Instar Drosophila Larvae Research Articles

A coumarin derived turn ‘off–on-off’ probe for selective sequential monitoring of Al3+ and Cu2+ ions with bio-imaging application

Herein, a coumarin-based chemosensor (E)-N'-(1-(2-oxo-2H-chromen-3-yl)ethylidene) thiophene-2-carbohydrazide (ACT) has been synthesized and thoroughly characterized by using various spectroscopic techniques. The molecular structure of the probe has been confirmed by single crystal X-ray diffraction technique. Hirshfeld surface analysis has also been carried out to investigate weaker interactions in the probe molecule. ACT shows a highly selective enhanced fluorometric response towards Al3+ in ethanol among different competing cations. Upon interaction with Al3+, a new complex ACT-Al3+ is formed which displays a strong yellow fluorescence due to chelation enhanced fluorescence process (CHEF). Furthermore, in situACT-Al3+ complex is exploited for sequential recognition of Cu2+ with fluorescence turn-off via paramagnetic quenching mechanism among different competing cations. This study presents an exceptionally low detection limits of ACT for Al3+ and Cu2+ ions (2.33 × 10−11 and 1.96 × 10−11 M, respectively). Furthermore, the binding constants of ACT for Al3+ and Cu2+ were found to be 7.97 × 104 and 9.79 × 104 M−1, respectively. The binding mechanism has been verified by 1H NMR titration and DFT calculations. Sensing behaviour of ACT towards Al3+ and Cu2+ have also been exploited in the development of paper strip kit and intracellular application in 3rd instar larvae of Drosophila.

Investigation of the Genotoxic Effect of Fluoxetine Hydrochloride in Drosophila melanogaster

This study aimed to determine the potential genotoxic effect of fluoxetine hydrochloride (FLX-HCl), an antidepressant commonly used for treating depression, using Somatic Mutation and Recombination Test (SMART). Third-¬instar Drosophila melanogaster larvae transheterozygous for the mutations multiple wing hair (mwh) and flare (flr3) were chronically fed in a medium containing different concentrations of FLX-HCl (0.1, 0.5, 1, and 2 mg/mL) in the experimental group. Distilled water, 0.1 mM ethyl methane sulfonate (EMS), and 2% dimethyl sulfoxide (DMSO) were used in negative, positive, and solvent control groups, respectively. The survival percentages were calculated by determining the number of individuals surviving when the larvae completed their development in the experimental and control groups. In all application groups, the wings of 40 individuals with both normal and serrate wing phenotypes were examined under a microscope, and genetic changes were evaluated by counting the mutant clones in the wings. The data obtained show that 1 and 2 mg/mL concentrations of FLX-HCl caused toxic effects in D. melanogaster individuals. Additionally, FLX-HCl showed a negative genotoxic effect at 0.1 mg/mL concentration, insignificant at 0.5 mg/mL concentration, and positive at 1 and 2 mg/mL concentrations in terms of total mutation evaluation and clone induction frequency in D. melanogaster individuals.

Evaluation of the toxic potential of Bisphenol-A glycidylmethacrylate (BisGMA) on the third instar larvae of transgenic Drosophila.

In the present study the cytotoxic and genotoxic effects of Bisphenol-A glycidyl methacrylate (BisGMA) was studied on the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. The concentration of BisGMA i.e. 0.005, 0.010, 0.015 and 0.020M were established in diet and the larvae were allowed to feed on it for 24h. A dose dependent significant increase in the activity of β-galactosidase was observed compared to control. A significant dose dependent tissue damage was observed in the larvae exposed to 0.010, 0.015 and 0.020M of BisGMA compared to control. A dose dependent significant increase in the Oxidative stress markers was observed compared to control. BisGMA also exhibit significant DNA damaged in the third instar larvae of transgenic D. melanogaster (hsp70-lacZ)Bg9 at the doses of 0.010, 0.015 and 0.020M compared to control. BisGMA at 0.010, 0.015 and 0.020 M was found to be cytotoxic for the third instar larvae of transgenic D. melanogaster (hsp70-lacZ) Bg9.

Colorimetric Synchronization of Drosophila Larvae.

The rapid succession of events during development poses an inherent challenge to achieve precise synchronization required for rigorous, quantitative phenotypic and genotypic analyses in multicellular model organisms. Drosophila melanogaster is an indispensable model for studying the development and function of higher order organisms due to extensive genome homology, tractability, and its relatively short lifespan. Presently, nine Nobel prizes serve as a testament to the utility of this elegant model system. Ongoing advancements in genetic and molecular tools allow for the underlying mechanisms of human disease to be investigated in Drosophila. However, the absence of a method to precisely age-match tissues during larval development prevents further capitalization of this powerful model organism. Drosophila spends nearly half of its life cycle progressing through three morphologically distinct larval instar stages, during which the imaginal discs, precursors of mature adult external structures (e.g., eyes, legs, wings), grow and develop distinct cell fates. Other tissues, such as the central nervous system, undergo massive morphological changes during larval development. While these three larval stages and subsequent pupal stages have historically been identified based on the number of hours post egg-laying under standard laboratory conditions, a reproducible, efficient, and inexpensive method is required to accurately age-match larvae within the third instar. The third instar stage is of particular interest, as this developmental stage spans a 48-hr window during which larval tissues switch from proliferative to differentiation programs. Moreover, some genetic manipulations can lead to developmental delays, further compounding the need for precise age-matching between control and experimental samples. This article provides a protocol optimized for synchronous staging of Drosophila third instar larvae by colorimetric characterization and is useful for age-matching a variety of tissues for numerous downstream applications. We also provide a brief discussion of the technical challenges associated with successful application of this protocol. © 2023 Wiley Periodicals LLC. Basic Protocol: Synchronization of third instar Drosophila larvae.

The Larval Plate Assay

The larval plate test has been used by researchers as a choice test to study the response of terrestrial larvae to various external cues. We describe here a laboratory exercise using third instar larvae of wild-type Drosophila to study the response toward multiple cues including olfactory as well as physical environmental cues such as light. The experiment can be easily carried out in an undergraduate lab and does not require any sophisticated instruments or equipment. The results of a series of experiments indicate the extent of response that each of the cue generates relative to the other. The highlight of the article however is the pre-lab discussion that can be done with students by putting forth various possible designs followed by discussion regarding the benefits and/or errors that could creep into the results obtained by using a particular design. Such a discussion would prove to be helpful in enhancing student understanding of experimental design, especially in designing experiments in animal behavior. The article also includes an extended theoretical problem set that can be used by facilitators in an undergraduate classroom activity.

Synthesis of Green Engineered Silver Nanoparticles through Urtica dioica: An Inhibition of Microbes and Alleviation of Cellular and Organismal Toxicity in Drosophila melanogaster.

Plant fractions have a diversity of biomolecules that can be used to make complicated reactions for the bioactive fabrication of metal nanoparticles (NPs), in addition to being beneficial as antioxidant medications or dietary supplements. The current study shows that Urtica dioica (UD) and biologically synthesized silver nanoparticles (AgNPs) of UD have antibacterial and antioxidant properties against bacteria (Escherichia coli and Pseudomonas putida) and Drosophila melanogaster (Oregon R+). According to their ability to scavenge free radicals, DPPH, ABTS, TFC, and TPC initially estimated the antioxidant potential of UD and UD AgNPs. The fabricated AgNPs were analyzed (UV-Vis, FTIR, EDS, and SEM) to determine the functional groups (alcohol, carboxylic acids, phenol, proteins, and aldehydes) and to observe the shape (agglomerated crystalline and rod-shaped structure). The disc diffusion method was used to test the antimicrobial properties of synthesized Ag-NPs against E. coli and P. putida. For 24 to 120 h, newly enclosed flies and third instar larvae of Drosophila were treated with UD and UD AgNPs. After exposure, tests for biochemical effects (acetylcholinesterase inhibition and protein estimation assays), cytotoxicity (dye exclusion), and behavioral effects (jumping and climbing assays) were conducted. The results showed that nanoparticles were found to have potent antimicrobial activity against all microbial strains tested at various concentrations. In this regard, ethno-medicinal characteristics exhibit a similar impact in D. melanogaster, showing (p < 0.05) significantly decreased cellular toxicity (trypan blue dye), enhanced biochemical markers (AChE efficacy and proteotoxicity), and improved behavioral patterns in the organism treated with UD AgNPs, especially in comparison to UD extract. The results of this study may help in the utilization of specific plants as reliable sources of natural antioxidants that may have been beneficial in the synthesis of metallic NPs, which aids in the production of nanomedicine and other therapeutic applications.

Application of the 3C Method to Study the Developmental Genes in Drosophila Larvae.

A transition from one developmental stage to another is accompanied by activation of developmental programs and corresponding gene ensembles. Changes in the spatial conformation of the corresponding loci are associated with this activation and can be investigated with the help of the Chromosome Conformation Capture (3C) methodology. Application of 3C to specific developmental stages is a sophisticated task. Here, we describe the use of the 3C method to study the spatial organization of developmental loci in Drosophila larvae. We critically analyzed the existing protocols and offered our own solutions and the optimized protocol to overcome limitations. To demonstrate the efficiency of our procedure, we studied the spatial organization of the developmental locus Dad in 3rd instar Drosophila larvae. Differences in locus conformation were found between embryonic cells and living wild-type larvae. We also observed the establishment of novel regulatory interactions in the presence of an adjacent transgene upon activation of its expression in larvae. Our work fills the gap in the application of the 3C method to Drosophila larvae and provides a useful guide for establishing 3C on an animal model.

Long-Term Observation of Locomotion of Drosophila Larvae Facilitates Feasibility of Food-Choice Assays.

Animal behavior is reflected by locomotor patterns. To decipher the underlying neural circuitry locomotion has to be monitored over often longer time periods. Here a simple adaptation is described to constrain movement of third instar Drosophila larvae to a defined area and use Frustrated total internal reflection based imaging method (FIM) imaging to monitor larval movements up to 1 h. It is demonstrated that the combination of FIM imaging and long analysis periods facilitates the conduction of food choice assays and provides the means to easily quantify food preferences.

Adverse biological effects of ingested polystyrene microplastics using Drosophila melanogaster as a model in vivo organism

ABSTRACT The abundant presence and extensive use of polystyrene microplastics (PSMPs) has recently become a serious environmental concern, as impact of exposure to these substances on human health remains unknown. While in vitro studies yield data on adverse effect of PSMPs, in vivo approaches are more relevant for risk assessment. Drosophila melanogaster is one of the most genetically and experimentally accessible model organisms used in biology as an in vivo model. D. melanogaster was selected as a representative in vivo model organism to examine the genotoxic potential of PSMPs at 5 concentrations of three different sizes namely 4, 10, or 20 µm. In particular, the wing somatic mutation and recombination test (SMART), a scalable, time-efficient in vivo assay developed to study genotoxicity of various compounds in a rapid manner at low costs was used. The third–instar Drosophila larvae were exposed to PSMPs through food at 5 concentrations ranging from 0.01-10 mM. Viability (lethality), larval length, morphological deformations, locomotor activity (climbing behavior), and genotoxic effects were the end-points measured. Exposure to PSMPs at 4, 10, or 20 µm produced significant morphological defects, impaired climbing behavior, and genotoxicity as evidenced by the SMART test demonstrating induction of somatic recombination. Significant increases were observed in the frequency of total spots, suggesting that PSMPs might induce genotoxic activity predominantly via initiation of somatic DNA recombination in a concentration-dependent manner.

Global chromatin organizer SATB1 acts as a context-dependent regulator of the Wnt/Wg target genes

Special AT-rich binding protein-1 (SATB1) integrates higher-order chromatin architecture with gene regulation, thereby regulating multiple signaling pathways. In mammalian cells SATB1 directly interacts with β-catenin and regulates the expression of Wnt targets by binding to their promoters. Whether SATB1 regulates Wnt/wg signaling by recruitment of β-catenin and/or its interactions with other components remains elusive. Since Wnt/Wg signaling is conserved from invertebrates to humans, we investigated SATB1 functions in regulation of Wnt/Wg signaling by using mammalian cell-lines and Drosophila. Here, we present evidence that in mammalian cells, SATB1 interacts with Dishevelled, an upstream component of the Wnt/Wg pathway. Conversely, ectopic expression of full-length human SATB1 but not that of its N- or C-terminal domains in the eye imaginal discs and salivary glands of third instar Drosophila larvae increased the expression of Wnt/Wg pathway antagonists and suppressed phenotypes associated with activated Wnt/Wg pathway. These data argue that ectopically-provided SATB1 presumably modulates Wnt/Wg signaling by acting as negative regulator in Drosophila. Interestingly, comparison of SATB1 with PDZ- and homeo-domain containing Drosophila protein Defective Proventriculus suggests that both proteins exhibit limited functional similarity in the regulation of Wnt/Wg signaling in Drosophila. Collectively, these findings indicate that regulation of Wnt/Wg pathway by SATB1 is context-dependent.

Candle soot derived carbon nanoparticles: An assessment of cellular and progressive toxicity using Drosophila melanogaster model

The biomedical demand of the nanomaterials is continuously increasing due to their wide range of applications in the field. However, before the implementation of these nanomaterials, toxicity assessment is essential for its safe usage. In the present study, the toxicity of carbon nanoparticles (CNPs) was investigated which was derived from candle soot and compared with commercially available multi-walled carbon nanotubes (CNTs) by using Drosophila melanogaster as a model system. First instar Drosophila larvae were exposed to CNPs as well as CNTs, and the toxic effects of these nanomaterials were compared. The result shows that both nanomaterials enhance the level of reactive oxygen species and oxidative stress in the Drosophila, which leads to the upregulation of heat shock proteins that may cause cytotoxicity in exposed Drosophila larvae. In contrast, exposure to CNPs and CNTs did not affect the developmental period of the larvae. Morphology of the internal organs, brain, gut and Malpighian tubules was also not altered in the exposed larvae. Similarly, no change observed in the cytoskeleton (F-actin) of these organs. Reproductive performance was slightly reduced in the case of CNPs compare to control. However, CNTs exposure did not show any significant effect on the reproductive performance of the flies that emerged from exposed larvae in comparison to control. Hence the study concludes that exposure to CNPs and CNTs cause a moderate level of cytotoxicity in Drosophila. The study also indicates that the inexpensive CNPs may use as an alternative to expensive CNTs for biomedical and biological applications.

Protective effect of Luteolin against methyl methanesulfonate-induced toxicity

The third instar larvae of transgenic Drosophila were allowed to feed on the diet having 10 µM of MMS alone and along with 10, 20 and 40 µM of Luteolin for 24 hrs. After the completion of 24 hrs, the larvae were subjected to ONPG assay, X-gal staining, trypan blue exclusion test, oxidative stress markers, apoptosis and comet assays. A dose-dependent increase in GSH content and decrease in the β-galactosidase activity, tissue damage, GST activity, LPO, PCC, Caspase-9/3 activities, apoptotic index, and DNA damage was observed in the larvae exposed to 10, 20 and 40 µM of Luteolin along with 10 µM of MMS.

A bioassay for monitoring the effect of diet on lipid accumulation in Drosophila melanogaster larvae

Drosophila is an emerging model organism in which to study the effects of diet on the regulation of metabolism and physiological function. In order to analyze the effects that different diets and food additives have on the accumulation of lipids in Drosophila larvae, we have established a control diet and optimized an established method to monitor the accumulation of lipids in third instar Drosophila larvae known as the floating larvae assay. We report on the simplicity and utility of using the floating larvae assay to access changes in lipid accumulation in flies raised on different diets.

The roles of V-type H+-ATPase and Na+/K+-ATPase in energizing K+ and H+ transport in larval Drosophila gut epithelia

We analyzed V-type H+-ATPase (VA) and Na+/K+-ATPase (NKA) along the caeca and midgut of third instar Drosophila larvae using immunohistochemistry and ATPase activity assays. Corresponding H+ and K+ fluxes were characterized using the Scanning Ion-Selective Electrode Technique (SIET), and the roles of transport ATPases in energizing ion transport across the larval gut were investigated by basal application of bafilomycin, a VA inhibitor, and ouabain, a NKA inhibitor. Addition of bafilomycin led to a decrease in H+ absorption along the caeca and midgut except at the copper cells and large flat cell zone of the middle midgut. H+ absorption was decreased by acetazolamide, consistent with carbonic anhydrase activity in all regions except at the large flat cell zone of the middle midgut. Bafilomycin or acetazolamide also led to decreased K+ absorption across the caeca and the anterior midgut. Our data show the dependence of K+ transport on H+ gradients established by the VA in the latter regions, consistent with the presence of a Cation-Proton Antiporter (CPA2) identified in other insect epithelia. Addition of ouabain led to the increase of K+ absorption along the anterior midgut and the large flat cell zone of the middle midgut, suggesting a role for the NKA in these regions. This study shows the importance of both ATPases in driving ion transport across the gut of larval Drosophila.

The Capacity to Act in Trans Varies Among Drosophila Enhancers.

The interphase nucleus is organized such that genomic segments interact in cis, on the same chromosome, and in trans, between different chromosomes. In Drosophila and other Dipterans, extensive interactions are observed between homologous chromosomes, which can permit enhancers and promoters to communicate in trans Enhancer action in trans has been observed for a handful of genes in Drosophila, but it is as yet unclear whether this is a general property of all enhancers or specific to a few. Here, we test a collection of well-characterized enhancers for the capacity to act in trans Specifically, we tested 18 enhancers that are active in either the eye or wing disc of third instar Drosophila larvae and, using two different assays, found evidence that each enhancer can act in trans However, the degree to which trans-action was supported varied greatly between enhancers. Quantitative analysis of enhancer activity supports a model wherein an enhancer's strength of transcriptional activation is a major determinant of its ability to act in trans, but that additional factors may also contribute to an enhancer's trans-activity. In sum, our data suggest that a capacity to activate a promoter on a paired chromosome is common among Drosophila enhancers.

Cardiac screening of intact Drosophila melanogaster larvae under exposure to aqueous and gaseous toxins in a microfluidic device

We present a semi-automated microfluidic chip for orientation, immobilization, chemical exposure, and cardiac screening of 3rd instar Drosophila melanogaster larvae.

An automated method to assay locomotor activity in third instar Drosophila melanogaster larvae

IntroductionThe purpose of these studies was to describe a novel application of an automated data acquisition/data reduction system, DanioVision™ by Noldus. DanioVision™ has the ability to detect changes in locomotor activity in third instar Drosophila melanogaster larvae. The noncompetitive GABAA receptor antagonist picrotoxin (PTX), was used as a pharmacologic agent to decrease locomotor activity. MethodsTwo strains of Drosophila were used in these studies; wild-type flies and flies with a mutation in the Rdl gene (RdlMD-RR). RdlMD-RRDrosophila are naturally occurring mutants that express an aberrant form of the GABAA receptor, which has a lower affinity for PTX, but not GABA itself. Larvae, extracted from food in 20% sucrose, were randomly placed into vials containing vehicle or PTX (0.03–3mM). After incubation of 2–24h, individual larvae were put in each well of a 6-well culture plate previously coated with 2% agar, the plate was then placed in the DanioVision™ apparatus. The activity of individual larva was recorded for 5min, digitized and analyzed using Ethovision® XT software. ResultsIncubation of third instar wild-type larvae in 1mM PTX for 4 or 24h decreased activity; whereas, a 2h incubation in PTX was without effect. PTX caused a concentration-dependent decrease in activity as demonstrated by consistently reduced locomotor activity with 1.0 and 3.0mM: 0.3mM resulted in variable decreases in locomotor activity and 0.03mM yielded no effect. By contrast, PTX did not affect activity in RdlMD-RR larvae even at the highest concentration, 3.0mM. DiscussionUsing an automated data acquisition system, it was found that PTX decreases activity in third instar Drosophila larvae due to a selective blockade of the GABAA receptor. The method will reduce the likelihood of human error and bias, as well as increase the speed and ease of data collection and analysis.

Do Late‐Instar Drosophila Larvae Experience Functional Oxygen Limitations?

Arthropods gain considerable mass between molts, increasing oxygen demand while parts of the respiratory system are fixed in size. We hypothesized that internal hypoxia could be a cue triggering molting in arthropods and/or be a functional reason that the animal needs to molt. In support of this hypothesis, hypoxic rearing (10 kPa) triggered an ecdysone peak and molting to adulthood at a smaller body size in Drosophila, and hyperoxia induced earlier pupariation (JEB 216:4334). We tested for evidence that late‐third instar Drosophila larvae show signs of functional oxygen‐limitation. The dorsal longitudinal tracheae increased in diameter through the instar, scaling with mass0.38, approximately as predicted to conserve oxygen delivery. Respiratory exchange ratios declined through the instar, indicating that metabolism remains aerobic and shifts away from carbohydrate catabolism. Lactate levels did not increase later in the instar and were unaffected by rearing in hyperoxia. However, hyperoxia (30 kPa) increased late‐instar metabolic rates, suggesting functional oxygen limitation. Late‐instar larvae had greater capacities to generate lactate in response to anoxia, suggesting past selection for coping with late‐instar oxygen limitation. Most late‐ but not early‐stage larvae unregulated HIF signaling. Together these data support the conclusion that Drosophila, and likely other arthropods, experience some functional oxygen limitations later in the instar that trigger HIF signaling, potentially linked to the molting cascade. This research was supported by NSF IOS 1256745.

Use of Daniovision™ to Measure Activity in 3rd Instar Drosophila melanogaster Larvae

The noncompetitive GABAA receptor antagonist picrotoxin (PTX) decreases activity in 3rd instar Drosophila larvae (Eur J Neurosci, 2006). The purpose of these studies was to replicate the data obtained by Stilwell et al. using an automated data acquisition/data reduction system, Daniovision™ by Noldus.Larvae were extracted from food (Carolina Biologic Supplies 4‐24) in 20% sucrose at the 3rd instar stage. The larvae were randomly placed into vials containing vehicle (20% sucrose + 0.3% DMSO) or PTX from 0.03‐3 mM: n = 20‐30 larvae/treatment. After incubation for 2‐24 hr, individual larvae were placed in separate wells of a 6‐well culture plate previously coated with 2% agar, then placed in the Daniovision™ apparatus. The activity of each larva was digitized and recorded for 5 minutes using Ethovision software.PTX reduces 3rd instar larvae activity in a concentration‐dependent manner, consistent with findings reported by Stilwell et al. Incubation in 1 mM PTX for 4 or 24 hours decreased activity, with consistent reduction produced by 3 mM, variably by 0.3 mM, and never by 0.03 mM. PTX, 0.03‐3 mM, did not alter activity in a mutant strain, Rdl, known to have a point mutation in the PTX binding site on the GABAA receptor conferring a lower affinity for PTX.We have shown that an automated system can be used to quantify activity in 3rd instar Drosophila melanogaster larvae. As human observation and review of videos is not required, and multi‐well plates can be used, we have refined an in vivo high‐throughput screen for anti‐seizure compounds.Supported by funds from the University of Saint Joseph.

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.