Gary B. Dunphy

Cellular immune responses of spruce budworm larvae to Entomophthora egressa protoplasts and other test particles

Abstract The protoplast stage of two isolates of Entomophthora egressa developed normally and eventually produced conidiophores when injected into larvae of the spruce budworm, Choristoneura fumiferana . The spruce budworm hemocytes never made long-term contact with the protoplasts either in vivo or in vitro. The protoplasts made active, short-term contact with spruce budworm granulocytes both in vivo and in vitro. Total larval hemocyte counts (THC) initially declined when larvae were injected with protoplasts, growth medium (MGM), or Escherichia coli . The recovery rate to THC control levels was similar for MGM and protoplasts and supports the concept of nonrecognition of protoplasts by the hemocytes. The granulocytes were important in both nodulation and phagocytosis of E. coli and Bacillus cereus , whereas the plasmatocytes were important in phagocytosis. In in vitro studies, spruce budworm granulocytes did not adhere to rod-shaped hyphal bodies, spherical hyphal bodies, or germinating spherical hyphal bodies of E. egressa , whereas the granulocytes readily encapsulated the hyphae. There was no evidence for the production by the protoplasts of metabolites which might interfere with hemocyte adhesion. When protoplasts contacted Tenebrio molitor granulocytes, the protoplasts reacted by increasing the number of protoplasmic extensions and by granule discharge. The process of granule discharge may be an active protoplast defense mechanism. The sporangiospores of Absidia repens and Rhizopus nigricans adhered to spruce budworm granulocytes; however, the number of A. repens spores per granulocyte and the level of granulocytes with spores decreased in the presence of phenylthiourea. The adhesion of A. repens spores to granulocytes was enhanced by N -acetylglucosamine, whereas glucosamine, sucrose, fucose, fructose, arabinose, and galactose either had no effect on or reduced spore adhesion. Thus, the chitin (or its subunits) in the hyphal wall may initiate the granulocyte response.

Response of eastern hemlock looper hemocytes to selected stages of Entomophthora egressa and other foreign particles.

Abstract Indirect evidence for the natural existence of the free-protoplast stage of the fungus Entomophthora egressa in the eastern hemlock looper, Lambdina fiscellaria fiscellaria, is presented. The protoplasts were viable after 72 hr postinjection and subsequent development in the host produced conidia characteristic of E. egressa. The hemocytes studied (plasmatocytes, granular cells, and spherule cells) did not adhere to the protoplasts either in vivo or in vitro. Cells of Escherichia coli and sporangiospores of Absidia repens adhered to the granular cells in vitro. The granular cells adhered to the hyphae of Rhizopus nigricans in vitro. The spherule cells strongly adhered to the hyphae and hyphal bodies of E. egressa in vitro. The protoplasts, hyphae, and conidia of E. egressa and the hemocytes of L. fiscellaria fiscellaria adhered to positively charged DEAE-Sephadex beads and not to negatively charged CM-Sephadex beads. Aspects of active and passive strategies for protoplast evasion of host hemocytes are discussed with some emphasis on hemocyte-protoplast electrostatic repulsion and active secretion of hemocyte inhibitors by the protoplasts.

A study of the surface proteins of Entomophthora egressa protoplasts and of larval spruce budworm hemocytes

Abstract Entomophthora egressa protoplasts either exposed to or not exposed to trypsin were not attacked by either trypsinized or non-trypsinized larval spruce budworm granulocytes. Granulocytes adhered to protoplasts exposed to papain, and this adhesion could be prevented by papainizing the hemocytes. Differences were observed in the responses of two E. egressa isolates when exposed to papain or to the papain-control solutions. Exposure of hemocytes to trypsin did not reduce either the number of Absidia repens sporangiospores per granulocyte or the percentage of granulocytes with spores, whereas, exposure to papain did. The role of surface proteins, particularly glycoproteins, in hemocyte-fungal cell interactions is briefly discussed.

Effects of Physical Factors on Protoplasts of Entomophthora Egressa

The effects of various physical factors on the growth of protoplasts of Entomophthora egressa were studied. In order to obtain growth in cultures shaken at 50 and 100 rpm using Graces liquid medium, a fetal-calfserum (FCS) supplement of 28 ml per liter (2.7%) was required. Concentrations of FCS up to 50 ml per liter failed to support growth at 150 rpm. Analysis of Graces medium indicated that it had an osmolality of 350 mOsM. This osmolality, except where designated, was maintained at the start of experiments in all media. The optimum sucrose concentration for protoplast stability (retention of spindle shape) was 350 mM (403 mOsM); for centrifugation, a 330-mM-sucrose-10-mM-MES buffer (pH 6.2) was optimum as based upon protoplast retention of shape and subsequent viability. The optimum temperature for growth (protoplast yield) was between 17 and 21 C. Of the three buffers tested (MES, MOPS and TES), MES at 10 mM was found to provide the best balance between buffering capacity and toxicity. The protoplasts grew over the pH range 5.2-8.2. The optimum pH for growth was 6.2.

Comparative Physiology of Two Isolates of Entomophthora Egressa

Isolate 458 of Entomophthora egressa from larvae of the eastern hemlock looper and isolate 521 from larvae of the spruce budworm differed in colony morphology on coagulated egg yolk medium, protoplast growth rate in modified Graces medium, regeneration sequence, effect on growth medium [pH, osmolality, individual and total ninhydrin-positive compound (NPC) levels, total protein and glucose concentration], rate of glucose uptake, protein synthesis, degree of amino acid utilization and growth response to CO2. The major NPC utilized included t.-aspartic acid, iL-glutamic acid, L-lysine, L-histidine, L-tyrosine, L-leucine, L-valine, I-glutamine, glycine, DL-serine and /3-alanine. The major endogenous protoplast NPC were L-glutamic acid, L.histidine, L-alanine, DLt.-serine and glycine with moderate levels of L.-aspartic acid, t-arginine, i.-proline, t.-glutamine, tL-asparagine and /f-alanine. As a result of the observed developmental and physiological differences, we conclude that isolates 458 and 521 are representatives of two distinct physiological races of E. egressa.

Effects of hormones on Entomophthora egressa morphogenesis

Abstract The morphological effects of a fungal sex hormone, trisporic acid (TA), and a synthetic insect juvenile hormone (JH) on mycelial cultures derived from protoplasts of the fungus Entomophthora egressa were determined. In comparison with the control treatment (no added hormone), only one treatment (JH and TA both added at 40 μg/50 ml) produced all of the control cell types. Under both experimental conditions, normal hypha (without swollen tips), spherical hyphal bodies, irregularly shaped hyphal bodies, and thick-walled spheres were present. This JH plus TA treatment differed from the control in that normal rod-shaped hyphal bodies were also present and the thick-walled structures tended to be variable in shape. Hyphal tip swelling which did not lead to conidium production was common to all treatments in which only JH was added. The results of the addition of various concentrations and combinations of JH and TA with regard to the production of thick-walled cell types and three forms of hyphal bodies are given and discussed.