Native Silk Fibers: Protein Sequence and Structure Influences on Thermal and Mechanical Properties.

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Published in final edited form as: Biomacromolecules. 2025 Mar 7;26(4):2043–2059. doi: 10.1021/acs.biomac.4c01781 Search in PMC Search in PubMed View in NLM Catalog Add to search Native silk fibers: protein sequence and structure influences on thermal and mechanical properties Elizabeth L Aikman Elizabeth L Aikman 1 Department of Chemical Engineering, University of Florida, Gainesville FL, 32611, USA Find articles by Elizabeth L Aikman 1, † , Lauren E Eccles Lauren E Eccles 1 Department of Chemical Engineering, University of Florida, Gainesville FL, 32611, USA Find articles by Lauren E Eccles 1, † , Whitney L Stoppel Whitney L Stoppel 1 Department of Chemical Engineering, University of Florida, Gainesville FL, 32611, USA Find articles by Whitney L Stoppel 1, * Author information Article notes Copyright and License information 1 Department of Chemical Engineering, University of Florida, Gainesville FL, 32611, USA † ELA and LEE contributed equally to the manuscript * Correspondence: Whitney L. Stoppel, PhD, 1030 Center Drive, PO Box 116005, Gainesville, FL 32611, USA, [email protected] Issue date 2025 Apr 14. PMC Copyright notice PMCID: PMC12155892  NIHMSID: NIHMS2082145  PMID: 40052735 The publisher's version of this article is available at Biomacromolecules Abstract Silk fibers produced by arthropods have inspired an array of materials with applications in healthcare, medical devices, textiles, and sustainability. Silks exhibit biodiversity with distinct variations in primary protein constituent sequences (fibroins, spidroins) and structures across taxonomic classifications, specifically the Lepidopteran and Araneae orders. Leveraging the biodiversity in arthropod silks offers advantages due to the diverse mechanical properties and thermal stabilities achievable, primarily attributed to variations in fiber crystallinity and repeating amino acid motifs. In this review, we aim to delineate known properties of silk fibers and correlate them with predicted protein sequences and secondary structures, informed by newly annotated genomes. We will discuss established patterns in repeat motifs governing specific properties and underscore the biological diversity within silk fibroin and spidroin sequences. Elucidating the relationship between protein sequences and properties of natural silk fibers will identify strategies for designing new materials through rational silk-based fiber design. Keywords: biopolymers, silk fibroin, major ampullate spidroin, protein composition, secondary structure mechanical properties Graphical Abstract 1. Introduction Natural fibers are products of years of evolutionary adaptation and development, bringing forth unique properties, structures, or functions that are specifically tailored to the needs and function of the organism. Thus, many groups have focused on natural biopolymers that comprise these fibers to elucidate the polymer structure to function relationships in nature, harness these advantageous properties in biomaterials, and fabricate materials that mimic the structure and properties of these natural polymers for applications in medicine, textiles, food, and agriculture. Natural biopolymers produced by plants or other organisms comprise many different classes of natural fibers currently in commercial use, such as cellulose, chitosan, collagen, and silk. 4 – 7 Silk fibers produced by species within the Araneae and Lepidopteran orders are most commonly investigated due to their superior mechanical strength and cultivation in textiles. Efforts to explore silks produced by Trichoptera (caddisflies), Hymenoptera (bees, wasps, ants), and other orders within the Insecta class 10 are strongly driven by the discovery and application of properties through the intersection of entomology and engineering. While there exist many organisms that have not been fully characterized due to challenges with organism cultivation or scale of polymer production, silk fibers are highly diverse, complex protein systems that have the potential to meet the required needs of robust and tunable biopolymers. The vast biodiversity of silk fiber composition, material properties, and abundance rises from the unique utilization of silk throughout an arthropod’s lifecycle. 10 , 11 Spiders can produce up to 7 types of silk, each with varying degrees of elasticity, strength, toughness, and adhesion tailored to distinct purposes of structural support and reinforcement, prey capture, attachment to surfaces, or reproduction. 12 Many species produce only a few types of silk throughout their lifecycle, though species in the family Araneidae (orb-weavers) are capable of producing all 7 types of silk in their webs ( Figure 1 ). 1 However, spider major ampullate (Ma, dragline) silk is primarily discussed in literature due to its high strength and elasticity relevant to biomaterial, industrial, and biomimetic material research 13 – 15 and numerous dedicated efforts towards understanding the dynamics of structure-function relationships within these silk fibers. 1 , 16 – 21 Figure 1: Open in a new tab The use of silk in nature depends on the arthropod and their environment. Araneae species can produce up to 7 different types of silk with varying strength, adhesion, or roughness tailored to a specific purpose. Trichoptera silk can be used to form nets to catch prey in streams or to generate protective cases by connecting small pebbles or rocks with silk fibers. Lepidopteran insects utilize silk to form protective and insulating cocoons, build nests, navigate across terrains, or mark trails to food sources. Silkworms produce one type of fiber, though the use of silkworm fibers vary across species depending on the insect’s environment and life cycle stage. Silk fiber morphology and production are variable within species strains or between insects of the same order ( Figure 2 ). The primary use of silkworm silk is in the construction of their cocoon to protect the insect during pupation, though cocoon silk fibers from Bombyx mori ( B. mori , Figure 2E ) have been observed to differ in color, porosity, and other structural characteristics across species strains. 22 , 23 B. mori and Saturniidae cocoon silks have also exhibited slight variability in physical properties as a function of the region in which the silk was collected, or rather the environmental conditions in which they were spun. 24 Some species, such as the Eastern tent caterpillar ( Malacosoma americanum ) 25 and the pantry moth ( Plodia interpunctella , P. interpunctella ), 26 , 27 will also produce large amounts of silk in late larval stages to aid in navigation to food sources or nesting to protect from predators or extreme temperatures, in addition to spinning cocoons ( Figure 1 ). Caddisflies, aquatic silk-spinning insects, also vary in their production of silk – case-builders use silk to form structural armor from small organic debris or pebbles ( Figure 2CD ), net-spinners spin small retreats to habitate or catch prey, and free-living caddisflies, which have neither a case nor retreat, primarily produce an underwater cocoon prior to pupation ( Figure 1 ). 28 – 30 Study of these insects has been focused on understanding how silk-spinning processes translate into wet environments, and how differences in silk protein composition influence the self-assembly and function of silk fibers underwater. 2 , 30 – 33 Expanding investigations into other silk-producing species opens opportunities to broaden the silk biomaterial field and improve the understanding of how differing silk protein structures translate to the structure and function of material performance. Figure 2: Open in a new tab Morphological variations of silk fibers within Arthropod orders. Representative SEM images of silk fibers from ( A ) Caerostris darwini , ( B ) Trichonephila clavata , ( C ) Hesperophylax occidentalis , ( D ) Limnephilus lunatus , ( E ) Bombyx mori , and ( F ) Antheraea yamamai . (A) and (B) are reprinted from the Spider Silkome database ( https://spider-silkome.org/ ) generated from Arakawa et al. 1 (C) is reprinted from Wang et al., 2 Copyright (2014), with permission from Elsevier. (D) is reprinted with permission from Rouhova et al. 3 (E) is reprinted from Eccles et al., 8 Copyright (2025), with permission from Elsevier. (F) is reprinted with permission from Zurovec et al. 9 Copyright 2016 American Chemical Society. 2. Structural and functional diversity of silk fibers Variations in specific purposes of silk fibers are supported by distinct differences in silk protein production, composition, and structure. Silk fibers are produced in arthropod silk glands, beginning as a heterogeneous solution of proteins secreted from cells in the silk gland lumen. 10 , 11 , 34 These highly diverse proteins are differentially expressed throughout silk glands and undergo sequential or combinatorial series of environmental gradients, water diffusion, and flow fields that induce structural transitions and lead to the formation of highly complex, hierarchical structures in Lepidoptera and Araneae silks ( Figure 3 ). 11 , 34 , 35 Figure 3: Open in a new tab Silk fiber composition and structure is analogous in Lepidoptera and Araneae. ( A ) Silk fiber formation and hierarchical structure in silkworms. ( B ) Spinning and hierarchical structure of major ampullate silk in spiders. Components of primary structural silk proteins (blue) and functional coating proteins (orange) are labeled corresponding to their relative location in the fiber structure. 2.1. Lepidoptera Lepidopteran silk fiber structure is primarily comprised of a structural fibroin core surrounded by an adhesive, protective coating which aids in securing the two fibroin brins into a single fiber and inferring unique properties to the silk ( Figure 3 ). Silk fibroin can be composed of up to 3 subunits secreted in the posterior region of the silk gland (PSG), a heavy chain, light chain, and a small glycoprotein, fibrohexamerin (P25/Fhx), that associates with the two chains and aids in fibroin stability. 36 FibH proteins are characterized by non-repetitive N- and C- termini and a highly repetitive region that correlates to the crystalline and amorphous structures that give rise to strength, elasticity, extensibility, and rigidity of silkworm silk fibers. 37 – 43 Proteins in the outer coating are less conserved or investigated than fibroins in Lepidoptera, though they include sericins, seroins, and other diverse proteins (mucins, enzymes, protease inhibitors) with varied function. 44 – 46 Sericins are a large family of proteins secreted in the middle of the silk gland (MSG) that comprise a large portion of the outer coating and aid in adhesion and antimicrobial activity. 9 , 45 – 47 Sericin types (Sericin A, B, C, and sericin-like) and ratios of sericins to other proteins and are highly variable across species, 9 , 46 though many groups have worked to utilize sericin proteins in biomaterials (recently reviewed by Veiga et al . 48 ). Small glycoproteins, termed seroins, produced in the MSG and PSG are classified into 5 subtypes, varying in activity, composition, and expression patterns within the silk gland. 44 , 49 , 50 Seroins have exhibited antiviral and antibacterial activity in addition to possible roles in the secretion and assembly of fibroin proteins due to their presence in both the outer coating and fibroin core. 44 , 49 While sericins, seroins, and other proteins present in the coating layer have functional properties that grant insects advantageous protection and capabilities, the physical properties of Lepidopteran silk fibers are primarily driven by silk fibroin and more specifically FibH. We focus on fibroin heavy chain as a major protein component of silk fibers in Lepidoptera and highlight the repeat motifs, repeat regularity, and amino acid composition of these proteins within Lepidopteran insects in the context of observed material properties. 2.2. Araneae Similar to Lepidopteran silks, major ampullate (Ma) spider silk consists of an inner structural protein core surrounded by a protective, functional coating or skin ( Figure 3 ). Major ampullate spider silk is single stranded, often produced in duplicate from paired silk glands, with a structural core contained within a skin layer and surrounded by glycoprotein-rich and lipid-rich layers. 34 , 51 The structural core is primarily composed of major ampullate spidroin (MaSp) proteins and can be further separated into an inner and outer core, defined by the presence of MaSp subtypes. 51 MaSp are large, semi-crystalline proteins with highly repetitive domains that primarily drive physical properties of major ampullate spider silk and non-repetitive N- and C- termini, analogous to FibH in Lepidoptera. Components that form the skin, glycoprotein, and lipid layers are not well known, but contain a variety of unidentified glycoproteins, carbohydrates, and lipids sometimes denoted as spider silk-constituting elements (SpiCE). 34 , 35 , 52 The utility of these layers to overall silk fiber function has been examined previously by Sponner et al . in Trichonephila clavipes ( T. clavipes ). 51 The skin offers resistance against chemical agents and microbial activities, in addition to adding plasticity and mechanical support under environmental stressors. The glycoprotein-rich coating serves as a barrier between the fiber and the environment. 51 As such, the glycoprotein coating is suggested to aid in maintaining water balance, contributing to the contraction state of the fiber (pliancy) and therefore function, as well as antimicrobial and lubricating properties. 51 In T. clavipes , the outermost layer of spider silk, the lipid coat, is believed to primarily function as a carrier for pheromones and pigments, aiding in sex or species recognition. 51 , 53 While the structural core and coating layers contribute to the functional utility of major ampullate spider silk, the self-assembly and secretion of these proteins is not yet understood in many species. Sonavane et al. provide an extensive, multi-level investigation into the structure and composition of the major ampullate silk gland of the Lariniodes sclopetarius orb-weaving spider, gaining insights into the biological organization of these complex processes that depend on protein compositional differences and structures. 34 We highlight the compositional and structural differences of major ampullate spidroin proteins and their implications to fiber properties of major ampullate silk. Through comparison of core structural protein repetitive regions and physical properties of insect and spider silks, we aim to provide insights into its structural organization as well as identify potential applications for these proteins for the fabrication of bioinspired materials, looking for strategies to employ rationale design of new materials through a better understanding of the connection between protein sequence and physical properties of natural silk materials. A better understanding will support future endeavors in the generation of artificial arthropod silks and synthetic proteins fabricated to mimic or match native silk fiber properties. 3. Silk Fibroin Heavy Chain (FibH) Conservation of repeat motifs and trends in FibH protein size are commonly observed within species of the same taxonomic family, as discussed further in this section and summarized in Table 1 . We primarily highlight complete FibH sequences in families in which physical properties of the native fibers are known to enable more thorough comparisons. However, we also acknowledge that many evaluations of diverse silks are limited by 1) scale of silk production/collection and the ability to evaluate fiber properties and/or 2) confirmation of the complete coding sequence for silk fibroin and other supporting proteins. For silks with incomplete sequence analysis, but reported properties, we describe the known repeat motifs of the partial repetitive region for relative analyses (noted by “-” in Table 1 ). The assembly of accurate, full-length gene and protein sequences of FibH has many hurdles, mainly due to their large size and extensive repeat regions, which makes them difficult to obtain through traditional short-read sequencing techniques. Thus, advancement of long-read, low-error sequencing technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore Technology (ONT), as well as genome assembly tools/algorithms has enabled many groups to make great strides in deciphering the complete genome and FibH sequence of more diverse insect silks (long-read sequencing and technologies are reviewed more in depth by Espinosa et al . 54 ). We highlight a few of these recent works 29 , 43 , 55 – 57 and others that have built upon the high quality genomes generated through the Darwin Tree of Life project 46 , 58 to bring attention to their unique FibH protein structures and their utility in a material discovery and development fields. Table 1: Repetitive amino acid motifs of Lepidoptera silk fibroin heavy chain (FibH) proteins. a , b Species Family FibH MW [kDa] GX (A) n (S) n /(A) n GGX Reference Bombyx mandarina Bombycidae 477.8 • 1 Lu 2023, 2 Zhang 2024 Bombyx mori Bombycidae 391.4 • 3 Zhou 2000 Acentria ephemerella Crambidae 535.1 • • • 4 Wu 2022, 5 Heckenhauer 2024 Chilo suppressalis Crambidae 383.1 • • • 4 Wu 2022 Elophila obliteralis Crambidae 528.8 • • 5 Heckenhauer 2024 Nymphula nitidulata Crambidae 494.5 • • • 5 Heckenhauer 2024 Parapoynx stratiotata Crambidae 614.6 • • • 5 Heckenhauer 2024 Bambalina sp. Psychidae - • • 6 Kono 2021 Canephora pungelerii Psychidae - • • 6 Kono 2021 Eumeta variegata Psychidae - • • 7 Yoshioka 2019, 8 Kono 2019 Acrobasis suavella Pyralidae 534.5 • • • • 4 Wu 2022 Endotricha flammealis Pyralidae 685.4 • • • • 4 Wu 2022 Ephestia kuehniella Pyralidae 493.7 • • • • 4 Wu 2022 Galleria mellonella Pyralidae 487.8 • • • • 4 Wu 2022 Plodia interpunctella Pyralidae 413.3 • • • 4 Wu 2022, 9 Kawahara 2022 Hypsopygia costalis Pyralidae 522.0 • • • • 4 Wu 2022 Actias luna Saturniidae 234.8 • • • 10 Marke 2024 Antheraea assamensis Saturniidae 229.5 • • • 11 Malay 2016 Antheraea pernyi Saturniidae 216.1 • • • 11 Malay 2016 Antheraea yamamai Saturniidae 234.6 • • • 11 Malay 2016 Samia cynthia ricini Saturniidae 227.4 • • • 11 Malay 2016 Open in a new tab a (−) represents sequences described as partial in NCBI or by the reference authors. b (•) denotes the presence of the repeat motif within the FibH protein. 3.1. Bombycidae Silkworm moth larva, family Bombycidae, produce silk throughout larval development for navigation and stability, though the greatest silk production occurs in the final life stage to construct a cocoon for protection during pupation. Bombycidae cocoon silk, specifically one of the most well-known and researched silkworm species, Bombyx mori ( B. mori , the domesticated silkworm), is extensively studied due to its abundance over other growth stage and wild silks and its applications in textiles and biomaterial research. 14 , 60 The molecular weight of B. mori FibH is 391 kDa, almost 90 kDa less than its common ancestor, Bombyx mandarina ( B. mandarina , 477.8 kDa) ( Table 1 ). 37 , 43 , 59 Bombycidae repeat units consist largely of (GX) n motifs in the form of the hexapeptide (GA) n GX where X is S, Y, or V with interspersed amorphous linker sequences (approximately 30–40 residues) ( Table 1 ). 37 , 38 , 43 Most of the crystalline region is comprised of (GAGAGS) n repeats, resulting in the formation of tightly packed, anti-parallel β-sheet structures through inter- and intra-molecular hydrogen bonding between adjacent amino acid chains. 37 , 61 , 62 Other motifs such as GAGAGY, GAGAGV, and GAGAGVGY containing large side chains (Y) and hydroxyl groups (V) are less common in the repetitive region of B. mori and B. mandarina FibH, yet still contribute to the formation of semi-crystalline domains. 62 The advantageous protein characteristics of Bombycidae FibH rise from its large β-sheet secondary structures, giving high rigidity to the folded protein, in addition to elasticity and movement in the protein chain imparted by the short, flexible amorphous sequences. 3.2. Saturniidae Silkworms in Saturniidae, some of the largest moth species in the world, primarily utilize silk fibers to form cocoons, sometimes incorporating leaves or other plant debris of their host trees as a camouflage strategy against predators. Saturniidae FibH proteins range between 216.1 kDa ( Antheraea pernyi ) to 234.8 kDa ( Actias luna ) with the repetitive domain generally consisting of alternating polyA [(A) n ] and glycine-rich (non-polyA) regions ( Table 1 ). PolyA motifs vary in length across species, averaging 12–13 alanine residues, while the glycine-rich region comprises of smaller motifs (GX) n or (GGX) n (X=A, S, Y, D, L, R) that varies largely across species ( Table 1 ). 24 , 56 Single glycine repeat motifs (GX) n could be structurally similar to the dominant, highly crystalline (GX) n motifs of Bombycidae FibH, though the stringent combination of polyA and glycine-rich repeats more closely resemble repetitive regions of major ampullate spider silk, 1 , 16 , 17 , 63 , 64 exhibiting similar properties of elasticity, extensibility, and strength in mechanical evaluation. 24 Alanine-alanine interactions within polyA regions are hypothesized to defer rigidity to the polymer structure through forming β-sheet structures. 24 As such, the proportion of polyA motifs within the repetitive region has been shown to positively correlate with thermal degradation, wherein silks with increased polyA content have higher degradation temperatures. 24 Additionally, diversification of amino acids interspersed within the glycine-rich repeats could impact the modularity of the protein chain, such as larger hydrophobic residues in wild species, Rhodinia fugax and Actias selene , leading to observed trends in physical properties across eight Saturniidae species. 24 3.3. Pyralidae Pyralids are a largely diverse, small moth family in which larva are commonly concealed feeders, frequently deemed as pest species of stored food products and agricultural crops. Pyralids spin silk for cocoons for pupation and throughout earlier life stages as they infest their food source or wander to locate spaces to spin their cocoon. FibH in pyralids ranges between 413.3 kDa ( P. interpunctella ) to 685.4 kDa ( Endotricha flammealis, E. flammealis ) with high proportions of G, S, and A residues in the protein sequence ( Table 1 ). 46 , 55 The large repetitive regions of Pyralid FibH have been found to be highly species-specific with varying arrangements and length of repetitive sequences, influencing the regularity of the sequence, which has been correlated previously to mechanical properties of Galleria mellonella ( G. mellonella ), P. interpunctella , and B. mori . 8 , 39 Despite the large diversity in sequence, known FibH proteins possess the general form of 4 hydrophobic residues (V or I), 1–4 hydrophilic residues (E, N, Q, R, D), and a serine and alanine-rich ((S) n /(A) n ) crystalline motif followed by a terminal repeat section comprised largely of glycine alternating with bulky amino acid residues (Y, L, W) ( Table 1 ). 39 , 46 Crystalline sequences include SSAAAAASSSS ( Ephestia kuehniella ), SSAASAAAA ( G. mellonella ), and SSAAAAAAAAAAA ( E. flammealis ), though the length of the polyA sequence is shorter on average than polyA sequences in Saturniidae. 24 , 46 Terminal repeat sections contribute to the high variability in repeat sequence regularity to a great extent, though it is hypothesized that fewer, longer homogenous repeats correlate to higher strength than shorter erratic ones. 8 , 39 3.4. Psychidae Bagworm species (family Psychidae) spin silk to build a “bag” constructed of silk and plant materials to protect larva from predators and attach this cocoon-like structure to surfaces throughout their development. Recent studies have explored the unique functional properties of bagworm silk and the structural similarities of Psychidae FibH when compared to other FibH and MaSp proteins. 40 – 42 In known Psychidae FibH proteins, the repetitive domain is characteristic of a polyA motif with a central glutamic acid residue (E), a pure (GA) n motif, an amorphous linker sequence, and a (GA) n region containing serine ( Table 1 ). 40 – 42 The polyA motifs in Psychidae have a similar utility to Saturniidae FibH polyA, contributing to the crystalline content of the protein through formation of β-sheet structures. Additionally, uninterrupted (GA) n repeats correlate to crystalline β-sheet structures, while the latter linker sequence and (GA) n GS contribute to amorphous or semi-crystalline structures within Psychidae FibH. 40 – 42 Lengths of the flanking (GA) n GS region varies in Eumeta variegata ( E. variegata ), Canephora pungelerii ( C. pungelerii ), and Bambalina sp. bagworms, with C. pungelerii comprising about half the length of the regions in Bambalina sp. and E. variegata . 40 , 41 Recent work by Kono et al . has proposed that the balance between crystalline (polyA and pure (GA) n ) and amorphous (linker, (GA) n GS) regions influences the tensile strength and modulus of silk fibers, wherein C. pungelerii silk exhibited values almost double those of Bambalina sp. and E. variegata . 41 3.5. Other Lepidoptera Investigations into the FibH of other Lepidoptera families are expanding as technologies to sequence the highly repetitive regions of FibH with high fidelity improve and as insects are cultivated and studied. Crambidae, the grass moth family, mostly consists of terrestrial silkworms, though a few species are semi-to fully aquatic throughout their life. Closely related to Pyralidae (both in the superfamily Pyraloidea), FibH from Crambid species is between 383.1 kDa ( Chilo suppressalis ) and 614.6 kDa ( Parapoynx stratiotata ) and contains characteristic polyA, (S) n /(A) n , and (GA) n domains with distributed amorphous sequences of terrestrial Lepidoptera ( Table 1 ). 46 , 57 However, Crambidae FibH sequences contain higher percentages of charged residues (P, L, R, D, E) than terrestrial Lepidoptera, exhibiting amino acid compositions that also trend to the FibH of aquatic silk-spinning Trichoptera. 57 Additionally, FibH from Hepialidae ( Hepialus californicus ) 65 and Endromidae ( Andraca theae ) 66 families have been characterized to provide insights on secondary structures within the protein, possessing similar crystalline motifs to other Lepidoptera (polyA, (GX) n , (GA) n ). Despite the increase in fully annotated FibH proteins in Lepidoptera, correlation of physical properties to known sequences are limited to native silk fibers where these material properties are reported. Thus, an essential aspect of understanding the sequence-structure-function relationship is evaluating the properties of these fibers through methodologies that yield comparable assessments across diverse silks. 3.6. Trichoptera Caddisflies (order Trichoptera) spin and utilize their silk underwater, forming protective cases, retreats, nets, and cocoons. Trichoptera silk has numerous advantageous properties as a natural material, namely underwater adhesive properties, strain recovery and hysteresis in stress response, and reversible assembly crystalline β-sheet structures, 32 , 30 , 31 which likely stem from the unique composition and structure of caddisfly FibH proteins. Still, comparisons of physical properties of caddisfly silk to other native silk fibers are difficult, as caddisfly silk is often not produced at high scales and many mechanical assessments are evaluated in water or ionic buffers to study the silk in conditions that more closely resemble its natural system. Nevertheless, we highlight the unique repetitive regions of Trichoptera FibH here to discuss hypotheses of caddisfly silk stability, strength, and the potential utility of caddisfly FibH sequences to inspire biomimetic materials with their structures and functions. Standring et al . provides a more comprehensive discussion of caddisfly silk diversity, silk fiber protein composition and structure, and perspectives on the properties and sequence-structure-function relationships within aquatic silk fibers in a recent review. 30 Trichoptera silks are composed of some of the largest silk fibroins to be reported, ranging from 628.3 kDa ( Himalospyche tibetanaa ) to up to almost 1000 kDa ( Limnephilus lunatus ). 3 , 28 , 29 , 55 Trichoptera FibH repetitive regions are also distinctly different from terrestrial Lepidoptera, with (SX) n E motifs (X is often V or I) followed by a glycine and proline-rich region (GX, GGX, GPGXX) that is variable in length. 28 , 29 (SX) n and glycine-proline interactions are predicted to form β-sheet and β-turn structures, respectively, representing a secondary structure formation not previously observed in Lepidoptera or Araneae silks. Trichoptera FibH have more abundant serine and charged amino acid residues (D, E, R, L), with considerably low alanine content, compared to many Lepidoptera. 29 , 57 These compositional differences enable phosphorylation of the protein chain (S), hypothesized to promote interfacial adhesion, 33 and stabilization of crystalline structures, promoting strength and mechanical response, in the presence of common multivalent cations in streams (Ca 2+ , Mg 2+ , Fe 3+ ). 31 , 32 While the evolutionary adaptation and roles in self-assembly of these unique silk proteins are yet to be fully understood, 30 Trichoptera FibH and other aquatic silk proteins call attention to a vast field of untapped potential as new functional materials or components for recombinant protein design and production. 4. Major Ampullate Spidroins (MaSps) Major ampullate silk is primarily composed of two main subtypes, MaSp1 and MaSp2, that differ in repeat motif composition and presence within the silk fiber ( Figure 3 ), suggesting these proteins have distinct structural roles within spider silk. Stemming from the advancement of sequencing and assembly technologies, many recent works have also identified additional MaSp subtypes in some species such as Trichonephila clavipes (8 subtypes, MaSp-a to MaSp-h), 67 Caerostris darwini ( C. darwini ; MaSp3, MaSp4, and MaSp5), 20 , 68 and Araneus ventricosus ( A. ventricosus ; MaSp3 and MaSp4). 64 , 69 Several groups have comparatively analyzed protein sequences and silk fibers across and within species to identify links between sequential differences and measured properties. A recent publication by Arakawa et al . provided the largest collection of spider silk transcriptomes (over 1000 species) and fiber properties (over 400 species) that links genotype to phenotype in major ampullate silk, in addition to other spider silk types illustrated in Figure 1 (provided in a searchable database: https://spider-silkome.org/ ). 1 Similar to Lepidoptera FibH, the complete protein sequence for these highly repetitive MaSp proteins is difficult to obtain, with much literature reporting partial N-terminus or C-terminus sequences for evolutionary or lineage analyses. As such, we highlight general trends in MaSp1 and MaSp2 sequence and variability (summarized in Table 2 ), other observed MaSp subtypes and their distinctions, and the hypothesized role of these structural proteins in major ampullate silk fibers. Table 2: Repetitive amino acid motifs of Araneae major ampullate spidroins (MaSp) proteins. a , b Species Family MaSp Subtype MaSp MW [kDa] GX (A) n GGX GPGXX Reference Araneus diadematus Araneidae MaSp1 - • • • 1 Arakawa 2022 MaSp2 - • • Araneus ventricosus Araneidae MaSp1 219.8 • • • 2 Kono 2019 MaSp2 245.4 • • • MaSp3 225.3 • • • MaSp4 211.8 • • 3 Wen 2023 Argiope argentata Araneidae MaSp1 - • • • 4 Baker 2022 MaSp2 - • • • • MaSp3 - • • • • Argiope aurantia Araneidae MaSp1 - • • • 4 Baker 2022 MaSp2 - • • • MaSp3 - • • • • Argiope trifasciata MaSp1 - • • • 4 Baker 2022 Araneidae MaSp2 - • • • MaSp3 - • • • • Caerostris darwini Araneidae MaSp1 246.2 • • • 5 Kono 2021, 6 Garb 2019 MaSp2 - • • • MaSp3 194.4 • • MaSp4 251.7 • MaSp5 99.4 • • Caerostris extrusa Araneidae MaSp1 310.5 • • • 5 Kono 2021 MaSp2 350.2 • • • MaSp3 200.0 • • • MaSp4 159.5 • MaSp5 120.6 • • Nephila pilipes Araneidae MaSp1 211.7 • • • 7 Kono 2021 MaSp2 254.8 • • • MaSp3 223.0 • • Trichonephila clavata Araneidae MaSp1 238.5 • • • 7 Kono 2021 MaSp2 305.1 • • • MaSp3 219.3 • • Trichonephila clavipes Araneidae MaSp1 221.7 • • • 7 Kono 2021 MaSp2 296.6 • • • MaSp3 177.8 • • Latrodectus geometricus Theridiidae MaSp1 - • • • 1 Arakawa 2022 MaSp2 - • • • • Latrodectus hesperus Theridiidae MaSp1 250.26 • • • 8 Ayoub 2007 MaSp2 311.5 • • • • Open in a new tab a (−) represents sequences described as partial in NCBI or by the reference authors. b (•) denotes the presence of the repeat motif within the MaSp protein. 4.1. Major Ampullate Spidroin 1 (MaSp1) MaSp1 comprises the majority of the core of major ampullate silk fibers ( Figure 3 ) and plays a large role in conferring strength to the fiber through specific arrangements and composition in its repetitive region. 1 , 51 The repetitive domain of MaSp1 proteins are dominated by motifs of polyA, (GX) n , and GGX (where X is often A, S, Y, L, Q) ( Table 2 ). 16 , 17 , 52 , 63 , 64 , 70 PolyA repeats are highly important in MaSp1 as the interlocked, hydrophobic interactions between consecutive alanine residues form extensive hydrogen bond-rich β-sheet regions that are critical to high strength, 16 , 17 as observed in some Lepidoptera silks. 24 , 40 – 42 , 56 Similarly, (GA) n motifs also comprise β-sheet crystalline structures, though they are hypothesized to have fewer hydrophobic interactions within the protein due to glycine abundance and lower binding energy. 16 Thus, the presence of both large polyA and (GA) n /(GX) n motifs versus primarily (GA) n /(GX) n abundance could be important in comparisons between Lepidoptera and Araneae silks, as (GA) n motifs would correlate to a lower tensile strength due to decreased interactions within and between structural proteins. 16 The elastic (semicrystalline to amorphous) regions of MaSp1 proteins are rich in GGX repeats which primarily form 3 10 -helices or β-turn structures. 16 The function of these motifs likely serves as a linker between crystalline domains, acting as spacers to aid crystalline structure alignment and playing a fundamental role in the extensibility of the protein under stress. 16 Comparative analyses of MaSp1 proteins between spider species revealed the strict uniformity of internal repeats within MaSp1 repetitive regions, with divergence between species primarily appearing in repeat length, composition, and frequency. The percent of the repetitive region covered by recognized structural motifs, or motif coverage, between Latrodectus hesperus ( L. hesperus ) and Argiope aurantia ( A. aurantia ) was comparable in total (94.2% versus 86.2%, respectively), though the individual contributions of polyA, (GA) n , GGX, and GXG motifs varied considerably. 19 The lengths, periodicity, and presence of charged and hydrophobic residues of these internal repeats were distinctly different between the two species, with L. hesperus displaying more periodicity in MaSp1, shorter polyA and uninterrupted GGX motifs, and lower levels of hydrophilic and negatively charged residues. 19 , 63 Furthermore, within 5 species studied by Malay et al ., all MaSp1 proteins displayed varying arrangements of (GX) n and (GGX) n motifs, though the only conserved motif across all species was GGY. 18 As observed in Lepidoptera, these differences of motif types contribute to the ratio of crystalline to elastic/amorphous structures and further suggest the importance of sequence regularity and the roles of certain motifs in silk protein function and fiber properties. Increasing polyA lengths were negatively correlated to supercontraction, owing to the rigid crystalline structure of interacting polyA chains in the presence or absence of humidity fluctuations. 1 Higher level periodicity may contribute to toughness properties of silk fibers, 19 possibly allowing the repeating motifs to form consistent short- and long-range interactions that bring about high mechanical strength. The abundance of tyrosine in repeat motifs (GGY) in MaSp1 proteins has also brought about hypotheses of its function in protein self-assembly, possibly playing a role in chain packing, similar to silkworm silks. 1 , 18 4.2. Major Ampullate Spidroin 2 (MaSp2) MaSp2 is the second major subtype of major ampullate spidroins, present in only the inner core of major ampullate spider silk ( Figure 3 ). In contrast to the role of MaSp1, MaSp2 is attributed to deferring elasticity and extensibility to silk fibers due to differences in the arrangement and composition of repeat motifs within the spidroins. MaSp2 proteins have been found to contain repeat motifs of polyA, GGX (X is often A, Y, S, Q), and GPGXX (X is often G, P, A, Y, Q, S) and are more proline-rich when compared to MaSp1 ( Table 2 ). 16 , 17 , 52 , 63 , 64 , 70 PolyA chains correspond to crystalline β-sheet regions as in other structural proteins, while the unique GGX and GPGXX motifs are responsible for forming 3 10 -helices and β-turns/β-spiral structures, respectively, within the protein. The flexibility and contractive properties of the spring-like GPGXX structures underlies the elastic mechanism of MaSp2 silk, contributing to dragline silk fiber extensibility and supercontraction in response to humidity changes. Many groups also note the frequent occurrence of di-glutamine (QQ), di-serine (SS), and SQ doublet motifs within some MaSp2 proteins and propose functions in the hierarchical organization of dragline silk, 18 , 19 , 35 , 63 , 64 , 70 possibly correlated to the observed compositional differences between MaSp1 and MaSp2 within the inner and outer regions of the structural core ( Figure 3 ). Variability in MaSp2 proteins among Araneae species exist primarily in the abundance or length of certain motifs that have been shown to correlate to observed fiber function. Malay et al . found a higher degree of interspecific sequence conservation in comparison to MaSp1 proteins in the family Araneidae, with the maintenance of a ~32 residue sequence containing polyA, GGY, QQ, and GPGXX motifs. 18 Alternatively, across families, differences in the abundance of GP, QQ, and GGY motifs in MaSp2 were correlated to differing web morphologies of orb weavers (Araneidae, Nephilidae, Deinopoidea), cobweb builder (Theridiidae), and sheet web builder (Pisauridae) species. 18 Relative to physical properties, the inclusion of longer or more abundant polyA chains was shown to negatively correlate with supercontraction and extensibility (strain at break) by Arakawa et al . 1 Likewise, the degree of supercontraction and extensibility was positively correlated to the abundance of glycine-rich motifs which are responsible for elastic or spring-like secondary structures. 1 The extensibility of dragline silk fibers has also been hypothesized to correspond to the relative length of GPGXX motifs, with longer, more uninterrupted motifs aligning with greater extensibility. 16 , 19 This trend further underlines the importance of sequence regularity (or periodicity) in structural proteins to bring about a desired function, which was observed in mechanical strength comparisons of Lepidoptera species B. mori , G. mellonella , and P. interpunctella . 8 , 39 Higher degrees of interruption, or irregularity, within repeat motif types in Lepidoptera FibH and Araneae MaSp2 align with decreased tensile strength and extensibility, 39 the respective structural roles of the proteins, possibly due to inability to maintain the inter- and intrachain interactions as in more ordered fibroins and spidroins. Overall, observed trends in variable repeat motifs and silk fiber physical properties speak further to the balance of crystalline and amorphous domains within structural proteins. Depending on the desired function of the protein, a trade-off between high strength and low flexibility (crystalline) and extensibility and low stiffness (amorphous/elastic) is contingent on the intricate divide between repeat motifs. 4.3. Other Major Ampullate spidroins Major ampullate spidroins subtype 3 (MaSp3) has distinct differences in repeat motif composition than MaSp1 and MaSp2. MaSp3 has been distinguished previously by the lack of characteristic polyA motifs (MaSp1 and MaSp2) and GPGXX (MaSp2) and higher percentages of large and polar amino acids. 71 Although, exceptions to this trend have been observed in six species of Araneidae MaSp3 ( Table 2 ). 52 , 64 , 70 In general, MaSp3 repetitive regions are dominated by GGX (X is primarily R and Y) motifs, identified in up to 90 species in the Araneidae family. 1 , 20 , 52 , 64 , 70 , 71 The presence of MaSp3 in major ampullate silk is correlated to silks with exceptional high toughness, possibly achieved through the increased extensibility granted from abundant GGX 3 10 -helical structures. 16 , 20 , 68 Major ampullate subtype 4 (MaSp4) proteins have also been found to lack crystalline polyA regions, containing primarily GPGPQ motifs (unique variants of GPGXX observed in MaSp2) with interspersed hydrophobic motifs ( Table 2 ). 20 , 69 MaSp4 have been identified in Araneidae species A. ventricosus , C. darwini , and Caerostris exuga ( C. exuga ) dragline silks, further supporting the high extensibility of these silks as MaSp4 repeat motifs are characteristic of elastic, β-turn structures. 20 , 68 , 69 Major ampullate subtype 5 (MaSp5) is also possessed by bark spiders C. darwini and C. exuga , rich in GGX (X is L, S, R) motifs and lacking polyA chains ( Table 2 ). 20 , 68 The expression of these unique, primarily elastic spidroins aid in deferring high extensibility to silk fibers, supporting hypotheses that ratios of structural protein types, and by extension, roles (i.e., strength vs. elasticity), are essential to the interactions that drive physical properties of silks. These protein-level trends speak further to the importance of the primary driving factors of silks, amino acid repeat motifs, and the delicate balance that dictates physical properties. 5. Sequence-structure-function relationships of native silk fibers Differences in functional ability of FibH and MaSp are hypothesized to be due to the protein structure differences between arthropods, with varying crystallinity, charge, and stability that could be directly translated to or inspire artificial production of materials with tailored elasticity and strength, thermal stability, or interactions with molecules or substrates. It is important to acknowledge that the correlation of silk protein sequence and structure to fiber physical properties is limited in terms of species, primarily due to either incomplete deduced protein sequences or lack of comparable physical properties and/or methodologies. As described through Table 1 and Table 2 , we denoted the major repeat motifs present in Lepidopteran FibH and Araneae MaSp. To better compare the effect of the repeat motifs on silk physical properties, the overall protein sequence was used to quantify the percentage of amino acid composition, crystalline regions, and repeat motifs (accession numbers of proteins are listed in Table S1 ). Amino acid composition and isoelectric points of FibH and MaSp proteins were exported from Geneious ® Biologics software. Repeat motifs (GX, (A) n , GGX, GPGXX) were identified in FibH and MaSp sequences in Geneious ® Biologics software and total motif counts were recorded for each sequence in the order of (A) n , GPGXX, GGX, and GX to avoid double counting glycine-rich motifs (illustrated in Figure 4 ). Table S1 summarizes these values for a selection of silk producing species that have full or partial sequences available. We focus on comparing the tensile modulus, tensile strength, and decomposition temperature reported for only native silk fibers, rather than degummed fibers, as degumming agents and temperatures can inflict changes on the fibers and structural proteins outside of just removing the outer protein coating. 72 , 73 Figure 4: Open in a new tab Repeat motif coverage within structural proteins ( A ) FibH, ( B ) MaSp1, and ( C ) MaSp2. Percent coverage values of the five motifs and accession numbers of proteins analyzed is available in Table S1 . 5.1. Mechanical Properties It is difficult to predict the success of existing materials and new silk sources due to the complexity of the proteins involved. Mechanical properties are typical characterization parameters for determining applications for a given material. For fibers and fiber-like materials, extensional mechanical properties are most relevant to the application of the fiber. Tensile modulus describes the elastic nature of a material and its ability to return to its original state after deformation. 74 , 75 Tensile strength is the maximum force that a sample can be displaced before fracture in the plastic regime. 75 Highlighting what is known both about a specific organism’s silk fiber properties, but also relating these properties back to the predicted protein sequence and secondary structure, based on newly annotated genomes, is not abundantly clear in literature. Fiber mechanical properties are highly dependent on fiber diameter, strain rate, fiber length, environmental conditions, and experimental method. 76 , 77 Sample size and instrument type can also affect the quality of data collected. Table 3 provides tensile modulus, tensile strength, and strain rates for many Lepidoptera and Aranea species. Another parameter to note is the strain response of silk fibers, as many researchers in literature report maximum strain values or extensibility. Fibers are typically able to reach maximum strains around 20% to 50% of their original resting length, however this parameter is greatly dependent on strain rate. 76 , 77 The mechanical properties provided in Table 3 are calculated with dimensions (cross-sectional area), so a better comparison can be made across fiber types. The strain rate is also reported to compare common experimental methods and demonstrate the effect of these contributing factors and differences among experimental techniques. There is good agreement of a consistent strain rate (10 mm/min) in the Lepidoptera mechanical properties reported, 8 , 24 , 40 , 41 which makes comparing these fibers across species easier. We also want to point out differences in comparing strain rates written as a percentage of overall length or as a millimeter distance as silk fibers are generally viscoelastic and highly dependent on strain rates. Table 3: Tensile modulus, tensile strength, and extensional strain rate reported for native silk fibers of Lepidoptera and Araneae silks. a , b Species Family Tensile Modulus [GPa] Tensile Strength [MPa] Extensional Strain Rate Reference Lepidoptera Silks B. mandarina Bombycidae 14.37 ± 2.87 556.95 ± 116.92 1 mm/min 1 Lu 2023 B. mori Bombycidae 11.7 ± 2.2 635 ± 108 50 %/min 2 Guo 2018 B. mori (Jp) Bombycidae 5.13 ± 1.6 400 ± 110 10 mm/min 3 Malay 2016 B. mori (Cn) Bombycidae 7.23 ± 1.68 460 ± 80 10 mm/min 3 Malay 2016 B. mori (In) Bombycidae 6.66 ± 1.06 550 ± 110 10 mm/min 3 Malay 2016 B. mori (Th) Bombycidae 8.61 ± 2.19 570 ± 120 10 mm/min 3 Malay 2016 Eumeta variegata Psychidae 5.67 ± 0.66 636 ± 55 10 mm/min 4 Kono 2019 Bambalina sp. Psychidae 8 700 10 mm/min 5 Kono 2021 * C. pungelerii Psychidae 11.9 1250 10 mm/min 5 Kono 2021 * G. mellonella Pyralidae 3.2 – 12.6 308.8 – 1270.4 6 %/min # 6 Glasper 2019 P. interpunctella Pyralidae 0.536 ± 0.127 24.2 ± 6.7 10 mm/min 7 Eccles 2025 A. pernyi Saturniidae 5 ± 0.6 426 ± 55 50 %/min 2 Guo 2018 A. aliena (wild) Saturniidae 4.09 ± 0.28 430 ± 10 10 mm/min 3 Malay 2016 A. assamensis Saturniidae 4.31 ± 1.15 360 ± 100 10 mm/min 3 Malay 2016 A. assamensis Saturniidae 5.3 ± 0.4 495 ± 48 50 %/min 2 Guo 2018 A. pernyi (Cn) Saturniidae 4.76 ± 1.09 430 ± 80 10 mm/min 3 Malay 2016 A. pernyi (Jp) Saturniidae 4.72 ± 1.73 340 ± 80 10 mm/min 3 Malay 2016 A. yamamai Saturniidae 4.58 ± 0.58 390 ± 70 10 mm/min 3 Malay 2016 R. fugax (wild) Saturniidae 4.69 ± 1.98 390 ± 140 10 mm/min 3 Malay 2016 S. c. pryeri Saturniidae 4.71 ± 1.26 530 ± 150 10 mm/min 3 Malay 2016 S. c. ricini Saturniidae 4.1 ± 0.6 284 ± 88 50 %/min 2 Guo 2018 S. c. ricini (In) Saturniidae 4.69 ± 1.26 470 ± 110 10 mm/min 3 Malay 2016 S. c. ricini (Jp) Saturniidae 4.61 ± 2.01 380 ± 120 10 mm/min 3 Malay 2016 S. jonasii (wild) Saturniidae 7.1 ± 2.6 370 ± 100 10 mm/min 3 Malay 2016 Araneae Major Ampullate Silks A. diadematus Araneidae 1.2 ± 0.2 824 ± 10 0.4 mm/min 8 Kohler 1995 A. diadematus Araneidae 10.4 ± 1.5 1700 ± 200 50 %/min 9 Liu 2007 A. ventricosus Araneidae - 906.9 ± 100.9 600 mm/min # 10 Kono 2019 A. ventricosus Araneidae 6.36 1050 10 mm/min 11 Arakawa 2022 A. argentata Araneidae 8.21 1463 6 %/min # 12 Swanson 2006 A. argentata Araneidae 8.0 ± 0.8 1217 ± 56 6 %/min # 13 Blackledge 2006 A. trifasciata Araneidae 11.2 ± 0.7 1192 ± 61 1.2 %/min # 14 Guinea 2005 C. darwini Araneidae 11.5 1652 600 %/min # 15 Agnarsson 2010 C. darwini Araneidae 9.54 1190 10 mm/min 11 Arakawa 2022 C. darwini Araneidae 8.73 ± 0.76 1170 ± 140 10 mm/min 16 Kono 2021 C. extrusa Araneidae 8.91 ± 1.29 1310 ± 190 10 mm/min 16 Kono 2021 N. pilipes Araneidae 8.54 1250 10 mm/min 11 Arakawa 2022 T. clavata Araneidae 12.18 1610 10 mm/min 11 Arakawa 2022 T. clavipes Araneidae 13.8 ± 0.8 1215 ± 60 6 %/min # 12 Swanson 2006 T. clavipes Araneidae 10.4 ± 2.86 1560 ± 240 10 mm/min 11 Arakawa 2022 L. geometricus Theridiidae 9.77 ± 1.25 970 ± 30 10 mm/min 11 Arakawa 2022 L. hesperus Theridiidae 17.4 ± 1.9 1400 ± 100 50 %/min 9 Liu 2007 L. hesperus Theridiidae 10.2 ± 0.75 1441 ± 60 6 %/min # 12 Swanson 2006 Open in a new tab a (#) denotes a standardized unit conversion from seconds to minutes. b (*) denotes values that were approximated from figures. Firstly, comparisons between Lepidoptera silks ( Figure 5A ) show good agreement within a given taxonomic family (expanded graphs in Figure S1 ) for the properties reported in Table 3 . Most tensile modulus values sit within the same order of magnitude (0 – 10 GPa), but tensile strength values span a smaller range (0 – 1000 MPa). In general silks within the same family have similar tensile properties. Saturniidae silks show the most clustering and overlap between mechanical property values as shown in Figure 5A . The impact of environmental conditions on silk properties is best shown through the reported Bombycidae values. B. mori sourced from four different countries shows an order of magnitude spread of tensile modulus (5 – 12 GPa) within the same species. 24 However, none of these values are outside the range of those given for Lepidoptera silks. With the lowest number of species reported, Pyralidae silks show a large range in variability. From all reported values, P. interpunctella has lower mechanical and thermal properties 8 than all other species reported here. Figure 5: Open in a new tab Silk fiber mechanical properties in Lepidoptera and Araneae from Table 3 . ( A ) Silk fiber tensile strength versus tensile modulus in silkworms. ( B ) Silk fiber tensile strength versus tensile modulus in spiders. Spider silks are most notable for their high mechanical strength. For example, C. darwini has the highest tensile properties reported in Table 3 , with a tensile strength ten times stronger than Kevlar. 78 Due to spider’s usage of silk and their cannibalistic nature, acquiring spider silk on scalable quantities to undergo tensile testing with many replicates is not always feasible. Additionally, for those spiders with fiber mechanical properties, the spiders are often randomly chosen from their proximity to researchers and not their affinity to a potential function. 78 There is less agreement in spider silk literature of a common strain rate among mechanical assessments and less available literature for single fiber extensional testing among different research groups. However, it should be noted that conventional strain rates described in Table 3 are generally much slower than the native function of spider silks in nature, such as stretching and catching flying insects. Due to the high strain rate dependence of silk fibers, Agnarsson et al . has suggested testing spider silks at a variety of strain rates due to the variety of natural uses of silks. 78 From the silk sources reported here, Araneae silks (from Table 3 ) shown in Figure 5B highlight this high strength where the reported tensile values are all greater than 500 MPa. Comparisons between Lepidoptera and Araneae silks further demonstrate the high strength of spiders as many Lepidoptera mechanical property values are an order of magnitude lower than Aranea silks. For traditional natural fiber uses (textiles, manufacturing, etc.) this high strength is preferred, but there are instances (tissue engineering, biologics, etc.) where lower strength materials would be required. From the spread in mechanical properties available across species and within a given family, connections can be made about the structure-mechanical function relationship in silks. In silk literature it has been hypothesized that the mechanical response of the material is directly related to the protein content and resulting secondary structure, with emphasis given to the frequency of the repeat unit structure correlating to mechanical strength. 1 , 39 , 55 Short motifs with irregular spacing are not able to have as many interactions as compared to a longer more repetitive repeat unit, resulting in possibly a poorly organized and weak material. 39 More repetitive units that contribute to β-sheet formation can generate large crystalline structures that are rigid but very strong. 24 , 39 Comparisons can be drawn between extensional mechanical properties and the percentage of repeat units present in the protein structure for Lepidoptera FibH. More rigid and crystalline structures tend to have higher tensile strength values while more elastic and flexible structures will show lower tensile values. Protein structures and repeat motifs that contribute to β-sheet formation should lead to higher mechanical property values as β-sheet regions are more crystalline and have better packing efficiency leading to more rigid structures. From a comparison of frequency of each amino acid present within the silk protein sequence and repeat motif sequence and frequency to mechanical properties, a select few amino acids and motifs showed common trends. Figure 6 shows linear best fit correlations for a few amino acids and repeat motifs that showed some dependence on mechanical property values. In the analysis of the linear correlation, the Pyralidae family was not included due to their deviation from the trends as Pyralid FibH is highly species-specific as discussed previously. P. interpunctella and G. mellonella data are still plotted in Figure 6 . Figure 6: Open in a new tab Silk protein parameters correlated to tensile modulus on the left y-axis and tensile strength on the right y-axis in a selection of Lepidoptera. Species were selected based on those which had partial or complete sequences as well as mechanical properties from Table 3 . Linear best fit lines were calculated without the inclusion of Pyralidae. R 2 values are given for each linear best fit line ( A ) Percentage of aspartic acid (Asp, D) ( B ) Percentage of glycine (Gly, G) ( C ) Percentage of (GGX) n repeat motif ( D ) Percentage of polyA repeat motif ( E ) Average polyA length ( F ) Percent coverage of repeat motifs. The first correlation found was between the quantity of aspartic acid to mechanical properties ( Figure 6A ). While the content of aspartic acid is small (0.3% – 5.2%) in the overall composition of Lepidoptera FibH, there is a fairly linear trend to tensile modulus (0.8460 R 2 ). As aspartic acid increases, the tensile modulus decreases. This correlation from aspartic acid likely comes from contributions due to its negative charge. Glycine also shows a linear trend with tensile modulus (0.9029 R 2 ) as well as tensile strength (0.5466 R 2 ) to some extent. As glycine content increases, so do the tensile mechanical properties ( Figure 6B ). This trend is expected as glycine makes up a major repeat unit in Lepidoptera silks in the form of (GX) n . This repeat unit is known to form β-sheet structures and thus is highly influential on the overall mechanical strength of the silk fibers. Another influential correlation was found between the (GGX) n repeat motif ( Figure 6C ) and tensile modulus (0.9326 R 2 ) and tensile strength (0.8419 R 2 ). As the amount of (GGX) n motifs go up, the mechanical property values go down. This is expected due to the contributions of (GGX) n to elasticity, which typically trends opposite to mechanical strength. The polyA motif, known to contribute to β-sheet structures, showed an inverse correlation to mechanical properties, as well as the average length of the polyA motif. For increasing percentages of polyA in the protein sequence ( Figure 6D ) and the increase in average length of the polyA motif ( Figure 6E ), both the tensile modulus and tensile strength decrease. Finally, the overall coverage of the FibH sequence by known repeat motifs was compared to mechanical property values ( Figure 6F ). Through these comparisons, it was found that as the number of repeat motifs increases throughout the protein sequence, both tensile modulus (0.9528 R 2 ) and tensile strength (0.6653 R 2 ) increase. As silks are highly crystalline biopolymers, the repeat motifs are dominated by those that make of β-sheet structures rather than amorphous regions. Thus, as the number of repeat motifs increases, the β-sheet structures are generally increasing as well and contributing to high rigidity and increasing mechanical properties. Additionally, a component of the silk protein structure that is more difficult to assess is the frequency and size of the repeat unit structures. While total motif content is influential on mechanical properties, the overall size and packing ability can affect the physical properties as well. Recently, it has been shown by Aikman et al. , through molecular dynamics simulations of silk fibroin from B. mori , that larger crystalline regions result in more robust materials, while smaller crystalline regions with more irregularities of interrupting flexible side chains provide more elastic properties. 85 Quantification of total amounts of (GX) n in the protein sequence does not clearly indicate size of the β-sheet structures formed during protein folding. Further investigations and application of computational tools, such as those by Moreno-Tortolero et al., 86 will advance the understanding of packing and crystal organization as a function of β-sheet content, improving our understanding of these dynamic material properties. 5.2. Thermal Properties Thermal properties are typically used for determining composition and stability of a given material, where transition temperatures can give information about the amorphous and crystalline content. However, transition temperatures are not easily identifiable in the thermal analysis of silks due to the presence of the outer coating proteins (sericins, seroins, etc.) discussed previously. 24 While degumming may remove the outer proteins, it may also degrade the core FibH protein. 24 , 72 , 73 Thus, it is difficult to assess thermal transition temperatures in silks due to their varying compositions of the raw fibers. According to data in literature, the thermal degradation temperature (T d ) is the more commonly available parameter measured in Lepidopteran silks. 8 , 24 , 87 , 88 The degradation temperature is a measure of when the material begins to decompose and break down its crystalline structures (inflection point of mass loss 87 or endothermic peak 24 ), just before full melting. Much of the silk literature that does report glass transition temperatures do so on processed biomaterials rather than native fibers. 89 , 90 Additionally, in major ampullate silk, the degradation temperature is not commonly reported. This may be due to the amount of raw material necessary for thermal measurements (mg scale), as spider silk is typically limited in sample size compared to the scale of material from Lepidopteran cocoons. The recent publication by Arakawa et al . provided thermal properties in the form of onset of degradation at 1%, 5%, and 10% mass loss for several hundred different spider species. 1 One group produced mass loss curves for T. clavipes , which we were able to estimate the midpoint degradation temperature from. 92 Table 4 shows the available degradation temperatures from literature for Lepidopteran silk sources (and T. clavipes ), as well as the technique used to collect thermal properties. Table 1: Thermal degradation temperatures and method of thermal analysis reported for native silk fibers. a Species Order Td [°C] Method Ramp Rate Reference B. mandarina Bombycidae 329.5 TGA 20 °C/min 1 Deng 2021 B. mori Bombycidae 314.3 TGA 5 °C/min 2 Mazzi 2014 B. mori (Jp) Bombycidae 335.36 ± 0.45 TGA/DSC 20 °C/min 3 Malay 2016 B. mori (Cn) Bombycidae 338.04 ± 0.85 TGA/DSC 20 °C/min 3 Malay 2016 B. mori (In) Bombycidae 327.07 ± 0.18 TGA/DSC 20 °C/min 3 Malay 2016 B. mori (Th) Bombycidae 328.42 ± 0.62 TGA/DSC 20 °C/min 3 Malay 2016 P interpunctella Pyralidae 293.9 ± 0.2 TGA 10 °C/min 4 Eccles 2025 A. aliena (wild) Saturniidae 371.04 ± 1.17 TGA/DSC 20 °C/min 3 Malay 2016 A. assamensis Saturniidae 372.99 ± 0.47 TGA/DSC 20 °C/min 3 Malay 2016 A. assamensis Saturniidae 345.4 TGA 5 °C/min 2 Mazzi 2014 A. mylitta Saturniidae 349.7 TGA 5 °C/min 2 Mazzi 2014 A. mylitta Saturniidae 362 DSC - 5 Darshan 2017 A. pernyi (Cn) Saturniidae 376.16 ± 0.09 TGA/DSC 20 °C/min 3 Malay 2016 A. pernyi (Jp) Saturniidae 375.96 ± 1.50 TGA/DSC 20 °C/min 3 Malay 2016 A. yamamai Saturniidae 376.31 ± 0.18 TGA/DSC 20 °C/min 3 Malay 2016 P. ricini Saturniidae 350.7 TGA 5 °C/min 2 Mazzi 2014 R. fugax (wild) Saturniidae 369.79 ± 0.20 TGA/DSC 20 °C/min 3 Malay 2016 S. c. pryeri Saturniidae 379.19 ± 1.66 TGA/DSC 20 °C/min 3 Malay 2016 S. c. ricini (Jp) Saturniidae 381.51 ± 0.48 TGA/DSC 20 °C/min 3 Malay 2016 S. c. ricini (In) Saturniidae 382.14 ± 1.22 TGA/DSC 20 °C/min 3 Malay 2016 S. jonasii (wild) Saturniidae 368.78 ± 0.35 TGA/DSC 20 °C/min 3 Malay 2016 T. clavipes Araneidae 250–350 TGA - 6 Agnarsson 2009 Open in a new tab a (*) denotes values that were approximated from figures. Surprisingly, minimal thermal analysis data on Lepidopteran silks exists, with most values in Table 4 sourced from only a few groups. 24 , 87 It is more difficult to draw conclusions across common techniques and experimental setup from limited silk thermal analysis sources. For thermal analysis techniques such as thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) slower ramp rates typically lead to better resolution for thermal transitions that are nearby. In general, more amorphous materials will have lower T d and more crystalline materials will have higher T d values as rigid β-sheet structures require more energy to break apart whereas flexible regions more easily rearrange and breakdown. Thus, for semi-crystalline silk, the T d should increase as more crystalline repeat motifs are present. We expect that the repeat motifs that form β-sheet structures, polyA and (GX) n , should produce higher degradation temperatures. Whereas groups with more amorphous repeat motifs, (GGX) n , should have weaker thermal stability and lower T d . Comparisons can be drawn between thermal properties and the percent coverage of repeat units present in the protein structure for a given silk. From a comparison of frequency of each amino acid present within the silk protein sequence and repeat motif coverage to degradation temperature, a few amino acids and motifs showed common trends. Malay et al. performed extensive thermal analysis, at 20 °C/min, to highlight the variability in location dependence on silk sources among the same species. 24 Their work found a relationship between polyA coverage, where lower amounts of polyA content correlated to lower thermal degradation temperatures. 24 This trend holds for other silk sources found in Table 4 . For example, Eccles et al . performed thermal analysis of P. interpunctella silk, which was found to have the lowest degradation temperature from literature, 8 as well as one of the lowest polyA motif coverages ( Figure 4 ). Malay et al. also found that the average polyA length did not have a positive correlation to degradation temperature demonstrating that the overall coverage of crystalline regions and the ratio between crystalline and non-crystalline regions may be more influential in predicting thermal stability. 24 Expanding on this analysis, Figure 7 shows the linear best fit correlations for amino acid, isoelectric points, and elastic repeat motifs that showed dependence on degradation temperature. Similar to the mechanical property analysis of the linear correlations, the Pyralidae family was not included as discussed previously, although the P. interpunctella thermal properties are still graphed in Figure 7 . The first correlation found was between the quantity of valine compared to degradation temperature ( Figure 7A ). While the coverage of valine is small (0.6% – 3.5%) in the overall composition of Lepidoptera FibH, there is a linear trend to T d (0.9284 R 2 ), where degradation temperature increases as valine content decreases. This is an interesting find as valine is typically found in the crystalline regions of FibH, such as (GA) n GV or GAGAGV in B. mori 37 , 38 , 62 or (SV) n E in Trichoptera, 28 , 29 and greater crystalline content can increase thermal stability. There is a weaker trend (0.5115 R 2 ) in degradation temperature increasing as isoelectric point (pI) increases ( Figure 7B ), but continued collection of data across organisms is necessary to make strong conclusions. Finally, a strong correlation exists between (GGX) n repeat motifs (0.9647 R 2 ). As (GGX) n repeat motifs increase the degradation temperature increases ( Figure 7C ). This is a surprising find as the (GGX) n repeat motif is expected to contribute to elasticity, which typically leads to less thermally stable materials. Perhaps these surprising correlations speak to the trend found by Malay et al ., 24 that the overall ratio of crystalline and amorphous regions may be the most influential contributor to degradation temperature predictions. Figure 7: Open in a new tab Silk protein parameters correlated to degradation temperature in a selection of Lepidoptera. Species were selected based on those which had partial or complete sequences from Figure 4 as well as thermal degradation temperatures from Table 4 . Linear best fit lines were calculated without the inclusion of Pyralidae. R 2 values are given for each linear best fit line ( A ) Percentage of Valine (Val, V) ( B ) Isoelectric point (pI) ( C ) Percent coverage of (GGX) n repeat motif. 6. Future Perspectives There exist many silk-producing species that generate useful natural biopolymer-based silk fibers. With a large distribution of functional proteins and protein compositions across species, an increasingly diverse array of materials and applications can be inspired by these biopolymers. This review summarizes the protein types that constitute insect and spider silk fibers, the characteristics of core structural proteins, and the influences of repeat motif composition, abundance, and regularity on silk fiber properties. Altogether, it serves to emphasize how the precise balance of crystalline and amorphous structures at the protein level can tune physical properties of hierarchically structured silk fibers. These studies have assisted in understanding natural silk fibers, in addition to the development of silk-based, biomimetic, or bio-inspired materials. Despite recent advancements and initiatives to standardize the characterization of silk proteins and fibers, understanding the sequence-structure-function relationship within silk proteins and fibers faces many challenges. Many gaps exist in the reporting of full length FibH and MaSp protein sequences and in the analysis of silk fiber properties through comparable methodologies, preventing large-scale comprehensive analyses across taxonomic families and orders. Leveraging the improvement of long-read sequencing techniques 29 , 54 – 57 and initiatives like the Spider Silkome Project 1 can further enable these comparisons, in addition to the identification of prospective applications. As experimental data are collected and tabulated, these data can be leveraged in computational material design 93 – 96 and development. Additionally, while this review focuses on the core structural proteins of silk fibers, the identity and role of supporting proteins in silk properties is less known. The characterization of these additional silk fiber-constituting elements ( e.g., sericins, seroins, SpiCE) and their purpose in fiber self-assembly, fiber dynamics, and fiber physical performance, remains a limitation to their utility in material applications. Although there are considerable efforts in the synthesis and use of silk-based biomaterials and artificial production of silk fibers, the expansive biodiversity of silks remains an untapped opportunity for material discovery and design. Silks have numerous tunable properties outside of the ones described here, namely biodegradability, cytocompatibility, adhesiveness, and ease of processing into various material structures. 14 , 35 Through the use of silk or silk-inspired polymers and peptides, the performance or production of materials can be tailored and improved for the desired applications in healthcare, 14 , 48 materials science, 97 synthetic biology, 98 or sustainability. 15 , 35 The ability to expand the uses of silks and diversify these applications is contingent upon the abundance of characterized silks and silk-producing organisms, understanding protein composition correlations to physical function, and multidisciplinary approaches to material design. Supplementary Material Supplemental Materials NIHMS2082145-supplement-Supplemental_Materials.pdf (652.5KB, pdf) 7. Supporting Information To generate the graphs in Figures 5 , 6 , and 7 , data from published literature or gene and protein sequence databases were analyzed. Number values for repeat motif coverage and accession numbers of structural proteins ( Table S1 ), amino acid composition of structural proteins ( Tables S2 , S3 , and S4 ), extensional mechanical properties organized by taxonomic family ( Figure S1 ). 8. Acknowledgements and Funding All Stoppel Lab members would like to acknowledge support from the National Institutes of Health National Institute of General Medical Sciences Maximizing Investigators’ Research Award (NIH NIGMS R35-GM147041) and an Integrative Biology Award from the Molecular and Cellular Biology Division of the National Science Foundation (NSF MCB-2217159). All Stoppel Lab members acknowledge support from the Dr. and Mrs. Frederick C. Edie Term Professorship at the University of Florida. ELA acknowledges support from the National Science Foundation Graduate Research Fellowship (DGE-2236414). Any opinions, findings, and conclusions or recommendations expressed in this manuscript are those of the authors and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health. All authors would like to acknowledge support from the University of Florida and the University of Florida Herbert Wertheim College of Engineering and Department of Chemical Engineering. Footnotes 9. Conflict of Interest The authors declare no conflict of interest at this time. 10. References 1. 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