Abstract
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.
Highlights
It is well known that insects are the most prosperous animal groups on the earth and distributed in almost every corner of the world [1]
Spinning and cocooning are the instincts of many insects, providing a shelter to the residing pupae to resist adverse factors
We have bioinformatically identified that cocoonase is specific to Lepidoptera, and demonstrated that it is the determining factor for breaking the sealed cocoon experimentally for the first time
Summary
It is well known that insects are the most prosperous animal groups on the earth and distributed in almost every corner of the world [1]. This is benefit to their adaptabilities to the environment during the long-term evolution process, such as the small size conducive to hiding themselves, competitive flight ability, amazing reproduction ability, short life cycle and so on. The construction of cocoon is one of the effective strategies for some holometabolous insects to protect the immobile pupa from mechanical damage, natural predators, parasites and other adverse factors before adult emergence [6,7,8,9]. To the best of our knowledge, insect adults escape from the cocoon through the following way: reserving an emergence valve or weak places on the cocoon [16], utilizing special structure of the head to break the cocoon [17], or secreting alkaline fluid that can soften the cocoon [18, 19]
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