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A characterization of the salivary gland proteins of the blood-sucking blackflies, Simulium vittatum and Simulium Decorum (Diptera: simuliidae)

University of British Columbia

Microscale protein assays, gradient polyacrylamide gel microelectrophoresis, and guinea-pig skin sensitivity tests, were employed to investigate changes occuring in the quantities and certain properties of salivary gland proteins of the females of two species of haematophagous blackflies. These changes in salivary gland protein properties were postulated to reflect the changes occuring in the nature and severity of the host response. A rapid dissection method was developed for the handling of the large numbers of laboratory-reared larvae, pupae and post-emergence adults. A progressive increase in protein content of macerated salivary glands was detected, beginning with pupae, and extending through the first 4 days of post-emergent adults. The protein content of glands from S. decorum increased from 1.5 μg in the pupa to 4.5 μg in the day 4 adult, and for the smaller species, S. vittatum, it increased from 0.9 μg to 2.6 μg. The water-soluble protein averaged 64% of the total protein for S. decorum and 76% for S. vittatum, these values being relatively constant throughout all ages. Polyacrylamide gel extending as a concentration gradient from 1 to 40% in 10 μL capillaries, and acting as a graded sieve, was used to separate the various proteins according to progressively smaller molecular size. Protein loads of between 0.2 μg and 1.0 μg were resolved into as many as 32 individual bands. The protein patterns of larval salivary glands differ quantitatively and qualitatively from those of pupae or adults. Pupal salivary glands are smaller than those of post-emergent stages, but structurally resemble those of adults and display a similar sequence of electrophoretic separation. Three protein bands from S. decorum in the MW range of 50,000 to 80,000 show a progressive increase in relative predominance from day 0 through day 4, the stage presumed to be the most prepared for blood-feeding. There is also a concentration of more rapidly migrating protein of ca. 10,000 MW in the day 4 salivary glands. Most of the protein in S. vittatum post-emergence salivary glands occurs in the range of 50,000-100,000 MW. The glands contain a progressively increasing concentration of ca. 90,000 MW protein which culminates in the day 4 glands. Day 1 salivary glands are the only stage to reveal three bands of rapidly migrating .low MW protein in the 2,000-5,000 MW range. Skin tests with S. vittatum salivary gland preparations in guinea-pigs, previously sensitized with whole fly tissue, gave presumptive evidence of a combination of antigenic and toxic factors in the glands. Salivary glands from all age classes induced delayed reactions, with day 4 yielding the strongest antigenic response. Both control and sensitized guinea-pigs showed a strong cutaneous response to the injection of day 1 salivary glands, implicating a toxic factor in the salivary glands at this stage. The recognition of two essentially different categories of noxious substances, namely toxins and sensitizers, paves the way for improved prognosis, prophylaxis involving antigens and therapy.

Text

2010-03-29

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http://hdl.handle.net/2429/22878

Forestry

University of British Columbia

Graduate