An Optimized Small-Scale Rearing System to Support Embryonic Microinjection Protocols for Western Corn Rootworm, Diabrotica virgifera virgifera

Abstract

Western corn rootworm (WCR), a major pest of corn, has been reared in laboratories since the 1960s. While established rearing methods are appropriate for maintaining WCR colonies, they are not optimal for performing germline transformation or CRISPR/Cas9-based genome editing. Here we report the development of an optimized rearing system for use in WCR functional genomics research, specifically the development of a system that facilitates the collection of preblastoderm embryos for microinjection as well as gathering large larvae and pupae for downstream phenotypic screening. Further, transgenic-based experiments require stable and well-defined survival rates and the ability to manipulate insects at every life stage. In our system, the WCR life cycle (egg to adult) takes approximately 42 days, with most individuals eclosing between 41 and 45 days post oviposition. Over the course of one year, our overall survival rate was 67%. We used this data to establish a quality control system for more accurately monitoring colony health. Herein, we also offer detailed descriptions for setting up single-pair crosses and conducting phenotypic screens to identify transgenic progeny. This study provides a model for the development of new rearing systems and the establishment of highly controlled processes for specialized purposes.

Keywords: CRISPR; function genomics; insect rearing; molecular biology; rootworm.

Figure 1

Egg rearing containers. Eggs are placed in the middle of a 30 mL cup and covered with soil.

Figure 2

Primary rearing containers. (A) Each 475 mL container possesses soil and newly sprouted corn; lids have holes to allow air exchange. (B) Minimum level of root growth required for larvae to feed.

Figure 3

Secondary rearing containers. (A) The half box set up: the empty half of a 475 mL container will be used to hold the contents of a primary rearing container following transfer. The intact lid should be used to cover the growing corn prior to adding insects. (B) After adding the contents from a primary rearing container, use a lid with a fine screen for air exchange.

Figure 4

Tertiary or pupal rearing containers (adult collection containers). Adult collection containers can be either tertiary rearing containers (with plants) or pupal rearing containers (without plants). Most adults exit the soil and can be seen through the lid (arrows point to newly eclosed adults). Once pupae start eclosing into adults, all plants can be cut back and removed to increase visibility, thereby aiding the collection of adults.

Figure 5

Containers required for a single WCR colony. The minimum space and assortment of containers required per colony using this rearing system. Upper level, left to right: two egg cups, two primary rearing containers, two secondary rearing containers, and one post-screening container. Lower level, left to right: adult cage and two adult collection containers.

Figure 6

WCR development time. Each bar indicates how many insects had eclosed by each of the given total days of development time (days). The average is 43 days.

Figure 7

Quality control assessment. This quality control system is based on the survival rates (black dots) for WCR (see Table S1). Survival rates between the upper red line (UCL) and lower red line (LCL) are within quality control standards, but those outside would be considered to be out of control. UCL (Upper control limit) = x + 3σ (x = mean, σ = standard deviation). LCL (Lower control limit) = x − 3σ.