Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon

Autoři: Tingting Gai aff001;  Xiaoling Tong aff001;  Minjin Han aff001;  Chunlin Li aff001;  Chunyan Fang aff001;  Yunlong Zou aff001;  Hai Hu aff001;  Hui Xiang aff002;  Zhonghuai Xiang aff001;  Cheng Lu aff001;  Fangyin Dai aff001
Působiště autorů: State Key Laboratory of Silkworm Genome Biology, Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Southwest University, Chongqing, China aff001;  Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China aff002
Vyšlo v časopise: Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon. PLoS Genet 16(9): e32767. doi:10.1371/journal.pgen.1009004
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009004


Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.

Klíčová slova:

Animal behavior – Guide RNA – Homozygosity – Insects – Moths and butterflies – Phylogenetic analysis – Pupae – Silkworms


1. Stork NE. How many species of insects and other terrestrial arthropods are there on Earth?. Annu Rev Entomol. 2018; 63:31–45. doi: 10.1146/annurev-ento-020117-043348 28938083

2. Koštál V. Eco-physiological phases of insect diapause. J Insect Physiol. 2006; 52(2), 113–127. doi: 10.1016/j.jinsphys.2005.09.008 16332347

3. Ruxton GD, Sherratt TN, Speed MP. Avoiding attack: the evolutionary ecology of crypsis, warning signals and mimicry. Oxford University Press, Oxford, UK; 2004.

4. Dingle H. Migration Strategies of Insects. Science. 1972; 175 (4028), 1327–1335. doi: 10.1126/science.175.4028.1327 17813822

5. Chapman JW, Reynolds DR, Wilson K. Long-range seasonal migration in insects: mechanisms, evolutionary drivers and ecological consequences. Ecol Lett. 2015; 18(3), 287–302. doi: 10.1111/ele.12407 25611117

6. Bartell DP, Sanborn JR, Wood KA. Insecticide Penetration of Cocoons Containing Diapausing and Nondiapausing Bathyplectes curculionis, an Endoparasite of the Alfalfa Weevil. Environmental Entomology. 1976; 5:659–61.

7. Halpern M, Gasith A, Broza M. Does the tube of a benthic chironomid larva play a role in protecting its dweller against chemical toxicants? Hydrobiologia. 2002; 470:49–55.

8. Stevens DJ, Hansell MH, Freel JA, Monaghan P. Developmental trade-offs in caddis flies: increased investment in larval defence alters adult resource allocation. Proc Biol Sci. 1999; 266(1423):1049.

9. Danks HV. The roles of insect cocoons in cold conditions. European Journal of Entomology. 2004; 101(3):433–7.

10. Jenkins MF. Cocoon building and the production of silk by the mature larva of Dianous coerulescens Gyllenhal (Coleoptera: Staphylinidae). Trans R entomol Soc London. 1958; 110:287–301.

11. Trouvelot L. The American Silk Worm. Am Nat. 1867; 1:30–8.

12. Donald LJ, Shaw MR, Takahashi M, Yanechin B. Cocoon silk chemistry of non-cyclostome Braconidae, with remarks on phylogenetic relationships within the Microgastrinae (Hymenoptera: Braconidae). Journal of Natural History. 2010; 38:2167–81.

13. Rudall KM, Kenchington W. Arthropod Silks: The Problem of Fibrous Proteins in Animal Tissues. Annual Review of Entomology. 1971; 16:73–96.

14. Sutherland TD, Young JH, Weisman S, Hayashi CY, Merritt DJ. Insect silk: one name, many materials. Annu Rev Entomol. 2010; 55:171–88. doi: 10.1146/annurev-ento-112408-085401 19728833

15. Ishii S, Inokuchi T, Kanazawa J, Tomizawa C. Studies on the cocoon of the oriental moth, Monema (Cnidocampa) flavescens, (lepidoptera: limacodidae). III. Structure and composition of the cocoon in relation to hardness. Japanese Journal of Applied Entomology and Zoology. 1984; 28(4), 269–273.

16. Harcourt DG. Biology of the Diamondback Moth, Plutella maculipennis (Curt.) (Lepidoptera: Plutellidae), in Eastern Ontario. II. Life-History, Behaviour, and Host Relationships. The Canadian Entomologist. 1957; 89(12), 554–564.

17. Yu R, Shi M, Huang F, & Chen X. Immature Development of Cotesia vestalis (Hymenoptera: Braconidae), an Endoparasitoid of Plutella xylostella (Lepidoptera: Plutellidae). Annals of the Entomological Society of America. 2008; 101(1), 189–196.

18. Latter OH. XVIII. The secretion of potassium hydroxide by Dicranura vinula (imago), and the emergence of the imago from the cocoon. Transactions of the Royal Entomological Society of London. 2009; 40(4), 287–292.

19. Latter OH. XIV. Further Notes on the Secretion of Potassium Hydroxide by Dicranura vinula (imago), and similar Phenomena in other Lepidoptera. Transactions of the Royal Entomological Society of London. 2009; 43(3), 399–409.

20. Duspiva F. The enzymatic processes when the silk spinner (Bombyx mori L.) breaks through the cocoon shell. Journal of Natural Science B. 1950; 5b:273–81.

21. Kafatos FC, Williams CM. Enzymatic Mechanism for the Escape of Certain Moths from Their Cocoons. Science. 1964; 146(3643):538–40. doi: 10.1126/science.146.3643.538 17806809

22. Kafatos FC, Tartakoff AM, Law JH. Cocoonase. I. Preliminary characterization of a proteolytic enzyme from silk moths. The Journal of Biological Chemistry. 1967; 242:1477–87. 6023217

23. Wu Y, Wang W, Wang BLD and Shen W, Cloning and expression of the cocoonase gene from Bombyx mori. Sci Agric Sin. 2008; 41:3277–3285.

24. Yamazaki Y, Ogawa K and Kanekatsu R, Isolation of cocoonase from the silkworm, Bombyx mori, by a high performance liquid chromatography and catalytic specificity. J Seric Sci Jpn. 1992; 61:228–235.

25. Hidetoshi T, Keiji K and Mitsuhiro M, Proteolytic characterization of cocoonase from the domestic silk moth, Bombyx mori. Pept Sci 42:479–482 (2005).

26. Fukumori H, Teshiba S, Shigeoka Y, Yamamoto K, Banno Y and Aso Y, Purification and characterization of cocoonase from the silkworm Bombyx mori. Biosci Biotechnol Biochem. 2014; 78:202–211. doi: 10.1080/09168451.2014.878215 25036672

27. Eguchi M, Iwamoto A. Proteases in the pupal midgut of the silkworm, Bombyx mori L. II. Hydrolysis of the solubilized fibroin and native silk. J Sericult Sci Japan. 1973; 42(2):144–50.

28. Eguchi M, Iwamoto A. Rôle of the midgut, crop, and maxillae of Bombyx mori in the production of cocoon-digesting enzyme. J Insect Physiol. 1975; 21(7):1365–82.

29. Wang H, Zhang C, Cui W, Liu X, Zhou Y, Cai Y, et al. Studies on Secretory Organs of Cocoonase and Silkmoth-vomiting Fluid of Silkworm, Bombyx mori. Acta Sericologica Sinica. 2005; 31(2):136–41.

30. Zhang C, Cui W, Guo Y, Wang Y, Mu Z. Ultrastructure changes and function of the midgut and salivary glands in Bombyx mori during the pupal-adult metamorphism. Acta Entomologica Sinica. 2007; 50(8):769–74.

31. Prasad BC, Pandey JP and Sinha AK, Study of Antheraea mylitta cocoonase and its use in cocoon cooking. Am J Food Technol. 2012; 7:320–325.

32. Geng P, Lin L, Li Y, Fan Q, Wang N, Song L, et al. A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): purification and characterization. Biochem Biophys Res Commun. 2014; 445:64–67 doi: 10.1016/j.bbrc.2014.01.155 24491553

33. Yang J, Wang W, Li B, Wu Y, Wu H and Shen W, Expression of cocoonase in silkworm (Bombyx mori) cells by using a recombinant baculovirus and its bioactivity assay. Int J Biol. 2009; 1:107–112.

34. Rodbumrer P, Arthan D, Uyen U, Yuvaniyama J, Svasti J and Wongsaengchantra PY, Functional expression of a Bombyx mori cocoonase: potential application for silk degumming, Acta Biochim Biophys Sin. 2012; 44:974–983. doi: 10.1093/abbs/gms090 23169343

35. Unajak S., Aroonluke S., & Promboon A. An active recombinant cocoonase from the silkworm Bombyx mori: bleaching, degumming and sericin degrading activities. J Sci Food Agr. (2014); 95(6), 1179–1189.

36. Zhu Y, Wang Z, He N, Li C. Enzymatic Activity and Protein Species Identification of Spit Liquid from Bombyx mori Moths. Science of Sericulture. 2014; 40(03):0452–57.

37. Harpel D, Cullen DA, Ott SR, Jiggins CD, Walters JR. Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene. Insect Biochem Mol Biol. 2015;63:7–13. doi: 10.1016/j.ibmb.2015.04.004 25958827

38. Smith G, Macias-Munoz A, Briscoe AD. Gene Duplication and Gene Expression Changes Play a Role in the Evolution of Candidate Pollen Feeding Genes in Heliconius Butterflies. Genome Biol Evol. 2016;8(8):2581–96. doi: 10.1093/gbe/evw180 27553646

39. Smith G, Kelly JE, Macias-Munoz A, Butts CT, Martin RW, Briscoe AD. Evolutionary and structural analyses uncover a role for solvent interactions in the diversification of cocoonases in butterflies. Proc Biol Sci. 2018; 285:2017–37.

40. Rubinoff D, Schmitz P. Multiple aquatic invasions by an endemic, terrestrial Hawaiian moth radiation. Proceedings of the National Academy of Sciences. 2010; 107(13), 5903–5906.

41. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 1997; 25(17):3389–402. doi: 10.1093/nar/25.17.3389 9254694

42. Altschul SF, Wootton JC, Gertz EM, Agarwala R, Morgulis A, Schaffer AA, et al. Protein database searches using compositionally adjusted substitution matrices. FEBS J. 2005; 272(20):5101–9. doi: 10.1111/j.1742-4658.2005.04945.x 16218944

43. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7):1870–1874. doi: 10.1093/molbev/msw054 27004904

44. Zuckerkandl E and Pauling L. Evolutionary divergence and convergence in proteins. In: Bryson V and Vogel HJ, editors. Evolving Genes and Proteins; 1965. pp. 97–166.

45. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985; 39(4):783–91. doi: 10.1111/j.1558-5646.1985.tb00420.x 28561359

46. Kimura M. A Simple Method for Estimating Evolutionary Rates of Base Substitutions Through Comparative Studies of Nucleotide Sequences. J Mol Evol. 1980; (16):111–20.

47. Gao F, Chen C, Arab DA, Du Z, He Y, Ho SYW. EasyCodeML: A visual tool for analysis of selection using CodeML. Ecol Evol. 2019; 9(7):3891–8. doi: 10.1002/ece3.5015 31015974

48. Sander J. D., & Joung J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. NAT BIOTECHNOL. 2014; 32(4), 347–355. doi: 10.1038/nbt.2842 24584096

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PLOS Genetics

2020 Číslo 9

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