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.
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.
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 plants3,4. The damage is caused by both the adults and the larval stages: mated females pierce the surface of the fruit for oviposition, allowing microorganisms to affect their commercial quality, whereas the larvae feed on the fruit pulp. After three larval stages, larvae emerge from the host and pupate into the soil. Ceratitis capitata displays an almost worldwide distribution, including Africa, the Middle-East, Western Australia, Central and South America, Europe, and areas of the United States5.
The most common strategies to limit medfly infestations involve the use of insecticides (e.g., Malathion, Spinosad) and the environmentally-friendly Sterile Insect Technique (SIT)6. The latter approach involves the release into the wild of hundreds of thousands of males rendered sterile by exposure to ionizing irradiation. The mating of such sterilized males to wild females results in no progeny, causing a reduction in population size, eventually leading to eradication. Although SIT has proven effective in multiple campaigns worldwide, its major drawbacks include the high costs of rearing and sterilizing millions of insects to be released. Marking of released individuals is necessary to distinguish sterile from wild insects captured in the field during monitoring activities and it is currently achieved using fluorescent powders. These procedures are costly and have undesirable side-effects7.
In order to optimize and/or to develop more effective approaches for the control of this pest, medfly biology and genetics have been widely explored by numerous researchers worldwide. The availability of the medfly genome sequence8,9, will facilitate novel investigations on gene functions. RNA interference is a powerful tool for such studies and it can be achieved through the microinjection of dsRNA (double-stranded RNA) or siRNA (small interfering RNA). This technique has been used, for example, to demonstrate that the sex determination molecular cascade in C. capitata is only partially conserved with respect to that of Drosophila10.
The development of protocols to microinject medfly embryos allowed C. capitata to be the first non-Drosophilid fly species to be genetically modified. As its eggs are similar to those of Drosophila, both in terms of morphology and resistance to desiccation11, the protocol to deliver plasmid DNA into pre-blastoderm embryos first developed for D. melanogaster12,13 was initially adapted for use in C. capitata. These first experiments allowed medfly germ-line transformation based on the transposable element Minos11. Subsequently, the original system was modified14 using other transposon-based approaches. This is the case of piggyBac from the Lepidoptera Trichoplusia ni15. The protocol has since been further optimized and this has permitted the transformation of other tephritid species16-21 and also of many other Diptera22-31. All these systems rely on the use of a typical binary vector/helper plasmid transformation system: artificial, defective transposons containing desired genes are assembled into plasmid DNA and integrated into the genome of the insect by supplying the transposase enzyme32. A number of transgenic medfly lines have been generated, with multiple features including strains carrying a conditional dominant lethal gene that induces lethality, strains producing male-only progeny and thus not requiring additional sexing strategies, and strains with fluorescent sperm, which may enhance the accuracy of the SIT monitoring phase33-37. Although the release in the wild of transgenic organisms has occurred in pilot tests against mosquitoes only38,39, at least one company is evaluating a number of transgenic medfly strains for their use in the field40.
Embryo microinjection can also favor the development of new genome-editing tools, such as transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated protein 9 nuclease (Cas9) and homing endonucleases genes (HEGs), which will enable novel evolutionary and developmental studies, as well as expanding the available biotechnological toolbox. Genome-editing approaches already allowed the generation of gene-drive systems in mosquitoes41, and their transfer to the medfly is imminent. Here we describe a universal protocol for microinjecting nucleic acids in medfly embryos that can be useful for all the above mentioned applications.
1. Experimental Set-up
2. Embryo Preparation
3. Embryo Microinjection
4. Post-injection Procedures
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 female gonads43,47,48. Based on the information available for closely related species, the dsRNA-induced gene silencing was expected to result in the ablation of the medfly female and male germ-line. A total number of 2,400 embryos were injected with a 2 µg/µl dsRNA mixture, from which 548 larvae hatched and 216 adults survived (unpublished data). In about 75% of the adults, testes and ovaries appeared to be either under-developed or totally absent (Figure 2). Quantification of innexin-5 transcript abundance in the abdomens of individuals from both sexes confirmed the significantly lower expression of the gene, as compared to the controls.
Generation of transgenic strains with fluorescent spermatozoa.
Medfly transgenic strains with fluorescently labeled sperm were generated by Scolari and colleagues34 by injecting embryos with plasmid DNA. The mixture contained two plasmids mixed in fixed relative percentages: one, the "Helper plasmid", encoded the piggyBac transposase; the other, the "Donor plasmid", contained the artificial transposon and carried two fluorescent markers, one expressed in the soma of males and females, the other specifically in the testes. The promoter of the beta 2-tubulin gene, responsible for the testes-specific expression, was fused at the ATG with the coding sequences of the fluorescent proteins turboGFP (construct #1260) or DsRedExpress (construct #1261), respectively. A total number of 821 embryos were injected, from which 205 larvae hatched and 37 adults survived. 9 female and 8 male crossings were set-up, 6 of which gave fluorescent progeny34 (License to reuse these data obtained from Elsevier – License Number 3796240759880). The successful transformation led to the development of strains with green and red fluorescent testes, respectively (Figure 3).
Figure 1. Insectary equipment and embryos. (A) Standard rearing cage containing 1,500-2,000 adults. The females lay eggs through the brass mesh at the front of the cage. Eggs are collected in water. On the left side of the cage, the strainer used to filter the eggs is visible. (B) Two boxes of standard larval food containing larvae. The boxes are placed in a bigger clear plastic box containing bran to facilitate pupation; the net covering the box has been removed. (C) Embryos arranged in rows. The edges of the double-coated tape on the slide have been marked with a white china marker. Eggs aligned on the tape will be covered with chlorotrifluoroethylene oil. (D) Microinjection apparatus (left) connected to a stereomicroscope equipped with a micromanipulator (right). (E) Embryo poles; the red arrow indicates the posterior pole (the injection site), whereas the black arrow indicates the anterior pole (where the micropyle is located). Scale bar = 500 µm. (F) Hatched larva moving in the oil. Scale bar = 500 µm. Please click here to view a larger version of this figure.
Figure 2. Male and female with under-developed gonads. Female (left) and male (right) dissected reproductive tracts. Above: wild-type individuals with normal gonads. Below: interfered individuals with under-developed gonads. MAGs: Male Accessory Glands; Sp: spermathecae. Scale bar = 500 µm. Please click here to view a larger version of this figure.
Figure 3. Transgenic males with fluorescent testes and sperm. Adult males transformed with construct #1260 and #1261, respectively. Arrows indicate the fluorescent testes. #1260 males show red body and green testes, whereas #1261 males show red testes and green bodies. Scale bar = 2 mm. Please click here to view a larger version of this figure.
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 facilitates these studies.
Injection of plasmid DNA can be used to modify insect genomes using transposase-mediated gene insertion, and, more recently, genome-editing tools (e.g., TALEN, CRISPR/Cas9, HEGs). These techniques have already allowed the development of strains of multiple insects species that might be used for pest control programs, aiming both at the eradication or at the replacement of wild population with insects with modified biological features. In this protocol, we describe an optimized method for medfly embryo microinjection with either plasmid DNA or dsRNA.
The availability of well-established and cost-effective semi-dry larval medium for medfly rearing, as well as the extensive molecular information achieved over the years by researchers studying the sex determination and cellularization process in this species, greatly facilitate the establishment of a reliable embryo microinjection protocol. In particular, the rearing protocol has been optimized to ensure the maximum fertility and vitality of the embryos. This is important to maximize the probability of obtaining viable adults with the least number of injected embryos possible. Different protocols are available for medfly rearing, such as the carrot-based larval food that we describe to rear the injected larvae. However, this method is more expensive and its preparation is more time-consuming than other media used for routine rearing.
An important barrier to the use of microinjection is the non-specific damage caused by the mechanical manipulation of the embryos. This includes multiple variables influencing survival of embryos, such as the piercing of the embryo membranes with the paintbrush used to orientate the embryos, the injected volumes, the injection site and buffer, and the type of needle used50. Some of these parameters have been optimized, such as the injected volumes and the buffer. In the case of needles, they can also be produced in-house using a puller, and this requires an optimization step to determine the ideal protocol.
Among the major drawbacks in the microinjection procedure is also dechorionation: although this step is essential to make the eggs easier to handler (i.e., less slippery) and to facilitate injection, prolonged exposure to bleach can heavily affect embryonic vitality. For this reason, the protocol we describe here reports the use of a very short dechorionation time (5 sec), which has been established as a good compromise between removal of the chorion and survival rate.
The protocols used to rear the subsequent life stage are also relevant. Larvae hatched from injected embryos have to be removed from the oil as soon as possible. Oil is indeed essential to prevent desiccation of the embryos, but can be noxious to the larvae. The method used to remove larvae, the time spent into the oil, and the type of food used are all important variables that need to be considered since they can compromise the final survival rate.
When screening adult and larvae, the immobilization methods can be also critical. Medfly adults can be immobilized using either ice or CO2, but prolonged exposure might be deleterious for adult survival. As an alternative to embryo microinjection, oral delivery has proven to be a less-invasive and potentially high-throughput method to perform RNAi assays. It can be particularly effective in species that are not amenable to microinjection, as well as for RNAi-mediated pest control in field populations.
In this paper, two cases have been reported as examples of the possible applications of the protocol described: first, the successful transformation through transposase-mediated gene insertion of the medfly genome, which led to the establishment of multiple strains with fluorescent testes. Using a similar microinjection procedure, it is also possible to inject dsRNA, with the effects of the knockdown being visible till the adult stage.
The establishment of a reliable microinjection protocol for the medfly embryos opened the way to consider genetically-modified strains as a tool to control flies in the wild, as alternative or complementary strategies to classical approaches, including the Sterile Insect Technique. Finally, recent advances in the technology for automated microinjection in zebra fish embryos might potentially help the development of high throughput delivery systems with important relevance for creating transgenic strains of the medfly and other relevant pests51.
The authors have nothing to disclose.
The authors would like to thank all the members of the “Insect Genetics and Genomics” Laboratory, in particular to Lorenzo Ghiringhelli who has worked at developing, adapting and maintaining the rearing of the medfly over the past thirty years. Part of the representative results of this paper have been reprinted from N. Biotechnology, 25(1) by Scolari F. et al., Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae), 76-84, 2008, with permission from Elsevier (License number 3796240759880). This work received support from Cariplo-Regione Lombardia “IMPROVE” (FS).
1 x injection Buffer | Buffer | 0.1 mM phosphate buffer pH 7.4, 5mM KCl | ||
Construct Plasmid | DNA | |||
Helper Plasmid | DNA | |||
dsRNA | RNA | Phenol-Chloroform purified | ||
Standard Larval food | Rearing 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 | Rearing 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 1L | ||
Adult Food | Rearing 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 | ||||
Bleach | Generic reagent | Diluite 1:2 before use | ||
Paintbrush (000) | Generic tool | |||
Micromanipulator | Instrument | Narishige | MN-153 | |
Microinjector | Instrument | Eppendorf | Femtojet | |
Adult cages | Generic tool | |||
Halocarbon oil 700 | Reagent | Sigma-Aldrich | H8898 | |
Ceratitis capitata | Animal | The strain used is ISPRA |