Morphology

The morphological features of the silks investigated in this study are presented in Fig. 1a. Variations in cocoon appearance, fibre packing density, degree of bonding and cross-sectional shape were observed. The B. mori cocoons exhibited a high degree of porosity and relatively loose bonding with sericin gum29, which correlates with the relative ease of unraveling the fibres from the cocoons. Antheraea cocoons featured thicker fibres that were densely packed, whereas S. c. ricini generated more loosely packed fibres. In A. aliena cocoons, the low porosity and brittle consistency of the silk made unraveling silk of extended fibre lengths difficult, whereas in R. fugax, fibres were densely packed and almost uninterruptedly coated with sericin. The cocoon from S. jonasii featured a lattice-like morphology with large apertures (>1 mm diameter), with fibres frequently occurring in thick bundles heavily bonded with sericin.

Figure 1 Morphologies of the different silks used in this study. (a) First row, representative photographs of cocoons (not to scale); second row, representative photomicrographs showing cross-sections of silk fibres embedded in cyanoacrylate glue (scale bar = 20 μm); third row, representative SEM images of fibre surfaces (scale bar = 10 μm). The phylogenetic assignment is based on Sima et al.47. Branch lengths are arbitrary and the position of R. fugax is tentatively assigned. (b) SEM image showing the presence of calcium oxalate crystals on the surface of S. c. pryeri silk fibre. (c) Cross-sectional photomicrograph showing the common occurrence of silk fibres stuck together with gum material in S. jonasii cocoons. (d) SEM image showing an example of abrupt changes in the morphology and fibre diameter in A. aliena. Full size image

Light microscopy of cross-sections and SEM surface visualization showed details of the fibre dimensions and morphology (Fig. 1a). In all cases, the native fibres (baves) consisted of two filaments or brins surrounded by an outer layer of sericin. The B. mori silk samples typically displayed triangular cross-sections, with considerable variations in surface areas. SEM images revealed a mostly intact sericin layer surrounding the fibres, occasionally with thin cracks or locally abraded regions (not shown). In Antheraea, Samia and R. fugax, the silk fibres had flattened or wing-shaped cross-sections, although variations were also common (e.g., as shown for A. yamamai). Under SEM, cubic crystals were frequently observed on the surface of the saturniid silks (shown in Fig. 1b for S. c. pryeri, with crystal dimensions of 0.5–2 μm on a side), corresponding to the secretion of calcium oxalate by the larvae during cocoon construction29. Overall, the A. aliena fibres showed the greatest structural heterogeneity, with frequent splitting and drastic changes in fibre diameter and morphology (Fig. 1c), which may help account for their poor mechanical properties (see below). In S. jonasii, the fibres were especially rounded and thick (typically >20 μm in diameter) and were typically encountered in bundles that were firmly bonded together (Fig. 1d).

Mechanical properties

The results of tensile deformation tests, which measured the ultimate tensile strength, extensibility (elongation to break), Young’s modulus and toughness (energy to break) of individual silk fibres are summarized in Table S1; representative stress-strain curves of each of the samples are shown in Fig. S81. The overall results fall within the range of expected values, although with a large data spread and a generally low level of reproducibility even between fibres from the same cocoon, as previously noted9,10,27,30,31. Certain trends were noted, e.g. the higher initial elastic modulus observed for Indian B. mori silk (with a mean value of 8.6 GPa) compared to the other B. mori samples, which may reflect differences in the local rearing environments. However, taken as a whole, no obvious correlations could be made between phylogeny and either the tensile strength or elastic modulus of the different samples, with mean values falling between 0.34–0.57 GPa and 4–8.6 GPa, respectively. On the other hand, the samples could be classified into two groups according to extensibility: those exhibiting higher strain to break included B. mori, Antheraea and Samia silks, with an overall mean range of 23–36%. The remainder of the samples, namely A. aliena, R. fugax and S. jonasii, displayed inferior extensibility values, ranging on average from 14–22%. As a consequence of the differences in extensibility, the calculated toughness values were also divergent between the two groups, which ranged from 0.06–0.1 GJ m−3 for the high extensibility silks and 0.03–0.06 GJ m−3 for the low extensibility silks.

Analysis of the tensile deformation curves showed that the different silk types followed different stress-strain paths, with qualitative features correlating with phylogeny (Fig. 2). Typical results of the B. mori tests are shown in Fig. 2a, which exhibited either simultaneous or separate breaking of the two strands upon the extension of force. The overall tensile path can be described as an initial elastic region of high modulus followed by an indistinct yielding region at approximately 5 to 10% strain and leading to a region with a nonlinearly decreasing slope toward eventual failure, consistent with previous reports13,32. Saturniid silks, in contrast, displayed stress-strain paths with generally more well-defined transitions. Figure 2b shows typical deformation curves for A. pernyi, representative of other Antheraea samples, which feature an initial elastic region and a prominent yield point at approximately 4% strain, followed by a post-yield region with significant differences in modulus among individual samples and frequently exhibiting sigmoidal strain hardening. A second yield point was typically observed at approximately 10% strain, followed by a region of lower slope until tensile failure10,11,33. Samia silk produced somewhat similar yet distinctive tensile behaviours, with a prominent yield point at around 4% strain usually followed by a plateau region that leads to an extended, uninterrupted strain-hardening region, until failure (Fig. 2c). The silk fibres from R. fugax featured two well-defined yield points at approximately 4 and 10% strain, although the extensibility was relatively low, with average breaking strains at approximately 20% elongation (Fig. 2d).

Figure 2 Stress-strain analyses of native silk fibres. Representative results are shown for (a) B. mori Jp, (b) A. pernyi Jp, (c) S. c. ricini Jp, (d) R. fugax. Full size image

Thermal analyses

The cocoon materials were subjected to thermogravimetric (TG) analyses, where changes in sample mass were measured as a function of a temperature gradient from 30 to 500 °C (Fig. 3). The overall results showed that the saturniid silks had higher thermal stability compared to B. mori, as seen in the values for the thermal decomposition peaks (T d ), in agreement with previous reports34,35,36. In most samples an initial weight loss was also seen below 100 °C, corresponding to the evaporation of adsorbed water. The exception is R. fugax, which produced a water evaporation peak at around 105 °C, presumably due to the dense packing of fibres in the cocoon, which could impede the loss of water molecules.

Figure 3 Thermogravimetric analyses of the different cocoon samples. Each panel combines the percent mass loss data (TG: top curve, left axis) and the first derivative plots of the percent mass remaining (DTG: bottom curve, right axis) versus temperature. Temperatures corresponding to peaks or transitions are indicated. (a) B. mori, (b) Antheraea, (c) Samia, (d) A. aliena, R. fugax and S. jonasii. Full size image

In the B. mori samples, no significant changes were observed after the water peak until above 200 °C and an abrupt decrease occurred beyond 280 °C, producing a T d peak at around 335 °C in the differential TG plots (DTG). Approximately 40% of the initial weight remained at the end of the run at 500 °C. For the saturniid silks, Antheraea and Samia samples showed qualitatively similar TG profiles, although the latter produced higher T d values and had somewhat sharper overall features. In contrast to B. mori, the thermal degradation followed a two-step regime, producing a shoulder at approximately 330–340 °C in the DTG plots, followed by a more drastic decrease in mass. A similar multistep profile was reported for regenerated film derived from degummed A. pernyi fibres34. A small but distinct peak was also sometimes observed at approximately 160–170 °C in the derivative plots, presumably corresponding to the degradation of calcium oxalate crystals37,38. The wild silk samples from A. aliena, R. fugax and S. jonasii showed broader transitions and generally less distinct features compared to the other saturniid silks (Fig. 3).

Differential scanning calorimetry (DSC) results are shown in Fig. 4 and Table S2. Consistent with the TG data, the saturniid silks showed more highly defined profiles with a greater number of transitions compared to B. mori. In B. mori samples, aside from the T w and T d peaks, an indistinct endothermic peak was seen at approximately 230 °C (denoted as T en1 ). The saturniid silks, in contrast, typically produced two small, distinct endotherms (T en1 and T en2 ), which have been attributed to molecular motion within the amorphous or laterally ordered regions of fibroin39. Another small endotherm sometimes appeared between 160–165 °C, attributed to the decomposition of calcium oxalate crystals (T co )37. A. aliena, R. fugax and S. jonasii displayed less defined peaks compared to the other saturniid samples, possibly reflecting a reduction in concerted molecular motions during heating. The overall results of the thermal analysis are compiled in Table S2.

Figure 4 DSC measurements of the different cocoon samples. Temperatures corresponding to the peaks or transitions are indicated: T w = water evaporation peak; T en(1,2) = endothermic peaks; T co = peak attributed to the degradation of calcium oxalate crystals; T d = thermal degradation peak. (a) B. mori, (b) Antheraea, (c) Samia, (d) A. aliena, R. fugax and S. jonasii. Full size image

X-ray scattering and birefringence

Synchrotron wide-angle X-ray scattering (WAXS) experiments were performed on the cocoon material from the different samples (Fig. S2). The B. mori, Antheraea and Samia samples produced strong reflections, compared to A. aliena, R. fugax and S. jonasii, which produced weakly diffracting patterns. Bragg peaks corresponding to the (020), (210), (100) and (300)/(400) reflections were identified in most cases. The 1D intensity profiles derived from azimuthal integration along the equatorial direction were fitted using Gaussian functions and the resulting full width at half maximum (fwhm) of the (020) and (210) peaks were used to calculate crystallite sizes (Fig. S3). The results of the analysis are shown in Fig. 5, which shows average values for (020) and (210) of 2.6 nm and 4.5 nm, respectively, for the B. mori samples and around 3.6 nm and 4.5 nm, respectively, for the saturniid silk samples. The degree of crystallinity of the different silk samples was also estimated from the WAXS data, as summarized in Table 1. All of the samples had crystallinity values ranging between 25–35%, suggesting a similar overall abundance of nano-crystalline structures. Birefringence was also measured to probe the degree of orientation of the crystalline units along the fibre axis (Table 1). The results showed considerably higher values for birefringence in B. mori compared to the saturniid silks, as in previous reports36,37, which together with the crystallinity measurements indicate an overall greater degree of order in the b-sheet along the longitudinal axes. The retardance images occasionally featured local regions of heterogeneity, suggesting certain variations in orientation of the crystalline regions caused by the natural spinning process (Fig. S4).

Table 1 Calculated values for crystallinity and birefringence. Full size table

Figure 5 Calculated β-sheet crystallite sizes by applying Scherrer’s equation to the full width at half maximum (fwhm) of the (020) (red circle) and (210) peaks (blue square). Error bars correspond to standard errors (n = 3). Full size image

Sequence analyses

The sequences of the repetitive fibroin domains from the different samples relevant to this study were analysed (Fig. 6). In case of Actias aliena and Saturnia jonasii only sequences from the congeneric A. selene and S. japonica, respectively, were available; these were used for the analysis on the assumption that the composition of the repetitive regions has been conserved at the genus level, as seen among Antheraea species15,40,41. It should be noted that intraspecific polymorphisms are known to occur in the silk genes, with variations in the overall length of the coding regions and in the relative arrangement of the sequence subtypes within the repetitive domains. However, based on current knowledge, the composition of tandem repeats is conserved within each species42,43,44,45.

Figure 6 Fibroin amino acid sequence analysis. (a) Representative repetitive sequences of the different silk fibroin are shown: B. mori heavy chain (GenBank AF226688), demonstrating the hierarchical arrangement of motifs and with the conserved spacer sequence underlined, S. cynthia ricini (BAQ55621), A. pernyi (AAC32606), A. selene (deduced sequence)71, R. fugax (BAG84270), S. japonica (BAH02016) and MaSp1 from spider dragline silk of Nephila clavipes14 (M37137). (b) Relative proportion of poly(Ala) residues within the saturniid fibroin tandem repeats versus the decomposition temperature (T d ) of the different samples derived from TG analysis, with error bars corresponding to standard deviation values. (c) Average relative abundance of selected residues and motifs within the repetitive regions of the different fibroin samples. Full size image

All analysed sequences showed a preponderance of Gly, Ala, Ser and Tyr residues; however, the organization of the internal repeats are different between the bombycoid and saturniid fibroins. The B. mori sequence features repetitive arrays of (GA)nGX (where X = S, Y or V), forming large blocks of varying length and arrangement that are interrupted by spacer sequences comprising ~43 residues20,46. In contrast, the saturniid repetitive domains consisted of alternating tandem repeats of poly(Ala) and non-poly(Ala) regions. The poly(Ala) blocks comprised on average 12–13 contiguous Ala residues, while the non-poly(Ala) blocks are Gly-rich, featuring various combinations of GX and GGX (X = typically A, S, Y, D, L or R). Interspecific variations within the non-poly(Ala) blocks were noted. The S. c. ricini sequence carried four subtypes bearing GGX motifs, while Antheraea sequences (A. pernyi, A. yamamai and A. assama) featured three GGX-containing subtypes plus a shorter subtype rich in charged polar residues (e.g. RRAGHDRAA in A. pernyi). In A. aliena, R. fugax and S. jonasii, the non-poly(Ala) regions contained a relatively low abundance of GGX motifs, but a relatively high proportion of bulky, hydrophobic residues.

Quantification of the abundance of poly(Ala) residues revealed that S. c. ricini contained the highest level among the saturniid repetitive sequences, at approximately 44.5% the total length of each tandem repeat, versus 43% for A. yamamai, 42.2% for A. pernyi, 39.6% for A. assama, 37% for R. fugax, 36% for S. japonica and 35.8% for A. selene. Strikingly, plotting the values of abundance of poly(Ala) residues among the different species revealed an excellent agreement with the thermal degradation values observed from the TG experiments (Figs 6b and 3), with the highest stability corresponding to the Samia samples, with a T d at approximately 382 °C. Conversely, the lowest abundance of poly(Ala) residues, in the Actias, Rhodinia and Saturnia sequences, exhibited the lowest thermal degradation temperatures, at around 369–371 °C. These findings support a view that the poly(Ala) runs constitute the β-crystalline component of silk that imparts rigidity to the polymer structure. Interestingly, no such correlation was observed between the average lengths of the poly(Ala) stretches per se and degradation temperature (not shown), suggesting that the ratio of crystalline to non-crystalline fractions is the main determinant of thermal stability.

It is useful to compare the sequences of Antheraea with Actias as they are phylogenetically closely related47 yet produce silks with divergent material properties; the latter is particularly brittle and exhibits low extensibility, as reported here and elsewhere9. Both sequences harbour a similar subset of tandem repeats, although Actias selene carries a relatively higher proportion of residues with bulky, hydrophobic side chains (notably Leu) compared to Antheraea. Plotting the abundance of selected sequence elements within the repetitive regions of the different fibroins gives a clearer view of these differences (Fig. 6c). Comparing the different saturniid species, the greatest variations were seen in the relative abundance of large hydrophobic residues, namely Leu, Val, Ile, Phe and Trp and in the abundance of GGX motifs. S. c. ricini in particular and the three Antheraea species, showed high GGX levels but relatively few hydrophobic residues. Conversely, the wild silks of R. fugax, A. selene and S. japonica all contained much higher levels of the bulky hydrophobic residues but fewer GGX motifs48,49. These results suggest that the balance between the abundance of large hydrophobic residues and flexible glycine-rich motifs, both of which are situated within the fibre amorphous phase, plays an important role in determining the overall material properties of the fibres. A summary of the results from the sequence analyses is presented in Table 2.