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Changes In Hemolymph During the Antibacterial Response of M. sexta

--Introduction--

All organisms must monitor and protect themselves from parasites and pathogens. To that end, complex mechanisms (defense responses) for identifying and destroying non-self threats have evolved. We rarely consider how insects fight off invaders but insects do get infections and they do survive them. Unlike vertebrates, insects do not appear to have an antibody response. Rather, insects mount a generalized response to kill invading organisms. They have both cellular (phagocytosis, aggregation) and humoral responses (release of proteins into the circulating fluid). These responses are similar to the vertebrate innate immunity response. One of the best-studied responses is against bacteria.

The initial response to large numbers of invading microorganisms is mediated by the hemocytes, which eliminate bacteria by phagocytosis and nodule formation. Nodules are formed by aggregation of hemocytes that also entrap bacteria in an extracellular matrix. This mass then undergoes melanization that kills the trapped bacteria.

Following this rapid response, there is an increase in antibacterial activity in the plasma fraction of the hemolymph. This antibacterial activity is due to the appearance of numerous proteins in the hemolymph. This humoral response is thought to kill any bacteria that have escaped the cellular responses and to provide some measure of protection during the time it takes to rebuild the hemocyte population depleted by nodule formation.

The particulars of antibacterial responses differ among insect orders and life stages. The antibacterial proteins of M. sexta. include: cecropins, attacins, lysozyme, and hemolin. These proteins attack the invading bacteria in a number of ways. Bactericidins are peptides that kill Gram negative bacteria by forming ion channels in the bacterial membrane. Attacins prevent Gram negative bacteria from undergoing division. Lysozyme is an enzyme that degrades bacterial cell walls. Hemolin is a protein that has some similarity to vertebrate immunoglobulin molecules and is involved in recognizing bacteria as foreign. Lysozyme and cecropin activities are a focus of the investigations for this laboratory exercise.

Lysozyme is an ~14 kDa enzyme that degrades the peptidoglycan layer of bacterial cell walls. It is most effective against Gram positive bacteria and is also thought to participate in the post-infection degradation of bacterial fragments released during phagocytosis and nodule formation. Lysozyme may be present in the hemolymph of untreated M. sexta larvae at low levels. The cecropin-like family of peptides (~4 kDa) have bactericidal activity against a broad spectrum of both Gram-positive and Gram-negative bacteria. The primary sequence and investigations of the secondary structure of the peptides indicate that the amino terminal one-third of the protein folds into an amphipathic helix; the remaining two-thirds of the protein is very hydrophobic. Cecropins are thought to insert into bacterial cellular membranes, forming a pore that dissipates electrochemical ion gradients, resulting in the death of the targeted bacterium.

Investigating the antibacterial response:

Overview.

Protocols:

Bacterial treatment of M. sexta larvae.

Injection of M. sexta larvae.

Collection of M. sexta hemolymph.

Determination of protein concentration in M. sexta hemolymph.

Determination of lysozyme activity in M. sexta hemolymph.

Determination of cecropin activity in M. sexta hemolymph.

SDS polyacrylamide gel electrophoresis of M. sexta hemolymph.

  


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March 1999