Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species’ unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species

CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution

Despite previous developmental studies on basally branching wingless insects and crustaceans, the evolutionary origin of insect wings remains controversial. Knowledge regarding genetic regulation of tissues hypothesized to have given rise to wings would help to elucidate how ancestral development changed to allow the evolution of true wings. However, genetic tools available for basally branching wingless species are limited. The firebrat Thermobia domestica is an apterygote species, phylogenetically related to winged insects. T. domestica presents a suitable morphology to investigate the origin of wings, as it forms the tergal paranotum, from which wings are hypothesized to have originated. Here we report the first successful CRISPR/Cas9-based germline genome editing in T. domestica. We provide a technological platform to understand the development of tissues hypothesized to have given rise to wings in an insect with a pre-wing evolution body plan

A hemimetabolous wing development suggests the wing origin from lateral tergum of a wingless ancestor

祖先の背中の肥大化が昆虫の翅を生んだ --150年来の昆虫翅進化の謎に迫る--. 京都大学プレスリリース. 2022-02-28.The origin and evolution of the novel insect wing remain enigmatic after a century-long discussion. The mechanism of wing development in hemimetabolous insects, in which the first functional wings evolved, is key to understand where and how insect wings evolutionarily originate. This study explored the developmental origin and the postembryonic dramatic growth of wings in the cricket Gryllus bimaculatus. We find that the lateral tergal margin, which is homologous between apterygote and pterygote insects, comprises a growth organizer to expand the body wall to form adult wing blades in Gryllus. We also find that Wnt, Fat-Dachsous, and Hippo pathways are involved in the disproportional growth of Gryllus wings. These data provide insights into where and how insect wings originate. Wings evolved from the pre-existing lateral terga of a wingless insect ancestor, and the reactivation or redeployment of Wnt/Fat-Dachsous/Hippo-mediated feed-forward circuit might have expanded the lateral terga

What serial homologs can tell us about the origin of insect wings [version 1; referees: 2 approved]

Although the insect wing is a textbook example of morphological novelty, the origin of insect wings remains a mystery and is regarded as a chief conundrum in biology. Centuries of debates have culminated into two prominent hypotheses: the tergal origin hypothesis and the pleural origin hypothesis. However, between these two hypotheses, there is little consensus in regard to the origin tissue of the wing as well as the evolutionary route from the origin tissue to the functional flight device. Recent evolutionary developmental (evo-devo) studies have shed new light on the origin of insect wings. A key concept in these studies is “serial homology”. In this review, we discuss how the wing serial homologs identified in recent evo-devo studies have provided a new angle through which this century-old conundrum can be explored. We also review what we have learned so far from wing serial homologs and discuss what we can do to go beyond simply identifying wing serial homologs and delve further into the developmental and genetic mechanisms that have facilitated the evolution of insect wings

A Baculovirus Immediate-Early Gene, <em>ie1</em>, Promoter Drives Efficient Expression of a Transgene in Both <em>Drosophila melanogaster</em> and <em>Bombyx mori</em>

<div><p>Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, <em>ie1</em>, from the <em>Bombyx mori</em> nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a <em>piggyBac</em>-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV <em>ie1</em> promoter; we performed a detailed investigation of the promoter in transgene expression pattern in <em>Drosophila melanogaster</em> and in <em>B. mori</em>. <em>Drosophila</em> and <em>Bombyx</em> belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, <em>ie1</em> promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the <em>ie1</em> promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous <em>ie1</em> promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the <em>ie1</em> promoter can be used as an efficient expression driver in a diverse range of insect species.</p> </div

Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in silkworm pupa.

<p>(A–C) Two-day old pupa. EGFP expression was evident throughout the pupal body. (A, A’) Dorsal views under white light (A) and EGFP-excitation wavelength light (A’). Upper pupa is non-transgenic pupa. (B) Ventral view. (C) Lateral view. (D) A ventral view of the head and thorax of 4-day old pupa. DsRed expression was evident in the compound eyes, whereas EGFP was not. Abbreviations: an, antenna; ce, compound eye; sp, spiracle; wg, wing. Scale bar = 5 mm.</p

Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in tissues dissected from 5th instar silkworm larvae.

<p>(A, A’) A dissected non-transgenic 5th instar larva. (B, B’) A dissected transgenic 5th instar larva. Intense EGFP fluorescence was evident in the anterior and posterior midgut. (C–J’) A dissected 5th instar larva. (C) Prothoracic gland. (D) Merged image of the transmitted light and EGFP fluorescence in the suboesophageal body. (E–F’) Trichogen (or trichogen and tormogen) cells in the epidermis in a non-transgenic larva (E, E’) and a transgenic larva (F, F’). (G–H’) Ovary of a non-transgenic larva (G, G’) and a transgenic larva (H, H’). EGFP was evident in tracheolar cells that were attached to the ovary. (I–J’) Tissues surrounding dorsal vessel of a non-transgenic larva (I, I’) and a transgenic larva (J, J’). EGFP was evident in pericardial cells along dorsal vessel and on the alary muscle, and peritracheal athrocytes, but not in fatbody. (A, B, E–J) White light, (A’, B’, C, E’-J’) EGFP-excitation wavelength light. The images for the comparisons of non-transgenic and transgenic larvae and tissues were obtained exactly by the same conditions. Abbreviations: dv, dorsal vessel; fb, fatbody; mg, midgut; pa, peritracheal athrocytes. Scale bars = 5 mm in (A) and (B), 500 µm in (C, E, F, G, H), 1 mm in (I, J).</p

Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in silkworm larva at different stages.

<p>(A, A’) First instar larva just after hatching. BmNPV <i>ie1</i> promoter-driven EGFP expression did not overlap with 3xP3-driven DsRed expression in the ventral nerve cord. Ventral view. (B) Late 2nd instar larva. EGFP was expressed throughout the whole body and throughout all larval stages. Dorsal view. (C) Early 3rd instar larva. Dorsal view. (D, D’) 4th instar larvae. Dorsal view. Upper larva is a non-transgenic larva. (E) Head and thorax of 5th instar larva. Lateral view. Scale bars = 2 mm.</p

Genomic DNA sequences surrounding <i>piggyBac</i> insertions.

<p>The flanking sequences of <i>piggyBac</i> insertion in <i>D. melanogaster</i> and <i>B. mori</i> have 100% identity with the genome DNA sequences of chromosome 3L and Bm_scaf 21 in chromosome 17, respectively. Abbreviations: Dm, <i>Drosophila melanogaster</i>; Bm, <i>Bombyx mori</i>.</p