Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.
The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.
The jewel wasp, N. vitripennis, is one of the four species within the genus Nasonia that are ectoparasitoids of flesh eating flies such as Sarcophaga bullata1. Due to their fast-generational periods, ease of rearing in the laboratory, and a range of unique and important biological attributes, N. vitripennis has been a focus for the development of multiple experimental tools found in traditional model organisms. For example, some unique biological attributes include a haplo-diploid reproduction system2, a relationship with microbial and genetic parasites3,4, and a supernumary (B) chromosome5,6,7. Taken together, these make N. vitripennis an important experimental system for experiments aimed at elucidating the molecular and cellular aspects of these processes, in addition to others including venom production8,9, sex determination10,11, and evolution and development of axis pattern formation12,13,14. Moreover, the genetic toolkit to study the biology of N. vitripennis has dramatically increased over the last decade or so, with the sequencing of a high-resolution genome15, several gene expression studies16,17,18, and the ability to functionally disrupt gene expression relying on RNA interference (RNAi)19,20, which together have improved the tractability and capabilities of performing reverse genetics in this organism.
Despite the many important scientific advancements and expanded toolkits in this organism, as of present knowledge only one group has successfully performed embryonic microinjections to generate heritable genome modifications21. This is primarily due to the difficulties of working with embryos of N. vitripennis as they are quite fragile and small, being ~2/3 the size of Drosophila melanogaster embryos, making them generally difficult to manipulate. Additionally, N. vitripennis females deposit their eggs into pre-stung blowfly pupae, within which the entirety of embryogenesis, larval, and pupal development occurs. Therefore, for successful microinjections, pre-blastoderm stage, embryos must be efficiently collected from host pupae, quickly microinjected, and immediately transplanted back into their hosts for development. These steps require precision and dexterity to avoid damaging the microinjected embryos, or the pupal hosts, making the technique exceptionally challenging. Notwithstanding, there is one short protocol published over a decade ago that describes N. vitripennis embryo microinjection22. However, this procedure requires that the freshly laid embryos be desiccated, it uses sticky tape to anchor the eggs for microinjection, and does not include a visual demonstration of the technique. Therefore, described here is an updated and revised protocol, including a visual procedure, detailing an improved step-by-step protocol for N. vitripennis embryo microinjections that can be followed by any basic lab to generate heritable genome modifications in this important model insect.
The recent sequencing of the N. vitripennis genome has unleashed an important need for molecular tools to functionally characterize unknown genes within this species23. The CRISPR-Cas9 system, and many other gene editing tools, have proven to be valuable in investigating gene functions for a number of organisms24. However, to generate heritable mutations, these tools require performing embryo microinjections. Therefore, demonstrated here is a detailed visual technique that includes a number of innovations that allow for efficient N. vitripennis embryo microinjections.
Overall, this detailed technique offers a number of significant innovations, with respect to existing methods23, that allow for efficient N. vitripennis embryo microinjections. For example, to facilitate the rapid collection of embryos, an oviposition tool (foam stopper) is created and used to restrict egg laying entirely to the posterior end of the host (Figure 1), which greatly facilitates collection of numerous embryos within a short period of time. Techniques for rearing wasp colonies with large numbers to collect greater numbers of eggs are also improved and defined. Additionally, to accelerate embryo alignment, an embryo alignment device which allows for embryos to be efficiently aligned and injected without having to use double-sided sticky tape to secure the embryos in place (Figure 3) is developed.
Furthermore, it is found that by keeping the embryos moist with water during injection and not desiccating the embryos or covering them with oil improved survival rates. Additionally, several capillary glass types are tested and parameters to construct the perfect needles for N. vitripennis microinjection (Table 1, Figure 4) are determined. Moreover, following microinjection, embryo survival rates are able to significantly increase due to incubating injected eggs in pre-stung hosts placed in high-humidity (70%) chambers. These innovations allow for a more streamlined and successful microinjection procedure for N. vitripennis.
Minor modifications in terms of injection apparatus, and rearing procedures can be made to this protocol, depending on user preferences. However, a number of critical steps will be essential for successfully generating mutants in N. vitripennis. For example, working quickly so that injected embryos are > 1 h old, and ensuring that injection needles are sharp enough to minimize damage to the embryo will both be essential. While this protocol should be effective for generating mutations in many (if not all) genes, one major limitation is the CRIPSR/Cas9 requirement to target a PAM (NGG) sequence which will dictate the target sequence.
In conclusion, this improved technique can be used to generate many kinds of genome modifications, such as mutations, deletions, and possibly even insertions using CRIPSR/Cas9 technologies21, or even transgene insertions to generate transgenic N. vitripennis, which should greatly accelerate functional research in this organism.
The authors have nothing to disclose.
This work was supported by a generous University of California, Riverside (UCR) laboratory start-up fund to O.S.A, a USDA National Institute of Food and Agriculture (NIFA) Hatch project (1009509) to O.S.A.
Bugdorm | Bugdorm | 41515 | Insect Rearing Cage |
Glass Test Tubes | Fisher Scientific | 982010 | |
Flesh fly pupae, Sarcophaga bullata | Carolina Insects | 144440 | |
Microelectrode Beveler | Sutter Instruments | BV10 | |
Diamond abrasive plate (0.7u to 2.0u tip sizes) | Sutter Instruments | 104E | |
Micromanipulator | World Precision Instruments | Kite R | |
Femtojet Express programmable microinjector | Eppendorf | ||
Micropipette Puller | Sutter Instruments | P-1000 or P-2000 | |
Stereo Microscope | Olympus | SZ51 | |
Compound Microscope | Leica DM-750 | ||
Aluminosilicate glass capillary tubing 1mm(outside diameter) X 0.58mm (inner diameter) | Sutter Instruments | BF100-58-10 | Can also use Borosilicate or Quartz |
Identi-Plugs | JAECE | L800-B | Foam plugs |
Microscope Slides | Fisherbrand | 12-550-A3 | |
Micro Cover glass | VWR | 48366045 | |
Adhesive | Aron Alpha | AA471 | For glueing coverslip onto microscope slide |
Fine-tip paintbrush | ZEM | 2595 | |
Ultra-fine tip forcep | Fisher Scientific | 16-100-121 | |
Femtotips Microloader tips | Fisher Scientific | E5242956003 |