Changes In Hemolymph During the Antibacterial
Response of M. sexta
--Overview--
For this series of experiments, one set of Manduca sexta
larvae are treated with an injection
of bacteria. A second set of larvae serve as a control
and either receive no injection or an injection of saline
(0.85% NaCl). After at least 18 hrs, hemolymph
is collected from each of the larvae and assayed by one
or more of the techniques listed below.
The concentration of total protein in the hemolymph
samples can be determined by a Bradford
assay. This technique involves reacting the hemolymph
sample with a dye that binds protein. A reading is then made
of the absorbance of the sample using a spectrophotometer.
The concentration of total protein in the sample is
determined by comparing the absorbance of the hemolymph
sample against a standard curve. In the standard curve,
known amounts of protein are reacted with the dye and the
absorbance is taken of the samples. The amount of protein is
then plotted against the absorbance.
A lysozyme assay may also be
performed on the hemolymph samples. The lysozyme activity is
determined using a spectrophotometric assay that measures a
decrease in turbidity of a suspension of Micrococcus
lysodeikticus cells. The rate of decrease is used to
determine the enzyme units in the hemolymph.
The cecropin activity in the
hemolymph samples can be measured using a zone of inhibition
assay; several microliters of hemolymph are placed in a
small well in an agar plate seeded with E. coli that is then
incubated overnight. As cecropin peptides diffuse, the
bacteria in the surrounding agar are killed. The remainder
of the plate develops a cloudy appearance as the bacteria
multiply. The diameter of the clear area surrounding the
well is proportional to the amount of cecropin activity
present in the hemolymph.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
is a technique used to separate proteins in a mixture by
molecular weight. The proteins in the hemolymph sample are
denatured and coated with a negatively charged detergent.
The sample is then loaded into a matrix. When an electric
current is applied to the matrix, the proteins migrate to
the positive pole with the smallest proteins migrating the
fastest. The position of the proteins in the matrix is then
made visible using a dye that stains the proteins.
For all of these experiments, a comparison is made
between the results for the hemolymph samples from treated
and control insects. Ideally, the data from an entire class
is pooled for statistical analysis. If several of the
experiments are performed, students can integrate data
across the experiments. For instance, do both lysozyme and
ceropin activities increase in treated insects; if so, can
an increase in the amount of activity be correlated with an
increase in the amount of each protein present in the
hemolymph (as determined by SDS-PAGE); likewise, does this
increase in protein activity correlate with an increase in
total protein concentration in the hemolymph?
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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