A Technique to Establish a Bacterial Infection Model in Insect Larvae

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.