Hilda Raes

No reduction of energy metabolism in Clk mutants.

Mutation in any of the four clock genes (clk-1, clk-2, clk-3, gro-1) causes an average slowing down of many temporal processes, and an increase of mean life span. The latter effect has been linked to the slow phenotype, and it has been reasoned that any reduction of the rate of living would reduce the load of oxidative damage, which is thought to drive the ageing process. To test this model we measured several parameters describing metabolic output in wild type worms and all four Clk mutants. We found no gross changes in metabolic output, as assessed from oxygen consumption and heat production rates, lucigenin-mediated light production capacity, ATP content, and lipofuscin autofluorescence. Catalase and superoxide dismutase (SOD) were variably altered, but not cooperatively, as would be expected to enhance reactive oxygen species (ROS) scavenging activity. Thus we conclude that the prolonged life span of Clk mutants cannot be attributed to reduced metabolic rate or an increased activity of the major antioxidant enzymes catalase and SOD.

Heavy-metal toxicity in an insect cell line : effects of cadmium chloride, mercuric chloride and methylmercuric chloride on cell viability and proliferation in Aedes albopictus cells

We evaluated the toxicity of CdCl2, HgCl2, and MeHgCl on the C6/36 cell line of Aedes albopictus. This cell line proved to be a suitable tool for studying heavy-metal toxicity in insect cells. Since data on heavy-metal toxicity in invertebrate cell cultures are almost nonexistent, our results are discussed in relation to in vivo invertebrate and in vitro vertebrate studies. Viability and proliferation were assessed by dye exclusion and DNA quantification, respectively. Viability tests were carried out with and without 5% fetal calf serum in the medium. The three metal species decreased viability to different extents (MeHgCl>HgCl2>CdCl2), and fetal calf serum had a protective effect. In serum-deprived cultures, LD50 values were 140.20, 2.51, and 2.08 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. For cultures with fetal calf serum, LD50 values were 149.71, 12.01, and 5.47 µmol/L, respectively. The viability curve for CdCl2 under serum-free conditions suggests the induction of a cell defense system. The three metal species also inhibited cell proliferation (MeHgCl> CdCl2> HgCl2). The IC50 values were 1.75, 18.36, and 0.96 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. In summary, low MeHgCl concentrations caused both cell death and inhibition of cell proliferation; HgCl2 primarily disrupted the plasma membrane, whereas CdCl2 primarily inhibited cell proliferation.

Cadmium pathology in an insect cell line: ultrastructural and biochemical effects.

Cadmium (Cd) pathology was studied in an insect cell line (Aedes albopictusC6/36) at the ultrastructural level. The most prominent pathological changes occurred at the level of the nucleus: chromatin clumping, indentations, filling and dilatation of the perinuclear cisternae and an increased amount of bound ribosomes were observed. In the cytoplasm, condensation and swelling of mitochondria, increase of both free and membrane-bound ribosomes, filling and dilatation of the rough endoplasmic reticulum, and increase of the lysosomal system were the most conspicuous effects. The increased content of the perinuclear and cytoplasmic cisternae was probably due to an increased protein synthesis or a disturbance of the protein export system. This picture differed clearly from the osmotically swollen electron-lucent cisternae that have been described in other pathological situations. The enhancement of the lysosomal system was paralleled by a slight but significant stimulation of the acid phosphatase activity in the sublethal Cd concentration range. In vitro experiments suggested that Cd probably acts directly on this enzyme. Abnormal medium acidification in cultures treated with low Cd levels was correlated with an increased production of lactic acid. Together with the morphological data, this suggested a Cd-induced impairment of the aerobic metabolism.

EFFECT OF SUBLETHAL DOSES OF CADMIUM, INORGANIC MERCURY AND METHYLMERCURY ON THE CELL MORPHOLOGY OF AN INSECT CELL LINE (AEDES ALBOPICTUS, C6/36)

The effect of CdCl2(44μm), HgCl2(3.7μm), and MeHgCl (2μm) on the morphology ofAedes albopictusC6/36 cells was studied at the light microscopical level. Treatment times and metal concentrations were in the sublethal range as determined by a fluorometric dye exclusion test. The three metal species had profound effects on the cell morphology. MeHgCl treatment induced the development of a large number of short, actin‐supported, tangled filopodia. Both CdCl2and HgCl2induced long extensions. Pretreatment with colchicine but not with cyto‐chalasin B prevented formation of these extensions which suggests that they were supported by microtubules. This was confirmed by immunostaining for microtubules. The extensions were relatively stable towards colchicine post‐treatment. To authors’ knowledge, this effect has not yet been described for heavy metals. The similarity with 20‐hydroxyecdysone‐treated cells and the occurrence of cytoplasmic feet in insect cells is discussed.

Organisation and ultrastructure of the regenerative crypts in the midgut of the adult worker honeybee (L. Apis mellifera)

The midgut epithelium of the adult honeybee consists of columnar and endocrine cells, both originating from regenerative crypt cells. The regenerative crypt is composed of stem cells and differentiating cells. The stem cells generate two forms of endocrine cells along with differentiating enterocytes of two distinct stages. At first they can be seen as light crypt cells which are not secretory active; they then develop into more electron dense, active secretory crypt cells. The developing enterocytes are arranged as tetrads, each composed of cells with the same degree of differentiation. This can be explained by the occurrence of cytoplasmic bridges between the cells of each tetrad. These fusomes are probably responsible for the apparent intercellular coordination. This is a new example of intercellular bridges between somatic cells and, as far as we know the first description of fusomes in the insect midgut epithelium. The microvilli on top of the crypt cells develop within a spherical extracellular space where glycosaminoglycans are secreted. This occurs by the coordinated activity of the four surrounding electron dense crypt cells. Microvilli formation therefore seems to be the first in a series of successive functions of honeybee enterocytes during their ontogeny.

Light and electron microscopical study of two types of endocrines cell in the midgut of the adult worker honeybee (apis mellifera).

The occurrence, development and ultrastructure of two types of gut endocrine cell have been studied in the midgut of adult honeybees. These cells, one of a basal granular type and one of a vesicular type, are evenly distributed throughout the posterior three-quarters of the midgut. Each crypt complex contains one of each cell type, both of which may be derived from the same stem cells as the enterocytes. They already contain their respective secretory product while still in the nidus. Both reach the midgut lumen by a narrow apex and are therefore of the open type. The granular cells release their secretory granules at the cell base in a typical endocrine way. In young vesicular cells the secretory vesicles are released at the cell base and in the intercellular spaces. Old cells are still filled with vesicles when they are shed in the midgut lumen. This seems to indicate that these cells have both an endocrine (or paracrine) and an exocrine function, the latter apparently by holocrinc release.

Distribution, accumulation and depuration of administered lead in adult honeybees

In this study the Pb concentration in honeybees was determined by graphite furnace AAS after peroral administration of PbCl2. The Pb concentration, expressed on a dry weight base was determined in relation to the distribution over the body, the accumulation time, the clearance and the exposure dose. Pb is concentrated in the digestive system with the midgut accounting for 67% and the rectum for 27% of the accumulated metal. The barrier function of this system is thus corroborated in honeybees. Pb accumulation happens slowly in young bees which feed mainly on pollen; once they switch from pollen to nectar there is a sharp rise in their Pb content. After the 26th day a limit of tissue accumulation seems to be reached. Pb accumulation is equally efficient when the contamination starts at a forager age. Pb clearance is slower than expected; after 12 days only one third of the initial lead burden has been cleared. The Pb concentration in the animals increases significantly with the Pb concentration in the sugar syrup which they are fed, except at the highest dose of 50 mg/l. The possible use of honeybees as biomonitors for Pb pollution seems promising from these results.

Cultured mosquito cells Aedes albopictus C6/36 (Dip., Culicidae) responsive to 20-hydroxyecdysone and non-steroidal ecdysone agonist

Abstract: A cell line derived from an important mosquito, Aedes albopictus Skuse, was responsive to treatments with the insect moulting hormone 20‐hydroxyecdysone (20E) and two new insecticides, RH‐5849 and RH‐5992, by cell clumping and inhibition of cell proliferation. The Aedes C6/36 cells showed typical numerous long extensions, and fluorescent BODIPY®‐phalloidin staining demonstrated that they resulted from F‐actin polymerization. In addition, the ultrastructure of treated cells showed a high degree of vacuolarization that is in accord with protein synthesis. SDS‐PAGE demonstrated that three proteins were induced that may belong to the hsp70 family. In conclusion, an insect cell line that responds to 20E may be useful for screening and identifying hormone analogues and novel insecticides.

Copper induces apoptosis in Aedes C6/36 cells.

The Aedes albopictus C6/36 cell clone is used as a model system to study the effects of heavy metals on insect cells. Here we report on the effects of Cu(2+) on these cells. Similar to Cd(2+) and Hg(2+), Cu(2+) induces hyperpolymerization of the microtubules; moreover, with Cu(2+) this is followed by cell aggregation and massive apoptosis. This process, which is cell density dependent, is maximal between 0.75 and 1 mM; this is just under the LC(50) as determined by a membrane integrity test. At higher Cu(2+) concentrations, cell death occurs by necrosis. Apoptosis was ascertained by fluorescence and electron microscopy and by agarose gel electrophoresis. At 0.75 mM, apoptosis started at 18-hr exposure time and the amount of apoptotic cells increased almost linearly until 42 hr; then a plateau was reached with 70-80% apoptotic cells. This is the first report on Cu(2+)-induced apoptosis in insect cells. Possible induction mechanisms are discussed in the light of existing literature on vertebrate cells.