This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.
Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.
Delivering small biomolecules, such as nucleic acids, to adult dipterans has proven to be challenging in both Culicidae and Muscidae. Oral uptake of dsRNAs has been reported to produce sterility (when targeting genes exclusively expressed in testes of adults) and mortality (when targeting SNF7 and a steroid receptor coactivator) in larval Aedes aegypti1,2. Mortality has also been observed when targeting HSP70 in larval Musca domestica3. Such phenotypic effects, however, have not resulted after feeding dsRNA to adult Ae. aegypti in sugar meals4,5.
Microinjection has been used to circumvent the midgut when introducing pathogens or nucleic acids, thereby inducing a systemic response5,6,7,8,9,10. Several microinjection techniques exist, involving equipment ranging from in-house produced apparatuses that require visual measurement of injection volume to microprocessor-controlled injectors that allow for automated volume delivery as low as 2.3 nL5,9,10,11. RNA interference (RNAi) triggers targeting ribosomal mRNAs, disrupt ovarian development in arthropods as diverse as the cattle tick Rhipicephalus microplus, the mosquitoes Aedes aegypti and Culex pipiens, and the house fly Musca domestica5,9,12,13. In these studies, monitoring the disruption of oviposition was essential for determining the efficacy of the inoculant as the phenotype may manifest as termination or reduction of progeny. This is a form of multigenerational lethal phenotype that is a critical and desired effect in many of the non-traditional biocontrol methods like Wolbachia-infected males introduction (sexual incompatibility) and RNAi induced sterility5,9,14. Tracking both mortality and fecundity is necessary for the characterizing and development of highly specific biorationals (naturally occurring pathogens and/or natural derivatives)15.
This protocol presents detailed microinjection techniques for both adult mosquitoes and house flies; a process that is often not well described in the literature. In addition, oviposition bioassay methodologies to adequately evaluate dsRNA effects on adult dipterans are described. These protocols were developed specifically for Aedes aegypti and Musca domestica but can be modified for other species.
1. Insect Preparation
2. Insect Injection
3. Aedes aegypti mortality and oviposition bioassay
4. Musca domestica mortality and oviposition bioassay
Microinjection of dsRNA in mosquitoes
This microinjection method has been used in our laboratory to evaluate gene expression, mortality and oviposition response for over 80 dsRNA triggers across several mosquito genera (Aedes, Anopheles, Culex). Injecting females from the colony strain of Ae. aegypti (ORL1952) with 1 µg of dsRNA derived from the ribosomal transcripts RPS6 and RPL26 (see Estep et al.5 for dsRNA production methods and sequence data) showed significant reduction in relative expression (RE) across multiple oviposition cycles when compared to mosquitoes injected with a control dsGFP. Aedes aegypti RE measured following the 2nd gonotrophic cycle showed significant reduction of RPS6 expression (P < 0.05) in mosquitoes injected with dsRPS6 as determined by a Student's t-test between the dsGFP control of the same group (Figure 4)5.
Fecundity was assessed from individual females using the oviposition bioassay described here. After 3 days post-injection, mosquitoes were blood fed, transferred into individual oviposition containers, and allowed to lay to completion. Average clutch sizes for the first gonotrophic cycle were significantly reduced for both dsRPS6 treated Ae. aegypti (n = 102 females, 1.3 ± 0.8 (mean ± SE) eggs) and dsRPL26 treated (n = 86 females, 4.0 ± 1.1 eggs) when compared to dsGFP injections (n = 79 females, 49.3 ± 3.5 eggs, Figure 5A). Clutch assessment after a second gonotrophic cycle also showed significantly reduced oviposition for both dsRNA treated groups, but with reduced effect size. Group sizes of Ae. aegypti were variable and thus means separation was performed by Dunn's test5. Twenty-four-hour mortality was normally 3% or less.
A clear dose effect was observed when injecting dsRPS6 from 1 µg to 50 ng in female Ae. aegypti, and clutch size varied significantly when compared to 1 µg/female dsGFP injected controls in all but the 25 ng/female dsRPS6 dose (Figure 5B).
Microinjection of dsRNA in house flies
Colony Musca domestica (ORL normal) were injected with 5 µg of dsRNA constructs designed against the house fly RPS6 and RPL26 transcripts9. As was observed in Ae. aegypti, significant reduction of both specific transcript expression (RPS6 and RPL26) and reduction in clutch size was observed (Figure 4 and Figure 5A). Significant reduction in RE was determined by Student's t-test between the dsGFP control of the same group (P < 0.05) and the Holm-Sidak test was used to determine significance between clutch sizes for different M. domestica treatment groups. Ovarian dissections showed reduced oogenesis in dsRPS6 and dsRPL26 treatments, while dsGFP fed flies had normal vitellogenin deposition as rated on the Tyndale-Biscoe scale9.
Figure 1: Microinjection setup for delivering microvolumes to adult mosquitoes and house flies. Injections are performed on cold-anesthetized insects staged on microscope slides over a chill table. Please click here to view a larger version of this figure.
Figure 2: Microinjection sites for Ae. aegypti and M. domestica. (A) Adult Ae. aegypti are injected in the middle one-third of the mesokatepisternum. (B) Adult M. domestica are injected in the mesopleuron. Arrows indicate sites of injection. Please click here to view a larger version of this figure.
Figure 3: Oviposition bioassay setup. (A) Ae. aegypti oviposition assay cups with seed germination paper as an oviposition substrate, capped with tulle and 10% sucrose-soaked cotton. (B) Adult female M. domestica in an oviposition assay container with food (A), water (B), and larval media (C). Please click here to view a larger version of this figure.
Figure 4: Relative expression of RPS6 in Ae. aegypti and M. domestica injected with species-specific dsRNAs. Aedes aegypti and M. domestica were injected with 1 and 5 µg respectively of either control dsRNA (dsGFP) or dsRNA designed against RPS6 or RPL26 transcripts from each insect. Significant reduction (P < 0.05) of RPS6 expression was observed in mosquitoes and flies injected with dsRPS6 and by flies injected with dsRPL26 (indicated by asterisks). Error bars represent mean ± SE and number at column base indicates number of individuals examined. This figure has been modified from Estep et al.5 and Sanscrainte et al.9. Please click here to view a larger version of this figure.
Figure 5: Average clutch size of Ae. aegypti and M. domestica injected with species-specific dsRNAs. (A) Egg deposition was significantly reduced (P < 0.05) in both Ae. aegypti and M. domestica after injection of 1 and 5 µg respectively of either control dsRNA (dsGFP) or dsRNA designed against RPS6 or RPL26 transcripts from each insect. Asterisks indicate significant differences from the dsGFP control of the same group. Error bars represent mean ± SE and number at column base indicates number of individuals examined. This figure has been modified from Estep et al.5 and Sanscrainte et al.9. (B) Dose curve of Ae. aegypti injected with dsRPS6 from 50 ng/female to 1000 ng/female shows significant reductions in fecundity in comparison to dsGFP injected cohorts (50, 100, and 1000 ng/female). Error bars represent mean ± 95% CI from 36-81 individual organisms per dose. Points with letters represent significant difference between groups (P < 0.05) identified by ANOVA. This figure has been modified from Estep et al.5. Please click here to view a larger version of this figure.
Microinjection is a valuable laboratory technique to ensure delivery of dsRNA or other biorationals (i.e., pesticides, viruses, microsporidia). While many laboratories perform microinjection, the exact amount injected is often unclear due to technical limitations of the delivery system where delivered volume is not directly measured11. Delivered volume and concentration are critical parameters that allow calculation of standard toxicological measures such as EC50 or IC50 or to define minimal effective doses5,16. This is especially important in functional studies using RNAi in adult insects, where delivery by feeding does not always result in systemic exposure to the desired particles and the actual dose crossing the midgut is unclear5.
While the microinjection procedure can be initially challenging, rapid improvements in speed and delivery accuracy are achieved with practice and patience. Mastering the injection procedure itself is a time investment, but, once accomplished, the procedure produces repeatable results and injury mortality of <3%. The ratio of successful injections will increase as proficiency increases allowing injection of 300 mosquitoes or flies per day.
Many of the challenges of microinjection are due to the needle being used. Determining the proper capillary needle break point and tip angle is a challenging aspect of the procedure and requires some trial and error to determine the most effective opening size for a given species. The fine needle most useful for mosquito microinjection (~150 µm) is too small to quickly deliver the larger volume injected into flies while conversely, the larger needle opening that works on flies (~250 µm) causes extensive injury damage to the smaller mosquitoes and unacceptable injection mortality. A needle broken with a blunt tip is often difficult to get through the cuticle without tissue damage and increased mortality. Insect scales or tissue pieces can clog needles over the course of an experiment, so it is often helpful to have several needles pulled if replacement is required. We have found that it is easier to replace a difficult needle rather than spend time to try to clear a jammed or fouled needle.
Observing the injection solution entering the insect is critical so that unsuccessful injections can be removed (see steps 2.14-2.15). Rhodamine B addition to injection solutions provides a clear visual to ensure that cold-staged insects receive proper dosing and is especially helpful while practicing (see step 2.7).
The injection and oviposition bioassay methods presented here have successfully induced gene knockdown and measurable phenotypic effects in adult Ae. aegypti and M. domestica when using dsRNAs designed against species-specific ribosomal transcripts (Figure 4 and Figure 5A). Additionally, the ability of this method to accurately deliver microvolumes to individuals allows for dose response curves to be generated for small amounts of material (as low as 50 ng; Figure 5B). This is especially useful for methods such as screening siRNAs or dsRNAs, as producing large quantities of these molecules can be cost-prohibitive.
This method can be modified to inject biological agents (viruses, bacteria, microsporidia) or chemical pesticides, after ensuring that any delivery buffers are innocuous by injection. As delivery volume is limited by the size of the insect, producing concentrated testing solutions is of importance.
To adequately evaluate dsRNA efficacy, it is necessary to track oviposition as lethal phenotypes may only manifest in progeny. As presented here, holding female Ae. aegypti separately after blooding allows for individual clutch size determination and further testing on the same individuals can be done downstream of oviposition (i.e., gene expression studies, additional gonotrophic cycles, tissue specific knockdown) to correlate reproductive output with other measures such as gene expression. As M. domestica generally oviposit in a group response, inciting isolated gravid flies to lay is very labor intensive and not practical for large numbers of individuals17. Therefore, a method to determine mean clutch size is presented.
The microinjection method presented here provides consistent systemic delivery to mosquitoes and house flies. Coupled with mortality and oviposition tracking bioassays, these versatile tools can be used to assess the transcriptional and phenotypic effects of small RNA molecules, biological agents, and traditional pesticides where oral delivery is not viable.
The authors have nothing to disclose.
The authors thank Hanayo Arimoto (US Navy), Dana Johnson, Lucy Li, and Roxie White (USDA-ARS) for help with data acquisition and analysis. We also thank Drs. Pia Olafson (USDA-ARS) and Ke Wu (University of Florida) for critical review of the manuscript and Niklaus Hostettler (University of Florida) for filming the microscope footage. Funding was provided by the USDA, the WII-141C project of the Navy and Marine Corps Public Health Center, and the Deployed War Fighters Protection Program. The funders had no role in design or direction of the study or development of the manuscript.
needle pulling | |||
vertical pipette puller | Kopf | 720 | settings: heater = 15 units, solenoid = 4 amps |
glass capillaries, 3.5" long, ID = 0.530 mm ± 25 μm, OD 1.14 mm | World Precision Instruments | 504949 | capillaries for pulling glass needles |
Name | Company | Catalog Number | Comments |
microinjector station | |||
Nanoliter 2010 | World Precision Instruments | NANOLITER2010 | microinjector |
manual micromanipulator | World Precision Instruments | KITE-L | left hand KITE manipulator |
magnetic stand | World Precision Instruments | M9 | holds micromanipulator |
precision stereo zoom binocular microscope on boom stand | World Precision Instruments | PZMIII-BS | dissecting scope for microinjections |
1.5X objective | World Precision Instruments | 501377 | objective for microscope |
light LED ring | World Precision Instruments | 504134 | light ring for injection microscope |
laboratory chill table | BioQuip Products | 1431 | chill table for microinjections |
microscope slides | Fisher Scientific | 12-544-1 | for staging insects while injections |
Rhodamine B, 98+% | Acros Organics | AC296571000 | |
Name | Company | Catalog Number | Comments |
mosquito oviposition bioassays | |||
large Petri dish | Fisher Scientific | FB0875714 | for holding staging slides |
plastic cup – 3 1/2 oz. | Dart Container Corporation | TK35 | mosquito oviposition bioassay cups |
matte tulle fabric | Joanne Fabrics | 1103068 | caps for oviposition bioassays |
blood source | locally acquired | typically bovine or live chickens | |
1 x 30mm clear edible collagen casing | Butcher and Packer | 30D02-05 | |
heavy weight seed germination paper | Anchor Paper Co | SD7615L | |
oral aspirator with HEPA filter | John W. Hock Company | 612 | |
Name | Company | Catalog Number | Comments |
house fly oviposition bioassays | |||
4 L plastic food storage canister | Walmart | 555115143 | |
10-inch stockinette sleeve | Medonthego.com | FS15001H | |
wheat bran | locally acquired | typically found at feed stores | |
Calf Manna performance supplement | ValleyVetSupply.com | 16731 | pelleted livestock feed |
dried egg yolk | BulkFoods.com | 40506 | |
black cotton cloth | locally acquired | typically in craft supplies section | |
60 mL cup | Dart Container Corporation | P200-N |