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United States Department of Agriculture

Agricultural Research Service

Title: A Secretory Pla2 Associated with Tobacco Hornworm Hemocyte Membrane Preparations Acts in Cellular Immune Reactions

Authors
item Park, Youngjin - UNIV OF WI - MADISON
item Stanley, David

Submitted to: Archives of Insect Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 9, 2005
Publication Date: November 1, 2005
Citation: Park, Y., Stanley, D.W. 2005. A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions. Archives of Insect Biochemistry and Physiology. 60:105-115.

Interpretive Summary: Biological control is a broad term for exploiting various organisms to control pest insect populations. Biological control strategies represent a sustainable alternative to use of general toxins in many agricultural areas. However, for many cropping and urban pest management systems, biological control methods are not sufficiently understood. In particular, we do understand how some biological control agents kill insect pests. Information in this area is necessary to develop the best biological control strategies. Agents used in the biological control of pest insects include predators, parasites and various pathogenic microbes such as viruses, bacteria and fungi. Some nematodes, very small worms, are potential biological control agents because they are virulent insect pathogens, however, their insect-killing properties are due to a bacterium that resides in the host worm which first impairs insect immune reactions and then quickly overruns and kills the insect. The freshly-killed insect is an ideal environment for nematode reproduction and development. In the present study, work to understand the insect-killing mechanisms of the bacteria it was found that the bacterium secretes specific chemicals that inhibit a specific enzyme responsible for mediating insect immune reactions. The enzyme is new to insect science and additional work yielded a preliminary biochemical description of the enzyme, including where the enzyme is located (blood cells). Research such as this will impact scientists engaged in the study of specific insect-killing mechanisms of biological control agents. A more important impact will be seen in later development of more effective agents which are able to exert multiple killing actions in crucial points in the lives of pest insects.

Technical Abstract: We report on a secretory phospholipase A2 (sPLA2) associated with membrane-enriched fractions prepared from hemocytes of the tobacco hornworms, Manduca sexta. Virtually no PLA2 activity was detected in serum of immunologically naïve or bacterially challenged hornworms. PLA2 activity was detected in cytosolic and membrane-enriched fractions prepared from hemocytes. PLA2 activity in the cytosolic fraction (1.2 pmol/mg/h) was approximately 4-fold greater than in the membrane-enriched fraction. The cytosol-associated PLA2 activity was strongly inhibited in reactions conducted in the presence of the specific cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP) but not in the presence of the sPLA2 inhibitor p-bromophenacyl bromide (BPB). Conversely, the membrane-associated PLA2 activity was inhibited in reactions conducted in the presence of BPB but not in the presence of MAFP. While the cytosol-associated PLA2 was independent of calcium, the membrane-associated sPLA2 required calcium for full catalytic activity. Hornworms treated with either BPB, MAFP or the glucocorticosteroid dexamethasone were severely impaired (by 50% to 80% relative to controls) in their ability to form nodules in reaction to bacterial challenge. However, the immune-impairing influence of the inhibitors was reversed by treating larvae with arachidonic acid, a precursor for eicosanoid biosynthesis. We infer that the biological significance of the sPLA2 (as well as the previously characterized cytosolic PLA2) relates to hydrolysis of polyunsaturated fatty acids from cellular phospholipids. Moreover, this enzyme may be the target of immunity-impairing factors from the bacterium Xenorhabdus nematophila. The fatty acids serve as precursors for generation of eicosanoids responsible for mediating and coordinating cellular immune reactions to infection.

Last Modified: 8/22/2014
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