This study investigates the immune response of Galleria mellonella larvae to Mycobacterium bovis BCG lux, a bioluminescent bacterial vaccine. The research focuses on the interactions between the bacteria and the larval immune cells, particularly the formation of granuloma-like structures and the role of melanin in the immune response.
Place a larva of Galleria mellonella — the greater wax moth — onto an injection platform, exposing the ventral side.
Take a microsyringe containing a suspension of Mycobacterium bovis BCG lux — a live bacterial vaccine strain genetically modified to produce bioluminescence. Inject the suspension in the last proleg, or abdominal appendages — releasing bacteria into the hemolymph — circulatory fluid in the body cavity.
Transfer the larva to a controlled environment and incubate. The bacteria utilize the hemolymph as a rich source of nutrients and multiply.
Hemocytes — invertebrate immune cells in the hemolymph — cluster around the bacteria, forming a granuloma-like structure to prevent infection spread. The hemocytes release pro-phenoloxidase, which undergoes proteolytic cleavage to form phenoloxidase.
Phenoloxidase synthesizes melanin which sequesters the bacteria at the infection site. The deposition of melanin pigment causes systemic melanization, darkening larval color.
The hemocytes engulf the bacteria into phagosomes. The phagosomes fuse with lysosomes to acquire lytic enzymes, causing bacterial degradation. A subset of bacteria can evade phagolysosomal degradation and reside in hemocytes — leading to persistent infection.
Measure the bacterial bioluminescence at defined intervals. An initial decline in bioluminescence indicates an effective anti-bacterial response, while plateauing of the value indicates a persistent infection.
Maintain the purchased instar larvae in the dark at 18 degrees Celsius upon arrival and use within one week of purchase. Identify and select healthy larvae for experimentation based on uniform cream color, appropriate size and weight, high motility, and possessing the ability to right themselves when turned over. Using blunt-end tweezers, carefully count the healthy larvae into a Petri dish lined with a layer of filter paper, and store at room temperature in the dark until use.
Prepare the injection platform by taping a 94-millimeter diameter circular filter paper to a flat, raised surface. Aspirate three volumes of 70% ethanol into a 25-microliter micro-syringe to sterilize, discard, and further rinse with three volumes of sterile PBST. Then, resuspend the prepared BCG lux inoculum, and aspirate 10 microliters into the sterilized microsyringe. Use a separate syringe to aspirate PBST for a negative control.
Following 10 injections, resuspend the BCG lux inoculum to ensure uniform cell suspension. Use tweezers to pick up one larva and place onto the injection platform. On the platform, flip the larva onto its back and immobilize by securing the head and tail with the tweezers. Locate the last left proleg counting down from the head of the larva and carefully insert the tip of the needle 5 to 6 mm deep at a 10 to 20-degree angle to the horizontal plane. Inject the inoculum from the syringe.
After each infection, transfer the infected larva into a Petri dish lined with a layer of filter paper. A single 94-millimeter Petri dish can accommodate up to 30 larvae. Store the Petri dish in a vented dark box inside an incubator at 37 degrees Celsius with 5% carbon dioxide.
Monitor the survival of the larvae every 24 hours. Larvae are considered dead when they fail to move in response to touch. Record the survival and assess statistical significance with a Mantel-Cox test.
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