Name: embryo injections for crispr-mediated mutagenesis in the ant harpegnathos saltator 
Uploaded: 2021-02-09T13:00:00
Description: Watch this Scientific Journal Video about Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator at JoVE.com

The authors thank&#160;Danny Reinberg&#39;s and Claude Desplan&#39;s labs at New York University and J&#252;rgen Liebig&#39;s lab at Arizona State University for their support on ant genetics. Hua Yan acknowledges support from the National Science Foundation I/UCRC, the Center for Arthropod Management Technologies under Grant No. IIP-1821914 and by industry partners. Maya Saar was supported by the United States - Israel Binational Agricultural Research and Development Fund, Vaadia-BARD Postdoctoral Fellowship No. FI-595-19.

1. Regular maintenance of Harpegnathos saltator colonies
<ol>
	<li>Maintain wild-type colonies of H. saltator in transparent plastic boxes in an ant rearing room at 22-25 &#176;C and a photoperiod of 12 hours light: 12 hours dark (12L:12D) lighting schedule.
	<ol>
		<li>Use small boxes (9.5 x 9.5 cm2) to rear individual workers or small colonies. Use medium boxes (19 x 13.5 cm2) or large boxes (27 x 19 cm2) to rear larger colonies (Figure 1</strong...

<ol>
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The evolution of eusociality amongst insects, including ants, bees, wasps, and termites, has resulted in the appearance of novel behavioral and morphological traits, many of which are understood to be influenced by a combination of environmental and genetic factors1,2,3,4. Unfortunately, the attractiveness and usefulness of eusocial insects as research models in the field of genetics has been h...

The evolution of eusociality in insects, namely those of the orders Hymenoptera and Blattodea (formerly Isoptera), has resulted in unique and often sophisticated behavioral traits that manifest on both the individual and the colony levels. Reproductive division of labor, a trait characterizing the most advanced groups of social insects, often involves caste systems composed of several behaviorally and often morphologically distinctive groups. Such behavioral and morphological diversity between castes is controlled not only by their genetic system, but also often by the environment1,2,3,4, making eusocial insects attractive subjects for genetic and epigenetic research.
The ability to manipulate the genetic system of eusocial insects has proven to be challenging as many species do not mate and reproduce in laboratory settings. Most eusocial insects also have very few reproductive individuals in a colony, limiting the number of offspring that can be produced and consequently, limiting the sample size for genetic manipulation5. Additionally, many eusocial insects have long generation times compared to insects commonly used for genetic studies (such as Drosophila), adding to the difficulty of establishing genetic lines5. Some eusocial species, however, can generate a large proportion of reproductively active individuals in a colony, which alleviates the challenges and provides opportunities to establish mutant or transgenic lines.
In the case of the ponerine ant species, Harpegnathos saltator, all female workers can become reproductively active upon the death of a queen or social isolation. These workers are referred to as &#34;gamergates&#34; and can be used to generate new colonies6. Furthermore, there may be more than one gamergate present in a colony, thus increasing offspring production5,7,8. Thus far, mutant and/or transgenic lines have been developed in the European honeybee, Apis mellifera, and in the ant species, H. saltator, Ooceraea biroi, and Solenopsis invicta9,10,11,12,13,14,15. Genetic analyses in social bees and ants have paved the way toward a better understanding of eusociality, providing an array of opportunities to study genes and their impacts on eusocial insect behavior and caste-specific physiology.
Here, we provide a protocol for genetic modification via the CRISPR/Cas9 system in H. saltator. Specifically, this technique was used to generate a germline mutation in orco, the gene encoding the obligate co-receptor of all odorant receptors (ORs)10. OR genes have been remarkably expanded in hymenopteran eusocial insects16, and orco plays an essential role in insect olfaction; in its absence, ORs do not assemble or function normally. Mutations of the orco gene therefore disrupt olfactory sensation, neural development, and associated social behaviors9,10.
In this protocol, Cas9 proteins and small guide RNAs (sgRNAs) are introduced into ant embryos using microinjection for the purpose of inducing mutagenesis of a target gene. Here, we will describe the microinjection procedure in detail along with directions regarding the care of colonies and injected embryos. These methods are appropriate for inducing mutagenesis in a variety of different genes in H. saltator ants and may be applied to a broader spectrum of hymenopteran insects.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>ThermoFisher</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 protein with NLS, high concentration</td>
			<td>PNA Bio</td>
			<td>CP02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane Roll 20 inch X 5 feet</td>
			<td>Hypogloss Products</td>
			<td>B00254CNJA</td>
			<td>The product has many color variations. Purchase it in red for use in making ant nests.</td>
		</tr>
		<tr>
			<td>Eclipse Ci-S upright microscope&#160;</td>
			<td>Nikon</td>
			<td>Ci-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Featherweight forceps, narrow tip</td>
			<td>BioQuip</td>
			<td>4748</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet ll microinjector</td>
			<td>Eppendorf</td>
			<td>920010504</td>
			<td>This product is no longer sold or supported by Eppendorf. A comparable microinjector may be used instead.</td>
		</tr>
		<tr>
			<td>Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>NCBI database</td>
			<td>National Center for Biotechnology Information</td>
			<td>Gene ID: 105183395&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>P-2000 Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-2000/G</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic boxes (19 X 13.5 cm2)</td>
			<td>Pioneer Plastics</td>
			<td>079C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic boxes (27 X 19 cm2)</td>
			<td>Pioneer Plastics</td>
			<td>195C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic boxes (9.5 X 9.5 cm2)</td>
			<td>Pioneer Plastics</td>
			<td>028C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz glass without filament</td>
			<td>Sutter Instruments</td>
			<td>Q100-50-7.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors, 8.5 cm</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
		<tr>
			<td>Winsor &#38; Newton Cotman Water Colour Series 111 Short Handle Synthetic Brush - Round #000</td>
			<td>Winsor and Newton</td>
			<td>5301030</td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo injections for crispr-mediated mutagenesis in the ant harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Using the protocol provided here, genome editing in Harpegnathos saltator embryos was performed successfully. These results were validated via polymerase chain reaction and pGEM cloning of DNA extracted from injected embryos followed by DNA sequencing. Efficiency of somatic mutagenesis using this protocol reached approximately 40%. F1 mutant males were mated to wild type females to produce heterozygous F2 females which, if not mated, produced F3 males. Mutant F3 males were mated ...

Watch this Scientific Journal Video about Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator at JoVE.com

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

This protocol allows us to generate genetically modified ants to understand the function of genes in communication and behavior in a social environment. Manipulating the genetic system of eusocial insects is challenging, as many species do not mate and reproduce in laboratory settings. The striking phenotype plasticity in the ant hypernasal cell floor allows us to establish CRISPR-mediated mutant lines.Demonstrating the procedure will be Kayli Sieber, a graduate student from a laboratory. Maintain wild-type colonies of H.saltator in transparent plastic boxes in an ant rearing room at 22 to 25 degrees Celsius and a 12 hour light, 12 hour dark lighting schedule. Use small boxes to rear individual workers or small colonies and medium or large boxes to rear larger colonies.To create nest boxes, use plaster to make the floors. As the wet plaster is drying in the medium and the large boxes, press a foam block into the plaster a few centimeters deep and a few centimeters from the back of the box to designate a lower nest region. Once the plaster has dried, cover the designated nest region with a square piece of glass.Feed colonies with live crickets twice per week, and apply water regularly to the plastic nest box flooring using a wash bottle. Whenever feeding occurs, remove trash and dead individuals. Freeze all waste and dead ants overnight at minus 30 degrees Celsius before disposing of these materials as regular garbage.Periodically add a pinch of dried sawdust to the colonies to help larvae as they undergo pupation and to help workers keep the nest box clean. Use a micro pipette puller to pull glass microinjection needles. Ensure that the glass being used has been stored in a dust-free and clean environment.Use a two-step process to pull microinjection needles. For the first step, Set the heat to 575, filament to 3, velocity to 35, delay to 145, and the pull to 75. For the second step, set the heat to 425, filament to 0, velocity to 15, delay to 128, and the pull to 200.Ensure that the resulting needle has a 2-millimeter taper and a tip of 0.5 micrometers. After pulling the needles, keep them in a dust free and clean environment until use. Prepare the microinjection mixture of casein proteins and in-vitro synthesized small guide RNAs.Keep the mixture on ice until it is time to load a microinjection needle. When not in use, store the microinjection mixture at minus 80 degrees Celsius. Set the injection parameters to an injection pressure of 140 hectopascal, a constant pressure of 70 hectopascal, and a time of 0.4 seconds.Adjust constant pressure such that material only flows in one direction, and adjust the injection pressure and time only if no material is flowing from the needle into the embryo. Load a microinjection needle with two microliters of the mixture using microloader pipette tips. Do this slowly to ensure that no bubbles are formed in the mixture.Break just the tip of the needle along the edge of the tape, such that a narrow taper is still maintained. Ensure that the needle is broken just enough that the tip is opened, but not so much that the taper is broken off. Then mount the needle to the micro manipulator.Select embryos for microinjection from the syncytial stage, which is when nuclei divide without cytokinesis. Line the embryos on a plate of double-sided tape stuck to a glass microscope slide, making sure that they are secured well to the tape to prevent movement during injection. Lay the embryos in a vertical orientation such that the lateral side of each embryo is at the edge of the tape.Place the slide and lined embryos onto the stage of the microscope at a designated microinjection workstation. Align the needle with the first embryo to be injected using the micromanipulator, and laterally puncture the first embryo along its dorsal or ventral axis under a microscope. Inject the microinjection mixture, and look for slight movement of the embryo, indicating an increase in internal pressure due to the injected liquid.Additionally, watch for the formation of a small droplet containing visible traces of tissue or lipid on the outer membrane of the embryo. Gently remove the needle from the embryo and proceed to the next embryo by adjusting the position of the microscope slide. Repeat until all embryos have been injected.Once all embryos on the slide have been successfully injected, transfer the slide to a humid box for one hour to give the embryos time to recover before being removed from the slide. After the incubation, wash the slide with ethanol, gently remove the injected embryos from the tape using featherweight forceps, and transfer them to a tube filled with a small amount of 70%ethanol. Invert the tube several times.Using a small and soft paintbrush, transfer all injected embryos to 1%agar plates with 2%antibiotic antimycotic. Incubate the plates at 25 degrees Celsius for approximately four weeks, checking regularly for hatching. Once the first embryo has hatched into a larva, return all embryos and larvae to a nest box with a few young nurse workers to care for the hatchlings.Maintain the colony following the previously demonstrated methods. This protocol was used to successfully perform genome editing in Harpegnathos saltator embryos. Results were validated via PCR and pGEM cloning of DNA extracted from injected embryos, followed by DNA sequencing.Efficiency of somatic mutagenesis using this protocol reached approximately 40%F1 mutant males were mated to wild type females to produce heterozygous F2 females which, if not mated, produced F3 males. Mutant F3 males were mated to heterozygous females to produce F4 homozygous mutant females. As a result of successful mutagenesis, unusual behaviors that correlated with the loss of the target gene were observed.The loss of Orco is associated with a loss of pheromone sensing, inability to detect prey, impaired fecundity, and wandering from the colony. When performing this protocol, embryo damage during injection needs to be minimized. This can be done by ensuring the needle tip is not opened too wide, and by moving the needle slowly when injecting.Similar techniques can be used to generate and maintain transgenic ants, which will provide more sophisticated tools for probing neuronal activity and behavior. CRISPR-mediated neurogenesis has been used in other eusocial insect species, such as honeybees, clonal raider ant, and fire ants. Genetic modifications will facilitate the functional studies on development, physiology, and social behavior in eusocial insects.

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