Allele‐specific knockouts reveal a role for apontic‐like in the evolutionary loss of larval melanin pigmentation in the domesticated silkworm, Bombyx mori

Abstract

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in melanin pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9‐targeted knockouts of pigment‐related genes as a tool to understand their potential contributions to domestication‐associated melanin pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9‐targeted knockout of yellow‐y. In yellow‐y knockout mutants, black body colour became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in melanin pigment formation. Further, we performed allele‐specific CRISPR/Cas9‐targeted knockouts of the pigment‐related transcription factor, apontic‐like (apt‐like) in B. mori × B. mandarina F1 hybrid individuals which exhibit B. mandarina‐like larval pigmentation. Knockout of the B. mandarina allele of apt‐like in F1 embryos results in white patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt‐like to the evolution of reduced melanin pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9‐targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.

Disruption of a BTB-ZF transcription factor causes female sterility and melanization in the larval body of the silkworm,Bombyx mori

“…The molecular mechanism by which Bmmamo contributes to pigmentation variation remains to be elucidated. For example, the apt-like gene associated with differential pigmentation of B. mori and B. mandarina larvae (Tomihara et al, 2022) is expressed in the larval integument, and its level reaches an earlier peak than that of the Bmmamo gene at the fourth molting stage (Figure S4). In consequence, we propose that the Bmmamo gene may be regulated by the apt-like gene.…”

“…In the past decade, B. mori mutants have been used to identify candidate genes involved in color variation as a result of selection during domestication. Yoda et al (2014) used positional cloning to identify the gene responsible for diverse coloration pattern in larvae of B. mori strains (apontic like; apt-like), which was afterward found to be involved in the pigmentation pattern variation between B. mori and B. mandarina larvae (Tomihara et al, 2022). Wang et al (2022) also performed positional cloning and found that the abnormal expression of the transcription factor gene SoxD is responsible for B. mori larval color mutation Ursa (U).…”

Sericin Ser3 Ectopic Expressed in Posterior Silk Gland Affects Hemolymph Immune Melanization Response via Reducing Melanin Synthesis in Silkworm

“…The problem of random transgenic integration of piggybac transposons has been solved using site-specific recombination technology [ 16 , 17 ], a GAL4/UAS dual expression system mediated by a tissue-specific promoter [ 18 , 19 ], and CRISPR-Cas9 gene editing technology [ 20 , 21 , 22 ]. Although some studies have recently begun to pay attention to biological security problems, such as developmental barriers and the low vitality of transgenic silkworms, there is still a lack of studies on the effects on biological security such as metabolism and immunity [ 10 , 23 ], despite reports that silkworm resistance was improved by the overexpression of antiviral or antimicrobial peptide-related genes specific to non-silk gland tissue when breeding silkworm varieties with high disease resistance [ 24 , 25 , 26 ].…”