Habitat selection by Drosophila melanogaster larvae

Drosophila melanogaster larvae are used to examine habitat choice behavior and its effect on a component of preadult fitness (pupal survivorship). We established strains of flies by collecting pupae from two microhabitats from an orchard. Strain differences in pupation site choice (on versus off fruit) persisted in a field-like laboratory assay without artificial selection. To produce heterogeneous environments, air temperature and soil water content were varied in these assays. A habitat suitability difference measure was used to determine for each environment, which microhabitat (on or off fruit) resulted in greater pupal survivorship. We found 1) that habitat choice behavior had both plastic and heritable components, 2) that strain-by-environment interactions influenced habitat choice behavior and pupal survivorship and, 3) a significant positive correlation between habitat suitability and larval habitat choice behavior.     

Proteomics of immune-challenged Drosophila melanogaster larvae hemolymph

In the last decade, the fruit fly Drosophila melanogaster has emerged as a promising invertebrate model for the investigation of innate immunity, in part because of its well characterised genetics. The information provided by the innumerous reports on Drosophila's immune response indicates that a large number of genes, in addition to the well-known antimicrobial peptide genes, are both up- and down-regulated upon immune challenge. Nevertheless, their contribution to fighting off infection has not been seriously addressed. With the application of recent advances in proteomics, the effects of an immune challenge in the overall modification of Drosophila 2-DE protein patterns were investigated. The aim of this study was to investigate hemolymph proteins differentially expressed between control and immunised larvae sets, which could be related solely to the Drosophila immune response. The list of immune-related protein spots included heat shock proteins and other proteins with chaperone properties, serine proteases, phenol oxidase, and Drosophila antioxidant system components, which accounted for 21% of the total of 70 identified proteins, metabolic enzymes implicated in pathways such as cellular respiration, fatty-acid oxidation, protein biosynthesis, and structural proteins.     

Serial polymers in the epidermis of Drosophila melanogaster larvae

The paper reports the existence of peculiar polymers (e-polymers) obtained from the epidermis of Drosophila melanogaster larvae. E-polymers result from the assembly of two components held together by alkali-labile bonds. Such components can be separated by CsCl density gradients and by DEAE-cellulose chromatography after controlled alkaline hydrolysis. One of the components contains predominantly neutral sugars and a phenolic substance (S-fraction). The other contains predominantly amino acids, aminosugars and a phenolic substance. This fraction can be visualized as serial multimers of a monomer subunit. It is suggested that e-polymers are continuous tridimensional structures which might have morphogenetic significance.     

Decarboxylases for polyamine biosynthesis in Drosophila melanogaster larvae.

Ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17) and S-adenosyl-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lase, EC 4.1.1.50) were assayed in Drosophilia melanogaster larvae. The highest enzyme activities were detected in 24 and 48 h larvae, with diminishing activities in subsequent larval stages. Stimulation of S-adenosylmethionine decarboxylase by putrescine was demonstrable in late but not in early stages of larval development.     

Isolation and characterization of lipophorin from Drosophila melanogaster larvae

The hemolymph lipoprotein lipophorin has been isolated from third-instar Drosophila melanogaster larvae by a technique that involves homogenization of whole larvae in a medium containing protease inhibitors and purification of the lipoprotein by density gradient centrifugation. Drosophila lipophorin has a density of 1.16 g/ml and is composed of 62.5% protein, 23.1% phospholipid, 7.4% diacylglycerol, 5.4% triacylglycerol, 0.9% hydrocarbon, and 0.7% sterol. As is the case with other insect lipophorins, Drosophila lipophorin contains two apolipoproteins, apolipophorin-I (M_r ≈ 275,000) and apolipophorin-II (M_r ≈ 76,000). Drosophila apolipophorin-I does not crossreact with antibodies prepared against apolipophorin-I from Manduca sexta.     

The involvement of catalase in alcohol metabolism in Drosophila melanogaster larvae

The involvement of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in the metabolism of alcohols was investigated by comparing Drosophila melanogaster larvae in which catalase was inhibited by dietary 3-amino-1,2,4-triazole (3AT) to larvae fed a diet without 3AT. 3AT inhibited up to 80% of the catalase activity with concordant small increases in the in vitro activities of sn-glycerol-3-phosphate dehydrogenase, fumarase, and malic enzyme, but with a 16% reduction in the in vivo incorporation of label from [14C]glucose into lipid. When the catalase activity was inhibited to different degrees in ADH-null larvae, there was a simple linear correlation between the catalase activity and flux from [14C]ethanol into lipid. By feeding alcohols simultaneously with 3AT, ethanol and methanol were shown to react efficiently with catalase in wild-type larvae at moderately low dietary concentrations. Drosophila catalase did not react with other longer chain alcohols. Catalase apparently represents a minor pathway for ethanol degradation in D. melanogaster larvae, but it may be an important route for methanol elimination from D. melanogaster larvae.     

Identification of vitelline membrane proteins in Drosophila melanogaster

In Drosophila melanogaster, proteins involved in vitelline membrane production are secreted by ovarian follicle cells during stages 9 and 10 of oogenesis. We have used SDS-PAGE and two-dimensional electrophoresis to identify six major size classes of radiolabeled components in purified vitelline membrane preparations. Analyses of in vivo labeled proteins from egg chambers of different developmental stages and stage 10 follicle cells show that components of five of these size classes are synthesized by follicle cells during the period of vitelline membrane deposition. Immunological analysis of eggshell antigens utilizing complex antisera raised to purified eggshell fragments has confirmed the identity of components of three size classes.     

Identification of chromosome inheritance modifiers in Drosophila melanogaster

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.     

Identification and characterization of Drosophila melanogaster paramyosin

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, alpha-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a lambda gt11 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.     

UCP4C mediates uncoupled respiration in larvae of Drosophila melanogaster

Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F‐ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva‐to‐adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.     

Hardening trumps acclimation in improving cold tolerance of Drosophila melanogaster larvae

Abstract Chill‐susceptible insects are able to improve their survival of acute cold exposure over both the short term (i.e. hardening at a relatively severe temperature) and longer term (i.e. acclimation responses at milder temperatures over a longer time frame). However, the mechanistic overlap of these responses is not clear. Four larval stages of four different strains of Drosophila melanogaster are used to test whether low temperature acclimation (10 °C for 48 h) improves the acute cold tolerance (LT₉₀, ～2 h) of larvae, and whether acclimated larvae still show hardening responses after brief exposures to nonlethal cold or heat, or a combination of the two. Acclimation results in increased cold tolerance in three of four strains, with variation among instars. However, if acclimation is followed by hardening pre‐treatments, there is no improvement in acute cold survival. It is concluded that short‐term thermal responses (e.g. hardening) may be of more ecological relevance to short‐lived life stages such as larvae, and that the mechanisms of low temperature hardening and acclimation in D. melanogaster may be antagonistic, rather than complementary.     

Observation of tyrosine-O-phosphate in Drosophila melanogaster larvae by 31P-NMR spectroscopy

31P-NMR spectra of intact larvae and pupae of Drosophila melanogaster have been obtained at 109.3 MHz. A major resonance in these samples has been identified as tyrosine-O-phosphate. Its chemical shift reflects the hemolymph plasma pH. Upon disruption of the organisms (necessary for chemical analyses of tyrosine-O-phosphate), phosphatases rapidly hydrolyze this phosphate ester, generating inorganic phosphate and free tyrosine.     

Image enhancement for tracking the translucent larvae of Drosophila melanogaster

Drosophila melanogaster larvae are model systems for studies of development, synaptic transmission, sensory physiology, locomotion, drug discovery, and learning and memory. A detailed behavioral understanding of larvae can advance all these fields of neuroscience. Automated tracking can expand fine-grained behavioral analysis, yet its full potential remains to be implemented for the larvae. All published methods are unable to track the larvae near high contrast objects, including the petri-dish edges encountered in many behavioral paradigms. To alleviate these issues, we enhanced the larval contrast to obtain complete tracks. Our method employed a dual approach of optical-contrast boosting and post-hoc image processing for contrast enhancement. We reared larvae on black food media to enhance their optical contrast through darkening of their digestive tracts. For image processing we performed Frame Averaging followed by Subtraction then Thresholding (FAST). This algorithm can remove all static objects from the movie, including petri-dish edges prior to processing by the image-tracking module. This dual approach for contrast enhancement also succeeded in overcoming fluctuations in illumination caused by the alternating current power source. Our tracking method yields complete tracks, including at the edges of the behavioral arena and is computationally fast, hence suitable for high-throughput fine-grained behavioral measurements.     

Location and connectivity of abdominal motoneurons in the embryo and larva of Drosophila melanogaster

The soma location and peripheral connectivity of motoneurons in abdominal segments of the embryo and larva of the fruitfly, Drosophila melanogaster are described as an initial step in determining the mechanisms by which motoneurons make connections with their target muscles in a genetically accessible organism. Embryonic motoneuron somata were retrogradely labelled by application of the fluorescent dye, DiI, to the whole peripheral nerve or to its separate anterior or posterior fascicles in segments A5-A7 of late stage 15/early stage 16 embryos. This technique reveals a stereotyped, segmentally repeated population of 34 motoneurons per hemisegment, several of which can be individually identified from their soma position. The same set of motoneurons was revealed in third instar larvae of D. melanogaster by cobalt backfilling of abdominal peripheral nerves, although the positions of some of these neurons change during larval development. The peripheral connectivity and axon morphology of several of the abdominal motoneurons was determined by intracellular injection with Lucifer Yellow in stage 16 embryos. For the motoneurons with axons in the anterior fascicle there is no clear relationship between somata groupings and the muscle targets innervated: contrary to earlier claims, these motoneurons arborize over both ventral and dorsal muscles. Individual motoneurons possess a stereotyped pattern of terminal arborization.     

The function of anal papillae in salt adaptation of Drosophila melanogaster larvae

The anal papillae of Drosophila melanogaster larvae showed strong developmental plasticity in media with different osmotic pressure. The size of the papillae decreased with increasing salt content of the medium, which supports the hypothesis of its absorptive function. We showed that CI-transport at the site of the anal papillae decreased with increasing salt concentration, which also supports the hypothesis, that the function of anal papillae in osmoregulation is absorption at low salt content and not excretion at high salt content. The inactive area between the anal papillae increased with increased salt concentration, and thus contributed to the decreasing size of the anal papillae. Remnants of this area in pupae were mistakenly measured as anal papillae in a study on salt adaptation by Waddington (1959).