VENOM FROM THE ECTOPARASITIC WASP Habrobracon hebetor ACTIVATES CALCIUM‐DEPENDENT DEGRADATION OF Galleria mellonella LARVAL HEMOCYTES

Ectoparasitoids inject venom into hemolymph during oviposition. We determined the influence of envenomation by the parasitoid, Habrobracon hebetor, on the hemocytes of its larval host, Galleria mellonella. An increase in both intracellular Са²⁺ content and phospholipase C activity of the host hemocytes was recorded during 2 days following envenomation by the parasitoid. The decreased hemocyte viability was detected 1, 2, and 24 h after the envenomation. Injecting of the crude venom (final protein concentration 3 μg/ml) into the G. mellonella larvae led to the reduced hemocyte adhesion. The larval envenomation caused a decrease in transmembrane potential of the hemocytes. These findings document the suppression of hemocytic immune effectors in the parasitized host larvae.     

Skin toxins and external parasitism of coral-dwelling gobies

Toxic ( Gobiodon spp.) and non-toxic ( Paragobiodon xanthosomus ) gobies became infected with external parasites (gnathiid isopods) at equal rates in a laboratory experiment. Parasites were evenly distributed over the body of P. xanthosomus but were mostly confined to the fins of Gobiodon spp., where toxin glands are less abundant. Skin toxins were not associated with the rate of infection but their distribution did appear to influence the site of parasite attachment.     

Reexamination of phenoloxidase in larval circulating hemocytes of the silkworm, Bombyx mori

We have developed a modified method to detect phenoloxidase activity on hemocytes by using freshly prepared l-DOPA (1 mg/ml in 35% ethanol) to fix and incubate larval hemocytes. This method is more sensitive than the common method, in which hemocytes were fixed in 4% formaldehyde and then incubated with 2 mg/ml l-DOPA in water separately. Phenoloxidase assayed using this modified method can be inhibited by phenyltiourea (phenoloxidase inhibitor). After incubation with l-DOPA solution in ethanol, most prohemocytes, all plasmatocytes and young granulocytes are stained brown due to oxidation of l-DOPA into pigments, indicating that they have phenoloxidase. Oenocytoids are dimly stained because many of their cell inclusions have been released during the treatment. Large propidium-iodide-negative prohemocytes have strong phenoloxidase activity and are easily misunderstood as propidium-iodide-positive oenocytoids if the fluorescent method is not used for identification. Thus, in addition to oenocytoids and plasmatocytes, some prohemocytes and granulocytes in the silkworm also have phenoloxidase.     

A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions

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 naive 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 the generation of eicosanoids responsible for mediating and coordinating cellular immune reactions to infection.     

Fat-body remodeling in Drosophila melanogaster

The remodeling of the larval fat body is observed in many insects during metamorphosis, but little is known about the physiological importance or the regulation of this process. In Drosophila melanogaster, fat-body remodeling involves the dissociation of the fat body into individual fat cells, which persist throughout pupal development but are later removed by cell death in the young adult. Inhibition of fat-body dissociation is associated with pharate adult lethality and thus is likely to be an essential developmental event. As a start toward understanding the role of fat-body remodeling in the life history of insects, we carried out a detailed study of fat-body disassociation in D. melanogaster using fluorescent microscopy, and tested whether this process is mediated by hemocytes as proposed for fat-body remodeling in Sarcophaga peregrina. We identified and correlated stereotypic events in fat-body dissociation with developmental changes during metamorphosis, and have demonstrated by cell ablation studies that fat-body remodeling in D. melanogaster is a hemocyte independent process.     

Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes: Screening of 23 molecules using flow cytometry

The shellfish industry is an important economic activity in France, occurring mostly in estuarine zones subject to pollution due to anthropogenic activities. The harmful effects of pollutants on species inhabiting these estuarine zones are not well known. Among marine species, bivalve mollusks---particularly Pacific oyster, Crassostrea gigas---may serve a model of interest. The species is sedentary and filter-feeding, which favors bioaccumulation of pollutants in their tissues. Oysters may be suitable for studies on disturbance by pollutants of physiological activities, among which defense mechanisms are poorly documented in bivalves. In this study, effects of pollutants on hemocyte functions were monitored in Pacific oyster, C. gigas. Hemocytes were exposed in vitro to selected pollutants. The strategy for investigating the effects of pollutants on hemocyte functions is based on several biomarkers, which is more relevant than that of published papers based on single-endpoint experiments. Pollutants belonging to the most important groups of xenobiotics (PAHs, PCBs, and pesticides) were selected and their effect on hemocyte activities was analyzed using flow cytometry. Twenty-three pollutants were tested and eight of them showed significant modulation of hemocyte activities. PAHs and PCB 77 induced a decrease of hemocyte activity after an incubation periods of 4 and 24 h at 200 μmol/L. Three pesticides (2,4D, paraoxon, and chlorothalonil) modulated hemocyte activities. A mixture of eight pesticides also decreased phagocytotic activity. This study is one of the first to investigate the effects of so many pollutants on hemocyte functions at the same time and therefore allows a real comparison of different pollutant effects     

Cytophysiological features of hemocytes in cockroaches

Summary The wide range of functional activities of circulating and sessile insect hemocytes expresses itself in highly specialized cytological terms. Electron microscopic studies carried out in five species of normal and experimentally manipulated cockroaches, in conjunction with light microscopic information, reveal a broad spectrum of structural organization and an apparent capacity for cellular modulation. In addition to conventional organelles, these hemocytes contain a class of unusual cytoplasmic inclusion bodies which seem to undergo striking transformations in response to specific functional demands. A variety of transitional forms attests to the existence of close links between tubule containing (type 1), electron dense (type 2), and large globular (type 3) inclusion bodies, and reveals the derivation of yet another special (lamellated, fusiform) inclusion from type 2 bodies. Confluence of the type 3 configurations into still larger lacunae may precede the release of their contents into the hemolymph, a process whose major effect seems to be the initiation of the clotting process.Another important activity of hemocytes concerns the programmed reorganization of the stromal framework of the various organs. The dominant feature of blood cells engaged in the deposition of connective tissue are greatly distended cisternae of the rough endoplasmic reticulum and accumulations of banded fibrils at the interface of cytoplasm and extracellular space. The engulfment of discarded stromal material can be visualized in fortuitous sections representing steps in its incorporation by hemocytes. Ultrastructural correlates of the breakdown of these and other disintegrating or noxious elements by certain hemocytes are prominent digestive vacuoles with heterogeneous contents and reaction products of hydrolytic enzymes. The capacity for the uptake of small particles by micropinocytosis is demonstrated by the localization of horseradish peroxidase activity at the cellular surface and within cytoplasmic vesicles.The diversity of structural appearances reflects a division of labor, while the many transitional features of hemocyte morphology favor the concept of functional flexibility of one basic cell type rather than a strict classification into distinctly separate cellular types.Supported by research grants NB-05219, NB-00840, and 5 P01 NS-07512 from the U.S.P.H.S.I am greatly indebted to Dr. Joseph Osinchak, City University, New York, who has generously permitted me to include some of his micrographs in the present study. I also want to express my gratitude to Mrs. Sarah Wurzelmann and Mr. Stanley Brown for their excellent technical assistance.     

Affiliation of large numbers of fibroblasts with larval muscle fibers

Muscle fibers from fourth and fifth instar caterpillars were examined with scanning and thin section electron microscopy. Scanning micrographs showed that early fifth instar specimens had a population of cells lying beneath the basal lamina over the surface of the muscle fiber and in conjunction with tracheoles and nerves. At least two cell types were present. One type could be categorized as tracheoblasts of their close association with the tracheoles and the presence of taenidia within the tracheoblast cytoplasm in sectioned material. A second cell type, characterized by long filamentous processes, contained extensive rough endoplasmic reticulum and cisternae swollen with an electron-dense substance similar in appearance to the basal lamina. This ultrastructural appearance is characteristic of vertebrate fibroblasts and certain types of insect hemocytes. Early and late fourth instar specimens had few cells on their muscle fiber surfaces. Measurements of the basal lamina thickness were taken from thin sections of nondigested muscle fibers of early fourth, late fourth, and early fifth instar animals. The results showed that the basal lamina underwent a large increase in thickness between the fourth and fifth instars. The proliferation of cells which appeared to be in an actively synthesizing state paralleled the increase in basal lamina thickness. This suggests the hypothesis that these cells are active in connective tissue formation, and contribute to the formation of the basal lamina that lies over both them and the muscle fiber.     

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.