Background

The silkworm Bombyx mori is an important industrial insect widely used for silk production and as a platform for producing foreign proteins and pharmaceuticals [1]. It has also served as a key model organism for lepidopteran insects, contributing to fundamental research in genetics, physiology, biochemistry, developmental biology, and pathology [2–4]. In recent years, the complete sequencing and annotation of the silkworm genome [5,6], along with advances in genetic engineering [7], have further established B. mori as a valuable model for functional genomics research. Germline transformation and genome editing in silkworms require the injection of vector solutions into early embryos [8,9]. However, the thick eggshell of silkworms presents a significant challenge for microinjection [10].

A previous microinjection protocol for silkworm eggs was reported by Tamura et al. [10]. Their protocol consists of three steps: 1) removing the eggs from the egg-laying sheet and arranging them on a glass slide with their ventral sides aligned for injection; 2) pre-piercing the eggshell using a tungsten needle mounted on a micromanipulator; and 3) the glass capillary mounted on the micromanipulator is precisely positioned to be inserted through the hole made in the eggshell and used to inject the solution. Although this method is effective, it requires a specialized manipulator and involves time-consuming procedures such as aligning the eggs and adjusting the micromanipulator. Additionally, it demands a high level of technical skill, making the overall workload substantial.

In contrast, our protocol is simpler and more efficient by eliminating all three steps. Eggs remain on the egg-laying sheet as laid, and injection is performed directly without removal or alignment. A thick-walled glass capillary, capable of penetrating the thick eggshell, is held by hand to directly pierce the eggshell at the desired position and inject the solution. By eliminating the need to align eggs and operate a micromanipulator, our protocol significantly improves work efficiency, enabling injections into a larger number of eggs in less time. In particular, injection into early embryos must be performed within a limited time window during the syncytial blastoderm stage, before the cellular blastoderm forms. Therefore, the efficiency of this method is crucial. Moreover, since it does not require the use of a micromanipulator, the technical burden is reduced, making the technique more accessible to a broader range of researchers.

The microinjection method described in this protocol is not limited to silkworm eggs but can be broadly applied to other species with thick eggshells, such as the gypsy moth Lymantria dispar, the tea tussock moth Orgyia thyellina, the brown-winged green stink bug Plautia stali, and the bell cricket Meloimorpha japonica. By mitigating the technical challenges associated with microinjection, this approach has the potential to facilitate research in a wide range of fields, including functional genomics.