Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

Insect guts are colonized by diverse bacteria that can profoundly impact the host's physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle (Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

Similar to mammals, the insect digestive tract is a cavity for food digestion and absorption. Most insects harbor diverse commensal bacteria that thrive in their guts and live on nutrition supplied by hosts¹. The gut commensal community has a profound impact on multiple physiological processes in insects, including food digestion and detoxification^2,3,4, nutrition and development^5,6,7, defense against pathogens and parasites^8,

1. Insect rearing

1. Maintain the P. versicolora population in a growth chamber at the condition of 27 °C and 70 ± 5% relative humidity with a photoperiod of 16 h light/8 h dark. Place them in perforated plastic boxes with tiled wet absorbing paper and feed them fresh poplar branches. Spray clean water on absorbing paper to maintain moisture and change the branches every two days.
2. Isolate adults for oviposition after pupation. Feed them tender leaves to obtain m.......

The life stages of P. versicolora are shown in Figure 1. The adult male is smaller than the adult female (Figure 1A). In the field, the beetle clusters its eggs on a leaf; here, four eggs were detached from a leaf (Figure 1B). The poplar stem segments and seedlings used for axenic insect rearing are shown in Figure 2. The gut of a 3^rd instar larva is shown in Fig.......

Preparation of germ-free larvae and obtaining gnotobiotic larvae by reintroducing specific bacterial strains are powerful methods to elucidate the mechanisms underlying host-microbe interactions. Newly hatched larvae obtain gut microbiota in two main ways: vertical transmission from the mother to the offspring or horizontal acquisition from siblings and the environment³⁴. The former can be fulfilled by parental transfer to the offspring through contamination of the egg surface^35<.......

This work was funded by the National Natural Science Foundation of China (31971663) and the Young Elite Scientists Sponsorship Program by CAST (2020QNRC001).