Fly Strains Research Articles

185

Introduction: Prolonged exposure to hyperoxia leads to the generation of reactive oxygen species (ROS) with subsequent cellular damage and multiple organ dysfunctions in humans. ROS mediate both direct and indirect modulation of signaling mechanisms. The human equivalent of the evolutionarily conserved innate immunity receptors, Toll-like receptors (TLR)-3 and 4, have been implicated in mediating hyperoxia-associated lung injury. Flies lack adaptive immunity and have been useful candidates in studying innate immunity. In fly strains that are highly resistant to high O2 exposure, it was reported that antimicrobial peptides play an important role in tolerance to oxidant stress. Hypothesis: We hypothesized that hyperoxic injury evokes innate immunity pathways in Drosophila. Methods: Flies (Berlin strain) were grown with standard cornmeal media and male flies younger than 7 days were selected for experiments. Hyperoxia chamber was designed and supplied with 1 L/min flow of 100% oxygen with humidification. Four groups (n=30/group) were designated for 1) gene expression assay with hyperoxic injury, 2) survival analysis with hyperoxic injury, 3) gene expression assay with normoxic control and 4) survival analysis control. At the time when 10% of flies of hyperoxic injury were dead, group 1) and 2) were homogenized for gene expression assay. Real-time PCR for Gram(-) pattern recognition receptor of IMD pathway, PGRP-LC (peptidoglycan recognition protein-LC) and end-product of Toll pathway, Def (defensin) were performed using 7300 Real time PCR system with TaqMan universal PCR Master Mix with TaqMan Gene Expression Assay primers. Relative gene expression was calculated after normalization to?-actin 5C. Results: Eleven flies (33.7%) were dead at 5th day after hyperoxic exposure and 28 flies (93.3%) were dead at 8th day. Among 30 flies with normoxia, 5 flies (10.0%) were dead at 8th day (p< 0.05). At 3rd day after hyperoxic exposure, gene expressions for innate immunity, PGRP-LC and Def were increased in flies with hyperoxic injury than in flies with normoxia; PGRP-LC 3.253:1(p<0.05) and Def 2.071:1(p<0.05). Conclusions: Hyperoxic injury caused fatal influences for survival of flies. Hyperoxic injury induces members of Drosophila innate immunity PGRP-LC and Def, suggesting a conserved pattern of adaptive changes against infection and ROS.

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis

In eukaryotes, the ubiquitin-proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 (+/+) (wild-type) and Dmp53 (-/-) Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis. Interestingly, Dmp53 gene seems to play a role in fly longevity and climbing ability. Moreover, Bortezomib proved to induce endoplasmic reticulum (ER) stress that was able to result in the engagement of unfolded protein response (UPR) signaling pathway, as respectively indicated by fly Xbp1 activation and Ref(2)P-containing protein aggregate formation. Larva salivary gland and adult brain both underwent strong ER stress in response to Bortezomib, thus underscoring the detrimental role of proteasome inhibition in larval development and brain function. We also propose that the observed upregulation of autophagy operates as a protective mechanism to "counterbalance" Bortezomib-induced systemic toxicity, which is tightly associated, besides ER stress, with activation of apoptosis, mainly mediated by functional Drice caspase and deregulated dAkt kinase. The reduced life-span of exposed to Bortezomib flies overexpressing Atg1_RNAi or Atg18_RNAi supports the protective nature of autophagy against proteasome inhibition-induced stress. Our data reveal the in vivo significance of proteasome functional integrity as a major defensive system against cellular toxicity likely occurring during critical biological processes and morphogenetic courses.

Drosophila EYA Regulates the Immune Response against DNA through an Evolutionarily Conserved Threonine Phosphatase Motif

Innate immune responses against DNA are essential to counter both pathogen infections and tissue damages. Mammalian EYAs were recently shown to play a role in regulating the innate immune responses against DNA. Here, we demonstrate that the unique Drosophila eya gene is also involved in the response specific to DNA. Haploinsufficiency of eya in mutants deficient for lysosomal DNase activity (DNaseII) reduces antimicrobial peptide gene expression, a hallmark for immune responses in flies. Like the mammalian orthologues, Drosophila EYA features a N-terminal threonine and C-terminal tyrosine phosphatase domain. Through the generation of a series of mutant EYA fly strains, we show that the threonine phosphatase domain, but not the tyrosine phosphatase domain, is responsible for the innate immune response against DNA. A similar role for the threonine phosphatase domain in mammalian EYA4 had been surmised on the basis of in vitro studies. Furthermore EYA associates with IKKβ and full-length RELISH, and the induction of the IMD pathway-dependent antimicrobial peptide gene is independent of SO. Our data provide the first in vivo demonstration for the immune function of EYA and point to their conserved immune function in response to endogenous DNA, throughout evolution.

Abstract 179: Mechanical Assessment of the Drosophila Heart Reveals Conservation of Human Heart Failure Mechanisms

Rationale: Current models for investigating the mechanisms of heart failure are limited by low throughput or genetic intractability. Therefore, we have developed a nanoindentation method to measure the intrinsic stiffness of the intact, living Drosophila heart tube to assess mechanical function in this rapidly aging, genetically malleable system which has been shown to experience age-related decline in cardiac function. Objective: Fruit fly strains universally experience decline in fractional shortening with age due to decreased diastolic diameter, though severity varies between strains. We compared a strain with significant decline in diameter with age and one without to examine the hypothesis that diastolic decline is the consequence of increase passive tension due to myocardial stiffening with age. Quantitative PCR was employed targeted towards mechanosensitive molecules known to be overexpressed in failing human hearts. Methods and Results: Female yw flies experience ∼18% reduction in diastolic diameter with age (59.3 at 1 week to 48.4 µm at 5 weeks of age, p &lt; 10-3) as calculated from m-mode kymographs of beating heart tube. Nanoindentation reveals 111% increase in stiffness (1.82 to 3.85 kPa, p &lt; 10-10) proximal to the cardiac cell-cell junctions. In contrast, w1118 flies experience ∼6% decline in diastolic diameter (74.1 to 69.5 µm, p = 0.049) and do not stiffen with age (p = 0.1923). qPCR reveals increased expression of cell-cell adhesion molecules in yw, such as vinculin and cadherin. Expression levels remain steady or decline in w1118. Further analysis reveals that w1118 experiences separation between juxtaposed muscle layers (410 nm at 1wk, 448 nm at 5wks) indicating matrix adhesion defects not observed in yw (0 nm, p &lt; 10-7 compared to 1wk w1118; 0 nm, p &lt; 10-8 compared to 5wk w1118). Conclusion: These data indicate structural changes in the fly are a consequence of adherens-junction-mediated mechanical remodeling similar to that which is observed in the failing human heart. Matrix defects and the lack of remodeling in w1118 suggest a link between costamere and adherens signaling during aging. Cardiac matrix content and adhesion signatures likely direct cardiac remodeling and function.

Characterization and evaluation of microsatellite markers in a strain of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae), with a genetic sexing character used in sterile insect population control

The oriental fruit fly, Bactrocera dorsalis, is a key economic insect pest reducing fruit yield and generating constraints in the international market. The application of the sterile insect technique (SIT) continues to reveal areas where new technologies can improve the effectiveness of fruit fly control. One such advancement concerns insect strains. In the present study, a mass-reared strain of the fly with a translocation-based genetic sexing character (Salaya1) based on a brown-white pupal colour dimorphism was genetically characterized using 11 microsatellite DNA markers. Subsequently, these markers were used to evaluate the maintenance of genetic variability in the strain under mass-rearing conditions. Mating competitiveness of this strain was also tested in field cages. Two of the newly characterized Y-pseudo-linked microsatellite markers were used for strain identification in field monitoring traps. The strain was also validated in a pilot integrated pest management (IPM) programme using male-only SIT in a fruit orchard. The programme resulted in the suppression of the fruit fly population.

Control of the olive fruit fly using genetics-enhanced sterile insect technique

BackgroundThe olive fruit fly, Bactrocera oleae, is the major arthropod pest of commercial olive production, causing extensive damage to olive crops worldwide. Current control techniques rely on spraying of chemical insecticides. The sterile insect technique (SIT) presents an alternative, environmentally friendly and species-specific method of population control. Although SIT has been very successful against other tephritid pests, previous SIT trials on olive fly have produced disappointing results. Key problems included altered diurnal mating rhythms of the laboratory-reared insects, resulting in asynchronous mating activity between the wild and released sterile populations, and low competitiveness of the radiation-sterilised mass-reared flies. Consequently, the production of competitive, male-only release cohorts is considered an essential prerequisite for successful olive fly SIT.ResultsWe developed a set of conditional female-lethal strains of olive fly (named Release of Insects carrying a Dominant Lethal; RIDL®), providing highly penetrant female-specific lethality, dominant fluorescent marking, and genetic sterility. We found that males of the lead strain, OX3097D-Bol, 1) are strongly sexually competitive with wild olive flies, 2) display synchronous mating activity with wild females, and 3) induce appropriate refractoriness to wild female re-mating. Furthermore, we showed, through a large proof-of-principle experiment, that weekly releases of OX3097D-Bol males into stable populations of caged wild-type olive fly could cause rapid population collapse and eventual eradication.ConclusionsThe observed mating characteristics strongly suggest that an approach based on the release of OX3097D-Bol males will overcome the key difficulties encountered in previous olive fly SIT attempts. Although field confirmation is required, the proof-of-principle suppression and elimination of caged wild-type olive fly populations through OX3097D-Bol male releases provides evidence for the female-specific RIDL approach as a viable method of olive fly control. We conclude that the promising characteristics of OX3097D-Bol may finally enable effective SIT-based control of the olive fly.

Monitoring of Five Different Insecticide Resistance Status in Turkish House Fly Musca domestica L. (Diptera: Muscidae) Populations and the Relationship Between Resistance and Insecticide Usage Profile

The objective of this study is to assess the resistance status and yearly changes involved for house fly populations from six cities in Turkey. Field strains of house fly (Musca domestica L. Diptera: Muscidae) were collected in 2004-2006 from cow farms (Antalya, İzmir) and garbage dumps (Adana, Ankara, İstanbul, Şanlıurfa) in Turkey. The resistance levels of first and two generation offspring were determined against five insecticides (cypermethrin, cyphenothrin, deltamethrin, permethrin and fenitrothion). While the highest resistance level for pyrethroid was determined for Antalya 2005 strain (851.97 Cypermethrin) and lowest resistance level for Şanlıurfa 2004 strain (2.06 Permethrin), the highest fenitrothion resistance was found in Şanlıurfa 2004 strain (50.37) and lowest fenitrothion resistance was found in Adana 2004 strain (6.45). Our results showed that pyrethroid resistance levels were very high and determined a decreasing trend for Antalya and İstanbul strains and an increasing trend for Adana and Şanlıurfa strain for all tested pyrethroid insecticides from 2004 to 2006. Although cypermethrin and cyphenothrin resistance showed a decreasing trend, deltamethrin and permethrin showed an increasing trend for the Izmir strain from 2004 to 2006. The same trend was also determined for the Ankara strain except for permethrin. Fenitrothion resistance was determined to be lower than pyrethroids, but these levels were still high. Flies from cow farms were generally more resistant than those from garbage dumps. Our results also revealed the presence of strong selective pressure on the populations.

An Introduction to Parasitic Wasps of &lt;em&gt;Drosophila&lt;/em&gt; and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites. The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids. Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited. Strains of L. boulardi also produce virus-like particles although they differ significantly in their ability to succeed on D. melanogaster. Some of these L. boulardi strains are difficult to grow on D. melanogaster as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols. In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections. Wasp infection activates immune signaling (Fig. 4). At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5). Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D). We present representative data with Toll signal transduction pathway components Dorsal and Spätzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

Pyriproxyfen and House Flies (Diptera: Muscidae): Effects of Direct Exposure and Autodissemination to Larval Habitats

Pyriproxyfen is an insect growth regulator with juvenile hormone-like activity that has potential uses for dipterans that are difficult to manage with conventional insecticides, such as house flies (Musca domestica L.). The objectives of this study were to determine the efficacy of this insect growth regulator against house flies using variety of delivery systems and target life stages, including an evaluation of the potential for autodissemination by female flies to larval development sites. Adult female house flies exposed to filter paper (3.75% active ingredient) or sugar treated with pyriproxyfen (0.01-0.1%) produced significantly fewer F1 pupae than untreated flies. Adult emergence from pupae was unaffected. In contrast, treatment of larval rearing medium with 0.35 ml/cm2 of a 12 mg pyriproxyfen/liter preparation had no effect on the number of pupae developing from eggs but markedly inhibited adult emergence from those pupae. There was little difference in susceptibility between an insecticide-susceptible and a wild strain of house fly. The LC50 for inhibiting fly emergence of dust formulations in diatomaceous earth incorporating commercial pyriproxyfen products ranged from 8 to 26 mg/liter, with little difference among products. Compared with untreated flies, significantly fewer pupae were produced at concentrations > 0.5% and no adults were produced at concentrations > 0.05% pyriproxyfen. When gravid females were exposed for 1 h to treated fabric (6 mg pyriproxyfen/cm2) and allowed to oviposit in rearing media containing eggs, sufficient pyriproxyfen was autodisseminated to reduce adult emergence from those eggs by > 99%. Intermittent contact with treated fabric over 2 d reduced adult emergence by 63-76%.

In Vivo Imaging of the Drosophila Larval Neuromuscular Junction

In the past decade, a significant number of proteins involved in the developmental assembly and maturation of synapses have been identified. However, detailed knowledge of the molecular processes underlying developmental synapse assembly is still sparse. We have developed an approach that makes extended in vivo imaging of selected proteins in live Drosophila larvae feasible at a single-synapse resolution. This protocol describes the repetitive, noninvasive imaging of the neuromuscular junction (NMJ) of a live, intact, anesthetized Drosophila larva over extended periods of time with confocal microscopy. Proteins of interest must be tagged with a fluorescent label and have to be expressed in transgenic fly strains. The method has proven highly useful for the study of synaptic assembly and the trafficking of proteins. These data contribute to our understanding of synaptic assembly under in vivo conditions.

Transgenic Expression and Purification of Myosin Isoforms Using the Drosophila melanogaster Indirect Flight Muscle System

We report a novel method for the generation of extremely pure transgenic myosin to carry out crystallization screening for x‐ray crystallography. We are optimizing a novel method for the use of genetically engineered Drosophila melanogaster to express histidine‐tagged recombinant muscle myosin isoforms. This method relies on several facts. First, that the Drosophila genome contains only one gene encoding all skeletal muscle myosin heavy chain isoforms. Second, that wild‐type or mutagenized versions of the gene can then be inserted into a specific location in the Drosophila genome. Third, that the Mhc10 fly line has endogenous myosin knocked out in the indirect flight muscles. Fourth, that the Actin88F promoter facilitates high‐level expression of accessible myosin in the thoracic indirect flight muscles. Finally, that cost of fruit fly colony propagation is relatively low. Using these advantages we devised a method for expressing and purifying two isoforms of recombinant histidine‐tagged myosin from an engineered fly strain. The myosin shows ATPase activity and is of sufficient purity and homogeneity for crystallization. This technique may prove useful for the expression and isolation of mutant myosins linked with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization. Supported by NIH R01 GM032443 to SIB.

Frequency-dependent and density-dependent larval competition between life-history strains of a fly, Lucilia cuprina

1. Competition was created between the larvae of two life-history strains of the blowfly Lucilia cuprina (Wiedemann) that have different requirements for larval resource acquisition. Adult females of one strain had the ability to mature eggs in the absence of adult feeding (autogeny) whereas the other strain lacked this ability. Autogeny shifts the burden of resource acquisition from adults to larvae, potentially leading to greater competition at this earlier life history stage. 2. A replacement series was used to determine the per-capita competitive effect between strains relative to the intra-strain effect, and density- and frequency-dependent variation in this per-capita effect was then evaluated. Evidence was found of competitive superiority of autogenous larvae when they occurred at a low frequency and low density, but their competitive ability was lost or reversed at higher frequencies and densities. 3. A dynamic competitive environment created by frequency and density dependence can account for the maintenance of genetic diversity for major life-history traits. Such competition may explain why autogeny is rare in field populations of L. cuprina even although underlying genetic variation for the trait seems to be present.

Tandem Affinity Purification in Drosophila Heads and Ovaries.

Tandem affinity purification (TAP) (Pugi et al., 2001; Rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three components: a calmodulin-binding peptide, a Tobacco etch virus (TEV) protease cleavage site and Protein A which is an immunoglobulin G (IgG)-binding domain. This protocol was modified from the original methodology used in yeast cells (Pugi et al., 2001; Rigaut et al., 1999) for isolation of protein complexes from Drosophila heads and ovaries expressing a TAP tagged protein of interest. To determine in vivo binding partners of the Drosophila fragile X protein (dFMR1), we developed a transgenic strain of flies expressing a recombinant form of dFMR1 with a carboxy-terminal TAP tag (Tsai and Carstens, 2006). To ensure that the construct was expressed at wild-type levels, we engineered this form of the tagged protein in the context of a genomic rescue construct that rescued a mutant sterility phenotype. The purification process was performed using mild conditions to maintain native protein interactions. For TAP methods in Drosophila S2 cell culture, we have successfully used a protocol previously published by Tsai and Carstens (Tsai and Carstens, 2006; Bhogal et al., 2011).

Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift.

This paper describes a new program SnpSift for filtering differential DNA sequence variants between two or more experimental genomes after genotoxic chemical exposure. Here, we illustrate how SnpSift can be used to identify candidate phenotype-relevant variants including single nucleotide polymorphisms, multiple nucleotide polymorphisms, insertions, and deletions (InDels) in mutant strains isolated from genome-wide chemical mutagenesis of Drosophila melanogaster. First, the genomes of two independently isolated mutant fly strains that are allelic for a novel recessive male-sterile locus generated by genotoxic chemical exposure were sequenced using the Illumina next-generation DNA sequencer to obtain 20- to 29-fold coverage of the euchromatic sequences. The sequencing reads were processed and variants were called using standard bioinformatic tools. Next, SnpEff was used to annotate all sequence variants and their potential mutational effects on associated genes. Then, SnpSift was used to filter and select differential variants that potentially disrupt a common gene in the two allelic mutant strains. The potential causative DNA lesions were partially validated by capillary sequencing of polymerase chain reaction-amplified DNA in the genetic interval as defined by meiotic mapping and deletions that remove defined regions of the chromosome. Of the five candidate genes located in the genetic interval, the Pka-like gene CG12069 was found to carry a separate pre-mature stop codon mutation in each of the two allelic mutants whereas the other four candidate genes within the interval have wild-type sequences. The Pka-like gene is therefore a strong candidate gene for the male-sterile locus. These results demonstrate that combining SnpEff and SnpSift can expedite the identification of candidate phenotype-causative mutations in chemically mutagenized Drosophila strains. This technique can also be used to characterize the variety of mutations generated by genotoxic chemicals.

A Method for the Transgenic Expression and Purification of Skeletal Muscle Myosin II Isoforms using Drosophila Melanogaster

There are many biologically interesting myosin isoforms that are expressed at levels too low for direct purification from in vivo sources. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We are optimizing a novel method for the use of Drosophila melanogaster that has been genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method relies on several facts. First, that the Drosophila genome contains only one gene encoding all skeletal muscle myosin isoforms. Second, that this DNA can be manipulated with molecular biology techniques and then be inserted into a specific location in the Drosophila genome. Third, that the Mhc10 fly line has endogenous myosin knocked out in the indirect flight muscles. Fourth, that the Actin88F promoter facilitates high-level expression of accessible myosin in the thoracic indirect flight muscles. Finally, that cost of fruit fly colony propagation is relatively low. Employing these advantages, we have created a system for the production of extremely pure skeletal muscle myosin. We demonstrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. This myosin shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. Our technique may prove useful for the expression and isolation of mutant myosins linked with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

Genetic Variation Results in Differing Age-Related Drosophila Myocardial Stiffness

Rapid aging of Drosophila melanogaster makes it suitable for studying age-related mechanical changes such as impaired heart function. The Drosophila heart consists of a cardiomyocyte tube bonded to a thin ventral muscle layer. It has previously been observed that there is a decrease in the diastolic heart tube diameter with age (>20%) in the control fly strain yellow-white (yw), which we hypothesized was due to an increase in passive tension due to myocardial stiffening. We sought to investigate this through nanoindentation in both yw and other control lines. Using a modified Hertzian analysis method, we probed the stiffness of juvenile and geriatric female yw flies and found age-related stiffening, a finding consistent with myocyte stiffening in other cardiac systems over time. Stiffness at the cardiac cell-cell junctions was 1.8± 0.1 vs. 3.8 ± 0.3 kPa in 1 week old flies which increased to 3.8 ± 0.3 kPa in 5 week flies. In contrast, white (w1118) flies were found to have no age-related stiffening. Consistently, w1118 also have a less significant decrease in diastolic diameter with age (<10%). Moreover, our analysis method is capable of detecting mechanical separation of the muscle layers, which was found to occur more frequently in w1118 than yw (0.41± 0.04 vs. 0.00 ± 0.02 μm separation, respectively, 1 week flies). Detection of mechanical separation between muscle layers via nanoindentation was modeled and verified in a microfabricated polydimethylsiloxane system. This first in situ mechanical analysis of a living myocardium revealed differences in cardiac mechanics due to age and suggest that aspects of the mechanical properties of the aging phenotype differ between Drosophila strains. We investigation on other laboratory Drosophila wildtype strains to assess the impact of diverse genetic backgrounds or mutations on age-related myocardial stiffening and cardiomyopathy.

Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

Neural Network Recognizes Fruit Fly's Wing Vibration Sound Based on Hilbert-Huang Transform

The paper applies Hilbert-Huang Transform to process the fruit fly’s wing vibration sound. Firstly, it gets Hilbert-Huang spectrum and marginal spectrum of the wing vibration sound of two different strains of fruit fly in the same species. Then, it analyzes differences of the energy size and the energy distribution, and extracts features including the relative energy, time-frequency entropy and the cumulated amplitude value of marginal spectrum. Finally, recognizes two different strains of fruit fly by BP neural network and obtains a satisfying result. The experiment result verifies effectiveness and feasibility of the feature extraction, and the paper offers a new evidence for the study of discrimination in intraspecies relationship of fruit fly.

Social synchronization of circadian locomotor activity rhythm in the fruit fly Drosophila melanogaster

Circadian clocks regulate the physiology and behaviour of organisms across a wide range of taxa. To keep track of local time, these clocks use a variety of time cues such as light-dark, temperature, food availability and social interaction cycles. This study assessed the role of social cues in modulating circadian clocks of the fruit fly Drosophila melanogaster. Using pair-wise interactions, we first estimated the percentage contribution of each interacting partner on the cumulative rhythmic behaviour of the pairs. Subsequently, we studied the effects of multi-individual (group-wise) interactions on the rhythmic behaviour of the group by estimating phase synchrony between individuals of different strains (having different circadian periods) maintained in both homogeneous and heterogeneous groups. Although it is known that social interactions improve synchrony between interacting individuals, we asked whether such interactions are able to synchronize the circadian rhythms of highly phase-desynchronized flies. We found that, although interactions between fly strains possessing different circadian periods failed to produce synchrony, social interactions among phase-desynchronized flies did enhance the phase synchrony of the interacting individuals. Differently phased individuals living in social groups displayed significantly greater phase synchrony than those living solitarily. Social synchronization is olfaction mediated as group-wise interactions among phase-desynchronized flies possessing compromised olfactory ability (Or83b(0)) did not improve phase synchrony. These results suggest that social cues synchronize the circadian clocks of Drosophila provided that the interacting individuals have similar clock periods.

Adaptation to a seasonally varying environment: a strong latitudinal cline in reproductive diapause combined with high gene flow in Drosophila montana

Adaptation to seasonal changes in the northern hemisphere includes an ability to predict the forthcoming cold season from gradual changes in environmental cues early enough to prepare for the harsh winter conditions. The magnitude and speed of changes in these cues vary between the latitudes, which induces strong selection pressures for local adaptation.We studied adaptation to seasonal changes in Drosophila montana, a northern maltfly, by defining the photoperiodic conditions leading to adult reproductive diapause along a latitudinal cline in Finland and by measuring genetic differentiation and the amount of gene flow between the sampling sites with microsatellites. Our data revealed a clear correlation between the latitude and the critical day length (CDL), in which half of the females of different cline populations enter photoperiodic reproductive diapause. There was no sign of limited gene flow between the cline populations, even though these populations showed isolation by distance. Our results show that local adaptation may occur even in the presence of high gene flow, when selection for locally adaptive life-history traits is strong. A wide range of variation in the CDLs of the fly strains within and between the cline populations may be partly due to gene flow and partly due to the opposing selection pressures for fly reproduction and overwinter survival. This variation in the timing of diapause will enhance populations’ survival over the years that differ in the severity of the winter and in the length of the warm period and may also help them respond to long-term changes in environmental conditions.