Insights into the structure and composition of mineralized hard cocoons constructed by the oriental moth, Monema (Cnidocampa) flavescens Walker

Insect Biochemistry and Molecular Biology 151 (2022) 103878 Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb Insights into the structure and composition of mineralized hard cocoons constructed by the oriental moth, Monema (Cnidocampa) flavescens Walker Lixia Qin a, b, 1, Jing Li c, 1, Kaiyu Guo a, b, Mengyao Lu a, b, Yan Zhang a, b, Xiaolu Zhang a, b, Yanqiong Zeng a, b, Xin Wang a, b, Qingyou Xia a, b, Ping Zhao a, b, Ai-bing Zhang c, **, Zhaoming Dong a, b, * a State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing, 400715, China Chongqing Key Laboratory of Sericultural Science, Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, 400715, China c College of Life Sciences, Capital Normal University, Beijing, 100048, China b A R T I C L E I N F O A B S T R A C T Keywords: Oriental moth cocoon Silk protein Calcium oxalate Asn-rich protein Animals widely use minerals and organic components to construct biomaterials with excellent properties, such as teeth, bones, molluscan shells and eggshells. The larvae of the oriental moth, Monema (Cnidocampa) flavescens Walker, secrete silk proteins that combine closely with calcareous minerals to construct a hard cocoon, which is completely different from the mineral-free Bombyx mori cocoon. The cocoons of oriental moths are likely to be the hardest among the cocoons constructed by insect species. The cocoons of oriental moths were found to be mainly composed of calcium oxalates and Asx/Ser/Gly-rich cocoon proteins, but the types of calcium oxalates and cocoon proteins remain to be elucidated. In this study, we provide an in-depth explanation of the inorganic and organic components in the oriental moth cocoon. Microscopy and imaging technologies revealed that the cocoon is composed of mineral crystals, silk fibers and other organic matter. X-ray diffraction and infrared spectral analyses showed that the mineral crystals in the oriental moth cocoon were mainly CaC2H2O4⋅H2O. ICPOES analysis suggested that the mineral crystals in the cocoons were mainly CaC2H2O4⋅H2O. LC-MS/MS-based proteomics allowed us to identify 467 proteins from the oriental moth cocoon, including 252 uncharacterized proteins, 87 enzymes, 36 small molecule binding proteins, and 5 silk proteins. Among the uncharacterized proteins, 25 of which were Asn-rich proteins because they contained a high proportion of Asn residues (19.1%– 41.4%). Among the top 20 cocoon proteins with the highest abundance, 9 of which were Asn-rich proteins. The qPCR was used to investigate the expression patterns of the major cocoon protein-coding genes. Three fibroins and three Asn-rich proteins were expressed only in the silk gland but not in other tissues. The expression of Asnrich proteins in the silk gland gradually increased from the anterior silk gland to the posterior silk gland. These findings provide important references for understanding the formation mechanism and mechanical properties of mineralized hard cocoons constructed by oriental moths. 1. Introduction eggshells. For example, mollusks use silk fibroin proteins, acidic mac­ romolecules, chitin, and calcium carbonate crystals to construct molluscan shells (Sudo et al., 1997; Marin et al., 2008; Furuhashi et al., 2009), while vertebrates use collagen, proteoglycans, and Animals widely use minerals and organic components to construct biomineralization materials, such as teeth, bones, molluscan shells, and Abbreviations: LC-MS/MS, liquid chromatography with tandem mass spectrometry; ICP-OES, inductively coupled plasma optical emission spectroscopy; FTIR, fourier transform infrared spectroscopy; XRD, X-ray diffraction; qRT-PCR, quantitative real-time PCR; TEF2, translation elongation factor 2; COM, calcium oxalate monohydrate; COD, calcium oxalate dihydrate; CF, calcium formate. * Corresponding author. State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing, 400715, China. ** Corresponding author. E-mail addresses: zhangab2008@cnu.edu.cn (A.-b. Zhang), dongzhaoming@swu.edu.cn (Z. Dong). 1 Authors contributed equally to this work. https://doi.org/10.1016/j.ibmb.2022.103878 Received 4 October 2022; Received in revised form 10 November 2022; Accepted 15 November 2022 Available online 19 November 2022 0965-1748/© 2022 Elsevier Ltd. All rights reserved. L. Qin et al. Insect Biochemistry and Molecular Biology 151 (2022) 103878 hydroxyapatite crystals to construct bones (Betts et al., 1981; Murshed 2018). Mineral crystals have high strength and hardness, whereas organic components are considered the key to maintaining toughness (Smith et al., 1999). The larva of the oriental moth, Monema (Cnidocampa) flavescens Walker, secretes silk proteins and other organic matter that combine closely with calcareous mineral crystals to construct a very hard cocoon, which is completely different from the mineral-free silk cocoon of the silkworm, Bombyx mori. The cocoon construction process of the oriental moth is as follows (Wu et al., 1983; Ishii 1984): the larva chooses a cocooning position and bites off the bark, then spin a layer of silk along the biting position to form a cocoon mattress. Next it spins the silk to form a cup-shaped reticulated cocoon with a round hole at one end, and finally spins silk to seal the cocoon. When the construction of the silk net is complete, the larva excretes a white calcareous muddy substance from the anus and brown mucus from the mouth, and then plasters them in­ side the silk net. Finally, the larva spins a small amount of silk to form a smooth inner silk layer. The cocoon of the oriental moth is oval in shape, similar to that of a sparrow egg, with a long axis of about 1.2 cm, short axis of about 0.9 cm, and cocoon layer thickness of 0.2–0.5 mm (Ishii 1984). Additionally, brown and white longitudinal stripes are visible on the surface of the cocoon (Ishii 1984). Research has found that the long axis could with­ stand 7.7 kg of pressure, while the short axis could withstand 6.4 kg of pressure (Ishii et al., 1984). It is known that the cocoons of oriental moths are the hardest among the cocoons constructed by Japanese insect species (Ishii et al., 1984). The reasons why the oriental moth cocoon is so hard can be analyzed from two aspects: structure and composition. From a structural point of view, the cocoon is oval-shaped, and can be divided into 4–5 layers (Ishii et al., 1984); from the perspective of chemical composition, inorganic ash and protein account for approximately 35% and 34% of the cocoon, respectively (Ishii et al., 1984). The inorganic matter is found to be mainly calcium oxalate, which comes from the Malpighian tube, while the proteins in the cocoon are thought to be produced by the silk gland and salivary gland (Ishii 1984). Amino acid analyses showed that the oriental moth cocoon proteins are rich in Asx (25%), Ser (18%) and Gly (17%) (Ishii et al., 1984). However, due to technical limitations and the lack of a sequenced genome, the types of calcium oxalates and protein components in the M. flavescens cocoon remain unknown. Here, we report an overall description of the cocoon composition of the oriental moth, M. flavescens. Microscopy and imaging revealed the presence of mineral crystals and silk fibers. Fourier-transform infrared (FTIR) and X-ray diffraction (XRD) analyses showed that the mineral crystal in the oriental moth cocoon consist mainly of CaCO3⋅H2O. Inductively coupled pPlasma optical emission Spectroscopy (ICP-OES) analysis revealed the proportions of inorganic and organic matter in the cocoons. Liquid chromatography with tandem mass spectrometry (LCMS/MS) based proteomics allowed us to identify new types of silk pro­ teins. These findings provide an important reference for understanding the composition and formation of the oriental moth cocoon. scraped and dried overnight at 65 ◦ C. FTIR spectroscopy was performed using a slide-on attenuated total reflectance (ATR) objective lens with a Thermo Scientific Nicolet iN10 spectrometer. Spectra were collected in the 400–4000 cm− 1 range at a resolution of 32 cm− 1 with 256 co-added scans. XRD measurements were performed using an X’Pert3 Powder Xray diffractometer (PANalytical, Almelo, Netherlands) with Cu-Kα ra­ diation from a source operated at 45 kV and 40 mA. 2.3. Determination of chemical elements in the cocoon The cocoons were weighed, digested with aqua regia, and then diluted to 25 mL with ultrapure water to determine the elemental con­ tent. The chemical composition was measured using inductively coupled plasma optical emission spectroscopy (ICP-OES, Agilent 730, Agilent Technologies, USA). 2.4. Identification of cocoon proteins The cocoons were ground using a SPEX SamplePrep 8000 Series Mixer/Mill and dissolved in 9 M LiSCN. The solubilized proteins were recovered by centrifugation at 12,000g for 10 min. Silk proteins were digested using the filter-aided sample preparation method. The 200 μg cocoon proteins were reduced with 10 mM DTT for 120 min at 37 ◦ C and then alkylated with 50 mM iodoacetamide for 60 min at 25 ◦ C in the dark. After washing twice with 8 M urea and four times with 50 mM NH4HCO3 in an ultrafiltration tube, proteins were incubated with 2 μg trypsin for 36 h at 37 ◦ C. Tryptic peptides were subsequently recovered by centrifugation in an ultrafiltration tube, lyophilized, and resuspended in 50 μL of 0.1% formic acid. Tryptic peptides were separated using a Thermo Fisher Scientific EASY-nLC 1000 system and an EASY-Spray column (C18, 2 μm, 100 Å, 75 μm × 50 cm) and analyzed using a Thermo Scientific Q Exactive mass spectrometer. Raw mass spectra data were analyzed using MaxQuant software (version 1.3.0.1). MaxQuant searches were performed against the M. flavescens genome database (unpublished data). Peptide searches were performed using Andromeda search algorithms with previously reported search parameters (Dong et al., 2016a). The identified peptides and proteins are listed in Supplementary Tables S1 and S2. 2.5. Annotation of cocoon proteins SignalP 5.0 Server was used to predict the presence of the signal peptides (Almagro et al., 2019). Amino acid sequences were aligned using ClustalX (Thompson et al., 1997), GeneDoc (Nicholas et al., 1997), and ESPript (Gouet et al., 2003). Molecular weight and isoelectric point were predicted using Compute pI/Mw tool (web.expasy. org/compute_pi/). 2.6. Expression analysis of cocoon proteins Seven tissues, including the head, mid gut, spine, integument, fat body, Malpighian tubule, and silk gland were collected on day 1 of wandering. Silk glands were further divided into five compartments. Total RNA was isolated using TRIzol reagent (Invitrogen, USA). The contaminating genomic DNA was digested with RNase-free DNase I (Promega, USA) for 30 min at 37 ◦ C, and the total RNA was reversetranscribed into cDNA using M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Pre­ mix Ex Taq (TaKaRa, Japan) in a qTOWER 2.2 qRT-PCR instrument (Analytikjena Biometra, Germany). The amplification reaction was performed at the following cycling conditions: initial denaturation at 95 ◦ C for 30 s, followed by 40 cycles of denaturation at 95 ◦ C for 10 s, annealing at 60 ◦ C for 30 s, and extension at 72 ◦ C for 35 s. Translation elongation factor 2 (TEF2) was used as the housekeeping gene. The expression values of target genes and housekeeping genes were auto­ matically obtained by the system software and recorded as Ct and Ch, 2. Materials and methods 2.1. Observation of the cocoons Larvae of the oriental moth, which construct hard cocoons in the last instar, were reared on hawthorn leaves at 25 ◦ C. The overall appearance of the cocoons was viewed using a Zeiss Stemi 2000C stereomicroscope, whereas the micromorphology was viewed using a Hitachi SU3500 Scanning electron microscope (SEM) with an accelerating voltage of 10 kV. 2.2. Characterization of minerals in the cocoon The white mineral crystals on the cocoon surface were carefully 2 L. Qin et al. Insect Biochemistry and Molecular Biology 151 (2022) 103878 Fig. 1. Morphology and structure of the oriental moth cocoon. Photographs showed four life stages of the oriental moth, including the egg (A), larva (B), pupa (C), and adult (D). White and brown stripes are clearly visible on the surface of the cocoon constructed by the last instar larva (E). The white and brown stripes were observed using the stereomicroscope (F) and SEM (G). Magnified SEM images showed mineral crystals in the white stripes of the cocoon (H) and silk fiber in the brown stripes of the cocoon (I). respectively. The relative expression value of each gene was calculated using the following formula: 2− Δ(Ct− Ch). The primers used for qRT-PCR are listed in Supplementary Table S3. a width of 4–5 μm and organic matter filling the gap of the silk fiber was observed (Fig. 1G and I). 3.2. Calcium oxalate monohydrate is the main inorganic matter in the oriental moth cocoon 3. Results 3.1. The oriental moth cocoon is composed of mineral crystals, silk fiber, and other organic matter The FTIR spectra were obtained from the mineral crystals scraped off white stripes on the cocoon surface (Fig. 2A–C). The peaks at 1628 cm− 1 and 1315 cm− 1 are the main antisymmetric carbonyl stretching bands – O) specific to the oxalate family and the metal carboxylate stretch (C– for calcium oxalate monohydrate (COM), respectively (Kesavan et al., 2012) (Fig. 2C). The XRD patterns were then obtained from the mineral crystals on the cocoon surface (Fig. 2D). The major diffraction peaks detected at 2θ = 15.2◦ , 24.6◦ , 30.4◦ , 36.2◦ , 38.4◦ and 46.1◦ were well assigned to the previously reported COM peaks (Polat and Sayan 2020). Minor peaks indicated that calcium formate (CF) was also detected in the mineral crystals (Fig. 2D). Overall, COM accounted for 99.03% of the mineral crystals and CF accounted for 0.97%. The elemental distribution in the oriental moth cocoons was quantitatively investigated using ICP-OES (Fig. 2E). Calcium was the most abundant element in the cocoon, reaching 18.67% (186.7 g/kg), and phosphorus, magnesium, potassium, and sulfur occupied 0.64%, 0.34%, 0.13%, and 0.10%, respectively, whereas other elements were below 0.10%. As a complete metamorphosis insect, the oriental moth goes through four life stages, including the egg, larva, pupa, and adult stages (Fig. 1A–D). Like many other lepidopteran species, the larvae of oriental moths construct cocoons in the last instar. The cocoon of the oriental moth is an ellipsoid shape, similar to that of a bird egg, with a long axis of 1.1–1.3 cm and a short axis of 0.7–0.9 cm (Fig. 1E). Longitudinal stripes of brown alternating with white are visible on the surface of the cocoon (Fig. 1E). The cocoon is very dense and smooth, and it is difficult to see silk fibers on the cocoon surface with the naked eye, even with a stereomicroscope (Fig. 1E and F). However, under the electron micro­ scope, we could clearly see the microstructures of the brown and white stripes (Fig. 1G–I). The cuboid mineral crystals in the white stripes are clearly visible with lengths of 5–10 μm and widths of 0.6–1 μm, most of which were arranged disorderly, and only a few were arranged in par­ allel (Fig. 1G and H). In the brown stripes, cross-arranged silk fiber with 3 L. Qin et al. Insect Biochemistry and Molecular Biology 151 (2022) 103878 Fig. 2. Inorganic matter in the oriental moth cocoon. (A) White and brown stripes are clearly visible on the cocoon surface. (B) White mineral powder was scraped from the cocoon surface. (C) FTIR spectra of white mineral powder obtained from the cocoon surface. (D) XRD patterns of white mineral powder from the cocoon surface. (E) ICP-OES analysis of the elemental distribution in the cocoon. Fig. 3. Classification and quantification of cocoon proteins of the oriental moth. All the identified cocoon proteins were classified into twelve groups: uncharac­ terized protein, silk protein, small molecule binding protein, enzyme, bioactive peptide, protease inhibitor, cuticle protein, transcription and translation factor, extracellular matrix protein, heat shock protein, membrane protein, and cytoskeleton protein. The iBAQ intensities were used to show the protein abundance. 4 L. Qin et al. Insect Biochemistry and Molecular Biology 151 (2022) 103878 Fig. 4. Sequence alignments of fibroin heavy chain (A), fibroin light chain (B), and fibroin p25 (C). The same conserved amino acids are labeled with dark red, and the amino acids with similar properties were labeled with light red. Mf, Monema flavescens; Bm, Bombyx mori; Fib_H: fibroin heavy chain; Fib_L: fibroin light chain; Fib_P25: fibroin p25. 3.3. Silk proteins and uncharacterized proteins are the major proteins in the oriental moth cocoon uncharacterized proteins, enzymes, small molecule binding proteins, protease inhibitors, cuticle proteins, transcription and translation fac­ tors, extracellular matrix proteins, membrane proteins, bioactive pep­ tides, heat shock proteins, cytoskeleton proteins, and silk proteins (Fig. 3 and Supplementary Table S2). A total of 287 uncharacterized proteins were identified in the oriental moth cocoon, followed by 87 enzymes and 36 small molecule binding proteins (Fig. 3 and Supplementary Table S2). For the abundance of proteins, the uncharacterized protein in the cocoon was also ranked first, followed by silk protein and small molecule binding protein (Fig. 3 and Supplementary Table S2). LiSCN was used to dissolve the cocoon proteins, and the solution was brown. After centrifugation, it was found that there were still many insoluble substances. LC-MS/MS was performed to identify soluble cocoon proteins in triplicate. Combining these data, 1934 peptides were identified and assembled into 468 protein groups (Supplementary Tables S1 and S2). These oriental moth cocoon proteins could be clas­ sified into 12 categories based on their annotated molecular functions: Table 1 Asn-rich proteins in the oriental moth cocoon. Protein name Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein Asn-rich protein 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Residue No. of Signal peptide Residue No. of mature protein MW pI Proportion of Asn Proportion of Gly Proportion of Ser 21 19 18 21 21 18 21 18 21 23 18 20 18 21 18 20 21 21 18 21 21 23 18 21 16 140 110 162 144 139 137 140 143 134 129 138 144 141 126 135 127 136 147 139 134 126 131 116 140 68 14.6 11.6 16.8 14.7 14.5 15.1 14.5 14.3 13.7 13.3 13.8 14.6 14.2 13.2 14.2 13.5 13.9 15.1 14.2 13.8 13.3 14.1 12.4 14.4 7.7 9.5 10.6 7.3 8.4 9.8 8.7 10.0 9.5 7.3 11.0 6.5 9.2 6.5 8.9 5.2 8.3 8.4 9.8 6.4 6.9 9.4 10.0 11.3 8.3 9.5 38.6% 38.2% 32.7% 36.8% 36.7% 19.0% 38.6% 31.5% 37.3% 40.3% 39.1% 37.5% 37.6% 35.7% 20.7% 37.0% 37.5% 39.5% 35.3% 35.8% 37.3% 36.6% 41.4% 37.1% 19.1% 20.7% 10.9% 24.7% 20.1% 20.9% 8.0% 20.7% 22.4% 21.6% 19.4% 23.2% 21.5% 23.4% 20.6% 8.9% 17.3% 22.1% 23.1% 21.6% 20.2% 19.8% 12.2% 12.9% 22.9% 1.5% 3.6% 10.0% 5.6% 10.4% 3.6% 8.0% 4.3% 13.3% 11.2% 3.1% 5.1% 8.3% 5.0% 5.6% 10.4% 5.5% 5.2% 4.8% 9.4% 8.2% 5.6% 10.7% 8.6% 5.7% 10.3% 5 L. Qin et al. Insect Biochemistry and Molecular Biology 151 (2022) 103878 Fig. 5. Sequence alignments of Asn-rich proteins. Asn, Gly, and hydrophobic amino acids are labeled blue, orange, and gray, respectively. Signal peptides were marked with a black box. Four regions are shown with black or gray lines, which are Gly-rich, Gly/Asn-rich, Asn-rich, and Gly-rich motifs. 3.4. Fibroins, sericins and seroins in the oriental moth cocoon associated with disulfide-linked heavy and light chains by noncovalent interactions (Tanaka et al. 1993, 1999b; Inoue et al., 2000). Sericin 1 is a glue protein rich in Ser (49%) and Gly (35%) (Supplementary Tables S4 and S5), which wraps and sticks the silk fiber (Garel et al., 1997). Seroin 1 is rich in Pro (13%) (Supplementary Tables S4 and S5), and might play dual roles in the silk, acting as an antimicrobial protein or as a molecular chaperone that interacts with fibroins and sericins (Dong et al., 2016b). By comparing the fibroins of the silkworm and oriental moth, we found that the homology of fibroin heavy chains was very low, except for the N-terminal 100 residues (38%) (Fig. 4A). In contrast, the fibroin light chains and fibroin p25 had relatively high similarity (34% and 49%) for the full sequence (Fig. 4B and C). Although only five proteins that make up silk were identified, their abundance was second only to the uncharacterized proteins in the ori­ ental moth cocoon (Fig. 3 and Supplementary Table S2). These five silk proteins were fibroin heavy chain (457 kDa), fibroin light chain (27 kDa), fibroin p25 (24 kDa), sericin 1 (973 kDa) and seroin 1 (27 kDa) (Supplementary Tables S4 and S5). According to studies of another lepidopteran insect silkworm, fibroin heavy chain, fibroin light chain, and fibroin p25 constitute the core of the silk fiber (Inoue et al., 2000). Fibroin heavy chains and light chains are rich in Ser (14% and 10%) and Ala (11% and 11%) (Supplementary Tables S4 and S5), and are linked by a single disulfide bond (Tanaka et al., 1999a), whereas fibroin P25 6 L. Qin et al. Insect Biochemistry and Molecular Biology 151 (2022) 103878 mature proteins were predicted to be 7.7–16.8 kDa, and the isoelectric points were widely distributed from 5.2 to 11.3 (Table 1). Multiple sequence alignments indicated that 21 Asn-rich proteins have high sequence homology except for Asn-rich proteins 2, 6, 15, and 25 (Fig. 5). Most Asn-rich protein sequences can be divided into four regions: Glyrich, Gly/Asn-rich, Asn-rich, and Gly-rich motifs (Fig. 5). Notably, Asn-rich proteins were highly abundant in the oriental moth cocoon. Among the top 20 cocoon proteins with the highest abundance, 9 were Asn-rich proteins (Fig. 6 and Supplementary Table S2), implying that they may play important roles in the cocoon. 3.6. Expression profile of fibroins and Asn-rich proteins Fig. 6. Twenty of the most abundant proteins in the oriental moth cocoon. The protein abundance was estimated by the iBAQ intensity value. The yellow and black colors show the Asn-rich proteins and silk proteins, respectively. The qPCR was used to investigate the expression patterns of major cocoon protein-coding genes (Fig. 7). Six genes expressed only in the silk gland but not in other tissues. On the premise of equal length, the silk gland was cut into five segments at the bend, including regions i, ii, iii, iv, and v (Fig. 7A). The last segment (region v) was longer than the other segments because its shape was very similar to that of the posterior silk gland of the silkworm. The qPCR results showed that the fibroin heavy chain and fibroin light chain were exclusively expressed in the posterior region v of the silk gland (Fig. 7B and C), whereas fibroin P25 was mainly expressed in region v of the silk gland, and slightly expressed in region iv of the silk gland (Fig. 7D). The expression of Asn-rich proteins 1, 3, and 4 in the silk gland increased gradually from anterior silk gland region i to posterior silk gland region v (Fig. 7E–G). The expression of Asn-rich proteins 1, 3, and 4 was significantly higher than that of the fibroin heavy chain, fibroin light chain, and fibroin p25. 3.5. Asn-rich proteins in the oriental moth cocoon 4. Discussion Among 287 uncharacterized proteins, 25 of which were named as Asn-rich proteins because they contained 19.1%–41.4% asparagine (Asn) (Table 1 and Supplementary Table S5). In addition, Asn-rich proteins also contain relative high proportion of glycine (1.5%–24.7%) and serine (3.1%–13.3%). All 25 Asn-rich proteins were predicted to contain signal peptides (Fig. 5), indicating that they were secretory proteins. After removing signal peptides, the molecular weights of the The cocoon of the oriental moth is very hard, providing perfect protection for the pupa. The hardness of the cocoon was thought to be due to the calcification process and the fine silk net filled with sclero­ tized proteins (Ishii et al., 1984). This study revealed that the main mineral in the cocoon was COM, and the main protein components included fibroins, sericins and Asn-rich proteins. The white and brown stripes on the cocoon surface of the oriental moth are clearly visible to the naked eye. When we enlarged the white Fig. 7. Expression patterns of the major cocoon protein-coding genes of the oriental moth. Seven tissues were collected from day 1 of wandering, including head (he), midgut (mi), spine (sp), integument (in), fatbody (fa), Malpighian tubule (ma), and silk gland (sg). The silk gland was further divided into five compartments, region i, ii, iii, iv, and v (A). The qRT–PCR was performed for six major cocoon protein-coding genes, including fibroin heavy chain (B), fibroin light chain (C), fibroin p25 (D), and Asn-rich protein 1 (E), Asn-rich protein 3 (F), and Asn-rich protein 4 (G). 7 L. Qin et al. Insect Biochemistry and Molecular Biology 151 (2022) 103878 patches by SEM, long rod-like mineral crystals were observed, which were thought to be calcium oxalate crystals from the Malpighian tube (Ishii et al., 1984). There are two common types of calcium oxalate crystals, monohydrate form (COM) and dihydrate form (COD), which belong to monoclinic and tetragonal systems of crystallization, respec­ tively (Franceschi and Horner 1980). This study suggests that the crys­ tals on the cocoon surface were mainly COM (99.03%) by XRD and FTIR. COM is commonly found in the Malpighian tubules of lepidopteran in­ sects (Teigler and Arnott 1972). However, some species, such as M. flavescens and Malacosoma neustria testacea, secrete COM into co­ coons (Ohnishi et al., 1968), while other species, such as B. mori, do not (Teigler and Arnott 1972). The proportion of calcium in the oriental moth cocoon was measured to be 18.67% by ICP-OES. Except for mineral crystals, the remaining matter in the cocoon was approximately 31.9%. A previous study found that the organic matter in the cocoon is mainly protein, accounting for approximately 34% (Ishii et al., 1984), which is consistent with the results of this study. Using LC-MS/MS to identify the protein components of the cocoons, 467 proteins were identified. The most abundant proteins are the unchar­ acterized proteins and fibroins. Fibroins are thought to be the main components of the cocoon in another Lepidopteran insect silkworm, accounting for more than 70% of cocoon weight (Mondal et al., 2007). However, the fibroin heavy chain, fibroin light chain, and fibroin p25 contents in the cocoon of the oriental moth were found to be very low in this study, ranking 20th, 54th, and 4th, respectively. This may be due to sclerotization and tanning of silk proteins (Ishii et al., 1984), resulting in insoluble brown cocoons. This process is very common in lepidoptera insect cocoons (Brunet and Coles 1974). Silk proteins are oxidatively conjugated with tanning phenols and cross-linked to form polymers. In the silkworm, fibroin heavy chain and fibroin light chain have higher abundance than fibroin p25 in the cocoon (Inoue et al., 2000). In the oriental moth, we also found that fibroin heavy and light chain are more abundant than fibroin p25 in the silk gland. Thus, the fact that fibroin heavy chain and light chain have lower abundance than p25 in the oriental moth cocoon means that the fibroin heavy chain and light chain might be cross-linked. Interestingly, uncharacterized proteins were found to be the most abundant in the oriental moth cocoon. In particular, 21 Asn-rich pro­ teins constitute a large family of proteins with possible common origins. These Asn-rich proteins have no homology to proteins from other spe­ cies. They are expressed only in the silk gland but not in other tissues, implying that they might be family/genus/species-specific silk proteins. Asn-rich proteins were found to be expressed in the whole silk gland, which is different from fibroins (only in the posterior silk gland) or sericins (only in the middle silk gland). Since that fibroins are in the core silk fiber and sericins are outside the fiber, Asn-rich proteins may be distributed throughout the silk fiber. Previous studies have found that some silk proteins are widely distributed in both the middle and pos­ terior silk glands, such as the protease inhibitor BmSPI51 and two seroin proteins (seroin 1 and seroin 2) (Singh et al., 2014; Zhang et al. 2020, 2021; Zhu et al., 2020). BmSPI51 and seroins have antimicrobial func­ tions in the cocoons. Whether Asn-rich proteins have antimicrobial ac­ tivity needs further study in the future. In this study, Asn-rich proteins were found to have a high proportion of Asn, Gly and Ser, which is consistent with previous results on the detection of amino acids in the oriental moth cocoon (Ishii et al., 1984). This result also suggests that Asn-rich proteins are indeed the main components of the oriental moth cocoon. Many adhesive glue proteins are also rich in Asn, Gly, and Ser, such as the sericins of silkworms (Guo et al., 2022), egg glue proteins of silkworm (Lei et al., 2021), and silk glue proteins of glow-worms (Walker et al., 2015). Therefore, Asn-rich proteins may play roles like sericins, the glue proteins in the silk. We noticed that Asn-rich proteins have higher expression in the silk gland than fibroins on day 1 of wandering. Why Asn-rich proteins require such high expression levels remains unknown. One possible reason is that Asn-rich proteins may form glue droplets to fill the gap between silk filaments, thus forming an impermeable hard cocoon different from other Lepidopteran insects. In addition, Asn-rich proteins may be related to the formation of calcium oxalate crystals. In conclusion, we speculate that the possible roles of Asn-rich proteins include antimicrobial activ­ ity, adhesion, and/or interaction with calcium oxalate crystals. Data availability Data will be made available on request. 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