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Histochemical and biochemical investigations concerning the function of larval oenocytes of Tenebrio molitor L. (Coleoptera, Insecta).

Larval oenocytes of Tenebrio molitor were investigated histochemically. In contrast to the lipid droplets of the fat body, they did not stain wit Sudan black. A positive reaction for lipoproteins appeared only after destructive oxidation with sodium hypochlorite. These lipoproteins are the remnants of degenerated membranes, as revealed by ultrastructural analysis. Polyphenols could be identified in th exocuticle of exuvia, and in the newly formed procuticle. Endocuticle, epidermis and oenocytes showed no staining reaction. In oenocytes a great amount of lipase is also present which could be detected with several Tweens as substrates. The significance of these lipases remains unclear, since only few glycerides are synthesized in the cells, as shown below. They may play a role in the extended membrane turnover observed in this cell type. In vitro incubation of oenocytes of the larval generation demonstrated that 14C-labeled acetate was only incorporated into the paraffin fraction. A negligible amount of the label was found in glycerides; wax esters were free of label. Larval epidermis is also capable of paraffin formation, but only to a small degree. Oenocytes of the imaginal generation located between the sternal epidermis cells of pupae and adults do not synthesize paraffins, but other more polar compounds not yet identified. Labeled waxes in cuticular lipids were detected only when 14C-acetate was injected into whole larvae, and the lipids extracted some hours later. Autoradiographs demonstrated that 14C-acetate was intensively incorporated into larval oenocytes, the rate varying in different cells. Incorporation into the epicuticle, probably into the wax layer, was clearly shown. Cuticulin and dense layer do not show an intensive label. The lamellated cuticle also seems to be impregnated with acetate derivatives.

Acetates

Toxicity of four common pollutants to the freshwater macroinvertebrates Chironomus riparius Meigen (Insecta:Diptera) and Gammarus pulex (L.) (Crustacea: Amphipoda).

The lethal toxicities of the four pollutants 3,4-dichloroaniline (DCA), atrazine, copper, and lindane were determined for the 2nd larval instar of the insect Chironomus riparius Meigen and the juvenile stage (2nd or 3rd moult) of the crustacean Gammarus pulex (L.). Median lethal concentrations (LC50s) were determined over a 240 h test period. The order of toxicity of the test chemicals is different for each species. For C. riparius, lindane was the most toxic, followed by copper, DCA, and atrazine. During the first 96 h of exposure, the order for G. pulex was copper, lindane, then DCA and atrazine with similar LC50 values. However, at 240 h lindane replaced copper as the most toxic chemical to G. pulex. The relative sensitivity of the two species was dependent on both the toxicant and the exposure period. The lethal concentrations determined for the four chemicals are compared to the results of other toxicity studies and discussed with respect to current standard test methods.

Aniline Compounds

Correlations between glucose-inhibition and control parameters of alpha-glucosidase kinetics in Apis mellifica haemolymph (Hymenoptera: Insecta).

Kinetics studies of haemolymph alpha-glucosidase inhibition by D. glucose led to general correlations between inhibition and control parameters, all over the honeybee development. Maximum velocities are not affected by the inhibition, while affinity constants are always significantly increased, and Hill coefficients tend to decrease, especially in for-aging adults and in prenymphs. In this later case, the 'n-type' effect tends to break the manifestation of the 'K-type' mechanism.

Animals

Genetic structure and phyletic relationships of eastern Mediterranean Bacillus atticus brunner (Insecta Phasmatodea): a biochemical study.

The allozymic characterization of several new Croatian, Greek, and Turkish samples thought to belong to different subspecies of Bacillus atticus or to atticus-like taxa is given. Several allelic combinations (zymotypes) were observed among both diploid and triploid samples; the occurrence of highly different levels of heterozygosity for the same locus among populations is also common. The biochemical-genetic features of the numerous zymotypes are interpreted on the basis of the recently assessed cytology of their parthenogenetic reproduction. Biochemical and meiotic features also allow one to suggest that both diploid and triploid cytotypes of B. atticus are more likely interracial hybrids in origin. The new triploid Greek samples show only small genetic distances from the Turkish triploid and diploid ones; also, they do not show clear-cut morphological differences, so that all triploids and Turkish diploid samples are together referred to as B. a. carius. On the other hand, all Croatian, Greek, and Italian diploids appear to belong to the same electrophoretic cluster, biochemically differentiated at a subspecific level from B. a. carious. This newly defined comprehensive group of diploid samples, which also morphologically show gradual patterns of variation, is referred to as B. a. atticus.

Animals

Effects of heavy metals (Hg2+, cd2+, Pb2+) during the embryonic development of acridid grasshoppers (Insecta, caelifera)

Heavy metals present in the soil get differently accumulated in organisms and show different rates of toxicity at different stages of the life cycle of any organism living there. To see whether such toxicants get accumulated during the embryonic development and/or change the normal developmental processes of organisms exposed to heavy metals, freshly laid eggs and egg pods of two acridids, Aiolopus thalassinus and Eyprepocnemis plorans, were tested against different concentrations of Hg2+, Cd2+, and Pb2+. At increasing Hg2+ concentration in the substrate, an increased egg mortality and reduced nymphal hatching in A. thalassinus were observed, yet no change in the normal duration for embryonic development was noticed. A higher hatching rate of E. plorans nymphs than that of A. thalassinus could possibly be due to the higher tolerance, contributed by larger egg volume and thicker foam around the egg pods of the former. Treated concentrations of Hg2+ could be of sublethal (0.121 &mgr;g Hg2+/g substrate) to lethal (0.605 &mgr;g and more Hg2+/g substrate) doses. The mortality of eggs did not always increase with increasing substrate concentration of Cd2+ and Pb2+, and the hatching rates of both grasshopper species in many cases were even higher than that in the control. Still, lower accumulation factors of Cd2+ and Pb2+ than that of Hg2+ were found coupled with not increased mortality. The tested concentrations of Cd2+ and Pb2+ in the substrates, thus, could be of subacute doses. The embryonic development was found prolonged due to Cd2+ and Pb2+. During parallel egg and egg pod treatments, higher metal concentrations were found in not developed eggs than that in fresh nymphs hatching from the same substrates. This indicated the role of foam around the egg pods as "protective filter" against the toxic substances in the soil.

Journal Article

Subcellular effects and localization of binding sites of phytohemagglutinin in the potato leafhopper, Empoasca fabae (Insecta: Homoptera: Cicadellidae).

To identify the means by which phytohemagglutinin (PHA) exerts its toxicity on the potato leafhopper, four different methods (thick and semi-thin sectioning combined with immunofluorescent staining, in vitro receptor autoradiography, and immunoelectron microscopy) were used to elucidate the PHA target tissue, binding site, and its effects on this tissue. Sixteen 1- or 2-day-old female potato leafhoppers were fed for 36 h on each of three treatments: a control, diet or a diet containing either the PHA-E subunit or the PHA-L subunit. The PHA-E subunit, but not PHA-L, had previously been shown to be lethal. The insects were then prepared for both light and confocal microscopy. Analysis of images showed that PHA bound only to the surface of midgut epithelial cells of the potato leafhopper. PHA-E caused severe disruption, disorganization, and elongation of the brush border microvilli, and swelling of the epithelial cells into the lumen of the gut, leading to complete closure of the lumen. Furthermore, PHA-E stimulated the division of midgut epithelial cell nuclei, leading to two nuclei in each cell. Nuclei later elongated and degraded. In contrast, PHA-L had little effect on the epithelial cells of the midgut. It did not strongly bind to the surface of epithelial cells and caused much less disruption of brush-border microvilli, less disorganization of the cells and less elongation of nuclei. Strong binding of PHA occurred solely on the cell membrane of the brush border microvilli of epithelial cells. In contrast, the controls (i.e., midgut tissue, blocking agent, PHA, and antibodies) showed that midgut tissue was not autofluorescent and showed no fluorescent binding signal. Analysis of both bright- and dark-field images obtained by autoradiography and immunoelectron microscopy confirmed these findings.

Animals

Ecdysteroid resistant subclones of the epithelial cell line from Chironomus tentans (Insecta, Diptera). I. Selection and characterization of resistant clones.

Chironomus tentans cells were cultured in the presence of gradually increasing concentrations of 20-OH-ecdysone or a nonsteroidal molting hormone agonist, the benzoylhydrazine RH 5992, for a period of about 2 yr. From these cultures, subclones were selected, which are resistant to up to 25 microM 20-OH-ecdysone according to morphological (changes in cell shape and cell arrangement) and physiological criteria (acetylcholinesterase induction, secretion of chitinolytic enzymes, thymidine incorporation). Some subclones, selected in the presence of 20-OH-ecdysone, are resistant only to molting hormone, but still respond to RH 5992 morphologically and biochemically, whereas subclones selected in the presence of the benzoylhydrazine showed no reaction neither to 20-OH-ecdysone nor to the hormone agonist. Hormone resistance is stable; 3 mo. after hormone withdrawal, resistant clones still do not respond to renewed exposure to 20-OH-ecdysone or RH 5992, respectively. Because in all resistant subclones tested so far all hormonally regulated responses known from sensitive cells were no longer detectable, it is assumed that the hormone signaling pathway itself is interrupted. Possible mechanisms of hormone resistance were discussed.

Acetylcholinesterase

Forskolin-insensitive adenylate cyclase in cultured cells of Choristoneura fumiferana (Insecta).

Adenylate cyclase from a spruce budworm cell line, IBRI-Cfl, is activated by octopamine (Ka = 50 microM), guanine nucleotides and sodium fluoride but not by forskolin. In addition, forskolin does not potentiate the octopamine-sensitive response. To our knowledge, this is the first published report of a hormone-sensitive adenylate cyclase, with a functional guanine nucleotide regulatory protein, that is insensitive to forskolin both in intact cells and washed membrane preparations.

1-Methyl-3-isobutylxanthine

Purification of a protease inhibitor which controls prophenoloxidase activation in hemolymph of Locusta migratoria (insecta).

A protein which inhibits the prophenoloxidase----phenoloxidase (EC 1.14.18.1) proteolytic activation in hemocyte extracts of Locusta migratoria was isolated from the plasma of the same insect and partially characterized. It shows a molecular weight of 14,000, an inhibiting activity toward the cascade system in the insect hemocytes, which resulted in a lower production of phenoloxidase, a key enzyme for the defence mechanism in arthropods. To identify the specificity of the Locusta inhibitor and consequently the specificity of its target enzyme, inhibitory tests were performed against a number of known serine-proteases. A strong in vitro inhibiting activity toward chymotrypsin and, to a lesser extent, toward human leukocyte elastase was present, while trypsin, Carlsberg subtilisin, human thrombin and pancreatic elastase failed to react. The lack of trypsin inhibition by the isolated inhibitor suggested that the trypsin-catalysed activation of the system in the hemocyte extract takes place under different controls or at an earlier stage of the cascade. The N-terminal sequence of the inhibitor reveals that this molecule is different from the protease inhibitors isolated from other arthropods.

Amino Acid Sequence

Further experimental evidence for the involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta, Orthoptera).

Ecdysone has recently been shown to be able to trigger meiotic reinitiation in vitro in submature oocytes of Locusta. In the present study we have experimentally depressed (by 60-70%) ecdysone biosynthesis in the ovaries of adult females by rearing them on a diet with a modified sterol profile. Mature oocytes from such females fail to undergo normal reinitiation, but when incubated in vitro, can be induced to break their meiotic arrest by the addition of exogenous ecdysone. These results lend further support to the hypothesis that in Locusta, ovarian ecdysone is involved in the control of meiotic reinitiation.

Animals