Pressure-induced silk spinning mechanism in webspinners (Insecta: Embioptera).

The articulated appendages of arthropods are highly adaptable and potentially multifunctional, used for walking, swimming, feeding, prey capture, or other functions. Webspinners (Order Embioptera) are a paragon in this context. In contrast to other arthropods producing silk, they utilize their front feet for silk production. However, employing the same leg for alternative functions rather than for pure locomotion potentially imposes constraints and compromises. We here present morphological and experimental evidence for a "passive" pressure-induced silk spinning mechanism induced by external mechanical stimuli. Furthermore, we demonstrate that, as a consequence of the conflicting functions for their front feet, webspinners have evolved a unique style of walking that reduces the potentially problematic contact between silk ejectors and the substrate. Here we answer for the first time a long-term question within this enigmatic group of insects-how webspinners can use their front feet to spin their nanoscale silk. This knowledge may open the door for experimental studies on an artificial spinning process and for future utilization in applied fields of robotics or chemistry.

Surface and Wetting Properties of Embiopteran (Webspinner) Nanofiber Silk.

Insects of the order Embioptera, known as embiopterans, embiids, or webspinners, weave silk fibers together into sheets to make shelters called galleries. In this study, we show that silk galleries produced by the embiopteran Antipaluria urichi exhibit a highly hydrophobic wetting state with high water adhesion macroscopically equivalent to the rose petal effect. Specifically, the silk sheets have advancing contact angles above 150°, but receding contact angle approaching 0°. The silk sheets consist of layered fiber bundles with single strands spaced by microscale gaps. Scanning and transmission electron microscopy (SEM, TEM) images of silk treated with organic solvent and gas chromatography mass spectrometry (GC-MS) of the organic extract support the presence of a lipid outer layer on the silk fibers. We use cryogenic SEM to demonstrate that water drops reside on only the first layer of the silk fibers. The area fraction of this sparse outer silk layers is 0.1 to 0.3, which according to the Cassie-Baxter equation yields an effective static contact angle of ∼130° even for a mildly hydrophobic lipid coating. Using high magnification optical imaging of the three phase contact line of a water droplet receding from the silk sheet, we show that the high adhesion of the drop stems from water pinning along bundles of multiple silk fibers. The bundles likely form when the drop contact line is pinned on individual fibers and pulls them together as it recedes. The dynamic reorganization of the silk sheets during the droplet movement leads to formation of "super-pinning sites" that give embiopteran silk one of the strongest adhesions to water of any natural hydrophobic surface.

A history of susceptibility to parasites and divergence in solitary, gregarious and agonistic behaviors of embiopterans.

Two species of Haploembia Ramburi (Oligotomidae: Embioptera), nonnative detritivores found in the western USA, display solitary tendencies, not typical for webspinners that usually share silk galleries. Reports from the 1960s based on native populations in Italy highlighted the impact of a gregarine that depressed male sterility and female survivorship in Haploembia solieri (Rambur). Sympatric asexual Haploembia tarsalis (Ross) lives a normal lifespan when parasitized, albeit suffering from reduced fecundity. Our goal is to characterize behavioral repertoires as individuals interact and to develop methods for future investigations focused on the impact of the little-known parasite. We quantified individual responses to conspecifics or other species in 10-min dyadic interactions and, in a 3-day trial, determined whether they aggregated when given dispersed resources. Replicated groups of four adult female H. solieri or H. tarsalis settled away from each other over the 3-day trials. In 10-min bouts of same or different species pairs, focal insects bolted back, retreated and attempted to escape when they encountered one another, especially when the opponent was H. tarsalis. Males courted conspecific females, but were dramatically repelled by H. tarsalis. Serving as a positive control, Oligotoma nigra (Hagen) (Oligotomidae) adult females paired with conspecifics displayed typical webspinner behaviors by sitting together, sharing silk. Haploembia solieri males did not respond negatively to O. nigra, not known to be parasitized by the gregarine, but did so when paired with H. tarsalis. Results align with the prediction that susceptibility to parasitism may have led to antisocial behaviors observed in two Haploembia species.

Maternal territoriality achieved through shaking and lunging: an investigation of patterns in associated behaviors and substrate vibrations in a colonial embiopteran, Antipaluria urichi.

Substrate vibration communication is displayed by a variety of insects that rely on silk for shelter. Such signaling is often associated with territoriality and social interactions. The goal in this study was to explore the use of substrate vibration by subsocial insects of the little-studied order Embioptera (also known as Embiidina). Antipaluria urichi (Saussure) (Embioptera: Clothodidae) from Trinidad and Tobago, a large embiopteran, exhibits maternal care and facultatively colonial behavior. Previous observations suggested that they were aggressive while guarding eggs but gregarious when not. Egg-guarders in particular have been observed shaking and lunging their bodies, but to date these putative signals have not been recorded nor were their contexts known. Staged interactions were conducted in the laboratory using residents that had established silk domiciles enveloping piezo-electric film used to detect vibrations. Predictions from two competing hypotheses, the maternal territoriality hypothesis and the group cohesion hypothesis, were erected to explain the occurrence of signaling. Experiments pitted pre-reproductive and egg-guarding residents against female and male intruders, representing social partners that ranged from potentially threatening to innocuous or even helpful. Behavioral acts were identified and scored along with associated substrate vibrations, which were measured for associated body movements, duration, and frequency spectra. Signals, sorted by the distinct actions used to generate them, were lunge, shake, push up, and snapback. Egg-guarding females produced most signals in response to female intruders, a result that supported the maternal territoriality hypothesis. Female intruders generally responded to such signaling by moving away from egg-guarding residents. In contrast, pre-reproductive residents did not signal much, and intruders settled beside them. Theme software was used to analyze the behavioral event recordings to seek patterns over time and their association with signals. Long patterns of behavioral acts were associated with shakes, lunges, and push-ups, indicating that interactions were occurring between the residents and intruders as would be expected when communication occurs. The value of Theme software, as well as the relationship between signaling by A. urichi and the risks and benefits of coloniality, are discussed.

Morphological transformation from fibers to sheets in embiopteran silk.

Embioptera (webspinners) are insects that construct domiciles using silk produced from their front feet. This silk is the finest known with measured single fiber diameters in the 30-140 nm range. In the wild, some webspinner silk on trees is observed to have a clothlike or shiny sheetlike appearance. Both forms of silk shield the occupants from rain water effectively: presumably valuable in tropical environments. In this article we elucidate the mechanism by which silk fibers are transformed into these structures through interaction with water. We quantify the evaporation rates of single water droplets which have been suspended on unmodified as-spun silk for two Trinidadian arboreal species: Antipaluria urichi (Clothodidae) and Pararhagadochir trinitatis (Scelembiidae). These rates are compared to those of droplets suspended on rose petals due to similar wetting properties (both hydrophobicity and pinning). We observe that on sufficiently thick silk, droplet evaporation rates decrease with time. This behavior is a result of a thin soluble film developing on the drop surface that later becomes a solid residual film. Experimentally verified theoretical models are invoked to support the results.

Interpreting nature's finest insect silks (Order Embioptera): hydropathy, interrupted repetitive motifs, and fiber-to-film transformation for two neotropical species.

Silks produced by webspinners (Order Embioptera) interact with water by transforming from fiber to film, which then becomes slippery and capable of shedding water. We chose to explore this mechanism by analyzing and comparing the silk protein transcripts of two species with overlapping distributions in Trinidad but from different taxonomic families. The transcript of one, Antipaluria urichi (Clothodidae), was partially characterized in 2009 providing a control for our methods to characterize a second species: Pararhagadochir trinitatis (Scelembiidae), a family that adds to the taxon sampling for this little known order of insects. Previous reports showed that embiopteran silk protein (dubbed Efibroin) consists of a protein core of repetitive motifs largely composed of glycine (Gly), serine (Ser), and alanine (Ala) and a highly conserved C-terminal region. Based on mRNA extracted from silk glands, Next Generation sequencing, and de novo assembly, P. trinitatis silk can be characterized by repetitive motifs of Gly-Ser followed periodically by Gly-Asparagine (Asn-an unusual amino acid for Efibroins) and by a lack of Ala which is otherwise common in Efibroins. The putative N-terminal domain, composed mostly of polar, charged and bulky amino acids, is ten amino acids long with cysteine in the 10th position-a feature likely related to stabilization of the silk fibers. The 29 amino acids of the C-terminus for P. trinitatis silk closely resemble that of other Efibroin sequences, which show 74% shared identity on average. Examination of hydropathicity of Efibroins of both P. trinitatis and An. urichi revealed that these proteins are largely hydrophilic despite having a thin lipid coating on each nano-fiber. We deduced that the hydrophilic quality differs for the two species: due to Ser and Asn for P. trinitatis silk and to previously undetected spacers in An. urichi silk. Spacers are known from some spider and silkworm silks but this is the first report of such for Embioptera. Analysis of hydropathicity revealed the largely hydrophilic quality of these silks and this feature likely explains why water causes the transformation from fiber to film. We compared spun silk to the transcript and detected not insignificant differences between the two measurements implying that as yet undetermined post-translational modifications of their silk may occur. In addition, we found evidence for codon bias in the nucleotides of the putative silk transcript for P. trinitatis, a feature also known for other embiopteran silk genes.

A Multiscale Characterization of Two Tropical Embiopteran Species: Nano- and Microscale Features of Silk, Silk-Spinning Behavior, and Environmental Correlates of their Distributions.

Embioptera display the unique ability to spin silk with their front feet to create protective domiciles. Their body form is remarkably uniform throughout the order, perhaps because they all live within the tight confines of silken tubes. This study contributes to an understanding of the ecology of Embioptera, an order that is rarely studied in the field. We conducted a census to quantify the habitats of two species with overlapping distributions on the tropical island of Trinidad in a search for characteristics that might explain their distinct ecologies. One species, Antipaluria urichi (Saussure) (Embioptera: Clothodidae), lives in larger colonies with more expansive silk in habitats throughout the island, especially in the rainforest of the Northern Range Mountains. The other, Pararhagadochir trinitatis (Saussure) (Embioptera: Scelembiidae), was found only in lowland locations. We quantified silk-spinning behavior and productivity of the two species and found that A. urichi spins thicker silk sheets per individual and emphasizes spin-steps that function to create a domicile that is more expansive than that produced by P. trinitatis. Their silks also interact differently when exposed to water: the smaller-diameter silk fibers of P. trinitatis form more continuous films on the surface of the domicile after being wetted and dried than that seen in A. urichi silk. This tendency gives P. trinitatis silk a shiny appearance in the field compared to the more cloth-like silk of A. urichi. How these silks function in the field and if the differences are partially responsible for the distinct distributions of the two species remain to be determined.

Comparison of embiopteran silks reveals tensile and structural similarities across Taxa.

Embioptera is a little studied order of widely distributed, but rarely seen, insects. Members of this group, also called embiids or webspinners, all heavily rely on silken tunnels in which they live and reproduce. However, embiids vary in their substrate preferences and these differences may result in divergent silk mechanical properties. Here, we present diameter measurements, tensile tests, and protein secondary structural analyses of silks spun by several embiid species. Despite their diverse habitats and phylogenetic relationships, these species have remarkably similar silk diameters and ultimate stress values. Yet, ultimate strain, Young's modulus, and toughness vary considerably. To better understand these tensile properties, Fourier transformed infrared spectroscopy was used to quantify secondary structural components. Compared to other arthropod silks, embiid silks are shown to have consistent secondary structures, suggesting that commonality of amino acid sequence motifs and small differences in structural composition can lead to significant changes in tensile properties.

Phylogeny of embiopterans (Insecta).

A cladistic analysis of embiopterans, based on 157 species (representing 70% of the known genera) and 186 morphological characters, is presented, as well as a molecular analysis for 22 taxa using genes encoding 16S, 18S and 28S rDNA and COI. Species of all known families are included, except Andesembiidae Ross (specimens of which are in a private collection). The evidence presented supports the monophyly of four of the families (Australembiidae, Oligotomidae, Teratembiidae, and Anisembiidae). Notoligotomidae is paraphyletic and included within the Afro-neotropical family Archembiidae (which is also paraphyletic). The genera Embia, Cleomia, Macrembia, and Dihybocercus (Embiidae) form, together with Australembiidae, a group strongly supported by morphology; the position of the remaining genera of Embiidae has two quite different resolutions. Almost 80% of the genera of Anisembiidae recently described appear as either paraphyletic or polyphyletic. Contrary to the opinion of other specialists, the major groups as well as the monophyly of some families are supported by features which have been ignored in classical approaches to the systematics of Embioptera, such as the ovipositor and cephalic and leg structures, characters with an almost perfect fit.

Resolving two Haploembia (Embioptera: Oligotomidae) cryptic species: molecular data confirms parthenogenetic females can be distinguished by their antisocial behavior.

The names of two cryptic species of Haploembia found in California are resolved and methods for identification are summarized. Molecular data of the Histone III subunit was used to evaluate color and behavior as species identifiers, confirming that antisocial behavior is a good identifier for the parthenogenetic species (Haploembia tarsalis), whereas the more variable coloration patterns were helpful, but less so. A genome size ratio of 1.44 between the parthenogenetic H. tarsalis and the sexually reproducing H. solieri was observed, along with higher genetic variation within the asexual lineage. This, and the identification of what appears to be a putative hybrid, contributes to current work examining mutation rates and selective pressures on genome size in parthenogenetic populations.

Structural and wetting properties of nature's finest silks (order Embioptera).

Insects from the order Embioptera (webspinners) spin silk fibres which are less than 200 nm in diameter. In this work, we characterized and compared the diameters of single silk fibres from nine species-Antipaluria urichi, Pararhagadochir trinitatis, Saussurembia calypso, Diradius vandykei, Aposthonia ceylonica, Haploembia solieri, H. tarsalis, Oligotoma nigra and O. saundersii. Silk from seven of these species have not been previously quantified. Our studies cover five of the 10 named taxonomic families and represent about one third of the known taxonomic family-level diversity in the order Embioptera. Naturally spun silk varied in diameter from 43.6 ± 1.7 nm for D. vandykei to 122.4 ± 3.2 nm for An. urichi. Mean fibre diameter did not correlate with adult female body length. Fibre diameter is more similar in closely related species than in more distantly related species. Field observations indicated that silk appears shiny and smooth when exposed to rainwater. We therefore measured contact angles to learn more about interactions between silk and water. Higher contact angles were measured for silks with wider fibre diameter and higher quantity of hydrophobic amino acids. High static contact angles (ranging up to 122° ± 3° for An. urichi) indicated that silken sheets spun by four arboreal, webspinner species were hydrophobic. A second contact angle measurement made on a previously wetted patch of silk resulted in a lower contact angle (average difference was greater than 27°) for all four species. Our studies suggest that silk fibres which had been previously exposed to water exhibited irreversible changes in hydrophobicity and water adhesion properties. Our results are in alignment with the 'super-pinning' site hypothesis by Yarger and co-workers to describe the hydrophobic, yet water adhesive, properties exhibited by webspinner silk fibres. The physical and chemical insights gained here may inform the synthesis and development of smaller diameter silk fibres with unique water adhesion properties.

The spinning apparatus of webspinners--functional-morphology, morphometrics and spinning behaviour.

Webspinners (Insecta: Embioptera) have a distinctly unique behaviour with related morphological characteristics. Producing silk with the basitarsomeres of their forelegs plays a crucial role in the lives of these insects--providing shelter and protection. The correlation between body size, morphology and morphometrics of the spinning apparatus and the spinning behaviour of Embioptera was investigated for seven species using state-of-the-art methodology for behavioural as well as for morphological approaches. Independent contrast analysis revealed correlations between morphometric characters and body size. Larger webspinners in this study have glands with greater reservoir volume, but in proportionally smaller tarsi relative to body size than in the smaller species. Furthermore, we present a detailed description and review of the spinning apparatus in Embioptera in comparison to other arthropods and substantiate the possible homology of the embiopteran silk glands to class III dermal silk glands of insects.

Description of four new species of the genus Ptilocerembia Friederichs, 1923 (Embioptera: Ptilocerembiidae) from Thailand.

Four new species of webspinners in the genus Ptilocerembia Friederichs (Ptilocerembiidae) are described including Ptilocerembia thaidina sp. n., P. senathami sp. n., P. catherinae sp. n. and P.  rossi sp. n. . from Thailand. Illustrations of heads, genitalia and papilla of adult males, sternite pattern of adult females together with photographs of adult males, females and their galleries for each species are provided. Notes on field observations and egg mass structure are given for P. catherinae sp. n. A distribution map and a dichotomous key to the Thai species in the genus Ptilocerembia are also included. 

Structural characterization of nanofiber silk produced by embiopterans (webspinners).

Embiopterans produce silken galleries and sheets using exceptionally fine silk fibers in which they live and breed. In this study, we use electron microscopy (EM), Fourier-transform infrared (FT-IR) spectroscopy, wide angle X-ray diffraction (WAXD) and solid-state nuclear magnetic resonance (ssNMR) techniques to elucidate the molecular level protein structure of webspinner (embiid) silks. Silks from two species Antipaluria urichi and Aposthonia ceylonica are studied in this work. Electron microscopy images show that the fibers are about 90-100 nm in diameter, making webspinner silks among the finest of all known animal silks. Structural studies reveal that the silk protein core is dominated by ß-sheet structures, and that the protein core is coated with a hydrophobic alkane-rich surface coating. FTIR spectra of native embiid silk shows characteristic alkane CH2 stretchings near 2800-2900 cm-1, which decrease approximately 50% after washing the silk with 2 : 1 CHCl3 : MeOH. Furthermore, 13C ssNMR data shows a significant CH2 resonance that is strongly affected by the presence of water, supporting the idea that the silk fibers are coated with a hydrocarbon-rich layer. Such a layer is likely used to protect the colonies from rain. FTIR data also suggests that embiid silks are dominated by ß-sheet secondary structures similar to spider and silkworm silk fibers. NMR data confirms the presence of ß-sheet nanostructures dominated by serine-rich repetitive regions. A deconvolution of the serine Cß NMR resonance reveals that approximately 70% of all seryl residues exist in a ß-sheet structure. This is consistent with WAXD results that suggest webspinner silks are 70% crystalline, which is the highest crystalline fraction reported for any animal silks. The work presented here provides a molecular level structural picture of silk fibers produced by webspinners.

Comparison of fibroin cDNAs from webspinning insects: insight into silk formation and function.

Embiopterans (webspinning insects) are renowned for their prolific use of silk. These organisms spin silk to construct elaborate networks of tubes in which they live, forage, and reproduce. The silken galleries are essential for protecting these soft-bodied insects from predators and other environmental hazards. Despite the ecological importance of embiopteran silk, very little is known about its constituent proteins. Here, we characterize the silk protein cDNAs from four embiopteran species to better understand the function and evolution of these adaptive molecules. We show that webspinner fibroins (silk proteins) are highly repetitive in sequence and possess several conserved characteristics, despite differences in habitat preferences across species. The most striking similarities are in the codon usage biases of the fibroin genes, particularly in the repetitive regions, as well as sequence conservation of the carboxyl-terminal regions of the fibroins. Based on analyses of the silk genes, we propose hypotheses regarding codon bias and its effect on the translation and replication of these unusual genes. Furthermore, we discuss the significance of specific fibroin motifs to the mechanical and structural characteristics of silk fibers. Lastly, we report that the conservation of webspinner fibroin carboxyl-terminal regions suggests that fiber formation may occur through a mechanism analogous to that found in Lepidoptera. From these results, insight is gained into the tempo and mode of evolution that has shaped embiopteran fibroins.

Characterization of silk spun by the embiopteran, Antipaluria urichi.

Silks are renowned for being lightweight materials with impressive mechanical properties. Though moth and spider silks have received the most study, silk production has evolved in many other arthropods. One insect group that has been little investigated is Embioptera (webspinners). Embiopterans produce silk from unique tarsal spinning structures during all life stages. We characterize the molecular and mechanical properties of Antipaluria urichi (Embioptera) silk through multiple approaches. First, we quantify the number of silk secretory structures on their forelimbs and the tensile properties of Antipaluria silk. Second, we present silk protein (fibroin) transcripts from an embiopteran forelimb protarsomere cDNA library. We describe a fibroin that shares several features with other arthropod silks, including a subrepetitive core region, a non-repetitive carboxyl-terminal sequence, and a composition rich in glycine, alanine, and serine. Despite these shared attributes, embiopteran silk has several different tensile properties compared to previously measured silks. For example, the tensile strength of Antipaluria silk is much lower than that of Bombyx mori silk. We discuss the observed mechanical properties in relation to the fibroin sequence, spinning system, and embiopteran silk use.