UBC Theses and Dissertations
Some aspects of the sequestration of cardenolides in the large milkweed bug, Oncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae) Moore, Lynn Marie Vasington
Specific aspects of the selective sequestration, excretion and tolerance of cardenolides in the large milkweed bug, Oncopeltus fasciatus have been studied using spectrophotometry assays, thin-layer chromatography, tracer studies, in vivo tolerance assays, and enzyme inhibition techniques. The cardenolide content of the dorsolateral space, gut, wings and fat body of Oncopeltus fasciatus was examined. The results indicate that the majority of cardenolides sequestered in the insect are concentrated in the dorsolateral space, which confirms the basic pattern of quantitative distribution of cardenolides in O. fasciatus determined in earlier work. Large amounts of cardenolides were not found in the gut, wings and fat body. The female fat body contained 4-5% of the total cardenolide content of the insect. The cardenolide content of male fat body, and gut and wings of both sexes was below the detection limit of the cardenolide assay. Thin-layer chromatography was used to determine the cardenolide array of various tissues and secretions of O. fasciatus reared on seeds of a single species of milkweed (A. speciosa) and adult extracts and dorsolateral space fluid of O. fasciatus reared on seeds of two species of milkweed with different cardenolide arrays (A. speciosa and A. syriaca). The results indicate that cardenolides are not sequestered in the insect simply on the basis of polarity and that metabolism and differential excretion of cardenolides are involved in the sequestration of cardenolides in O. fasciatus.. The similarities in the cardenolide profiles of O. fasciatus reared on different food sources, and tissues of O. fasciatus reared on a single food source indicates that there is regulation of the cardenolide array in O. fasciatus. An in vitro preparation of Malpighian tubules was used to investigate the excretion of the polar cardenolide, ouabain, in O. fasciatus. Both segments of the tubules were found to metabolize ouabain. The distal Segment (Segment II) secreted primary urine and ouabain. Secretion of ouabain by Segment II was not observed to occur against a concentration gradient and increased with increasing fluid secretion. The proximal segment (Segment I) reabsorbed fluid and ouabain but not metabolites of ouabain. Ouabain was reabsorbed against a strong concentration gradient (23-fold), was independent of fluid reabsorption, and increased with increasing fluid secretion by Segment II. In rapidly secreting Malpighian tubules (a situation of high cardenolide secretion by Segment II), the presence of Segment I reduced the excretion of ouabain by 84 - 93%, mainly by reducing ouabain concentration. It appears excretory loss of cardenolides can be reduced in O. fasciatus and thus may be a factor in the sequestration of cardenolides in this insect. O. fasciatus tolerated 1954x and 7288x, respectively, the LD₅₀ ouabain dose of Schistocerca gregaria and Periplaneta americana when ouabain was injected into the hemocoel of these insects. The maximum ouabain dose that could be injected into 0. fasciatus (200 nmoles) resulted in no mortality; this dose is higher than the lethal ouabain doses recorded for vertebrates and invertebrates. The ouabain concentration resulting in 50% inhibition (I₅₀) of Na,K-ATPase activity was determined in lyophilates of nervous tissue of 0. fasciatus and brain and recta of S. gregaria and were 2.0 x 10⁻⁴, 2.0 x 10⁻⁶, and 1.0 x 10⁻⁶ M, respectively. The I₅₀ value for ouabain inhibition of Na,K-ATPase activity in the nervous tissue of O. fasciatus is higher than the I₅₀ values for nervous tissue in most other insects as well as many other invertebrate and vertebrate tissues. Thus, the presence of ouabain resistant Na,K-ATPases appears to be a factor in the tolerance and sequestration of plant cardenolides in O. fasciatus.