International Journal of Molecular Sciences Article Comparison of Silks from Pseudoips prasinana and Bombyx mori Shows Molecular Convergence in Fibroin Heavy Chains but Large Differences in Other Silk Components Michal Rindos 1,2 , Lucie Kucerova 1 , Lenka Rouhova 1,2, Hana Sehadova 1,2, Michal Sery 1, Miluse Hradilova 3, Peter Konik 2 and Michal Zurovec 1,2,* 1 Biology Centre of the Czech Academy of Sciences, Institute of Entomology, 37005 Ceske Budejovice, Czech Republic; [email protected] (M.R.); [email protected] (L.K.); [email protected] (L.R.); [email protected] (H.S.); [email protected] (M.S.) 2 Faculty of Science, University of South Bohemia, 37005 Ceske Budejovice, Czech Republic; [email protected] 3 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Praha, Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-387775267 Abstract: Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of Pseudoips prasinana has previously been studied using X-ray analysis and classified in the same category as that of Bombyx mori, suggesting that silks of both species have similar properties despite their considerable Citation: Rindos, M.; Kucerova, L.; phylogenetic distance. In the present study, we examined P. prasinana silk using ‘omics’ technology, Rouhova, L.; Sehadova, H.; Sery, M.; including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis Hradilova, M.; Konik, P.; Zurovec, M. of cocoon proteins. We found that although the central repetitive amino acid sequences encoding Comparison of Silks from Pseudoips prasinana and Bombyx mori Shows crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting Molecular Convergence in Fibroin fibers exhibit quite different mechanical properties. Our results suggest that these differences are Heavy Chains but Large Differences most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in P. in Other Silk Components. Int. J. Mol. prasinana silk. Furthermore, we show that whilst P. prasinana cocoons are predominantly made of silk Sci. 2021, 22, 8246. https://doi.org/ similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present 10.3390/ijms22158246 only at the escape valve. Academic Editors: Ilaria Armentano Keywords: Bena prasinana; Bombycidae; Nolidae; phylogeny; transcriptomics; fibrohexamerins and Klaus H. Hoffmann Received: 13 May 2021 Accepted: 27 July 2021 1. Introduction Published: 31 July 2021 Many lepidopteran larvae produce silk serving in different forms as a protection of Publisher’s Note: MDPI stays neutral their larval and pupal stages. The silk consists of a fibrous hydrophobic core formed by with regard to jurisdictional claims in fibroin molecules and a hydrophilic coating comprising adhesive proteins called sericins, published maps and institutional affil- as well as additional proteins with antimicrobial functions [1]. The amino acid composition iations. of silk proteins greatly varies among Lepidoptera. The fibroin core consists of three fibroin molecules. Fibroin heavy chain (Fib-H), and fibroin light chain (Fib-L) are attached via a disulphide bond and their assembly is crucial for the intracellular transport of nascent silk [2]. Fibrohexamerin or fibroin P25 (Fhx/P25), contributes to the formation of the high molecular mass fibroin elementary units via hydrophobic interactions with the Fib-H and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Fib-L complexes [3]. This article is an open access article In the 1960s, X-ray structural analysis was performed on a number of natural mate- distributed under the terms and rials, including silks from about 30 species belonging to different groups of Lepidoptera. conditions of the Creative Commons According to the results, silks were divided into four classes depending on the composition Attribution (CC BY) license (https:// of the so-called crystalline regions of Fib-H, formed by amino acid residues in the beta- creativecommons.org/licenses/by/ conformation. These regions are responsible for the mechanical strength of the silk [4,5]. 4.0/). The crystalline regions are characterized by the intersheet packing distance of β-sheet Int. J. Mol. Sci. 2021, 22, 8246. https://doi.org/10.3390/ijms22158246 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 8246 2 of 13 crystallites and their specific sequence motifs [5,6] as follows: class I, intersheet packing distance of 9.3 Å and alternating amino acids glycine and alanine or serine; class II, dis- tance of 10 Å and sequence ((Gly-Ala)x-Alay)n; class III, distance of 10.6 Å and polyalanine repeats; class IV (sometimes considered as atypical), distance of 15.0 Å, with a low amount of alanine residues and a high proportion of serine, glutamic acid, and aspartic acid [5,6]. The sequences of several class III fibroins have been elucidated and its previously predicted polyalanine or alanine-serine regions were confirmed [7–9]. Furthermore, a recent study describing silk from Eumeta (Clania) variegata, a class II silk, contained ((Gly-Ala)x-Alay)n chains as predicted [10]. The silk of Pseudoips prasinana (originally belonging to the genus of Bena) has been placed in class I together with that of Bombyx mori; it has been suggested that the silks of both species have a similar structure, both containing long stretches of alternating glycine and alanine residues. The class I-X-ray structure of B. mori and P. prasi- nana silks is rare among Lepidoptera and has so far only been found in these two unrelated species (Figure S1). P. prasinana is a nocturnal bivoltine moth from the family Nolidae belonging to the su- perfamily Noctuoidea. It has a widespread distribution, occurring throughout the Palearctic region, and inhabits various types of forest habitat, where it feeds mainly on beech and oak leaves. Fully grown larvae are green and approximately 4 cm long (Figure1A) and adults Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 14 have a wingspan 30–40 mm. Pupation takes place in a boat-shaped parchment-like cocoon (Figure1B) on the underside of the leaf [11,12]. Figure 1.Figure Pseudoips 1. prasinanaPseudoips L. larva, prasinana cocoon andL. silk. larva, (A) Last cocoon-instar larva. and (B silk.) Cocoon; (A) arrow Last-instar on larva. (B) Cocoon; arrow the right points to the exit valve. (C,D) Outer and (E,F) inner side of the cocoon escape valve, exam- ined usingon an the optical right microscope. points ( toF) Arrowheads the exit valve. indicate (theC, baseD) Outerof the newly and detected (E,F) innersilk type. side of the cocoon escape valve, (G,H) Scanningexamined electron using micrographs an optical of the escape microscope. valve from the (F outside.) Arrowheads Scale-bars: (C indicate,E) 0.5 mm; the base of the newly detected (D,F) 0.2 mm; (G,H) 100 μm. silk type. (G,H) Scanning electron micrographs of the escape valve from the outside. Scale-bars: 2. Results(C ,E) 0.5 mm; (D,F) 0.2 mm; (G,H) 100 µm. 2.1. Compact Structure of P. prasinana Cocoons Pseudoips prasinana cocoons are approximately 2.5 cm long and have the appearance of parchment; they are more compact and thinner than those of B. mori. They also differ from B. mori cocoons by having a boat shape with a wider anterior part ending in a pointed thinned edge. The edge structure, which resembles a seam, serves as an escape valve for the emerging adult (Figure 1B–H). Further examination using light and scanning electron microscopy revealed that P. prasinana silk filaments mostly form arched structures, indicating that the larva performs Int. J. Mol. Sci. 2021, 22, 8246 3 of 13 Since the studies of the 1960s, many new methodological approaches have been de- veloped, providing the opportunity to study physiology in much more detail, as well as enabling systematic comparisons of biological systems that were not previously possible. ‘Omics’ technologies have revolutionized silk research, and are able to generate massive datasets that cover transcribed genes and proteins of entire tissues, a significant improve- ment over the traditional time-consuming isolation and analysis of single molecules [13]. In the present study, we analyzed the protein composition of P. prasinana cocoons and detected and annotated 136 proteins, included the proteins of the fibroin fraction as well as four sericins, seroin, and protease inhibitors. Characterizing P. prasinana silk and comparing it with that of B. mori will help to understand the mechanism of silk protein changes during evolution as well as elicit the function of individual silk components. Our results show that fibrohexamerin and fibrohexamerin-like genes expanded in P. prasinana, and therefore likely cause the difference in mechanical properties between P. prasinana and B. mori silks. 2. Results 2.1. Compact Structure of P. prasinana Cocoons Pseudoips prasinana cocoons are approximately 2.5 cm long and have the appearance of parchment; they are more compact and thinner than those of B. mori. They also differ from B. mori cocoons by having a boat shape with a wider anterior part ending in a pointed thinned edge. The edge structure, which resembles a seam, serves as an escape valve for the emerging adult (Figure1B–H). Further examination using light and scanning electron microscopy revealed that P. prasinana silk filaments mostly form arched structures, indicating that the larva performs semi-circular movements with its head during cocoon spinning (Figure2A–H). In contrast, the silk architecture at the seam region appears different and delimits the escape valve opening.