Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Summary

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Abstract

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Introduction

In 1998, Fire and Mello reported that double-stranded RNA (dsRNA) can induce inhibition of gene function in Caenorhabditis elegans¹. This response triggered by dsRNA was named RNA interference (RNAi), and such RNAi-mediated gene silencing was reported to be conserved in animals, plants, and fungi^2-7. In plants and some animals, RNAi functions systemically, meaning that the effect can spread to other cells/tissues where dsRNA is not directly introduced (reviewed in^8-10). Scientists have made use of this endogenous cellular RNAi response by designing dsRNAs to target genes of interest, thereby knocking down gene function without....

Protocol

1. Rearing of D. maculatus

NOTE: A breeding colony of D. maculatus was set up in the authors' lab using adults and larvae purchased commercially. The species identity was verified using DNA barcoding⁵³.

1. To set up a new cage in the lab, spread a thin layer of wood shavings into a medium-size insect cage (30.5 x 19 x 20.3 cm³). Place a ~ 10 x 6 x 3 cm³ chunk of Styrofoam in the cage to let larvae hide for pupation. Add 20 - 50 beetles (either adults or late instar larvae). Beetles will hide in the wood shavings.
2. Add wet cat food in a Petri dish or a weighing boat. Cover ....

Results

The authors' lab has used RNAi technology to study the functional evolution of genes regulating segmentation in insects^53,55. While all insects are segmented, the genes regulating this process appear to have diverged during insect radiations^26,38,56-63. Genetic screens in Drosophila identified a set of nine pair-rule segmentation genes that are responsible for promoting the formation of body segments^64-70. Here, the ortholog of one of these genes,.......

Discussion

While a small number of sophisticated model systems (mice, flies, worms) were developed during the 20^th century, the 21^st century has seen a wave of new animal systems being developed in labs throughout the world. These new systems allow scientists to address comparative, evolutionary questions that cannot be probed using only the 'standard' model systems. This deployment of new models requires the rapid development of methods for lab culturing, gene identification, and functional approaches.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dermestes maculatus live beetles	Our lab or Carolina Biological Supply	#144168	Our lab strain was verified by COI barcoding; strain variation from Carolina cannot be ruled out
Wet cat food	Fancy Feast		Chunks of meat with gravy. Can buy at most pet food and grocery stores
Dry dog food	Purina Puppy Chow		Can buy at most pet food and grocery stores
Insect cage (size medium, 30.5 x 19 x 20.3 cm)	Exo Terra	PT2260	For colony maintenance. Can use larger cage if needed
Insect cage (size mini, 17.8 x 10.2 x 12.7 cm)	Exo Terra	PT2250	For embryo collection
Petri dish	VWR	89038-968
Cotton ball	Fisher	22-456-883
Megascript T7 transcription kit	Fisher	AM1334	For 40 reactions
Pneumatic pump	WPI	PV830
Capillary holder	WPI
Micromanipulator	NARISHIGE	MN-151
Black filter paper (90 mm)	VWR	28342-010
Food coloring (green)	McCormick
Borosilicate glass capillary	Hilgenberg	1406119
Needle puller (micropipette puller)	Sutter Instrument Co.	P-97
Microscope glass slide	WorldWide Life Sciences Division	41351157
Sealing film (Parafilm M)	Fisher	13-374-12
Model 801 Syringe (10 µl)	Hamilton	7642-01
Needle (32-gauge)	Hamilton	7762-05
Fixation Solution (Pampel's)	BioQuip Products, Inc.	1184C	Toxic, needs to be handled in fume hood
Forcep (DUMONT #5)	Fine Science Tools	11252-30
Cover slip (24 x 50 mm, No. 1.5)	Globe Scientific	1415-15
Eppendorf Femtotips Microloader pipette tip	Fisher	E5242956003
Dissecting microscopy for embryo injection	Leica	M420
Dissecting microscopy for larval phenotypic visualization	Zeiss	SteREO Discover. V12
DIC microscopy	Zeiss	AXIO Imager. M1