Background

Ladybird beetles belong to the order Coleoptera and encompass approximately 6,000 species worldwide, with around 190 species being described in Japan [1,2]. These beetles display diverse color patterns on their elytra, primarily black and red, which are not only species-specific but also display a high degree of diversity within species (intraspecific polymorphism) (reviewed in Ando and Niimi [3]). Of ladybird beetles, Harmonia axyridis has become an emerging model organism in diverse biological fields, such as color patterning [4,5], population genetics of intraspecific polymorphism [6,7], biological insecticides [8], and phenotypic plasticity [9]. H. axyridis possesses desirable characteristics as an experimental animal, such as the availability of an artificial diet, long-term storage of adults at low temperatures (up to 2 years and 8 months; personal observation), commercially available adults, high fecundity, easy access to fertilized eggs, and ease of sexing in adults based on morphology and at any stage by PCR-based techniques [10]. The ladybird beetle's lifecycle is relatively short—approximately 3 days for embryogenesis, around 11 days for larval development through three molts, 4.5 days for pupal development, and a lifespan of 2–3 months for adults (Figure 1). Furthermore, modern experimental techniques, including high-quality genome data [11,12], cryopreservation of the gonad [13], and numerous useful transgenic lines and mutant strains [14–16] including flightless mutants [17], and modern functional genomics, including RNAi, transgenics, and genome editing, have been developed for this species [4,14,15,18–20].

Figure 1. Life stages of Harmonia axyridis. The developmental period for each stage under laboratory conditions at 25 °C is indicated at the bottom. The period shown for adults represents the time during which they lay eggs after mating. It takes approximately one week after eclosion for adults to reach sexual maturity. Scale bar: 1 mm.

Fertilized egg microinjection is an essential approach for functional genomics including transgenesis and genome editing, being the most commonly used method in insects. While there are several methods for delivering reagents into eggs—including electroporation [21–23] and particle guns [24–26], which enable simultaneous treatment of multiple eggs—microinjection is the most widely used method for introducing nucleic acids, proteins, and chemicals into insect eggs. This method, however, has problems such as frequent clogging due to the backflow of egg contents, which prevents efficient injection. To overcome this problem, we developed an injection method using constant fluid flow (i.e., constant pressure during injection). This method not only prevents needle clogging but also contributes to increased injection throughput because it allows the delivery of the injection solution without egg-by-egg manual injection. However, this microinjection system is not able to control a precise injection volume, but the injection volume can be controlled by adjusting the time the needle is kept inside the egg. Our injection system is performed under a stereomicroscope with a micromanipulator and an injection needle holder, making the construction of an injection system more affordable than systems with compound microscopes. This paper describes the methodology of microinjection into the multicolored Asian ladybird beetle embryos using constant fluid flow injection with a stereomicroscope.