et al., 2025), the b parameter reflecting the intersheet dimensions of the nanocrystallites is larger in TU silk than in MA silk, which can be attributed to larger side-chain amino-acids in TU silk\u0026rsquo;s nanocrystals\u0026nbsp;(Tian \u0026amp; Lewis, 2006). This structural difference likely contributes to the greater extensibility of TU silk.\u003c/p\u003e\n\u003cp\u003eWe also performed statistical analysis (Figure S2, S3). We analyzed the behavior of the parameters upon extension using a linear weighted least-squares (WLS) regression fit (Figure S2). For the unit cell parameter b (Figure 4 b), the difference between TU and MA silk at 30% RH included significant decreases for all zones for TU silk and non-significant increases in case of MA silk. In TU silk, there is a trend of higher b values in the Edge zone, predominantly at high RH.\u003c/p\u003e\n\u003cp\u003eThe unit cell parameter a (Figure 4 a) remained fairly constant with only small absolute changes. However, statistical analysis showed that the parameter decreased slightly with increasing extension for both TU and MA silk, but the trend was less significant at higher RH levels (Figure S2). This suggests that strain-induced structural changes in the interchain direction upon extension are less pronounced under humid conditions (Figure 4 a). The unit cell parameter c (Figure 4 c), which corresponds to the fiber axis, increased with extension for both silk types, with limited statistical significance due to the weak (002) reflection and corresponding Fit uncertainty (Figure S4). This is consistent with findings from Gli\u0026scaron;ović et al. (2008), who observed a nearly linear correlation between the c value and strain in dragline silk (Gli\u0026scaron;ović et al., 2008).\u003c/p\u003e\n\u003cp\u003eThe Herman\u0026rsquo;s orientation parameter is a measure of the degree of molecular alignment within a fiber (De Volder et al., 2014), with values ranging from 0 (completely random orientation) to 1 (perfect alignment along the fiber axis). This parameter provides insights into how the crystalline and amorphous regions of the silk align under mechanical strain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor TU silk, the Herman\u0026rsquo;s orientation parameter increased with extension, with significant increases observed at 30% RH in the Transition and Edge zones. This indicates that molecular alignment is enhanced under low-humidity conditions. In contrast, MA silk exhibited mixed trends: a non-significant increase at 30% RH, a non-significant decrease at 45% RH, and variable trends at 65% RH. These findings align with previous studies on MA silk, such as Gli\u0026scaron;ović et al. (2008), who reported little to no dependency of crystalline orientation on strain for MA silk at moderate RH levels (Gli\u0026scaron;ović et al., 2008).\u0026nbsp;The significant increases in molecular alignment for TU silk highlight its dynamic structural response to strain, particularly in the Transition and Edge zones.\u003c/p\u003e\n\u003cp\u003eThe L\u003csub\u003e020\u003c/sub\u003e particle size did not exhibit significant trends for either silk type, consistent with previous findings for MA silk at 50% RH (Gli\u0026scaron;ović et al., 2008). However, L\u003csub\u003e210\u003c/sub\u003e showed decreasing trends, with significant decreases for TU silk at 30% and 45% RH and for MA silk in some zones at the same RH levels. This may reflect unfolding or splitting of \u0026beta;-sheet crystallites along the hydrogen bond direction under strain (Gli\u0026scaron;ović et al., 2008) or might be the effect of transverse contraction under applied strain. The lattice spacing d\u003csub\u003e210\u003c/sub\u003e generally decreased with extension for both silk types, indicating transverse contraction of the crystalline structure under strain. For TU silk, this trend was highly significant at 30% RH, less significant at 45% RH, and non-significant at 65% RH. For MA silk, d\u003csub\u003e210\u003c/sub\u003e showed a highly significant decrease at 30% RH, significant decreases in the Edge zone at 45% RH, and no significant trends elsewhere. Unlike previous studies that reported no change in peak positions under strain (Gli\u0026scaron;ović et al., 2008; Yazawa et al., 2020), our findings suggest densification\u0026nbsp;of the silks\u0026rsquo; crystalline structure under mechanical strain, which is less pronounced at higher humidity levels, i.e. mitigated by humidity. For TU silk this densification is accompanied by increased reorientation of crystallites.\u003c/p\u003e\n\u003ch2\u003eii. Ultrastructural parameters upon RH increase\u003c/h2\u003e\n\u003cp\u003eWhen\u0026nbsp;analyzing\u0026nbsp;the trends of structural parameters with increasing RH, distinct differences between TU and MA silk emerged, providing additional insights into their structural changes under varying environmental conditions (Figure S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor TU silk, the unit cell parameter b is highly dependent on changes in RH. The intersheet spacing (b) increases with increasing RH across all zones. In contrast, MA silk shows no clear trend (Figure S3). One possible explanation could be the distinct amino-acid profile of TU silk.\u0026nbsp;\u0026beta;-sheets are formed based on structural domains like AAQAASAA, AAQAA, and AASQAA (Tian \u0026amp; Lewis, 2006). This leads to larger spacing between the\u0026nbsp;\u0026beta;-sheets compared to the poly-A\u003csub\u003en\u003c/sub\u003e/(GA)\u003csub\u003en\u003c/sub\u003e model of MA silk and therefore to a more hydrophilic character of TU silk\u0026nbsp;(Hu et al., 2005). This fact may allow water molecules to enter and increase the distance between the\u0026nbsp;\u0026beta;-sheets even more.\u003c/p\u003e\n\u003cp\u003eFor MA silk, the crystallinity parameter (Figure S5) showed a general decreasing trend with increasing RH, with some significant decreases observed (Figure S3). This suggests that the crystalline regions of MA silk become less ordered under humid conditions, potentially due to water infiltration disrupting hydrogen bonds. In contrast, no clear trend was observed for TU silk, indicating that its crystallinity is less affected by RH. These findings differ from previous studies, which reported constant crystallinity for MA silk under varying RH conditions (Yazawa et al., 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe orientation parameter (Figure S6) for MA silk exhibited a trend of increasing values with increasing RH, with significant increases in the Bulk and Edge zones (Figure S3). These contrasts previous studies reporting a decrease in orientation with increasing RH for MA silk (Lef\u0026egrave;vre et al., 2009; Yazawa et al., 2020). For TU silk, the trends in orientation were mixed and non-significant, suggesting, that its molecular alignment is less sensitive to changes in RH. The increase in orientation parameter for MA silk with RH may reflect a structural adaptation to maintain alignment of nanocrystallites under humid conditions, which could contribute to its mechanical stability.\u003c/p\u003e\n\u003cp\u003eThe L\u003csub\u003e020\u003c/sub\u003e particle size (Figure S7) for MA silk exhibited an increasing trend with RH, with a significant increase in the Bulk zone at 2% extension (Figure S3). This suggests that the crystalline regions of MA silk expand slightly under humid conditions, possibly due to water-induced swelling. For TU silk, L\u003csub\u003e020\u003c/sub\u003e showed a decreasing trend in the Bulk zone, with significant decreases at 2% and 8% extension. This is noteworthy, since the b parameter significantly increases in TU-silk at higher RH, which would suggest a greater particle size in the intersheet direction. However, as the Scherrer width does not directly provide the particle size but the length of coherently scattering domains in a certain crystal direction, the decrease in L\u003csub\u003e210\u0026nbsp;\u003c/sub\u003epoints towards a loss of order upon increasing RH. This corresponds well with the observation of greater swelling in b direction in TU silk than in MA silk, which could cause disorder on the molecular scale. The L\u003csub\u003e210\u003c/sub\u003e particle size (Figure S8) for TU silk showed an overall increasing trend with RH, with significant increases at 10% extension in the Edge zone and at 0% extension in the Transition and Edge zones (Figure S3). This suggests that the\u0026nbsp;\u0026beta;-sheet crystallites in TU silk may reorganize under humid conditions. Previous studies reported no dependency of \u003cstrong\u003eL\u003csub\u003e210\u0026nbsp;\u003c/sub\u003e\u003c/strong\u003eparticle size on RH \u0026nbsp;for MA silk (Gli\u0026scaron;ović et al., 2008), which aligns with the mixed trends observed in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe lattice spacing d\u003csub\u003e210\u0026nbsp;\u003c/sub\u003e(Figure S9) for TU silk generally increased with RH, with significant increases at 0% extension in the Bulk zone and at 0%, 6%, and 10% extension in the Transition zone (Figure S3). This trend suggests that the crystalline structure of TU silk becomes less dense under humid conditions, likely due to water infiltration. This is also in line with a partly significant increase of unit cell parameter b in all zones with increasing RH for TU silk. For MA silk, d\u003csub\u003e210\u003c/sub\u003e exhibited mixed\u0026nbsp;trends, with some significant decreases at 0% extension, these findings support previous reports of limited dependency of d-spacings on RH for MA silk (Gli\u0026scaron;ović et al., 2008).\u003c/p\u003e\n\u003ch2 id=\"_Toc211011556\"\u003eiii. Trends between different zones\u003c/h2\u003e\n\u003cp\u003eThe analysis of structural parameters differing across the Bulk, Transition and Edge zones revealed that, for the majority of parameters and conditions, there were no statistically significant differences between the zones. Across all tested combinations of RH and extension steps, the p-values for most parameters exceeded the significance threshold of 0.05.\u0026nbsp;One exception to this overall trend was observed for TU silk and the parameter \u003cstrong\u003ed\u003csub\u003e210\u003c/sub\u003e\u0026nbsp;\u003c/strong\u003eat 30% RH and 0% extension, where a significant difference was found between the Edge and Bulk zones. The mean difference between these zones was 0.4758, with a p-value of 0.0205. This localized variation suggests that spatially dependent adaptations may occur under specific environmental conditions. The Edge zone, being more exposed to environmental factors, may experience greater water loss or structural rearrangement, leading to differences in lattice spacing compared to the Bulk zone. However, no significant differences were observed between the Bulk and Transition zones or between the Transition and Edge zones for d\u003csub\u003e210\u003c/sub\u003e or any other parameter. Furthermore, it seems that the crystallinity parameter is decreased in the edge zone compared to the rest for both silk types (Figure S5). Another edge effect we observe in TU silk is an increase in b parameter at higher RH in the edge zone compared to the other zones (Figure 4 b).\u0026nbsp;Despite these exceptions, the overall homogeneity of the studied parameters of the silk structure across the zones was consistent, even under varying RH and extension conditions for both silk types. These findings suggest a relatively homogeneous distribution of the studied structural features within the silk fibers at the measured length scales, aligning with the studies that emphasize the uniform hierarchical structure of silk (Lin et al., 2017; Qiu et al., 2019).\u003c/p\u003e\n\u003cp\u003eThis rather homogeneous structural organization contrasts with earlier\u0026nbsp;studies using experimental removal (Sponner et al., 2007; Yazawa et al., 2018), X-ray nanodiffraction (Riekel et al., 2017), imaging (Du et al., 2006), spectroscopic methods (Brown et al., 2011), and computational modeling (Giesa et al., 2011; Keten \u0026amp; Buehler, 2010), which suggest that dragline silk contains a lipid/glycoprotein coat, a skin, and at least one core region (often subdivided into outer and inner components) (Stadlmayr et al., 2025).\u0026nbsp;These layers serve distinct functions: the core (including crystalline \u0026beta;-sheet domains) usually provides strength, stiffness, and energy dissipation, whereas the skin acts as a protective or elastic barrier\u0026nbsp;(Yazawa et al., 2019). In detail, Sponner et al. reported based on biochemical analysis that MA silk consists of 5 different layers, from which the three layers described together as the shell were described to be no larger than 250 nm together\u0026nbsp;(Sponner et al., 2007). An outer spidroin protein core with mainly MaSp1 and a thickness of up to 400 nm was compared to an inner core with a mixture of MaSp1 and MaSp2\u0026nbsp;(Sponner et al., 2007). These differences in protein composition can be only hardly detected by X-ray nanodiffraction and glycoprotein and lipid layers are too small in dimension to be detected. However, by applying X-ray nanodiffraction Riekel et al. (2017) discovered a 1 \u0026micro;m thick skin layer composed of\u0026nbsp;nanofibrils. Our results indicate that such differentiation may not be as pronounced as previously suggested, at least for the structural parameters and length scales analyzed in this study. This suggests that at least for the silk type investigated in this study - the hierarchical structure is more uniformly distributed than generally assumed.\u0026nbsp;Nevertheless, a clear understanding of the hierarchical structure of silk is still lacking, since the observed features and length scales depend very much on the sample preparation and experimental techniques\u0026nbsp;(Wang \u0026amp; Schniepp, 2019).\u003c/p\u003e\n\u003ch2 id=\"_Toc211011557\"\u003eb. Comparative Insights Between TU and MA Silk\u003c/h2\u003e\n\u003cp\u003eThe differences observed between TU and MA silk in their response to extension and RH underscore the importance of silk type and environmental conditions in determining mechanical and structural behavior. The differences reflect the unique hierarchical organization and biological roles of the two silk types. 5 gives an overview of the major effects of strain and changes in RH on both silk types.\u003c/p\u003e\n\u003cp\u003eThe significant decrease in the b parameter with extension for TU silk at 30% RH contrasts with the non-significant positive trend for MA silk. This suggests that TU silk nanocrystals which are characterized by larger side-chain amino acids (Peter et al., 2025; Tian \u0026amp; Lewis, 2006), undergo compression and deformation under strain, leading to a reduction in b. In contrast, at higher RH (45% and 65%) we observe an increase in the b parameter upon extension for TU silk, but only in the Edge zone. This is remarkable, since we would normally expect transverse contraction upon longitudinal elongation. The transverse expansion suggests decreased interaction between the \u0026beta;-sheets upon hydration to the point where other deformation mechanisms may occur, such as inter-sheet sliding. We hypothesize that the occurrence of this effect at higher humidity and in the edge zone only might be due to increased exchange of water molecules with the environment in these regions. In contrast, MA silk\u0026rsquo;s b parameter remains more stable, reflecting its more compact and robust crystalline structure. This stability aligns with MA silk\u0026rsquo;s role as dragline silk, where high tensile strength and stiffness are prioritized over extensibility.\u003c/p\u003e\n\u003cp\u003eSimilarly, the increasing Herman\u0026rsquo;s orientation parameter with extension for TU silk especially at 30% RH in the Transition and Edge zones indicates enhanced molecular alignment under low-humidity conditions, which may contribute to its superior extensibility. In contrast, MA silk exhibited mixed trends in molecular alignment dependent on extension.\u003c/p\u003e\n\u003cp\u003eFurthermore, we observe generally larger values for the crystallinity parameter in MA silk than for TU silk at higher RH and therefore a relative reduction in the crystallinity parameter at higher RH for TU silk (Figure S5). \u0026nbsp;This might be one reason for the generally higher tensile strength of MA silk compared to TU silk (Peter et al., 2025).\u003c/p\u003e\n\u003cp\u003eAdditional differences were observed in particle size and lattice spacing. For example, \u003cstrong\u003eL\u003csub\u003e020\u003c/sub\u003e\u003c/strong\u003e showed a decreasing trend for TU silk in the Bulk zone, while MA silk exhibited an increasing trend with RH increase. Given the increase of the b parameter in TU silk, the decrease in \u003cstrong\u003eL\u003csub\u003e020\u003c/sub\u003e\u003c/strong\u003e points to a greater disorder in intersheet direction for this type of silk with increasing RH. The lattice spacing d\u003csub\u003e210\u003c/sub\u003e for TU silk generally increased with RH increase, reflecting a loosening of the crystalline structure, whereas MA silk showed mixed trends, consistent with its more stable crystalline organization.\u003c/p\u003e\n\u003cp\u003eThese comparative insights provide a deeper understanding of the hierarchical mechanisms underlying the mechanical behavior of spider silk and offer valuable guidance for the design of biomimetic materials. For instance, TU silk\u0026rsquo;s flexibility and structural changes could inspire the development of dynamic scaffolds for tissue engineering, while MA silk\u0026rsquo;s stability could inform the design of high-strength fibers for load-bearing applications.\u003c/p\u003e\n\u003ch2 id=\"_Toc211011558\"\u003ec.\u0026nbsp; Limitations and Future Directions\u003c/h2\u003e\n\u003cp\u003eWhile this study provides significant insights into the adaptive mechanisms of spider silk, it is limited by the length scales studied (100\u0026ndash;250 nm) and the range of environmental conditions tested. The focus on this specific length scale and the need to average data within pre-defined zones may have constrained our ability to capture finer structural details, such as molecular-level interactions within the crystalline and amorphous regions. Computational molecular modeling could help address this limitation by providing a more detailed understanding of these interactions. However, challenges remain, as the full composition of the amorphous matrix and the protein sequences may not yet be fully characterized, complicating the modeling process.\u003c/p\u003e\n\u003cp\u003eAdditionally, the environmental conditions tested in this study were limited to variations in RH at a constant temperature. Other factors known to influence spider silk\u0026rsquo;s properties, such as temperature fluctuations, UV exposure, and cyclic environmental stress, were not explored (Wharton et al., 2025). Investigating these effects could provide a more complete picture of spider silk\u0026rsquo;s adaptability and durability. Additionally, studying other silk types, such as flagelliform silk or aciniform silk, and their responses to dynamic environmental changes could further expand our understanding of the diverse mechanical and structural properties of spider silk. These limitations may have influenced the generalizability of the findings to other silk types or real-world conditions. \u0026nbsp;These limitations may have influenced the generalizability of the findings to other silk types and species or actual environmental conditions.\u003c/p\u003e\n\u003cp\u003eFuture research should address these limitations by exploring finer length scales to capture additional structural details. Combining nanobeam X-ray diffraction with complementary techniques, such as neutron scattering or Raman spectroscopy, could provide a more comprehensive understanding of silk\u0026rsquo;s hierarchical structure and its dynamic responses to environmental stimuli. For instance, neutron scattering could offer insights into hydrogen bonding and water interactions, while Raman spectroscopy could reveal molecular-level changes in silk\u0026rsquo;s chemical structure under strain or varying humidity.\u003c/p\u003e\n\u003cp\u003eExpanding the range of environmental conditions tested is another critical direction for future research. Investigating the effects of temperature, UV exposure, and cyclic environmental changes could provide a more complete picture of spider silk\u0026rsquo;s adaptability and durability. Additionally, studying other silk types, such as flagelliform, aciniform silk, or attaching silk, and their responses to dynamic environmental changes could further expand our understanding of the diverse mechanical and structural properties of spider silk.\u003c/p\u003e\n\u003cp\u003eAddressing the limitations of our study and pursuing these future directions will deepen our understanding of spider silk\u0026rsquo;s remarkable adaptability to environmental changes and enhance its potential for biomimetic applications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides new insights into the hierarchical structure and environmental adaptability of spider silk, with a focus on the distinct responses of tubuliform (TU) and major ampullate (MA) silk from \u003cem\u003eTrichonephila\u003c/em\u003e (\u003cem\u003eT.) inaurata\u0026rsquo;s\u003c/em\u003e egg sac to mechanical strain and relative humidity (RH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Using high-resolution nanobeam X-ray diffraction at synchrotron facilities, we revealed that increasing RH significantly reduces Young\u0026rsquo;s modulus, yield strain, and yield stress for both silk types, with TU silk exhibiting a slightly higher sensitivity to humidity. Structural analysis highlighted key differences, such as the pronounced adaptability of TU silk\u0026rsquo;s nanocrystals and molecular alignment under strain, compared to the more stable crystalline organization of MA silk. Another example is the intersheet spacing being highly sensitive to strain and RH for TU silk. These differences reflect the unique biological roles of TU silk in egg sac construction and MA silk as dragline silk, emphasizing their specialized mechanical and structural properties. Our findings expand the understanding of spider silk\u0026rsquo;s hierarchical mechanisms, particularly for the underexplored TU silk, and challenge some previously reported trends for MA silk. Notably, we observe predominantly homogeneous ultrastructural parameters across the diameter of the silk fiber. Therefore, no clear evidence of core-shell differences was observed at the studied length scales and for the studied parameters, suggesting that \u003cem\u003eT. inaurata\u0026rsquo;s\u003c/em\u003e MA and TU silk\u0026rsquo;s hierarchical structure might be more homogeneous than previously hypothesized. These findings challenge earlier models proposing distinct functional roles for the core and shell regions of spider silk fibers. Future studies should explore finer structural details and additional environmental factors, such as temperature and UV exposure, to further unravel the remarkable adaptability of spider silk.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ea. Preparation of Samples\u003c/h2\u003e \u003cp\u003eThe samples used in this study were prepared from egg sac silk of \u003cem\u003eT. inaurata\u003c/em\u003e spiders. The housing of the spiders, as well as the harvesting of the silk, were conducted following previous protocols (Peter et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Stadlmayr et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Two types of silk fibers were extracted from the egg sacs: MA silk and TU silk. The extraction process was performed under a digital microscope (Keyence VHX-5000) to ensure precision and minimize damage to the fibers as previously described (Peter et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIndividual silk fibers were carefully glued onto 3D-printed plastic support frames using superglue. To avoid pre-stressing the fibers, minimal force was applied during the manipulation and mounting process. The length and diameter of each fiber were measured using a digital microscope (Keyence VHX-5000). The diameter was determined by averaging fifteen measurements taken at different positions along the length of the fiber to ensure accuracy and account for any variability in thickness. The gauge length was approx. 4 mm for each sample. After preparation, the samples were stored in small plastic containers at ambient temperature and humidity until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eb. Experimental Setup and Measurement procedure\u003c/h2\u003e \u003cp\u003eTo investigate spatially-resolved ultrastructural changes in single threads of spider silk during stretching, two synchrotron experiments were conducted using X-ray nanodiffraction in transmission mode. The general experimental setup, including the incoming X-ray beam, the in-situ stretching setup, a flight tube, and detector, is shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The first experiment was performed at the NanoMAX beamline (MAX IV) (Carbone et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Johansson et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) under ambient conditions (conditions were measured to be 22\u0026deg;C, 30% RH), while the second was conducted at the nanofocus beamline ID13 (ESRF) using a custom-designed humidity cell to test samples at varying RH levels (30%, 45%, and 65% RH). The customised humidity controller uses a PID algorithm to dynamically mix dry and humidified nitrogen at room temperature, based on feedback from a Sensirion humidity sensor installed few millimeters below the silk fibres.In both experiments, high-speed mesh scans were employed, scanning the fibers line by line perpendicular to their long axis with continuously moving motors (fly scan) and a line spacing of 1 \u0026micro;m to minimize radiation damage while maintaining high spatial resolution. For this, we evaluated if the ultrastructural parameters changed upon increasing scanning time. There was no major change of parameters when comparing them from one scan line to the next one. Therefore, we assume that we scanned fast enough and chose a far enough distance between the scan lines (1 \u0026micro;m) so we could avoid major structural changes due to radiation damage.\u003c/p\u003e \u003cp\u003eAt NanoMAX, the fibers were mounted perpendicular to the monochromatic X-ray beam (λ\u0026thinsp;=\u0026thinsp;0.08856 nm, 14 keV, 5.57\u0026times;10\u003csup\u003e10\u003c/sup\u003e photons/s). The beam had a full width at half maximum (FWHM) of 100 nm, and nano-diffraction measurements were performed with exposure times of 10\u0026ndash;20 ms and step sizes of 100\u0026ndash;250 nm during continuous movement (fly scan). The sample-to-detector distance (0.1496 m) was calibrated using an LaB\u003csub\u003e6\u003c/sub\u003e (NIST 660C) standard and processed with the pyFAI software package. The detector used was a single photon counting pixel detector (Pilatus3X 1M, Dectris, Switzerland). The specific in-situ stretching setup, including a schematic sketch of the nanoindenter device (Alemnis AG, Thun, Switzerland)) used for stretching the fiber and the clamping mechanism for the 3D-printed sample support, is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. The sides of the plastic supports were cut using a hot thread cutter (TZ1300 Thread Zap II, The Beadsmith), allowing the load to be exclusively carried by the fiber.\u003c/p\u003e \u003cp\u003eOnce the load was exclusively carried by the fiber, alignment corrections were applied using the nanoindenter motors. Stretching was performed incrementally, starting with small 0.01 mm adjustments to detect a slight force increase. Afterwards the fibers were stretched in 2% extension steps, where time for stretching was calculated based on the initial length of each fiber. The stretching speed was set to 50 \u0026micro;m/s, and a mesh scan was initiated immediately after each extension. Stress relaxation was observed during scanning. New fibers were used for each extension step. Extensions were performed up to a maximum of 18%, and force-displacement data was recorded to calculate mechanical parameters such as Young\u0026rsquo;s modulus. Higher extensions were not possible since a large amount of fibers ruptured at higher extensions. Stress-strain curves of TU and MA silk are shown in Figure S10.\u003c/p\u003e \u003cp\u003eAt ID13, the fibers were mounted similarly, perpendicular to the monochromatic X-ray beam (λ\u0026thinsp;=\u0026thinsp;0.08157 nm, 15.2 keV, 1.01\u0026times;10\u003csup\u003e11\u003c/sup\u003e photons/s). Nano-diffraction measurements were performed with a beam FWHM of 100 nm, exposure times of 7\u0026ndash;20 ms, and step sizes of 100\u0026ndash;250 nm perpendicular to the fiber\u0026rsquo;s long axis and 1\u0026micro;m step size between lines. The sample-to-detector distance (0.1381 m) was calibrated using an Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e standard and processed with pyFAI. The detector used was a single photon counting pixel detector (EigerX 4M, Dectris, Switzerland). The setup including a custom-made stretching cell with a humidity-controlled chamber, as shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec, was used to test fibers at 30%, 45%, and 65% RH. The same 3D-printed sample supports and hot thread cutter were used as in the NanoMAX experiment. Stretching followed the same procedure as at NanoMAX, with incremental 2% extensions and mesh scans performed after each step. Force-displacement data was recorded to analyze mechanical behavior under varying humidity conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ec. Data processing and analysis\u003c/h2\u003e \u003cp\u003eThe mechanical properties of the silk fibers, including Young\u0026rsquo;s modulus, yield stress and yield strain, were determined from force-displacement data obtained during tensile testing. The force-displacement data was converted into stress-strain data by incorporating the diameter and length of the fiber. The original cross-sectional area of the fiber was calculated using the measured diameter, assuming a circular cross-section. In our previous research (Peter et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), we found, that TU silk shows an irregular shaped fiber surface with longitudinal grooves on the surface. For the analysis of the mechanical parameters, we assumed a circular cross section also for TU silk. True stress and true strain were also calculated, with true stress accounting for the reduction in cross-sectional area during deformation and true strain calculated as the natural logarithm of the engineering strain increment. The stress-strain data was filtered to isolate the linear elastic region, defined as the strain range between 0.2% and 1%. This range was chosen to ensure accurate determination of Young\u0026rsquo;s modulus while avoiding non-linear effects. Linear regression was applied to the filtered data to calculate the slope of the stress-strain curve, representing Young\u0026rsquo;s modulus. The standard error of the slope was calculated to estimate the uncertainty in Young\u0026rsquo;s modulus. The 0.2% offset method was used to determine the yield stress and yield strain. A parallel line to the linear fit, offset by 0.2% strain, was calculated, and the intersection of this offset line with the stress-strain curve was identified as the yield point. For statistical analysis a 1-way ANOVA (Origin 2025) and a mean value comparison by a Tukey test were performed.\u003c/p\u003e \u003cp\u003eScattering data was generated as HDF5 files, and integration was performed using the python libraries pyFAI (Kieffer et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) for ESRF data and azint (Jensen et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) for MAX IV data. The number of data points was set to 1000 in the q-range and 360 in the azimuthal range.\u003c/p\u003e \u003cp\u003eThe scattering patterns from each zone were averaged, and background intensity was calculated from areas without fiber. The background was subtracted, and the intensity was normalized against the incoming beam intensity. The resulting Intensity versus q plots were fitted using the lmfit library in Python. For the analysis of the (020), (210), (002) and (x)-peaks, the azimuthal range was selected based on the caked scattering intensity map. We named the (x)-peak according to our previous research on TU and MA silk (Peter et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The peaks were fitted using Gaussian functions, where some are referred to as Short-Range-Order (SRO) peaks, and the rest are referred to as Bragg peaks based on previous fits on experimental data of spider silk (Riekel et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sampath et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The lattice spacing d was calculated using Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, where q is the center position of the respective peak. Particle size was determined using the Scherrer formula with k\u0026thinsp;=\u0026thinsp;0.9, and a crystallinity parameter was estimated by fitting the overall azimuth-averaged Intensity versus q curve (Figure S11).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:d=2\\pi\\:/q$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHerman\u0026rsquo;s orientation parameter \u003cem\u003ef\u003c/em\u003e was derived by fitting the scattering Intensity versus the azimuth for specific q ranges corresponding to the (020) and (210) peaks (Figure S12). Information on the crystallite orientation about the fibers' long axis was obtained from the peaks\u0026rsquo; FWHM (full width at half maximum). Herman\u0026rsquo;s orientation function f (Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) ranges from 0 (no preferred orientation) to 1 (perfect alignment) for crystals along the measurement direction (Du et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The angle between the c-axis and the fiber axis is referred to as Φ (Blamires et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:f=(3\u0026lang;{cos}^{2}\\varphi\\:\u0026rang;-1)/2\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eDue to the orientation of the (020) and (210) reflections in the equatorial plane Grubb et al. defined the following relationship (3) with A\u0026thinsp;=\u0026thinsp;0.8 and B\u0026thinsp;=\u0026thinsp;1.2 since (210) is 65 \u0026deg; from (020) (Grubb \u0026amp; Jelinski, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Since the reflections are measured perpendicular to the fiber axis, φ can be referred to as 90\u0026deg; - Φ. Therefore, 〈cos\u003csup\u003e2\u003c/sup\u003eϕ〉 = 1 - 〈cos\u003csup\u003e2\u003c/sup\u003eφ〉. Since the fitted functions are Gaussians, 〈cos\u003csup\u003e2\u003c/sup\u003eφ\u003csub\u003e1\u003c/sub\u003e〉 can be approximated by {cos(0.4FWHM\u003csub\u003e210\u003c/sub\u003e)}x\u003csup\u003e2\u003c/sup\u003e and 〈cos\u003csup\u003e2\u003c/sup\u003eφ\u003csub\u003e1\u003c/sub\u003e〉 by {cos(0.4FWHM\u003csub\u003e020\u003c/sub\u003e)}\u003csup\u003e2\u003c/sup\u003e (Du et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\u0026lang;{cos}^{2}\\varphi\\:\u0026rang;=1-A\u0026lang;{cos}^{2}{\\varphi\\:}_{1}\u0026rang;-B\u0026lang;{cos}^{2}{\\varphi\\:}_{2}\u0026rang;$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eExemplary scattering data with fitted functions for all other parameters that are not shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e can be found in the supplementary information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ed. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed using Python (version 3.9). The analysis aimed to identify trends in key parameters as a function of extension, RH, and measurement zones (Bulk, Transition, and Edge). Parameters of interest included Herman\u0026rsquo;s orientation factor, lattice spacings, crystallinity parameter, and the unit cell parameters. For each parameter, error values originating from fitting were incorporated into the analysis.\u003c/p\u003e \u003cp\u003eTo analyze trends as a function of extension, weighted least-squares (WLS) regression was applied for each silk type, RH, and measurement zone. WLS regression was chosen because it accounts for heteroscedasticity in the data by weighting each data point according to the inverse square of its associated error. This ensures that data points with smaller errors contribute more to the regression model, improving the reliability of the results. The weights were based on the inverse square of the parameter\u0026rsquo;s error. For each regression, the slope, p-value, and direction of trend (increasing or decreasing) were extracted. Statistical significance was determined based on the p-value, with thresholds of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (significant), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (moderately significant), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (highly significant). Similarly, trends as a function of RH were analyzed for each silk type, extension, and measurement zone and WLS regression was applied.\u003c/p\u003e \u003cp\u003eTo assess potential differences between the zones for various parameters, a statistical analysis was conducted. The dataset included measurements for multiple parameters across different combinations of RH and extension steps. For each parameter, the data was grouped by zone, and statistical tests were performed to determine whether significant differences existed between the zones.\u003c/p\u003e \u003cp\u003eThe data was grouped by RH and extension step, and statistical tests were applied to each unique combination. A one-way ANOVA was used to test for differences in means between the zones when the data met the assumptions of normality and homogeneity of variance. For cases where these assumptions were not met, or where data was limited, a non-parametric Kruskal-Wallis test was used as an alternative. A significance level of 0.05 was applied, and p-values below this threshold were considered indicative of statistically significant differences between zones. For cases where significant differences were detected in the ANOVA, a post-hoc Tukey\u0026rsquo;s HSD test was performed to identify which specific pairs of zones differed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Major-ampullate\u003c/p\u003e\n\u003cp\u003eRH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Relative humidity\u003c/p\u003e\n\u003cp\u003eSAXS\u0026nbsp;\u0026nbsp;Small-angle X-ray scattering\u003c/p\u003e\n\u003cp\u003eSRO\u0026nbsp; \u0026nbsp;\u0026nbsp;Short-range order\u003c/p\u003e\n\u003cp\u003eT. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Trichonephila\u003c/p\u003e\n\u003cp\u003eTU\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tubuliform\u003c/p\u003e\n\u003cp\u003eWAXS\u0026nbsp;Wide-angle X-ray scattering\u003c/p\u003e\n\u003cp\u003eWLS \u0026nbsp; \u0026nbsp;Weighted least-squares\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank Maja Vasiljevic, Arno Frank, and Charbel Sakr for their assistance during the experiments conducted at the synchrotron facilities. Their support and expertise were instrumental in the successful completion of this work.\u003c/p\u003e\n\u003cp\u003ePart of the experiments were performed on beamline ID13 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France We acknowledge the ESRF for the provision of synchrotron radiation, and we would like to thank the beamline staff for assistance and support in using the beamline. We acknowledge the MAX IV Laboratory for beamtime on the NanoMAX beamline under proposal 20230683. Research conducted at MAX IV, a Swedish national user facility, is supported by Vetenskapsrådet (Swedish Research Council, VR) under contract 2018-07152, Vinnova (Swedish Governmental Agency for Innovation Systems) under contract 2018-04969 and Formas under contract 2019-02496.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded in whole by the Austrian Science Fund (FWF) [10.55776/P33613].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided online under DOI:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltman, G. H., Diaz, F., Jakuba, C., Calabro, T., Horan, R. L., Chen, J., Lu, H., Richmond, J., \u0026amp; Kaplan, D. L. (2003). Silk-based biomaterials. \u003cem\u003eBiomaterials\u003c/em\u003e,\u003cem\u003e 24\u003c/em\u003e(3), 401-416. https://doi.org/10.1016/s0142-9612(02)00353-8\u003c/li\u003e\n\u003cli\u003eBergmann, F., Stadlmayr, S., Millesi, F., Zeitlinger, M., Naghilou, A., \u0026amp; Radtke, C. (2022). The properties of native Trichonephila dragline silk and its biomedical applications. \u003cem\u003eBiomaterials Advances\u003c/em\u003e,\u003cem\u003e 140\u003c/em\u003e, 213089. https://doi.org/https://doi.org/10.1016/j.bioadv.2022.213089\u003c/li\u003e\n\u003cli\u003eBlackledge, T. A. (2012). Spider silk: a brief review and prospectus on research linking biomechanics and ecology in draglines and orb webs. \u003cem\u003eThe Journal of Arachnology\u003c/em\u003e,\u003cem\u003e 40\u003c/em\u003e(1), 1-12, 12. https://doi.org/10.1636/M11-67.1\u003c/li\u003e\n\u003cli\u003eBlamires, S. J., Nobbs, M., Martens, P. J., Tso, I. M., Chuang, W.-T., Chang, C.-K., \u0026amp; Sheu, H.-S. (2018). Multiscale mechanisms of nutritionally induced property variation in spider silks. \u003cem\u003ePLoS One\u003c/em\u003e,\u003cem\u003e 13\u003c/em\u003e(2), e0192005. https://doi.org/10.1371/journal.pone.0192005\u003c/li\u003e\n\u003cli\u003eBraxton, T., Holland, C., \u0026amp; Greco, G. (2025). The circular argument behind spider and silkworm silk mechanical properties. \u003cem\u003eMaterials \u0026amp; Design\u003c/em\u003e,\u003cem\u003e 260\u003c/em\u003e, 115224. https://doi.org/https://doi.org/10.1016/j.matdes.2025.115224\u003c/li\u003e\n\u003cli\u003eBrown, C. P., MacLeod, J., Amenitsch, H., Cacho-Nerin, F., Gill, H. S., Price, A. J., Traversa, E., Licoccia, S., \u0026amp; Rosei, F. (2011). The critical role of water in spider silk and its consequence for protein mechanics [10.1039/C1NR10502G]. \u003cem\u003eNanoscale\u003c/em\u003e,\u003cem\u003e 3\u003c/em\u003e(9), 3805-3811. https://doi.org/10.1039/C1NR10502G\u003c/li\u003e\n\u003cli\u003eCarbone, D., Kalbfleisch, S., Johansson, U., Bjorling, A., Kahnt, M., Sala, S., Stankevic, T., Rodriguez-Fernandez, A., Bring, B., Matej, Z., Bell, P., Erb, D., Hardion, V., Weninger, C., Al-Sallami, H., Lidon-Simon, J., Carlson, S., Jerrebo, A., Norsk Jensen, B., . . . Roslund, L. (2022). Design and performance of a dedicated coherent X-ray scanning diffraction instrument at beamline NanoMAX of MAX IV. \u003cem\u003eJournal of Synchrotron Radiation\u003c/em\u003e,\u003cem\u003e 29\u003c/em\u003e(3), 876-887. https://doi.org/doi:10.1107/S1600577522001333\u003c/li\u003e\n\u003cli\u003eCohen, N. (2023). The underlying mechanisms behind the hydration-induced and mechanical response of spider silk. \u003cem\u003eJournal of the Mechanics and Physics of Solids\u003c/em\u003e,\u003cem\u003e 172\u003c/em\u003e, 105141. https://doi.org/10.1016/j.jmps.2022.105141\u003c/li\u003e\n\u003cli\u003eCohen, N., Levin, M., \u0026amp; Eisenbach, C. D. (2021). On the Origin of Supercontraction in Spider Silk. \u003cem\u003eBiomacromolecules\u003c/em\u003e,\u003cem\u003e 22\u003c/em\u003e(2), 993-1000. https://doi.org/10.1021/acs.biomac.0c01747\u003c/li\u003e\n\u003cli\u003eDe Volder, M., Park, S., Tawfick, S., \u0026amp; Hart, A. J. (2014). Strain-engineered manufacturing of freeform carbon nanotube microstructures. \u003cem\u003eNature Communications\u003c/em\u003e,\u003cem\u003e 5\u003c/em\u003e(1), 4512. https://doi.org/10.1038/ncomms5512\u003c/li\u003e\n\u003cli\u003eDu, N., Liu, X. Y., Narayanan, J., Li, L., Lim, M. L., \u0026amp; Li, D. (2006). Design of superior spider silk: from nanostructure to mechanical properties. \u003cem\u003eBiophys J\u003c/em\u003e,\u003cem\u003e 91\u003c/em\u003e(12), 4528-4535. https://doi.org/10.1529/biophysj.106.089144\u003c/li\u003e\n\u003cli\u003eEwunkem, A. J., \u0026amp; Agee, K. (2022). Spider Parental Care and Awe-Inspiring Egg Sac (Cocoon). \u003cem\u003eInternational Journal of Zoology\u003c/em\u003e,\u003cem\u003e 2022\u003c/em\u003e, 6763306. https://doi.org/10.1155/2022/6763306\u003c/li\u003e\n\u003cli\u003eGiesa, T., Arslan, M., Pugno, N. M., \u0026amp; Buehler, M. J. (2011). Nanoconfinement of Spider Silk Fibrils Begets Superior Strength, Extensibility, and Toughness. \u003cem\u003eNano Letters\u003c/em\u003e,\u003cem\u003e 11\u003c/em\u003e(11), 5038-5046. https://doi.org/10.1021/nl203108t\u003c/li\u003e\n\u003cli\u003eGli\u0026scaron;ović, A., Vehoff, T., Davies, R. J., \u0026amp; Salditt, T. (2008). Strain Dependent Structural Changes of Spider Dragline Silk. \u003cem\u003eMacromolecules\u003c/em\u003e,\u003cem\u003e 41\u003c/em\u003e(2), 390-398. https://doi.org/10.1021/ma070528p\u003c/li\u003e\n\u003cli\u003eGosline, J. M., Guerette, P. A., Ortlepp, C. S., \u0026amp; Savage, K. N. (1999). The mechanical design of spider silks: from fibroin sequence to mechanical function. \u003cem\u003eJ Exp Biol\u003c/em\u003e,\u003cem\u003e 202\u003c/em\u003e(Pt 23), 3295-3303. https://doi.org/10.1242/jeb.202.23.3295\u003c/li\u003e\n\u003cli\u003eGrubb, D. T., \u0026amp; Jelinski, L. W. (1997). Fiber Morphology of Spider Silk:\u0026thinsp; The Effects of Tensile Deformation. \u003cem\u003eMacromolecules\u003c/em\u003e,\u003cem\u003e 30\u003c/em\u003e(10), 2860-2867. https://doi.org/10.1021/ma961293c\u003c/li\u003e\n\u003cli\u003eHu, X., Lawrence, B., Kohler, K., Falick, A. M., Moore, A. M. F., McMullen, E., Jones, P. R., \u0026amp; Vierra, C. (2005). Araneoid Egg Case Silk:\u0026thinsp; A Fibroin with Novel Ensemble Repeat Units from the Black Widow Spider, Latrodectus hesperus. \u003cem\u003eBiochemistry\u003c/em\u003e,\u003cem\u003e 44\u003c/em\u003e(30), 10020-10027. https://doi.org/10.1021/bi050494i\u003c/li\u003e\n\u003cli\u003eJensen, A. B., Christensen, T. E. K., Weninger, C., \u0026amp; Birkedal, H. (2022). Very large-scale diffraction investigations enabled by a matrix-multiplication facilitated radial and azimuthal integration algorithm: MatFRAIA. \u003cem\u003eJournal of Synchrotron Radiation\u003c/em\u003e,\u003cem\u003e 29\u003c/em\u003e(6), 1420-1428. https://doi.org/doi:10.1107/S1600577522008232\u003c/li\u003e\n\u003cli\u003eJohansson, U., Carbone, D., Kalbfleisch, S., Bjorling, A., Kahnt, M., Sala, S., Stankevic, T., Liebi, M., Rodriguez Fernandez, A., Bring, B., Paterson, D., Thanell, K., Bell, P., Erb, D., Weninger, C., Matej, Z., Roslund, L., Ahnberg, K., Norsk Jensen, B., . . . Vogt, U. (2021). NanoMAX: the hard X-ray nanoprobe beamline at the MAX IV Laboratory. \u003cem\u003eJournal of Synchrotron Radiation\u003c/em\u003e,\u003cem\u003e 28\u003c/em\u003e(6), 1935-1947. https://doi.org/doi:10.1107/S1600577521008213\u003c/li\u003e\n\u003cli\u003eKeten, S., \u0026amp; Buehler, M. J. (2010). Nanostructure and molecular mechanics of spider dragline silk protein assemblies. \u003cem\u003eJournal of the Royal Society Interface\u003c/em\u003e,\u003cem\u003e 7\u003c/em\u003e(53), 1709-1721. https://doi.org/10.1098/rsif.2010.0149\u003c/li\u003e\n\u003cli\u003eKieffer, J., Orlans, J., Coquelle, N., Debionne, S., Basu, S., Homs, A., Santoni, G., \u0026amp; De Sanctis, D. (2025). Application of signal separation to diffraction image compression and serial crystallography. \u003cem\u003eJournal of Applied Crystallography\u003c/em\u003e,\u003cem\u003e 58\u003c/em\u003e(1), 138-153. https://doi.org/doi:10.1107/S1600576724011038\u003c/li\u003e\n\u003cli\u003eKornfeld, T., Nessler, J., Helmer, C., Hannemann, R., Waldmann, K. H., Peck, C. T., Hoffmann, P., Brandes, G., Vogt, P. M., \u0026amp; Radtke, C. (2021). Spider silk nerve graft promotes axonal regeneration on long distance nerve defect in a sheep model. \u003cem\u003eBiomaterials\u003c/em\u003e,\u003cem\u003e 271\u003c/em\u003e, 120692. https://doi.org/10.1016/j.biomaterials.2021.120692\u003c/li\u003e\n\u003cli\u003eLechner, A., Trossmann, V. T., \u0026amp; Scheibel, T. (2022). Impact of Cell Loading of Recombinant Spider Silk Based Bioinks on Gelation and Printability. \u003cem\u003eMacromolecular Bioscience\u003c/em\u003e,\u003cem\u003e 22\u003c/em\u003e(3), 2100390. https://doi.org/https://doi.org/10.1002/mabi.202100390\u003c/li\u003e\n\u003cli\u003eLefevre, T., \u0026amp; Auger, M. (2016). Spider silk as a blueprint for greener materials: a review. \u003cem\u003eInternational Materials Reviews\u003c/em\u003e,\u003cem\u003e 61\u003c/em\u003e(2), 127-153. https://doi.org/10.1080/09506608.2016.1148894\u003c/li\u003e\n\u003cli\u003eLef\u0026egrave;vre, T., P\u0026eacute;zolet, M., Cooper, G., Cruz, D. H., West, M. M., Obst, M., Hitchcock, A. P., Karunakaran, C., \u0026amp; Kaznatcheev, K. (2009). Mapping molecular orientation in dry and wet\u0026lt;i\u0026gt;Nephila clavipes\u0026lt;/i\u0026gt;dragline spider silk. \u003cem\u003eJournal of Physics: Conference Series\u003c/em\u003e,\u003cem\u003e 186\u003c/em\u003e, 012089. https://doi.org/10.1088/1742-6596/186/1/012089\u003c/li\u003e\n\u003cli\u003eLi, J., Li, S., Huang, J., Khan, A. Q., An, B., Zhou, X., Liu, Z., \u0026amp; Zhu, M. (2022). Spider Silk-Inspired Artificial Fibers. \u003cem\u003eAdvanced Science\u003c/em\u003e,\u003cem\u003e 9\u003c/em\u003e(5), 2103965. https://doi.org/https://doi.org/10.1002/advs.202103965\u003c/li\u003e\n\u003cli\u003eLin, T.-Y., Masunaga, H., Sato, R., Malay, A. D., Toyooka, K., Hikima, T., \u0026amp; Numata, K. (2017). Liquid Crystalline Granules Align in a Hierarchical Structure To Produce Spider Dragline Microfibrils. \u003cem\u003eBiomacromolecules\u003c/em\u003e,\u003cem\u003e 18\u003c/em\u003e(4), 1350-1355. https://doi.org/10.1021/acs.biomac.7b00086\u003c/li\u003e\n\u003cli\u003eMarsh, R. E., Corey, R. B., \u0026amp; Pauling, L. (1955). The structure of tussah silk fibroin (with a note on the structure of [beta]-poly-l-alanine). \u003cem\u003eActa Crystallographica\u003c/em\u003e,\u003cem\u003e 8\u003c/em\u003e(11), 710-715. https://doi.org/doi:10.1107/S0365110X5500217X\u003c/li\u003e\n\u003cli\u003eNaghilou, A., Peter, K., Millesi, F., Stadlmayr, S., Wolf, S., Rad, A., Semmler, L., Supper, P., Ploszczanski, L., Liu, J., Burghammer, M., Riekel, C., Bismarck, A., Backus, E. H. G., Lichtenegger, H., \u0026amp; Radtke, C. (2023). Insights into the material properties of dragline spider silk affecting Schwann cell migration. \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e,\u003cem\u003e 244\u003c/em\u003e, 125398. https://doi.org/https://doi.org/10.1016/j.ijbiomac.2023.125398\u003c/li\u003e\n\u003cli\u003eNova, A., Keten, S., Pugno, N. M., Redaelli, A., \u0026amp; Buehler, M. J. (2010). Molecular and Nanostructural Mechanisms of Deformation, Strength and Toughness of Spider Silk Fibrils. \u003cem\u003eNano Letters\u003c/em\u003e,\u003cem\u003e 10\u003c/em\u003e(7), 2626-2634. https://doi.org/10.1021/nl101341w\u003c/li\u003e\n\u003cli\u003ePeter, K., Stadlmayr, S., Naghilou, A., Ploszczanski, L., Hofmann, M., Riekel, C., Liu, J., Burghammer, M., Gusenbauer, C., Konnerth, J., Schniepp, H. C., Rennhofer, H., Sinn, G., Radtke, C., \u0026amp; Lichtenegger, H. C. (2025). Exploring the Unique Properties and Superior Schwann Cell Guiding Abilities of Spider Egg Sac Silk. \u003cem\u003eACS Applied Bio Materials\u003c/em\u003e,\u003cem\u003e 8\u003c/em\u003e(2), 1307-1319. https://doi.org/10.1021/acsabm.4c01587\u003c/li\u003e\n\u003cli\u003ePlaza, G. R., Guinea, G. V., P\u0026eacute;rez‐Rigueiro, J., \u0026amp; Elices, M. (2006). Thermo‐hygro‐mechanical behavior of spider dragline silk: Glassy and rubbery states. \u003cem\u003eJournal of Polymer Science Part B: Polymer Physics\u003c/em\u003e,\u003cem\u003e 44\u003c/em\u003e(6), 994-999. https://doi.org/10.1002/polb.20751\u003c/li\u003e\n\u003cli\u003ePuranik, N., Tiwari, S., Kumari, M., Yadav, S. K., Dhakal, T., \u0026amp; Song, M. (2025). Advanced Bioactive Polymers and Materials for Nerve Repair: Strategies and Mechanistic Insights. \u003cem\u003eJournal of Functional Biomaterials\u003c/em\u003e,\u003cem\u003e 16\u003c/em\u003e(7), 255. https://www.mdpi.com/2079-4983/16/7/255\u003c/li\u003e\n\u003cli\u003eQiu, W., Patil, A., Hu, F., \u0026amp; Liu, X. Y. (2019). Hierarchical Structure of Silk Materials Versus Mechanical Performance and Mesoscopic Engineering Principles. \u003cem\u003eSmall\u003c/em\u003e,\u003cem\u003e 15\u003c/em\u003e(51), 1903948. https://doi.org/https://doi.org/10.1002/smll.201903948\u003c/li\u003e\n\u003cli\u003eRadtke, C., Allmeling, C., Waldmann, K. H., Reimers, K., Thies, K., Schenk, H. C., Hillmer, A., Guggenheim, M., Brandes, G., \u0026amp; Vogt, P. M. (2011). Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep. \u003cem\u003ePLoS One\u003c/em\u003e,\u003cem\u003e 6\u003c/em\u003e(2), e16990. https://doi.org/10.1371/journal.pone.0016990\u003c/li\u003e\n\u003cli\u003eRiekel, C., Burghammer, M., Dane, T. G., Ferrero, C., \u0026amp; Rosenthal, M. (2017). Nanoscale Structural Features in Major Ampullate Spider Silk. \u003cem\u003eBiomacromolecules\u003c/em\u003e,\u003cem\u003e 18\u003c/em\u003e(1), 231-241. https://doi.org/10.1021/acs.biomac.6b01537\u003c/li\u003e\n\u003cli\u003eRiekel, C., Burghammer, M., \u0026amp; Rosenthal, M. (2019). Nanoscale X-Ray Diffraction of Silk Fibers. \u003cem\u003eFrontiers in Materials\u003c/em\u003e,\u003cem\u003e 6\u003c/em\u003e. https://doi.org/10.3389/fmats.2019.00315\u003c/li\u003e\n\u003cli\u003eRising, A., \u0026amp; Johansson, J. (2015). Toward spinning artificial spider silk. \u003cem\u003eNat Chem Biol\u003c/em\u003e,\u003cem\u003e 11\u003c/em\u003e(5), 309-315. https://doi.org/10.1038/nchembio.1789\u003c/li\u003e\n\u003cli\u003eRuiz, V., Jiang, P., M\u0026uuml;ller, C., Jorge, I., V\u0026aacute;zquez, J., Ridruejo, \u0026Aacute;., Aznar-Cervantes, S. D., Cenis, J. L., Messeguer-Olmo, L., Elices, M., Guinea, G. V., \u0026amp; P\u0026eacute;rez-Rigueiro, J. (2019). Preparation and characterization of Nephila clavipes tubuliform silk gut [10.1039/C9SM00212J]. \u003cem\u003eSoft Matter\u003c/em\u003e,\u003cem\u003e 15\u003c/em\u003e(14), 2960-2970. https://doi.org/10.1039/C9SM00212J\u003c/li\u003e\n\u003cli\u003eSampath, S., Isdebski, T., Jenkins, J. E., Ayon, J. V., Henning, R. W., Orgel, J. P. R. O., Antipoa, O., \u0026amp; Yarger, J. L. (2012). X-ray diffraction study of nanocrystalline and amorphous structure within major and minor ampullate dragline spider silks. \u003cem\u003eSoft Matter\u003c/em\u003e,\u003cem\u003e 8\u003c/em\u003e(25), 6713. https://doi.org/10.1039/c2sm25373a\u003c/li\u003e\n\u003cli\u003eSch\u0026auml;fer, A., Vehoff, T., Gli\u0026scaron;ović, A., \u0026amp; Salditt, T. (2008). Spider silk softening by water uptake: an AFM study. \u003cem\u003eEuropean Biophysics Journal\u003c/em\u003e,\u003cem\u003e 37\u003c/em\u003e(2), 197-204. https://doi.org/10.1007/s00249-007-0216-5\u003c/li\u003e\n\u003cli\u003eSeydel, T., Knoll, W., Greving, I., Dicko, C., Koza, M. M., Krasnov, I., \u0026amp; M\u0026uuml;ller, M. (2011). Increased molecular mobility in humid silk fibers under tensile stress. \u003cem\u003ePhysical Review E\u003c/em\u003e,\u003cem\u003e 83\u003c/em\u003e(1), 016104. https://doi.org/10.1103/PhysRevE.83.016104\u003c/li\u003e\n\u003cli\u003eSponner, A., Vater, W., Monajembashi, S., Unger, E., Grosse, F., \u0026amp; Weisshart, K. (2007). Composition and hierarchical organisation of a spider silk. \u003cem\u003ePLoS One\u003c/em\u003e,\u003cem\u003e 2\u003c/em\u003e(10), e998. https://doi.org/10.1371/journal.pone.0000998\u003c/li\u003e\n\u003cli\u003eStadlmayr, S., Mautner, A., Bacher, M., Peter, K., Mentler, A., Schulz, S., Lichtenegger, H., Brecker, L., Bismarck, A., Naghilou, A., \u0026amp; Radtke, C. (2025). Holistic Analysis of Material Properties in Phylogenetically Diverse Spider Silks and Their Influence on Cell Adhesion. \u003cem\u003eAdvanced Functional Materials\u003c/em\u003e,\u003cem\u003e 35\u003c/em\u003e(15), 2415945. https://doi.org/https://doi.org/10.1002/adfm.202415945\u003c/li\u003e\n\u003cli\u003eStadlmayr, S., Peter, K., Millesi, F., Rad, A., Wolf, S., Mero, S., Zehl, M., Mentler, A., Gusenbauer, C., Konnerth, J., Schniepp, H. C., Lichtenegger, H., Naghilou, A., \u0026amp; Radtke, C. (2024). Comparative Analysis of Various Spider Silks in Regard to Nerve Regeneration: Material Properties and Schwann Cell Response. \u003cem\u003eAdvanced healthcare materials\u003c/em\u003e,\u003cem\u003e 13\u003c/em\u003e(8), 2302968. https://doi.org/https://doi.org/10.1002/adhm.202302968\u003c/li\u003e\n\u003cli\u003eTermonia, Y. (1994). Molecular Modeling of Spider Silk Elasticity. \u003cem\u003eMacromolecules\u003c/em\u003e,\u003cem\u003e 27\u003c/em\u003e(25), 7378-7381. https://doi.org/10.1021/ma00103a018\u003c/li\u003e\n\u003cli\u003eTian, M., \u0026amp; Lewis, R. V. (2006). Tubuliform silk protein: A protein with unique molecular characteristics and mechanical properties in the spider silk fibroin family. \u003cem\u003eApplied Physics A\u003c/em\u003e,\u003cem\u003e 82\u003c/em\u003e(2), 265-273. https://doi.org/10.1007/s00339-005-3433-8\u003c/li\u003e\n\u003cli\u003eVehoff, T., Gli\u0026scaron;ović, A., Schollmeyer, H., Zippelius, A., \u0026amp; Salditt, T. (2007). Mechanical Properties of Spider Dragline Silk: Humidity, Hysteresis, and Relaxation. \u003cem\u003eBiophysical Journal\u003c/em\u003e,\u003cem\u003e 93\u003c/em\u003e(12), 4425-4432. https://doi.org/https://doi.org/10.1529/biophysj.106.099309\u003c/li\u003e\n\u003cli\u003eWang, Q. J., \u0026amp; Schniepp, H. C. (2019). Nanofibrils as Building Blocks of Silk Fibers: Critical Review of the Experimental Evidence. \u003cem\u003eJom\u003c/em\u003e,\u003cem\u003e 71\u003c/em\u003e(4), 1248-1263. https://doi.org/10.1007/s11837-019-03340-y\u003c/li\u003e\n\u003cli\u003eWarwicker, J. O. (1956). The crystal structure of silk fibroins [10.1039/TF9565200554]. \u003cem\u003eTransactions of the Faraday Society\u003c/em\u003e,\u003cem\u003e 52\u003c/em\u003e(0), 554-557. https://doi.org/10.1039/TF9565200554\u003c/li\u003e\n\u003cli\u003eWarwicker, J. O. (1960). Comparative studies of fibroins. II. The crystal structures of various fibroins. \u003cem\u003eJ Mol Biol\u003c/em\u003e,\u003cem\u003e 2\u003c/em\u003e, 350-362. https://doi.org/10.1016/s0022-2836(60)80046-0\u003c/li\u003e\n\u003cli\u003eWharton, D., Weston, N., \u0026amp; Goodacre, S. (2025). Come rain or shine: effects of external conditions on the properties of linyphiid silk [Original Research]. \u003cem\u003eFrontiers in Arachnid Science\u003c/em\u003e,\u003cem\u003e Volume 4 - 2025\u003c/em\u003e. https://doi.org/10.3389/frchs.2025.1580992\u003c/li\u003e\n\u003cli\u003eWork, R. W., \u0026amp; Morosoff, N. (1982). A Physico-Chemical Study of the Supercontraction of Spider Major Ampullate Silk Fibers. \u003cem\u003eTextile Research Journal\u003c/em\u003e,\u003cem\u003e 52\u003c/em\u003e(5), 349-356. https://doi.org/10.1177/004051758205200508\u003c/li\u003e\n\u003cli\u003eYazawa, K., Malay, A. D., Masunaga, H., Norma-Rashid, Y., \u0026amp; Numata, K. (2020). Simultaneous effect of strain rate and humidity on the structure and mechanical behavior of spider silk. \u003cem\u003eCommunications Materials\u003c/em\u003e,\u003cem\u003e 1\u003c/em\u003e(1), 10. https://doi.org/10.1038/s43246-020-0011-8\u003c/li\u003e\n\u003cli\u003eYazawa, K., Malay, A. D., Masunaga, H., \u0026amp; Numata, K. (2018). Role of Skin Layers on Mechanical Properties and Supercontraction of Spider Dragline Silk Fiber. \u003cem\u003eMacromolecular Bioscience\u003c/em\u003e,\u003cem\u003e 19\u003c/em\u003e(3). https://doi.org/10.1002/mabi.201800220\u003c/li\u003e\n\u003cli\u003eYazawa, K., Malay, A. D., Masunaga, H., \u0026amp; Numata, K. (2019). Role of Skin Layers on Mechanical Properties and Supercontraction of Spider Dragline Silk Fiber. \u003cem\u003eMacromolecular Bioscience\u003c/em\u003e,\u003cem\u003e 19\u003c/em\u003e(3), 1800220. https://doi.org/https://doi.org/10.1002/mabi.201800220\u003c/li\u003e\n\u003cli\u003eYe, L., Liu, X., Li, K., Li, X., Zhu, J., Yang, S., Xu, L., Yang, M., Yan, Y., \u0026amp; Yan, J. (2023). A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials. \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e,\u003cem\u003e 253\u003c/em\u003e, 127125. https://doi.org/https://doi.org/10.1016/j.ijbiomac.2023.127125\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Synchrotron Radiation, In-situ Tensile Testing, X-ray Scattering, Ultrastructure, Humidity","lastPublishedDoi":"10.21203/rs.3.rs-8335511/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8335511/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpider silk’s exceptional mechanics and environmental adaptability stem from a hierarchical structure of crystalline β-sheet nanodomains embedded in an amorphous protein matrix. While major ampullate (MA) silk’s structural responses to strain and environment are well studied, tubuliform (TU) silk—critical for egg sac construction and promising for biomedical uses—remains poorly understood. We investigate TU and MA silk under varying relative humidity (RH) and mechanical strain using spatially resolved, high‑resolution nanobeam X‑ray diffraction. Increasing RH markedly reduces Young’s modulus, yield strain, and yield stress in both silks, with TU showing greater humidity sensitivity. Ultrastructural analysis reveals key contrasts: TU exhibits pronounced changes in nanocrystal dimensions, molecular alignment, and lattice spacing with strain and humidity, whereas MA maintains more stable crystalline organization, reflecting optimization for tensile strength. By mapping structural parameters across single-fiber diameters (edge, transition, bulk), we provide spatially resolved insights into TU’s ultrastructure and hierarchical mechanisms. Notably, structural parameters are predominantly homogeneous across the fiber diameter for both silks at the studied length scales. These distinct structural responses offer design templates for biomimetic materials. Future work should examine additional environmental variables (e.g., temperature, solvents) and finer length scales to further unlock spider silk’s potential as a model biomaterial.\u003c/p\u003e","manuscriptTitle":"Local changes in individual spider silk fibers from tubuliform and major-ampullate glands under varying humidity and tensile strain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 00:06:12","doi":"10.21203/rs.3.rs-8335511/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-materials","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmat","sideBox":"Learn more about [Communications Materials](https://www.nature.com/commsmat/)","snPcode":"","submissionUrl":"","title":"Communications Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cbb03075-f07f-498b-b04f-74b00f1cf12e","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60796835,"name":"Biological sciences/Structural biology/X-ray crystallography/Nanocrystallography"},{"id":60796836,"name":"Physical sciences/Materials science/Biomaterials/Biomaterials \u0026#x2013; proteins"},{"id":60796837,"name":"Biological sciences/Structural biology/X-ray crystallography/Nanocrystallography"},{"id":60796838,"name":"Physical sciences/Materials science/Biomaterials/Biomaterials \u0026#x2013; proteins"}],"tags":[],"updatedAt":"2026-03-06T10:46:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 00:06:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8335511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8335511","identity":"rs-8335511","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}