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

Summary

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

Abstract

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.

Introduction

Thermobia domestica belongs to one of the most basal insect orders, Zygentoma, which retains an ancestral ametabolous and wingless life cycle. Such basal phylogenetic position and ancestral characteristics set this species as an attractive model for studying the mechanisms underlying the success of insects on Earth, which cover over 70% of the described animal species¹. T. domestica has long been used mainly to study ancestral characteristics of insect physiology because of its suitable features as a laboratory model, such as a relatively short lifecycle (2.5–3.0 months from embryo to reproductive adult;

Protocol

1. Maintenance of laboratory colonies

1. For the maintenance of wildtype and mutant populations, use a large plastic container (460 mm x 360 mm x 170 mm) with regular artificial fish food, water in plastic cups with a ventilation hole on the top, a folded paper for hiding the insects, and layered cotton for laying eggs (Figure 2A). Keep all T. domestica cultures inside 37 °C incubators and set the relative humidity (RH) inside each container to 60%–80%.
   NOTE: Because T. domestica absorbs water vapor from the atmosphere¹¹, a direct water supply is not needed. The appropri....

Results

In our hands, about 100 eggs can be well injected with a single injection capillary when it has the adequate tip (Figure 3C). Injection of gRNA/Cas9 ribonucleoprotein complex in embryos within the first 8 h after egg laying results in indels at the gRNA targeted site. This causes biallelic mutations in some cells of the injected generation (G0) and thus mutant mosaic phenotypes are usually obtained in G0. For example, when this protocol was used to inject a gRNA that is designed to target th.......

Discussion

For the successful generation of the desired T. domestica mutant with CRISPR/Cas9, it is first important to collect a sufficient number of staged embryos for injection. For a constant collection of a sufficient number of T. domestica eggs, the key is to select an appropriate size of the container to have a lower population density because it would help the successful completion of a series of complex mating behaviors, which is repeated after every adult molt¹³. A male T. dome.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plate	Corning	83-3738
Alt-R S.p. HiFi Cas9 Nuclease V3, 100 µg	Integrated DNA Technologies	1081060
Anti-static cleaner	Hozan	Z-292	for removing static electricity from a 24-well plate
Barrier Box 20.7L	AS ONE	4-5606-01	Large container
FemtoJet 4i	Eppendorf	5252000013	Electronic microinjector
Femtotip II, injection capillary	Eppendorf	5242957000	Glass injection capillary
High Pack 2440mL	AS ONE	5-068-25	Middle-sized container
Incubator	Panasonic	MIR-554-PJ	for 37 °C incubation. No need to humidify inside the incubator.
KOD Fx Neo	Toyobo	KFX-101	PCR enzyme for genotyping. Optimized for an amplification from crude templates.
Magnetic stand	Narishige	GJ-8	for holding the micromanipulator
Microloader	Eppendorf	5242956003
Micromanipulator	Narishige	MM-3
Microscope	Olympus	SZX12	for microinjection. More than 35X magnification is sufficient for the microinjection
MultiNA	Shimadzu	MCE-202	Microchip electrophoresis system
NiceTac	Nichiban	NW-5	Double-sided tape to place eggs on a glass slide
Paint brush (horse hair)	Pentel	ZBS1-0
Plant culture dish	SPL Life Sciences	310100	Mating dish and water supplies for a large and middle-sized containers
Proteinase K, recombinant, PCR Grade Lyophilizate from Pichia pastoris	Roche	3115836001
SZX 12 microscope	Olympus	SZX 12	More than 35X magnification is sufficient for the microinjection
Talcum powder	Maruishi	877113
Tetra Goldfish Gold Growth	Spectrum Brands		Artificial regular fish food