Name: delivery of nucleic acids through embryo microinjection in the worldwide agricultural pest insect, ceratitis capitata
Uploaded: 2016-10-01T14:00:00
Description: Watch this Scientific Journal Video about Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata at JoVE.com

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. Experimental Set-up
<ol>
	<li>Insectary requirements
	<ol>
		<li>Maintain all C. capitata life stages at 25 &#176;C, 65% humidity and 12/12 hr light/dark photoperiod.</li>
		<li>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&#39;s base to provide flies with water by means of capillary action. Use a mixture of yeast and sugar (...

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...

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&#160;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.

<table border="0" cellpadding="0" cellspacing="0" width="791">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1 x injection Buffer&#160;</td>
			<td style="width:107px;">Buffer</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">0.1 mM phosphate buffer pH 7.4, 5mM KCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Construct Plasmid</td>
			<td style="width:107px;">DNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Helper Plasmid</td>
			<td style="width:107px;">DNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dsRNA</td>
			<td style="width:107px;">RNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Phenol-Chloroform purified</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Standard Larval food</td>
			<td style="width:107px;">Rearing Food&#160;</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">1.5 L H2O, 100 ml HCl 1%, 5 g broad-spectrum antimicrobial agent used in pharmaceutical products&#160; dissolved in 50 ml of ethanol, 400 g sugar, 175 g demineralized brewer&#8217;s yeast, 1 kg soft wheat bran</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Carrot Larval Food</td>
			<td style="width:107px;">Rearing food</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">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</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult Food</td>
			<td style="width:107px;">Rearing food</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td>yeast extract and sugar (1:10)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope slides</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>Z692247</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injection needles&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Eppendorf</td>
			<td align="right">5242956000</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloaders</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Eppendorf</td>
			<td align="right">5242956003</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double slided tape</td>
			<td style="width:107px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Whatman Black circle paper</td>
			<td style="width:107px;"></td>
			<td style="width:93px;"></td>
			<td></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:107px;">Generic reagent</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Diluite 1:2 before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paintbrush (000)</td>
			<td style="width:107px;">Generic tool</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator</td>
			<td style="width:107px;">Instrument</td>
			<td style="width:93px;">Narishige</td>
			<td style="width:119px;">MN-153</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microinjector</td>
			<td style="width:107px;">Instrument</td>
			<td style="width:93px;">Eppendorf</td>
			<td style="width:119px;">Femtojet</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult cages</td>
			<td style="width:107px;">Generic tool</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Halocarbon oil 700</td>
			<td style="width:107px;">Reagent</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8898</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ceratitis capitata</td>
			<td style="width:107px;">Animal</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">The strain used is ISPRA</td>
		</tr>
	</tbody>
</table>

delivery of nucleic acids through embryo microinjection in the worldwide agricultural pest insect, ceratitis capitata

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.

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...

Watch this Scientific Journal Video about Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata at JoVE.com

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

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.

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

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

The overall goal of this procedure is to deliver nucleic acids into the embryos of Ceratitis Capitata, an important agricultural pest, to ultimately expand the toolbox for its control. This method helps functionally correctorize genes in an immobil organism, as well as generate transgenic strains. The main advantage of this technique is that very low amount of nucleic acids are injected to achieve the desired effects.Maintain all the life-stages of the medfly colony at 25 degrees Celsius with 65%humidity and a 12 12-hour light/dark photo-period with a photo-phase starting at 8 a.m. Insert a sponge strip through a small opening in the cage's base to provide the flies with water by means of capillary action. You establish a six liter cage, load one with between 1, 500 and 2, 000 pupae.For food, load the cage with about 45 grams of a one to 10 mixture of yeast and sugar. To collect eggs, place a plate filled with water below the cage. Females will deposit eggs through the mesh, which will then collect in the water.Because of virgin females can ova-posit, ensure that the eggs are all fertilized by waiting until the flies are at least a week old, before collecting eggs. To collect eggs, filter the water with a fine-strainer. Then using a pasteural pipette, transfer the eggs into a plastic box loaded with about 0.5 liters of standard larval food.If not enough eggs were laid, then increase the yeast content of the adult's food up to 25%Place each box in a transparent container, containing a layer of bran to favor pupation. Cover the container with a net lid to allow air circulation. After about 10 days, check for third-instar larvae that have jumped down into the bran layer for pupation.Once found, prepare to check for pupae on the following days. To collect the pupa, use a soft brush to sweep them up from the container. Initially, collect the pupae in a small cup.After the collections are completed, transfer the pupae to a new adult cage. The adults should emerge in about 10 days. For the micro-injection, have slides prepared in advance.Attach a two centimeter strip of double-coated tape to a slide and mark the edges with a china marker to make a wall for an oil layer that keeps the embryos hydrated. To collect freshly laid embryos, allow young fertilized females to ova-posit for 30 minutes on a new tray and then collect the embryos. To remove the chorion from the embryos, dip the strainer and eggs into a commercial 50%bleach solution for five seconds.It is critical to not over bleach the embryos. Immediately wash the bleached embryos by carefully dipping the strainer into clean ultra-pure water. Do this at least four times.Then, keep the strainer in clear ultra-pure water and inject the embryos within two hours. Next, using a fine paintbrush, collect about 50 embryos and place them on a disc of black filter paper soaked with water. Under a dissecting stereo microscope, row the embryos onto the double-coated tape of a prepared microscope slide.Orient all the embryos in the same direction. It is important to manipulate the embryos carefully because the piercing of the embryo membrane with the paint brush may cause damage that reduce the embryo's survival. Now cover the embryos with a layer of chloro-trifloro etholene oil.The oil should be contained by the china marker borders. Don't overflow the borders. Using a micropipette, load the injection needle with a few microliters of nucleic acid solution at one to two micrograms per microliter.Then, connect the needle to the micro-injection apparatus and set it to provide a half second injection at 800 hecto-pascals with a back pressure of 150 hecto-pascals. To inject, use a foot-pedal trigger and use the micro-manipulator to control the needle while using the stage controls to move the slide. Confirm that the injection bolus is being ejected properly and then align the needle with the posterior pole of the first embryo in the row.By moving the stage, insert the needle into the embryo and inject the bolus of nucleic acids. The injected liquid will induce an increase in internal pressure resulting in a slight movement of the embryo. Release the pedal, and immediately but gently, remove the needle from the embryo.A little cytoplasm will leak at the injection site. This is normal. If embryos are bursting with the injection, then let the embryos desiccate for a few minutes before covering them in oil.Following the injection, transfer the slide to a petri dish. Place a disc of filter paper in the lid of the dish. Soak the filter with water and close the petri dish with paraffin film.Incubate the chamber at 25 degrees Celsius. Two days after the injection, check the slides several times throughout the day for hatched larvae. Look for larvae in the oil and using a fine paintbrush, transfer them to a petri dish with carrot-based larval food.Load each dish with up to 200 larvae. The larva cannot survive in the oil long, so search often. Incubate the larvae with the lid mostly closed, so there is still air-flow to the larvae.Check the dishes daily and after a week prepare to collect larva by placing the dishes in a container with bran. Collect the pupae and soon after expect them to emerge. Later anesthetize hatched adults using carbon dioxide and inspect them for transformance.When screening out, that's mobilization method can be critical. Medfly adults can be anesthetized using ice or CO2 but prolonged exposure might reduce other's survival. Check for the presence of red fluorescence using a DS red wide filter.Or check for green fluorescence using a EYFP filter. Transfer the transformed individuals into a small cage with wild-type adults of the opposite sex to establish a heterozygous strain. In one experiment, double-strained RNA was used to silence inx5 gene expression.The adult germ-line of uninjected controls appears to be normal as expected, while treated individuals showed underdeveloped testes and ovaries. Quantification of inx5 transcript abundance in the abdomens of individuals from both sexes confirmed the significantly lower expression of the gene as compared to the controls. In a second experiment, two strains with labeled sperm were generated using the beta-2 tubulin promoter to drive different fluorescent markers.Transgenic males showed a distinct fluorescence pattern in the body and in the testes. With this video, we provide our optimized protocols for the rearing and micro-injection of nucleic acids into medfly embryos. Once mastered, up to 1, 000 embryos can be injected over an eight hour working day.Following this procedure, other metals like thy-lino-crisp-er, cas-min-eta-geno-meta-tine can be performed to develop a new generation of medfly strains.

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