Drosophila melanogaster Larva Injection Protocol
October 19th, 2021
Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.
This protocol addresses the difficulties and challenges with microinjecting larva by describing a precise method that will allow the use of Drosophila melanogaster larva as a model for various immune and molecular studies. This is a simple injection technique that presents an efficient, cost-effective and rapid method to repeatedly deliver a variety of substances in a uniform manner, which allows for consistent and reliable results. Begin by selecting third instar stage larvae and washing them with Ringer's solution in a small Petri dish.
Place a filter paper in a Petri dish and moisten it with 5 ml of Ringer's solution. Then place the larvae on the filter paper. Prepare capillaries using 3.5"glass capillary tubes in a micropipette puller.
Open the glass capillary with the help of straight serrated medium point forceps. Fill the open capillary with mineral oil using a plastic disposable syringe. Then, eject the oil out of the capillary tube with a nanoliter injector.
Fill the capillary with the desired bacterial preparation for injection. Transfer the anesthetized larvae to a filter paper moistened with Ringer's solution. Using forceps, apply firm pressure on the dorsal side of the posterior end of the larvae, where the tracheal spiracles are apparent.
Then, insert the needle horizontally, towards the posterior end of the larvae, and inject the bacterial stocks. Remove forceps to avoid spillage of hemolymph or intestine. Then, withdraw the needle previously inserted into larvae.
Infect 20 larvae for each experimental condition. Using a paintbrush, gently transfer the injected larvae to a separate moist filter paper. Add Ringer's solution as necessary to prevent desiccation.
Incubate the Petri dish in an incubator at 25 C.Drosophila melanogaster larvae infected with Photorhabdus asymbiotica exhibited a 57%survival rate for 24 hours following injection, while larva injected with Escherichia coli showed an 85%survival rate at the same time point. The importance of this method is that the needle is held approximately parallel to the larva. The pressure applied to hold the larva is very little, which reduces the possibility of hemolymph leakage, fatal injury, or inadequate delivery of the desired substance.
This technique can be perfected with practice.
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Overview
This protocol describes a larval injection technique for Drosophila melanogaster, aimed at facilitating the use of larvae in immune and molecular studies. The method addresses challenges in microinjecting larvae and provides a reliable approach for substance delivery.
Key Study Components
Area of Science
- Neuroscience
- Microbiology
- Immunology
Background
- Drosophila melanogaster is a widely used model organism.
- Understanding antimicrobial innate immune responses is crucial.
- Larval stages offer additional insights into microbial infection strategies.
- Microinjection techniques are essential for experimental consistency.
Purpose of Study
- To promote D. melanogaster larvae as a model system.
- To provide a detailed method for larval microinjection.
- To enhance the reliability of immune and molecular studies.
Methods Used
- Select third instar larvae for injection.
- Wash larvae with Ringer's solution.
- Prepare capillaries using glass capillary tubes.
- Inject substances uniformly into the larvae.
Main Results
- The technique allows for efficient substance delivery.
- Results are consistent and reliable across experiments.
- Cost-effective method suitable for various studies.
- Facilitates exploration of immune responses in larvae.
Conclusions
- The larval injection technique is a valuable tool for researchers.
- It expands the utility of D. melanogaster in scientific studies.
- Future research can leverage this method for deeper insights.
