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Tobacco Hornworms

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Antibacterial response (hemolymph)

Changes In Hemolymph During the Antibacterial Response of M. sexta


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?


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