Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon

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 knockout 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.

Expansion of Targetable Sites for the Ribonucleoprotein-Based CRISPR/Cas9 System in the Silkworm Bombyx Mori

“…The delivery of CRISPR/Cas9 system into silkworms has been accomplished using the binary transgenic method [3,[5][6][7] and the Cas9 mRNA-or protein-based methods [4,10]. In the binary method, the RNA polymerase type III promoter U6 is widely exploited to express sgRNA in vivo.…”

“…In contrast, the silkworm U6 promoter effectively expresses sgRNAs initiated with any nucleotide [17], providing additional versatility. Unfortunately, even with this improvement, the transgene-based method is time consuming and di cult to implement [3,5].…”

“…show that our improved method provides researchers with an expanded target repertoire, and can reach some targets at high e ciency even if matching 5' GG residues are unavailable. Our method further increases the advantages of the Cas9 ribonucleoprotein (RNP)-based system over the widely used binary transgenic system [3,5].…”

“…However, di culties in designing and engineering ZFNs and TALENs have greatly limited the application of gene editing in silkworms. With the development of CRISPR/Cas9, gene editing in silkworms has become far more tractable [3,4].…”

“…Gene editing using CRISPR/Cas9 in silkworms has typically been performed with a binary transgenic system [3,[5][6][7]. This system requires two separate transgenic lines, one of which expresses universal Cas9 and the other a customized sgRNA.…”