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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