Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

Summary

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

Abstract

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.

In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Introduction

The Mediterranean fruit fly (medfly) Ceratitis capitata is a cosmopolitan species that extensively damages fruits and cultivated crops. It belongs to the Tephritidae family, which includes several pest species, such as those belonging to the genera Bactrocera and Anastrepha. The medfly is the most studied species of this family, and it has become a model not only for the study of insect invasions¹, but also for optimizing pest management strategies².

The medfly is a multivoltine species that can attack more than 300 species of wild and cultivated plants^3,4. The damage is caused by ....

Protocol

1. Experimental Set-up

1. Insectary requirements
   1. Maintain all C. capitata life stages at 25 °C, 65% humidity and 12/12 hr light/dark photoperiod.
   2. Place about 1,500-2,000 medfly pupae in a 6 L cage. Use a cage with a brass mesh on one side with holes small enough to stimulate oviposition⁴². Insert a sponge strip through a small opening in the cage's base to provide flies with water by means of capillary action. Use a mixture of yeast and sugar (1:10) as food source for the adults (Figure 1A). Increase the amount of yeast (up to 1:3, yeast:sugar) should the females fail to lay....

Results

Here we report two applications of embryo microinjection directed at the functional characterization of a gene of interest (Case 1), and at the generation of transgenic strains (Case 2), respectively.

Delivery of dsRNA into embryos to unravel gene function.

The innexin-5 gene encodes a gap-junction that, in insects, is expressed specifically in the male and fe.......

Discussion

Microinjection of nucleic acids in insect embryos is a universal technique that facilitates both the analysis of gene function and biotechnological applications.

The recent publication of the genome sequences from an increasing number of insect species leads to an urgent need for tools for the functional characterization of genes of yet unknown function. RNA-interference has proven to be one of the most valuable methods to infer molecular functions⁴⁹ and embryo microinjection facili.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 x injection Buffer			0.1 mM phosphate buffer pH 7.4, 5 mM KCl
Construct Plasmid
Helper Plasmid
dsRNA			Phenol-Chloroform purified
Standard Larval food			1.5 L H2O, 100 ml HCl 1%, 5 g broad-spectrum antimicrobial agent used in pharmaceutical products dissolved in 50 ml of ethanol, 400 g sugar, 175 g demineralized brewer's yeast, 1 kg soft wheat bran
Carrot Larval Food			2.5 g agar, 4 g sodium benzoate, 4.5 ml 37% HCl, 42 g yeast extract, 115 g carrot powder, 2.86 g broad-spectrum antimicrobial agent , water to 1 L
Adult Food			yeast extract and sugar (1:10)
Microscope slides	Sigma-Aldrich	Z692247
Injection needles	Eppendorf	5242956000
Microloaders	Eppendorf	5242956003
Double slided tape
Whatman Black circle paper	Sigma-Aldrich	Z695505
Bleach			Diluite 1:2 before use
Paintbrush (000)
Micromanipulator	Narishige	MN-153
Microinjector	Eppendorf		Femtojet
Adult cages
Chlorotrifluoroethylene oil	Sigma-Aldrich	H8898
Ceratitis capitata			The strain used is ISPRA