Egg Microinjection for the Silkworm Bombyx mori

Materials and reagents

Biological materials

1. Bombyx mori eggs, larvae, pupae, and adults can be purchased from the National BioResource Project (NBRP) at Kyushu University; eggs are also available from the National Agriculture and Food Research Organization (NARO). When rearing from eggs or larvae, mulberry leaves (available from NBRP at Kyushu University) or artificial diet (available from Nosan Corporation) can be used as feed

Note: Since some silkworm strains are not suitable for rearing on artificial diets, it is recommended to check the feeding ability of artificial diets using the comprehensive silkworm resource database, Silkworm Base (https://shigen.nig.ac.jp/silkwormbase/about_kaiko.jsp).

Reagents

1. Fast Green FCF (Sigma-Aldrich, catalog number: F7252)

Note: This dye was used in Video 1 as a visual marker at a final concentration of 0.1% to demonstrate the injection process.

Laboratory supplies

1. Micro-loading tip (Eppendorf, catalog number: 5242 956 003)

2. Glass capillary (Daiwa Union, catalog number: uMPm-02)

Note: This glass capillary is a commercially available product suitable for silkworm egg microinjection. It does not require shaping with a puller. The capillary has an outer diameter of 1.5 mm, an inner diameter of 0.6 mm, and a tip outer diameter of approximately 35 μm, which we define as a “fine capillary tip” in this protocol.

3. Egg-laying sheet (Ehime-Sanshu, catalog number: 125)

4. Clear plastic cup (used for mating and oviposition) (Ai’ris, catalog number: 414-9650-727)

5. Insect rearing case (Sanplatec, catalog number: 02322)

6. Petri dish (Ina Optica, catalog number: I-90)

7. Paper towel (placed at the bottom of the rearing case)

8. Humid chamber

Equipment

1. FemtoJet 4i (Eppendorf, catalog number: 2229001215)

2. Microcentrifuge (TOMY, model: MX-307 or equivalent)

3. Foot pedal switch (Eppendorf, included with the FemtoJet 4i)

4. Capillary holder (Narishige, model: HI-7)

5. Grip head for capillary holder (Narishige, model: HIC-1.5)

Note: Although the FemtoJet 4i includes an Eppendorf capillary holder and grip head, we used the Narishige HI-7 capillary holder in this protocol. This holder, when combined with the HIC-1.5 grip head, provides a smoother and more stable grip due to the absence of a joint gap. While the combination of the Eppendorf holder and grip head is also compatible with the same glass capillaries, we found the Narishige setup easier to handle during handheld injection.

6. Pipetman [GILSON, catalog number: F123600 (P20)]

7. Stereomicroscope (Olympus, model: SZX16 or equivalent)

Procedure

A. Egg collection

1. Determine the sex of Bombyx mori pupae and place ten males and ten females in separate insect rearing cases. Allow them to emerge under controlled conditions of 25 °C and a 12/12 h light/dark photoperiod.

Note: The sex of pupae can be visually determined based on body size and morphological differences in the posterior ventral region (Figure S1). Female pupae are generally larger than males due to egg development. Sex differentiation is possible from the onset of pupation until just before adult emergence.

2. In the evening of the day of eclosion, pair one adult male and one adult female in a clear plastic cup and allow them to mate overnight under light conditions (Figure 1A).

Figure 1. Management of silkworm oviposition. (A) Mating of adult silkworms. (B) Mated female silkworms on egg-laying sheets. (C) Oviposition by mated female silkworms.

3. The following morning, separate the mating pairs and cover each female with a clear plastic cup placed on an egg-laying sheet (Figure 1B).

4. Move the above oviposition setup to a dark environment to initiate egg-laying. Define this time point as 0 h post-oviposition initiation.

Note: A dark environment facilitates oviposition.

5. Check the oviposition status every 30 min. If a female is laying eggs, collect the egg-laying sheet (Figure 1C) for Section B.

6. For females that are still ovipositing or have not yet started laying eggs, repeat step A5 at 30-min intervals to collect eggs suitable for microinjection.

Notes:

1. Eggs are naturally attached to the egg-laying sheet due to an adhesive substance secreted by the female during oviposition.

2. The number and timing of egg-laying vary among individuals.

3. Collecting eggs at 30-min intervals ensures the acquisition of sparsely laid eggs suitable for microinjection.

B. Preparation of eggs

1. Cut out the egg-laying sheet to facilitate handling during injection (Figure 2A).

Note: Select sparsely laid eggs that are easier to inject. Do not use densely clustered eggs (e.g., lower left in Figure 2A).

Figure 2. Microinjection setup for Bombyx mori eggs. (A) Selection and cutting of the egg-laying sheet suitable for injection. (B) Thick-walled glass capillary used for injection. The boxed region is magnified to show the capillary tip. Scale bars: upper, 1 mm; lower, 200 μm. (C) B. mori egg. A: anterior; D: dorsal; P: posterior; V: ventral. An arrowhead at the anterior indicates the micropyle. Scale bar, 500 μm. (D) Overview of the microinjection system. (E) Injection process, showing manual handling of the capillary under a stereomicroscope. (F) Injected eggs are kept on Petri dishes in a humid chamber. Moist paper towels are on the bottom.

2. Immerse the cut egg-laying sheet in 70% ethanol for 2 min for egg surface sterilization, followed by air-drying on a clean bench for 5 min.

3. Keep the dried eggs in a sterilized insect rearing case treated with 70% ethanol to maintain aseptic conditions.

C. Preparation of the injection solution and loading it into the injection capillary

Note: This protocol focuses on the injection procedure itself and does not specify a particular injection solution. In Bombyx mori, various substances such as siRNA [2], dsRNA [11,12], mRNA [8], plasmid DNA [13], and proteins [14] have been successfully used for injection by our group and others, depending on the experimental objectives.

1. Centrifuge an injection solution at maximum speed at 4 °C for 3 min to remove debris and prevent capillary clogging.

2. Using a micro-loading tip and a Pipetman, carefully load 10 μL of the injection solution into the back of the injection capillary.

Notes:

1. A pre-shaped, commercially available capillary is used in this step; detailed specifications are described in the Materials and Reagents section.

2. For approximately 100 eggs, 10 μL of injection solution is sufficient.

3. Ensure that the capillary does not contain air bubbles. If present, remove air bubbles by gently flicking the injection capillary, taking care not to break its fine tip (Figure 2B). If the capillary tip is not fine enough, it may create a large hole in the egg during injection, potentially disrupting embryonic development.

3. Attach the capillary to the capillary holder (Figure 2D).

D. Egg microinjection

1. Set FemtoJet 4i to manual mode and adjust the injection parameters as follows: injection pressure (Pi) of 150 hPa and a constant pressure (Pc) of 50 hPa.

2. Place the cut and sterilized egg-laying sheet on the microscope stage and adjust the microscope focus to the surface of the egg.

3. Hold the capillary holder by hand and position the tip of the glass capillary at the center of the microscope field of view. Confirm that both the capillary tip and the egg surface are in focus.

4. Rotate and move the egg-laying sheet with one hand while holding the capillary holder with the other. Bring the capillary tip close to the position of the egg to be injected (Figure 2C, E; Video 1).

Note: For germline transformation, injection should be performed into the posterior ventral region, where germ cells are formed, to enhance transgenesis and genome editing efficiency. The posterior ventral region can be identified under a microscope based on the external features of the egg. The ventral side tends to be slightly more rounded, while the dorsal side appears relatively flat (Figure 2C). In addition, the anterior end of the egg can be recognized by the presence of a micropyle (indicated by an arrowhead in Figure 2C). By using these morphological criteria, the posterior ventral region can be consistently identified for injection.

Critical: Microinjection should be performed within 5 h post-oviposition, during the syncytial blastoderm stage, before cellular blastoderm formation. Once the embryo reaches the cellular blastoderm stage, where cell membranes have formed, macromolecules such as vectors can no longer penetrate. Additionally, the eggshell remains relatively soft immediately after oviposition, making it suitable for penetration with a glass capillary.

5. Insert the glass capillary just slightly into the egg by manually manipulating the capillary holder (Figure 2E; Video 1).

6. Press the foot pedal switch of the FemtoJet to inject the solution.

Note: Since this system does not allow precise control of the injection volume, it is crucial to accurately determine the appropriate amount to inject. A larger injection volume is generally preferable; however, the optimal volume is defined as the amount that does not leak out of the egg when the capillary is withdrawn after injection. To provide a more visual understanding of injection techniques and the appropriate injection volume, we recorded a video demonstrating the injection procedure using a dye solution (Video 1). While a slight variation in injection volume may occur depending on the operator’s technique, the use of visual confirmation and the injection pressure settings (Pi: 150 hPa; Pc: 50 hPa), as described in step D1, ensures consistent and reproducible injection performance.

7. Withdraw the capillary from the egg.

Note: When the capillary is withdrawn from the eggshell, some injection solution inside the capillary may be released outside the eggshell. However, this does not affect the injection process (Video 1).

8. Continue injecting as many eggs as possible using the same glass capillary. With proper injection technique, a single glass capillary can typically be used to inject 100 to 150 eggs.

Note: If the capillary clogs, remove it from the egg and immerse it in a drop of sterile water while pressing the foot pedal switch several times to clear debris. Alternatively, use the CLEAR function of the FemtoJet. If the debris cannot be cleared, replace the glass capillary. Additionally, if the tip of the glass capillary is damaged and the opening becomes enlarged, replace the capillary.

E. Post-injection care of eggs

1. Transfer the egg-laying sheet with the injected eggs to a humid chamber inside an incubator at 25 °C until just before hatching (Figure 2F).

Critical: Eggs pierced during injection are prone to dehydration, which may result in embryonic lethality during development. Therefore, immediately after injection, the egg-laying sheet should be transferred to a humid chamber. To maintain sufficient humidity and prevent dehydration, sterile water should be periodically added to the paper towel lining the bottom of the chamber, either daily or as necessary.

2. Just before hatching (approximately 10 days after oviposition), transfer the egg-laying sheet with the attached eggs to an insect rearing case and provide artificial diet or mulberry leaves.

Note: The hatched larvae can be reared in insect rearing cases on an artificial diet or mulberry leaves until the final instar.

Data analysis

This protocol aims to establish an efficient method for egg microinjection in Bombyx mori and does not involve new experimental data analysis. However, several established approaches for evaluating the effectiveness of egg microinjection have been reported and may serve as useful references for future applications.

For example, Masumoto et al. [11] injected dsRNA targeting the Ubx gene and assessed RNAi efficiency based on morphological abnormalities observed in larval segments. Kobayashi et al. [12] confirmed successful embryonic RNAi by detecting a loss of the target protein expression via western blotting. Masumoto et al. [13] injected a GFP expression vector into embryos and demonstrated successful transgenesis by identifying GFP-positive individuals. Additionally, other studies have confirmed the reduction of target mRNA levels by qRT-PCR following siRNA injection [2]. Silkworm genome editing has been validated by sequencing genomic DNA after injection with TALEN [8] or CRISPR/Cas9 [2].

These examples illustrate commonly used strategies for evaluating the success of egg microinjection in B. mori, depending on the experimental goals and molecular targets.

Validation of protocol

This protocol has been used and validated in the following research articles:

• Masumoto et al. [11]. Functional analysis of Ultrabithorax in the silkworm, Bombyx mori, using RNAi. Dev. Genes Evol. (Figure 3)

• Masumoto et al. [13]. A baculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori. PLoS One. (Figure 6)

• Kobayashi et al. [12]. Cloning of cDNA encoding a Bombyx mori homolog of human oxidation resistance 1 (OXR1) protein from diapause eggs, and analyses of its expression and function. J. Insect Physiol. (Figure 6)

Supplementary information

The following supporting information can be downloaded here:

1. Figure S1. Representative male and female pupae of the silkworm Bombyx mori.

Acknowledgments

T.Y., K.M., and T.N. conceived and developed the protocol. T.N. supervised the study. H.Y. and T.N. performed experiments to validate the protocol. H.Y. and T.N. wrote the manuscript with feedback from all authors. All authors reviewed and approved the final version of the manuscript. H.Y. acknowledges support from the Japan Society for the Promotion of Science (JSPS) (Grant No. 22K05451). We also appreciate the support of Dr. Kenichiro Itami (RIKEN), who facilitated the acquisition of research equipment through JSPS (Grant No. 19H05463). This protocol was adapted from Masumoto et al. [11,13] and Kobayashi et al. [12]. We thank the Emerging Model Organisms Facility of NIBB Trans-Scale Biology Center for technical assistance.

Competing interests

The authors declare no conflicts of interest.

References

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