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.
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.
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; Figure 1A) and an easy breeding. In the past three decades, its use has been expanded to investigate ancestral characteristics of various traits such as body plan, neural differentiation, and circadian rhythms2,3,4.
The application of advanced genetic tools in T. domestica could further accelerate such contributions in a wide research area. Successful RNA interference (RNAi)-mediated gene knockdown in embryos, nymphs, and adults has been reported in T. domestica4,5,6. The efficiency of systemic RNAi is still highly species-dependent—for example, it is generally high in coleoptera whereas it is low in the lepidoptera order7. The efficiency and duration of the RNAi knockdown in T. domestica is yet to be assessed. In addition to RNAi, we have previously reported a successful CRISPR/Cas9-mediated gene knockout in T. domestica8. The CRISPR/Cas system has been widely applied for genome editing in insects particularly for targeted gene knockout. Its use could be expanded for other applications such as gene reporter assay, cell lineage tracking, and manipulation of transcriptional activity by knocking-in exogenous constructs after the establishment of a protocol for delivering components of the CRISPR/Cas system into nuclei9. Combined with the published genome assembly10, the wide use and further development of the CRISPR/Cas-based genome editing in T. domestica would facilitate studies focusing on the evolutionary mechanisms behind the outstanding adaptive success of insects. Here, we describe a detailed protocol for embryo microinjection and for mating adult T. domestica to generate a mutant strain using CRISPR/Cas9. Considering this novel method, we discuss the importance of considering the unique biology of non-traditional model species for successful applications of these techniques.
1. Maintenance of laboratory colonies
2. Egg collection and microinjection
3. Mating
4. Genotyping
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 the white gene, 32.6% of G0 nymphs display partial loss of pigmentation in their compound eyes and dorsal regions (Figure 5)8.
Using the presently described dry injection method, when 80–120 eggs are injected the survival rate of the injected embryos is as high as 40%–60%. This is in contrast with the previous wet injection method, in which eggs are injected and maintained on an agarose plate, occasionally resulting in a survival rate of less than 10%.
Assessment of the germline transformation of G0 adults and mutated G1 individuals was done by genomic PCR followed by HMA. In HMA, wildtype and mutant alleles anneal in each possible combination, which typically results in four distinct bands on a gel electrophoresis (two homoduplexes and two heteroduplexes)14. In G1 samples, differential band patterns between wildtype and mutated samples are clearly distinguishable (Figure 4B). Germline transformation was found in 39.1% of the G0 adults when we targeted the white gene8. In our experience, the percentage of mutated individuals in G1 nymphs from a single G0 pair varies from 25% to 100%.
To evaluate the effect of our mating environment on the mating success, we crossed wildtype adults in a mating dish and obtained a success rate of 95.8% (23/24 pairs).
Figure 1: The lifecycle of T. domestica. (A) Approximate duration of each developmental stage in T. domestica. (B) Dorsal view of an adult female. Arrowhead indicates a well-developed ovipositor. Please click here to view a larger version of this figure.
Figure 2: Artificial environments used in this protocol. (A) A large container for laboratory colony, (B) a medium-size container for egg collection, (C) mating dishes with water supplies in a large container, (C’) a mating dish, (D) a 24-well plate with water supply in the medium-size container. Boxed region is magnified in (D’) to show a T. domestica individual. Please click here to view a larger version of this figure.
Figure 3: Dry injection of T. domestica eggs. (A) Eggs aligned on a glass slide. Black arrow indicates the point where a needle is inserted. (B) Shape of a glass needle tip used for injection. (C) The same glass needle before (top) and after (bottom) breaking the tip. Needles were filled with 1% neutral red. Arrowhead indicates the tip of the needle. Scale bar is 1 mm. (D) Uninjected egg. (E) A good example of an injection. Arrow indicates the point of injection. (F–H) The solution is overflowing from the injected site (F) or from the opposite side (G) of the injected egg; too much volume of injection caused a burst (H). Arrowhead indicates overflowed egg content. (I) Normally developed late embryo. Arrowhead indicates the colored compound eye. (J) Shrunken damaged egg 3 days after injection. Please click here to view a larger version of this figure.
Figure 4: Genotyping of G1 individuals. (A) PCR primers are designed to amplify a 120 bp genomic region that includes the gRNA target site. (B) Multiple bands of homoduplex and heteroduplex DNAs are detected in mutated samples while single bands appear in unmutated samples. L: DNA ladder, U: unmutated samples, M: mutated samples. (C) Representative result of direct sequencing of PCR products from wildtype and heterozygous mutant samples. Sequences of wildtype and mutant () allele are shown on top. The forward primer shown in (A) was used as a sequencing primer. The sequence of a heterozygous mutant (bottom) is indicated by two overlapping sequences from the predicted cleavage site (arrow). Please click here to view a larger version of this figure.
Figure 5: Mosaic loss of pigmentations in compound eyes and in the dorsal region after targeting the white gene with gRNA/Cas9 protein injection. (A) Wildtype and (B) white gRNA/Cas9 protein-injected first instar nymphs. Partial loss of black and pink pigmentations in eyes (arrowhead) and in the dorsal region (arrow) are indicated. Scale bar is 200 µm. Please click here to view a larger version of this figure.
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. domestica adult transfers its sperm indirectly to a female via a spermatophore. Sweetman (1938) reported that it takes male adults 20 to 35 min from the initiation of the mating behavior to the placement of a spermatophore12. It is likely that disturbance of the interaction between a mating pair by other individuals prevents successful fertilization, which could happen more frequently in a dense environment.
Although eggs are collected 8 h after replacing the cotton inside a container to collect enough eggs in our protocol, the higher efficiency of genome editing may be achieved by collecting eggs and injecting them within a shorter time (e.g., 4 h after egg laying) when injected materials have more chance to be delivered to the large proportion of nuclei. Injection to a sufficient number of eggs within a shorter time could be done by (1) increasing the number or the size of containers if space allows, or (2) repeating the same procedure to obtain a sufficient number of injected eggs.
The site of injection is generally considered to be important for successful germline transformation in insects. T. domestica eggs are usually ellipsoidal in shape and contain a germ band at one pole of their longitudinal axis. It is recommended to inject the gRNA/Cas9 mixture at the midpoint of the egg longitudinal axis because it is hard to identify the pole where a germ band is formed in early embryos due to the variable shape of T. domestica egg. Although the gRNA/Cas9 solution is not directly injected to the site of the germ band formation, we have achieved germline transformation in as high as 39.1% of G0 adults8.
During the microinjection of eggs, it is important to use a fine needle tip to obtain a high survival rate, as it is the case for other animal models. We obtained a higher survival rate after injection with ready-made needles than when using homemade glass needles in T. domestica eggs. However, a similarly high survival rate could be achieved by replicating the fine needle shape with homemade needles (as shown in Figure 3B,C). According to our experience, the dry injection method results in a higher survival rate than the previously reported wet injection method8. We found that egg incubation in a dry environment is key for this improvement, taking advantage of the fact that T. domestica eggs and early nymphs are resistant to desiccation. This is in accordance with previous reports suggesting that this species prefers a dry environment particularly during early developmental stages and that a wet environment can even be harmful16. Instead of a plastic plate, an agarose gel can be used for a quick alignment of the eggs; however, transferring the injected eggs to a dry surface leads to a higher survival rate.
In conclusion, our method underlines the importance of taking the unique biology of T. domestica into account for a successful genome editing: keeping a sparse environment for collecting a sufficient number of eggs and a dry environment for having a higher survival rate of injected embryos. The basic protocols for injection, mating, and culture maintenance reported here are used not only for generating mutant strains with genome editing, but also for applications of other genetic tools such as RNAi and transgenesis and will help to understand the mechanisms underlying the early evolution of insects.
The authors have nothing to disclose.
TO and TD were supported by JSPS KAKENHI grant numbers 19H02970 and 20H02999, respectively.
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 |