Filippi's glands (FGs), formerly also called Lyonet's glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, Bombyx mori. We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.

Biology Centre of the Czech Academy of Sciences Institute of Entomology 37005 Ceske Budejovice Czech Republic

Faculty of Education University of South Bohemia 37005 Ceske Budejovice Czech Republic

Faculty of Science University of South Bohemia 37005 Ceske Budejovice Czech Republic

See more in PubMed

Helm E. Über die Spinndrüsen der Lepidopteren. Wilhelm Engelmann; Lemgo, Germany: 1876.

Waku Y., Sumimoto K.I. Ultrastructure of Lyonet’s gland in the silkworm (Bombyx mori L.) J. Morphol. 1974;142:165–185. doi: 10.1002/jmor.1051420205. PubMed DOI

Patra S., Singh R.N., Raziuddin M. Morphology and histology of Lyonet’s gland of the tropical tasar silkworm, Antheraea mylitta. J. Insect Sci. 2012;12:1–7. doi: 10.1673/031.012.12301. PubMed DOI PMC

Paudel S., Mikó I., Deans A., Rajotte E., Felton G. Lyonet’s gland of the tomato fruitworm, Helicoverpa zea (Lepidoptera: Noctuidae) PeerJ. 2018;6:e26455v1. doi: 10.3897/rio.5.e35906. DOI

Vegliante F. Larval head anatomy of Heterogynis penella (Zygaenoidea, Heterogynidae), and a general discussion of caterpillar head structure (Insecta, Lepidoptera) Acta Zool. 2005;86:167–194. doi: 10.1111/j.1463-6395.2005.00198.x. DOI

Chi C., Drew W.A., Young J.H., Curd M.R. Comparative morphology and histology of the larval digestive system of two genera of Noctuidae (Lepidoptera): Heliothis and Spodoptera. Ann. Entomol. Soc. Am. 1975;68:371–380. doi: 10.1093/aesa/68.2.371. DOI

Victoriano E., Gregorio E.A. Ultrastructure of the Lyonet’s glands in larvae of Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae) Biocell. 2004;28:165–169. doi: 10.32604/biocell.2004.28.165. PubMed DOI

Drecktrah H.G., Knight K.L., Brindley T.A. Morphological investigations of the european corn borer. Iowa State. J. Sci. 1966;40:257–286.

Day M.F., Waterhouse D.F. In: Chapter 12 in Insect Physiology. Roeder K., editor. John Wiley and Sons; New York, NY, USA: 1953.

Wigglesworth V.B. The Principles of Insect Physiology. Chapman and Hall; London, UK: 1972.

Wang X., Li Y., Liu Q., Tan X., Xie X., Xia Q., Zhao P. GC/MS-based metabolomics analysis reveals active fatty acids biosynthesis in the Filippi’s gland of the silkworm, Bombyx mori, during silk spinning. Insect Biochem. Mol. Biol. 2019;105:1–9. doi: 10.1016/j.ibmb.2018.12.009. PubMed DOI

Foo C.W.P., Bini E., Hensman J., Knight D.P., Lewis R.V., Kaplan D.L. Role of pH and charge on silk protein assembly in insects and spiders. Appl. Phys. A-Mater. 2006;82:223–233. doi: 10.1007/s00339-005-3426-7. DOI

Wang X., Li Y., Peng L., Chen H., Xia Q., Zhao P. Comparative transcriptome analysis of Bombyx mori spinnerets and Filippi’s glands suggests their role in silk fiber formation. Insect Biochem. Mol. Biol. 2016;68:89–99. doi: 10.1016/j.ibmb.2015.11.003. PubMed DOI

Machida Y. Studies on the silk glands of the silkworm, Bombyx mori L. I. Morphological and functional studies of Filippi’s glands in the silkworm. Sci. Bull. Fac. Agric. Kyushu Univ. 1965;22:95–108.

Sehnal F., Akai H. Insect Silk Glands—Their Types, Development and Function, and Effects of Environmental-Factors and Morphogenetic Hormones on Them. Int. J. Insect Morphol. Embryol. 1990;19:79–132. doi: 10.1016/0020-7322(90)90022-H. DOI

Sehadova H., Zavodska R., Zurovec M., Sauman I. The Filippi’s Glands of Giant Silk Moths: To Be or Not to Be? Insects. 2021;12:1040. doi: 10.3390/insects12111040. PubMed DOI PMC

Van der Kloot W.G., Williams C.M. Cocoon construction by the cecropia silkworm. I. The role of the external environment. Behaviour. 1953;5:141–156. doi: 10.1163/156853953X00087. DOI

Chen F., Porter D., Vollrath F. Structure and physical properties of silkworm cocoons. J. R Soc. Interface. 2012;9:2299–2308. doi: 10.1098/rsif.2011.0887. PubMed DOI PMC

Chen F.J., Porter D., Vollrath F. Morphology and structure of silkworm cocoons. Mat. Sci. Eng. C Mater. 2012;32:772–778. doi: 10.1016/j.msec.2012.01.023. DOI

Guerra P.A., Reppert S.M. Dimorphic cocoons of the cecropia moth (Hyalophora cecropia): Morphological, behavioral, and biophysical differences. PLoS ONE. 2017;12:e0174023. doi: 10.1371/journal.pone.0174023. PubMed DOI PMC

Sehadova H., Guerra P.A., Sauman I., Reppert S.M. A re-evaluation of silk measurement by the cecropia caterpillar (Hyalophora cecropia) during cocoon construction reveals use of a silk odometer that is temporally regulated. PLoS ONE. 2020;15:e0228453. PubMed PMC

Hamilton C.A., St Laurent R.A., Dexter K., Kitching I.J., Breinholt J.W., Zwick A., Timmermans M.J.T.N., Barber J.R., Kawahara A.Y. Phylogenomics resolves major relationships and reveals significant diversification rate shifts in the evolution of silk moths and relatives. BMC Evol. Biol. 2019;19:182. doi: 10.1186/s12862-019-1505-1. PubMed DOI PMC

Victoriano E., Pinheiro D.O., Gregorio E.A. Histochemical and ultrastructural evidence of lipid secretion by the silk gland of the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) Neotrop. Entomol. 2007;36:707–711. doi: 10.1590/S1519-566X2007000500011. PubMed DOI

Engster M.S. Studies on silk secretion in the trichoptera (F. Limnephilidae): I. Histology, histochemistry, and ultrastructure of the silk glands. J. Morphol. 1976;150:183–211. doi: 10.1002/jmor.1051500109. PubMed DOI

Wang J., Xia Q., He X., Dai M., Ruan J., Chen J., Yu G., Yuan H., Hu Y., Li R., et al. SilkDB: A knowledgebase for silkworm biology and genomics. Nucleic Acids Res. 2005;33:D399–D402. doi: 10.1093/nar/gki116. PubMed DOI PMC

Lu F., Wei Z.Y., Luo Y.J., Guo H.L., Zhang G.Q., Xia Q.Y., Wang Y. SilkDB 3.0: Visualizing and exploring multiple levels of data for silkworm. Nucleic Acids Res. 2020;48:D749–D755. doi: 10.1093/nar/gkz919. PubMed DOI PMC

Armenteros J.J.A., Tsirigos K.D., Sonderby C.K., Petersen T.N., Winther O., Brunak S., von Heijne G., Nielsen H. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 2019;37:420–423. doi: 10.1038/s41587-019-0036-z. PubMed DOI

Domigan L.J., Andersson M., Alberti K.A., Chesler M., Xu Q., Johansson J., Rising A., Kaplan D.L. Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands. Insect Biochem. Mol. Biol. 2015;65:100–106. doi: 10.1016/j.ibmb.2015.09.001. PubMed DOI PMC

Terry A.E., Knight D.P., Porter D., Vollrath F. pH induced changes in the rheology of silk fibroin solution from the middle division of Bombyx mori silkworm. Biomacromolecules. 2004;5:768–772. doi: 10.1021/bm034381v. PubMed DOI

Azuma M., Ohta Y. Changes in H+-translocating vacuolar-type ATPase in the anterior silk gland cell of Bombyx mori during metamorphosis. J. Exp. Biol. 1998;201:479–486. doi: 10.1242/jeb.201.4.479. PubMed DOI

Akai H. The ultrastructure and functions of the silk gland cells of Bombyx mori. In: King R.C., Akai H., editors. Insect Ultrastructure. Volume 2. Plenum Press; New York, NY, USA: 1984. pp. 323–359.

Farkas R., Datkova Z., Mentelova L., Low P., Benova-Liszekova D., Beno M., Sass M., Rehulka P., Rehulkova H., Raska O., et al. Apocrine secretion in Drosophila salivary glands: Subcellular origin, dynamics, and identification of secretory proteins. PLoS ONE. 2014;9:e94383. doi: 10.1371/journal.pone.0094383. PubMed DOI PMC

Dong Z., Zhao P., Wang C., Zhang Y., Chen J., Wang X., Lin Y., Xia Q. Comparative proteomics reveal diverse functions and dynamic changes of Bombyx mori silk proteins spun from different development stages. J. Proteome Res. 2013;12:5213–5222. doi: 10.1021/pr4005772. PubMed DOI

Zhang Y., Zhao P., Dong Z., Wang D., Guo P., Guo X., Song Q., Zhang W., Xia Q. Comparative proteome analysis of multi-layer cocoon of the silkworm, Bombyx mori. PLoS ONE. 2015;10:e0123403. doi: 10.1371/journal.pone.0123403. PubMed DOI PMC

Kurioka A., Yamazaki M., Hirano H. Primary structure and possible functions of a trypsin inhibitor of Bombyx mori. Eur. J. Biochem. 1999;259:120–126. doi: 10.1046/j.1432-1327.1999.00030.x. PubMed DOI

Nirmala X., Kodrik D., Zurovec M., Sehnal F. Insect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor. Eur. J. Biochem. 2001;268:2064–2073. doi: 10.1046/j.1432-1327.2001.02084.x. PubMed DOI

Ozaki N., Ohmuraya M., Hirota M., Ida S., Wang J., Takamori H., Higashiyama S., Baba H., Yamamura K. Serine Protease Inhibitor Kazal Type 1 Promotes Proliferation of Pancreatic Cancer Cells through the Epidermal Growth Factor Receptor. Mol. Cancer Res. 2009;7:1572–1581. doi: 10.1158/1541-7786.MCR-08-0567. PubMed DOI

Jin H.J., Kaplan D.L. Mechanism of silk processing in insects and spiders. Nature. 2003;424:1057–1061. doi: 10.1038/nature01809. PubMed DOI

Stewart R.J., Wang C.S. Adaptation of caddisfly larval silks to aquatic habitats by phosphorylation of h-fibroin serines. Biomacromolecules. 2010;11:969–974. doi: 10.1021/bm901426d. PubMed DOI

Kamioka S., Mukaiyama F., Takei T., Ito T. Digestion and utilization of artificial diet by the silkworm, Bombyx mori, with special references to the efficiency of the diet at varying levels of dietary soybean meal. J. Sericultural Sci. Jpn. 1971;40:473–483.

Rouhova L., Kludkiewicz B., Sehadova H., Sery M., Kucerova L., Konik P., Zurovec M. Silk of the common clothes moth, Tineola bisselliella, a cosmopolitan pest belonging to the basal ditrysian moth line. Insect Biochem. Mol. Biol. 2021;130:103527. doi: 10.1016/j.ibmb.2021.103527. PubMed DOI

Cox J., Hein M.Y., Luber C.A., Paron I., Nagaraj N., Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteom. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591. PubMed DOI PMC

Cox J., Neuhauser N., Michalski A., Scheltema R.A., Olsen J.V., Mann M. Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment. J. Proteome Res. 2011;10:1794–1805. doi: 10.1021/pr101065j. PubMed DOI

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M.Y., Geiger T., Mann M., Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI