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.
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.
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 "gamergates" 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.
1. Regular maintenance of Harpegnathos saltator colonies
2. Preparation of quartz glass microinjection needles
3. Preparation of microinjector
4. Injection of embryos
5. Rearing of injected embryos
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 to heterozygous females to produce F4 homozygous mutant females. Absence of the target peptide was further confirmed via mass spectrometry of these F4 homozygous female individuals. Wings were clipped from males using microdissection scissors and used for genotyping purposes. As workers do not have wings, they are normally sacrificed and genotyped after experiments. As a result of successful mutagenesis, unusual behaviors were observed, which correlated with loss of the target gene. The loss of orco resulted in abnormal behaviors related to loss of pheromone sensing, inability to detect prey, impaired fecundity, and wandering from the colony. Furthermore, orco mutant ants exhibited a decreased number of odorant receptor neurons and antennal lobe glomeruli, suggesting that neuroanatomy in ants is dependent on odorant receptor functionality10.
Figure 1: Harpegnathos saltator nests. (A) External features of a 19 x 13.5 cm2 nest box containing an H. saltator colony. (B) Internal features of a 19 x 13.5 cm2 nest box containing an H. saltator colony. Note the presence of a lower nest region under a square piece of glass. (C) External features of a 9.5 x 9.5 cm2 nest box containing a small H. saltator colony. Such a nest box is also suitable for isolated workers and mutant colonies. (D) Internal features of a 9.5 x 9.5 cm2 nest box containing a small H. saltator colony. Please click here to view a larger version of this figure.
Figure 2: Needle for Harpegnathos saltator embryo microinjection. Note the thin taper of the needle (marked with an arrow). The needle may be opened by breaking the tip slightly. Please click here to view a larger version of this figure.
Figure 3: Alignment of embryos on double-sided tape. Embryos should be aligned so that their length is parallel with the long edge of the tape. This enables successive injections to be performed with ease by moving the slide on the stage. Please click here to view a larger version of this figure.
Figure 4: Embryo and needle as seen during microinjection. (A) Proper alignment of the needle with the embryo prior to injection. The needle rests perpendicular to the mid-point of the embryo's side, the typical site of injection. (B) The embryo and needle following a successful injection. A small droplet protrudes from the embryo's side (marked with an arrow). Please click here to view a larger version of this figure.
Figure 5: Diagram of basic mutant crosses. CRISPR may induce mutations on the target gene in F0 females, which are subsequently isolated upon adulthood to induce the gamergate transition. If mutations occur in germline cells, unmated gamergates may lay mutant male eggs. The F1 mutant adult males may be mated with wild-type females to generate heterozygous offspring, or they may be mated with heterozygous females to generate homozygous or heterozygous offspring. Please click here to view a larger version of this figure.
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 hindered by the difficulties associated with mutagenesis in this group. This hindrance occurs due to reproductive division of labor, a key trait of eusocial insects in which only a few members of a colony may reproduce. This trait places limitations on the number of mutant offspring and makes the development of genetic lines challenging5. The ponerine ant species, Harpegnathos saltator, provides a solution to this dilemma, as all females are capable of becoming reproductive gamergates when isolated5. Here, we provide methods for mutagenesis in H. saltator using the CRISPR/Cas9 system, delivered via embryo microinjection.
Proper colony maintenance and selection of embryos at the appropriate developmental stage for microinjection are critical. Previous work established that the syncytial stage in insect development is the ideal stage for genome editing21, but the timing of this stage in H. saltator embryonic development was previously unknown. Via nuclear staining of early embryo sections, we were able to determine that the syncytial stage in H. saltator embryos lasts until 36 hours after egg deposition, allowing us to determine when to select new embryos for microinjection10.
Selecting parameters for needle pulling and for microinjection can be challenging. Factors to consider when selecting parameters for needle pulling include (1) the type of glass used, (2) the desired purpose of the needle, (3) the desired tip size, (4) the amount of resistance the needle will experience during injection, (5) the desired taper length, and (6) the type of cell or organism that will be injected. Suggestions and guidelines for pulling various needle types can be found in various operation manuals17. Similarly, there are factors to consider when selecting parameters for microinjection including (1) size of the needle being used and (2) the embryos to be injected22. While this protocol focuses on microinjection in H. saltator embryos, the techniques may vary in other insects with modification to these parameters. In particular, the parameters for needle pulling and injection will differ if the embryo in question is soft or dechorionated. H. saltator embryos have a tough chorion, and injection results are best when a needle with a short taper is used (approximately 2 mm). Embryos that have less firm chorion may be injected with needles with longer tapers (approximately 10 mm).
In H. saltator, injected embryos cannot be returned immediately to a colony, as doing so will risk their destruction by nursing workers. If applying this protocol to other social insect species, this may not be the case. Determining the best post-microinjection rearing methods in other species may require some trial and error. In the case of H. saltator, embryos need to be reared on agar plates until hatching. Once hatched, they can be returned safely to a small colony with a few workers to care for the injected brood10. A similar method of embryo care is utilized in fire ants (Solenopsis invicta) and clonal raider ants (Ooceraea biroi) to increase the survival rate of injected embryos9,15. Upon adult eclosion, mature mutant females may be kept individually or in small colonies to begin their reproductive cycle. Directions for the care of H. saltator mutants are provided in this protocol, but if using a different species, directions for care of injected embryos and mutant adults should be modified to fit the needs of the species.
The protocol provided here is optimized for mutagenesis, in which specifically non-essential genes are targeted. In this case, the orco gene was targeted, and mutations in orco did not affect ant survival to adulthood. Similarly, this protocol may be used to target other non-essential genes, including the ones associated with other sensory receptors. If the target gene is essential, transgenic ants will have to be generated instead, either via CRISPR or transposon. Transposon-mediated transgenesis has been used in honeybees13 and may apply to ants. If the desired outcome is transgenic organisms, the injected materials will have to be different. However, post-injection processes will be similar, and therefore some aspects of this protocol will be beneficial despite the difference in the desired outcome.
Overall, H. saltator is ideal for use as a model organism and for performance of genetic crosses due to its plastic reproductive system in which workers begin reproducing after isolation10. This differentiates this ant species from other ants in which CRISPR/Cas9 technology has been established, for example, O. biroi, a species in which all females reproduce clonally. H. saltator presents unique research opportunities amongst organisms of its kind as genetic lineages can be established and mutations can be maintained across generations in this species. The novelty of this system allows researchers not only to generate mutant ants, but also to develop transgenic lines in the future. This provides opportunities for novel research to study genetic control of advanced eusociality.
The authors have nothing to disclose.
The authors thank Danny Reinberg's and Claude Desplan's labs at New York University and Jürgen Liebig'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.
Antibiotic-Antimycotic (100X) | ThermoFisher | 15240-062 | |
Cas9 protein with NLS, high concentration | PNA Bio | CP02 | |
Cellophane Roll 20 inch X 5 feet | Hypogloss Products | B00254CNJA | The product has many color variations. Purchase it in red for use in making ant nests. |
Eclipse Ci-S upright microscope | Nikon | Ci-S | |
Featherweight forceps, narrow tip | BioQuip | 4748 | |
FemtoJet ll microinjector | Eppendorf | 920010504 | This product is no longer sold or supported by Eppendorf. A comparable microinjector may be used instead. |
Microloader pipette tips | Eppendorf | 930001007 | |
NCBI database | National Center for Biotechnology Information | Gene ID: 105183395 | |
P-2000 Micropipette Puller | Sutter Instruments | P-2000/G | |
Plastic boxes (19 X 13.5 cm2) | Pioneer Plastics | 079C | |
Plastic boxes (27 X 19 cm2) | Pioneer Plastics | 195C | |
Plastic boxes (9.5 X 9.5 cm2) | Pioneer Plastics | 028C | |
Quartz glass without filament | Sutter Instruments | Q100-50-7.5 | |
Vannas scissors, 8.5 cm | World Precision Instruments | 500086 | |
Winsor & Newton Cotman Water Colour Series 111 Short Handle Synthetic Brush – Round #000 | Winsor and Newton | 5301030 |