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Method Article
Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.
The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.
CRISPR/Cas9 gene editing is a powerful technology for functional genetic studies, especially in many rising model organisms such as the jewel wasp, Nasonia vitripennis. The ease of rearing and the availability of a complete genome makes the jewel wasp an important experimental system for elucidating the molecular mechanisms of different biological processes. For example, N. vitripennis has recently been used to unravel the epigenetic basis of the haplodiploid sex determination system1,2, the biology of B-chromosomes3,4,5,6, and the genetic basis for circadian and seasonal regulation7,8. Some of the features that make N. vitripennis amenable to work with include short generation time (~2 weeks at 25 °C), high reproduction rates, easy sex separation at the pupal stage, and the ability to diapause and store strains at 4 °C. The life cycle begins with female wasps parasitizing the pupae of the blowfly, Sarcophaga bullata. Through their ovipositor, females lay up to 50 eggs in the pupal case of the blowfly. Eggs develop into larvae that feed on the S. bullata pupa, continue to develop over the next several days, and then pupate, followed by adult eclosion and emergence from the host puparium9.
Molecular tools to perform functional genetic studies in N. vitripennis, such as RNA interference (RNAi)10 and CRISPR/Cas911,12, are available, but are limited, primarily due to difficulties in performing embryonic microinjections13. As N. vitripennis eggs require a pupal host for development, egg manipulation is very challenging. Pre-blastoderm stage embryos must be collected from the host blowfly pupae, quickly microinjected, and immediately transferred back to the host for development13. These steps require precision and specialized training to avoid damaging the microinjected embryos or the pupal hosts13. Moreover, the eggs are very small and fragile, especially after microinjections, with a very viscous cytoplasm causing a continuous clogging of the injecting needle13. These features make embryonic microinjections exceptionally challenging, requiring highly trained operators and specialized equipment that is absent in most N. vitripennis laboratories.
The optimization of alternative injection methods for the delivery of CRISPR reagents would contribute to the consolidation of N. vitripennis as a model organism. The manipulation of pupae and adults is less challenging than manipulating embryos and can be accomplished with a basic injection setup. Here, two protocols are described for injection of pupae and adults: one involving specialized equipment for injections, and the other involving the use of an aspirator tube assembly fitted with a glass capillary needle. The use of an aspirator tube is particularly suited for laboratories that do not have access to specialized equipment for embryo microinjections. Efficient injections of different developmental stages of N. vitripennis, including white or black pupae and adult wasps, are demonstrated. Wasps at the white pupal stage are particularly suited for RNAi-mediated knockdown experiments. Although RNAi in Nasonia was first described by Lynch and Desplan in 200610, there is no visual procedure available for how RNAi injections are performed. RNAi was recently used to discover the haploidizer gene of the B-chromosome PSR (Paternal Sex Ratio)3 and to study the involvement of the clock gene, period, in N. vitripennis biological rhythms7.
Black pupae and adult wasps can be used to induce CRISPR/Cas9 germline gene editing using ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) and BAPC (Branched Amphiphilic Peptide Capsules) protocols. These two ovary delivery methods have been recently described to be effective in Nasonia for generating germline mutations in the target gene, cinnabar7,12. Here, a simplified protocol is provided for injections including a visual procedure of a step-by-step methodology for both pupal and adult injections that can be utilized to generate functional genetics studies in Nasonia and likely in other parasitoid wasps, without requiring specialized equipment and bypassing embryonic microinjections.
1. Nasonia rearing
2. Alignment of white and black pupae
3. Needle preparation
4. Pupal injection with femtojet
5. Pupal injection with aspirator tube
6. Adult injection with aspirator tube
7. Post-injection care and mutant screening
8. Post-injection crosses and rearing
Figure 1: Timeline for pupal and adult Nasonia vitripennis microinjection. (A) Male and female white (top) and black (bottom) pupae are collected, (B-C) aligned and glued to a glass slide for injection. (D) Capillary needle is prepared and opened, sliding it on two overlapping slides. (E) Needle is attached either to the Femtojet (top) or to an aspirator tube (bottom) for injections.(F) The injected pupae on the slide are transferred to a Petri dish with a wet tissue on the bottom to keep humidity. Upon emergence, (G) females are placed singularly in small glass tubes and allowed to oviposit on Sarcophaga pupa. (H) Screening of the offspring to detect mutants. Please click here to view a larger version of this figure.
Figure 2: Crossing scheme after injection. Schematic representation of crossing scheme procedure in the case of CRISPR/Cas9-mediated gene editing of genes (A) that induce a visible phenotype and (B) that do not confer visible phenotype. (C) Schematic representation of RNAi screening procedure. Abbreviations: cin = cinnabar; PCR = polymerase chain reaction. Please click here to view a larger version of this figure.
This paper presents two easy methods for pupal and adult microinjection, either using a femtojet or an aspirator tube. The first method allows a more precise injection of liquid, which is important for RNAi consistency, whereas the second one allows the injection of larger amounts of liquid into Nasonia pupae or adults.
Representative results presented in Table 1 show good survival rates (from 20% to 89...
With the recent increased use of Nasonia vitripennis as a model organism for various biological questions2,3,7,17, there is a need to develop and optimize injection methods to enable a simplified and efficient protocol for the functional analysis of N. vitripennis genes. The current methods involving embryonic microinjection of gene editing reagents are challenging
JLR and DCR have filed for provisional patent protection on ReMOT Control technology. O.S.A is a founder of Agragene, Inc., has an equity interest, and serves on the company's Scientific Advisory Board. All other authors declare no competing interests.
This work was supported in part by UCSD startup funds directed to O.S.A. and NSF/BIO grant 1645331 to J.L.R.
Name | Company | Catalog Number | Comments |
100 x 15 mm Stackable Petri Dishes, Polystyrene, Mono, Sterile | Sigma | 960-97693-083 | |
Aluminosilicate glass capillary tubing 1 mm(outside diameter) x 0.58 mm (inner diameter) | Sutter Instruments | BF100-58-10 | Can also use Borosilicate or Quartz |
Aspirator tube assemblies for calibrated microcapillary pipettes | Sigma | A5177-5EA | |
BAPC | phoreus biotech | BAPtofect-25 0.5 mg Kit | |
Cas9 Protein with NLS | PNABio | CP01 | |
Dissecting needle | VWR | 10806-330 | |
DNase/RNase-Free distilled Water | Invitrogen | 10977-015 | |
Femtojet Express programmable microinjector | Eppendorf | ||
Femtotips Microloader tips | Fisher Scientific | E5242956003 | |
Fine-tip paintbrush | ZEM | 2595 | |
Flesh fly pupae, Sarcophaga bullata | Ward's Science | 470180-392 | |
Food colorant dye | |||
Glass Test Tubes | Fisher Scientific | 982010 | |
Glue | Elmer's | washable, no toxic school glue | |
Micropipette Puller | Sutter Instruments | P-1000 or P-2000 | |
Microscope Slides | Fisherbrand | 12-550-A3 | |
Stereo Microscope | Olympus | SZ51 |
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