A comparative study of specific chromatic adaptation of the wildtype and trp mutant of Drosophila melanogaster

The trp mutant of Drosophila melanogaster was re-examined and compared with the wildtype using monochromatic blue and orange light to manipulate the bi-stable visual pigment states in the peripheral retinula cells R1-6 of white-eyed flies. Recovery of sensitivity by application of orange light either during or after blue-adaptation is different in w;trp flies from that in bw;cn flies and does not proceed as predicted from the trp genotype. Blue-adaptation by isolating the activity of the central retinula cells confirms that the trp lesion affects these receptors also.     

Effects of zinc on programmed cell death of Musca domestica and Drosophila melanogaster blood cells

Programmed cell death (PCD) and phagocytotic activity of immune cells play a pivotal role in insect development. We examined the influence of Zn²⁺, an important element to fundamental biological processes, on phagocytosis and apoptosis of hemocytes in two fly species: Musca domestica and Drosophila melanogaster. Hemocytes were isolated from the third instar larvae of both species and treated for 3 h with zinc chloride solutions, containing 0.35 mM or 1.7 mM of Zn²⁺, and untreated as control. Phagocytotic activity of hemocytes was examined by flow cytometry after adding latex fluorescent beads to the medium, while apoptosis was evaluated by application of annexinV-FITC and pan-caspase-FITC inhibitor. Mitochondrial viability was determined by measuring resazurin absorbancy in the cell medium. The obtained results showed that Zn²⁺ increases phagocytosis and affects PCD of both species hemocytes but each in a different way. Zinc decreases fraction of annexin-positive hemocytes in M. domestica but increases it in D. melanogaster. The pan-caspase analysis revealed low and high activity of caspases in hemocytes of M. domestica and D. melanogaster, respectively. Zn²⁺ also decreased the viability of hemocyte mitochondria but only in D. melanogaster. It suggests that flies use different pathways of PCD, or that Zn plays a different role in this process in M. domestica than in D. melanogaster.     

Membrane remodeling and glucose in Drosophila melanogaster: A test of rapid cold-hardening and chilling tolerance hypotheses

Insect cold tolerance varies at both the population and species levels. Carbohydrate cryoprotectants and membrane remodeling are two main mechanisms hypothesised to increase chilling tolerance in Drosophila melanogaster, as part of both long-term (i.e., evolutionary) change and rapid cold-hardening (RCH). We used cold-selected lines of D. melanogaster with and without a pre-exposure that induces RCH to test three hypotheses: (1) that increased cold tolerance would be associated with increased free glucose; (2) that increased cold tolerance would be associated with desaturation of membrane phospholipid fatty acids; and (3) that increased cold tolerance would be associated with a change in phospholipid head group composition. We used colourimetric assays to measure free glucose and a combination of thin layer chromatography-flame ionization detection and gas chromatography to measure membrane composition. We observed a consistent decrease in free glucose with RCH, and no relationship between free glucose and basal cold tolerance. Also, phospholipid head group ratios and fatty acid composition showed no change following an RCH treatment. Thus, we conclude that changes in free glucose and membrane composition are unlikely to be significant determinants of variation in cold tolerance of D. melanogaster.     

Longevity of seven species of cactophilic Drosophila and D. melanogaster on carbohydrates

Adults of 6 species of Drosophila that use decaying prickly pear cactus (Opuntia sp.) as breeding and feeding sites were compared to each other and to D. nigrospiracula, whose host is saguaro cactus, and to the cosmopolitan D. melanogaster, in their utilization of 21 sugars for longevity (time to 50% mortality). In general, the utilization of sugars by these flies for longevity followed the pattern observed with the other insects. None of the species were able to live very long on solutions of pentoses, uronic acids, inositol, rhamnose, sorbose or the β-linked disaccharides, lactose and cellobiose. Althogh all could use glucose, fructose, sucrose, maltose and melezitose well, their life spans on galactose, mannose, trehalose and raffinose were more variable. Two of the Opuntia feeders were also tested on a number of other carbohydrates. Ribitol, mannitol, sorbitol and xylitol significantly prolonged the life of D. arizonensis but not that of D. wheeleri. Neither species lived long on solutions of arabitol, galactitol, starch, inulin or on arabogalactan.     

Multiple function of pteridines in Drosophila: The fluorescence of the ejaculatory bulb in Drosophila melanogaster

It is demonstrated that the strong fluorescence of the ejaculatory bulb of Drosophila melanogaster males is caused by the presence of pteridines. The pteridine composition in the bulb is affected by the mutations ry² and ma-l^F1 in which isoxanthopterin has also been detected. Our results show that the bulbs of wild-type and white-eyed mutant males possess the same pteridines. Some data suggest that the bulbal pteridines originate from the testis region. Partly on the basis of former histochemical findings it is suggested that in the bulbal cavity the pH is high favouring the fluorescent dihydro-states of the pteridines present. All these and additional literature data on the ejaculatory bulb are discussed in connection with various biological processes. Some internal larval structures in which pteridines play or might play a functional role were found to present autofluorescence.     

Radioprotective effect of six chemicals against X-ray-induced genetic damage in D. melanogaster

In Drosophila melanogater six chemicals were tested for radioprotectiveeffect against X-ray-induced genetic damage such as sex-linked recessive lethals and autosomal translocations using Oster's ring-X chromosome stock. A 2-day brood pattern was followed to score the damage induced at different spermatogenic stages separately. In all cases the chemicals were injected before X-irradiation. 10-mM solution of reduced glutathione (GSH) provided statistically significant protection against sex-linked recessive lethals in all broods. In translocation tests this chemical reduced the frequency in all broods but the result is not statistically significant. Cysteamine (MEA) did not show any protective effect but the frequency of lethals was slightly reduced in the first and fourth broods. 2-Aminoethyl isothiuronium Br·HBr (AET) showed a statistically significant protective effect when the data of the replicate experiments were pooled. Negative results were obtained for 5-hydroxytryptamine (5-HT) in sex-linked lethal tests. Aminoethyl phosphorothioate (AEPT) reduced the frequencies of both sex-linked lethals and autosomal translocations in all broods consistently but the results are not statistically significant. In tests for both lethals and translocations the reduction was largest in the stages with highest radiosensitivity. N(3-Aminopropyl)aminoethyl phosphorothioate (3AP-AEPT) gave no protection.     

Protein and carbohydrate composition of larval food affects tolerance to thermal stress and desiccation in adult Drosophila melanogaster

Larval nutrition may affect a range of different life history traits as well as responses to environmental stress in adult insects. Here we test whether raising larvae of fruit flies, Drosophila melanogaster, on two different nutritional regimes affects resistance to cold, heat and desiccation as well as egg production and egg-to-adult viability. We raised larvae on a carbohydrate-enriched and a protein-enriched growth medium. We found that flies developed on the high protein medium had increased heat and desiccation tolerance compared to flies developed on the carbohydrate-enriched medium. In contrast, flies developed on the carbohydrate-enriched growth medium recovered faster from chill coma stress compared to flies developed on a protein-enriched medium. We also found gender differences in stress tolerance, with female flies being more tolerant to chill coma, heat knockdown and desiccation stress compared to males. Egg production was highest in females that had developed on the protein-enriched medium. However, there was a sex-specific effect of nutrition on egg-to-adult viability, with higher viability for males developing on the sucrose-enriched medium, while female survival was highest when developing on the protein-enriched medium. Our study indicates that larval nutrition has a strong impact on the ability to cope with stress, and that the optimal nutrient composition varies with the type of stress.     

A genetic and dietary study of the physiology of pyrimidine synthesis in Drosophila melanogaster

A combination of genetic and dietary manipulations have been utilized to investigate the physiology of pyrimidine metabolism throughout the Drosophila life cycle. We present evidence that the dietary sources of pyrimidines ingested at the larval stage are sufficient for all subsequent stages of the life cycle, except the process of oögenesis in adult females where a maternal supply of endogenously synthesized pyrimidines is normally required. Deprivation of dietary and de novo synthesis does not affect adult longevity; indicating that with the exception of oögenesis, all normal functions are carried out by recycling pyrimidines produced or ingested at the larval stage.In an enzymatic analysis, we have determined that Drosophila does not adjust for pyrimidine dietary deficiencies by significantly altering the level of synthesis of two tested enzymes encoded by the rudimentary locus, even though the pyrimidine deficiency is a rate limiting step in the completion of development.     

A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

The function of the gene gilgamesh (89B9-12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with γ-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.     

The brain can eat: Establishing the existence of a central pattern generator for feeding in third instar larvae of Drosophila virilis and Drosophila melanogaster

To establish the existence of a central pattern generator for feeding in the larval central nervous system of two Drosophila species, the gross anatomy of feeding related muscles and their innervation is described, the motor units of the muscles identified and rhythmic motor output recorded from the isolated CNS. The cibarial dilator muscles that mediate food ingestion are innervated by the frontal nerve. Their motor pathway projects from the brain through the antennal nerves, the frontal connectives and the frontal nerve junction. The mouth hook elevator and depressor system is innervated by side branches of the maxillary nerve. The motor units of the two muscle groups differ in amplitude: the elevator is always activated by a small unit, the depressor by a large one. The dorsal protractors span the cephalopharyngeal skeleton and the body wall hence mediating an extension of the CPS. These muscles are innervated by the prothoracic accessory nerve. Rhythmic motor output produced by the isolated central nervous system can simultaneously be recorded from all three nerves. The temporal pattern of the identified motor units resembles the sequence of muscle contractions deduced from natural feeding behavior and is therefore considered as fictive feeding. Phase diagrams show an almost identical fictive feeding pattern is in both species.     

Saxitoxin binding to sodium channels in head extracts from wild-type and tetrodotoxin-sensitive strains of Drosophila melanogaster

Extracts prepared from heads of Drosophila melanogaster show high-affinity binding ($\text{K}{}_{\mathrm{<mtext>D</mtext>}}\text{= 1.9}\text{nM}$) of [³H]saxitoxin, a compound known to bind to and block voltage-sensitive sodium channels in other organisms. The interaction between saxitoxin and the Drosophila saxitoxin receptor is non-cooperative and reversible with a half-life of 18.3 s for binding at 4°C. The saturable binding is specifically inhibited by tetrodotoxin with a $\text{K}{}_{\mathrm{<mtext>I</mtext>}}\text{= 0.30}\text{nM}$. The number of saturable binding sites in the extract is 97 fmol/mg protein. Since approx. 50% of the binding activity is recovered in the extract, the number of binding sites in the head is estimated to be 6.4 fmol/mg head. Nerve conduction in Drosophila larvae is completely blocked after 20 min in a bathing solution containing 200 nM tetrodotoxin. A comparison between the binding and the electrophysiological studies in Drosophila and other organisms suggests that the Drosophila saxitoxin receptor is part of the voltage-sensitive sodium channel involved in the propagation of action potentials. A mutant (ttx^s), which is abnormally sensitive to dietary tetrodotoxin, is shown to be indistinguishable from wild type with respect to [³H]saxitoxin-binding properties and physiological sensitivity to tetrodotoxin. These studies provide techniques which can be used to identify mutants with defects in the saxitoxin-binding component of the sodium channel.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.     

Cellular internalization and stress response of ingested amorphous silica nanoparticles in the midgut of Drosophila melanogaster

Background

Amorphous silica nanoparticles (aSNPs) are used for various applications including food industry. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the midgut cells of an organism. The study aims to examine cellular uptake of aSNPs (< 30 nm) in the midgut of Drosophila melanogaster (Oregon R⁺) owing to similarities between the midgut tissue of this organism and human and subsequently cellular stress response generated by these nanoparticles.

Methods

Third instar larvae of D. melanogaster were exposed orally to 1–100 μg/mL of aSNPs for 12–36 h and oxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bromide staining), cellular internalization (TEM) and apoptosis endpoints.

Results

A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in a concentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 along with caspases activation, membrane destabilization and mitochondrial membrane potential loss was also observed. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles and by direct membrane penetration.

Conclusion

aSNPs after their internalization in the midgut cells of exposed Drosophila larvae show membrane destabilization along with increased cellular stress and cell death.

General significance

Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thus their industrial use as a food-additive may raise concern to human health.     

Crystalline layer in Drosophila melanogaster eggshell: arrangement of components as revealed by negative staining and reconstruction

Eggshell formation in Drosophila melanogaster is used as a model system in studies of cellular differentiation. A detailed knowledge of eggshell structure is necessary for this purpose and also to permit correlation of eggshell structure with function. Unique among the eggshell layers, the innermost chorionic layer (ICL) was investigated by means of transmission electron microscopy of thin sections and whole mounts, utilizing conventional fixation. LaNO₃ impregnation and negative staining with uranyl acetate. Whole mount face views of negatively stained ICLs were processed by means of optical and computer reconstruction. The ICL, which almost fully covers the oocyte, consists of 4 5 bilaminar sublayers with a total thickness of 400–500 Å. It appears to be formed by crystallites 1– μm in size, containing roughtly spherical molecules which are 30 Å in diameter approximately. Each unit cell probably includes 8 molecules and also distinct vacant spaces, differing in size, ICL may be involved in the exchange of the respiratory gases during embryogenesis.