Locomotor Assays in Drosophila Larvae and Adult Flies.

Brain defects often lead to motor dysfunctions in humans. Drosophila melanogaster has been one of the most useful organisms in the study of neuronal biology due to its similarities with humans and has contributed to a more detailed understanding of the effects of genetic dysfunctions in the brain on behavior. We herein present modified protocols for the crawling assay with larvae and the climbing assay with adult flies that are simple to perform as well as a series of commands for ImageJ to automatically analyze data for the crawling assay.

Genomes of historical specimens reveal multiple invasions of LTR retrotransposons in Drosophila melanogaster during the 19th century.

Transposable element invasions have a profound impact on the evolution of genomes and phenotypes. It is thus an important open question how often such TE invasions occur. To address this question, we utilize the genomes of historical specimens, sampled about 200 y ago. We found that the LTR retrotransposons Blood, Opus, and 412 spread in Drosophila melanogaster in the 19th century. These invasions constitute second waves, as degraded fragments were found for all three TEs. The composition of Opus and 412, but not of Blood, shows a pronounced geographic heterogeneity, likely due to founder effects during the invasions. Finally, we identified species from the Drosophila simulans complex as the likely origin of the TEs. We show that in total, seven TE families invaded D. melanogaster during the last 200y, thereby increasing the genome size by up to 1.2Mbp. We suggest that this high rate of TE invasions was likely triggered by human activity. Based on the analysis of strains and specimens sampled at different times, we provide a detailed timeline of TE invasions, making D. melanogaster the first organism where the invasion history of TEs during the last two centuries could be inferred.

Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

Comparative Transcriptomic Analysis Revealing the Potential Mechanisms of Erythritol-Caused Mortality and Oviposition Inhibition in Drosophila melanogaster.

Erythritol has shown excellent insecticidal performance against a wide range of insect species, but the molecular mechanism by which it causes insect mortality and sterility is not fully understood. The mortality and sterility of Drosophila melanogaster were assessed after feeding with 1M erythritol for 72 h and 96 h, and gene expression profiles were further compared through RNA sequencing. Enrichment analysis of GO and KEGG revealed that expressions of the adipokinetic hormone gene (Akh), amylase gene (Amyrel), α-glucosidase gene (Mal-B1/2, Mal-A1-4, Mal-A7/8), and triglyceride lipase gene (Bmm) were significantly up-regulated, while insulin-like peptide genes (Dilp2, Dilp3 and Dilp5) were dramatically down-regulated. Seventeen genes associated with eggshell assembly, including Dec-1 (down 315-fold), Vm26Ab (down 2014-fold) and Vm34Ca (down 6034-fold), were significantly down-regulated or even showed no expression. However, there were no significant differences in the expression of three diuretic hormone genes (DH44, DH31, CAPA) and eight aquaporin genes (Drip, Big brain, AQP, Eglp1, Eglp2, Eglp3, Eglp4 and Prip) involved in osmolality regulation (all p value > 0.05). We concluded that erythritol, a competitive inhibitor of α-glucosidase, severely reduced substrates and enzyme binding, inhibiting effective carbohydrate hydrolysis in the midgut and eventually causing death due to energy deprivation. It was clear that Drosophila melanogaster did not die from the osmolality of the hemolymph. Our findings elucidate the molecular mechanism underlying the mortality and sterility in Drosophila melanogaster induced by erythritol feeding. It also provides an important theoretical basis for the application of erythritol as an environmentally friendly pesticide.

Mitochondrial Differentiation during Spermatogenesis: Lessons from Drosophila melanogaster.

Numerous diseases can arise as a consequence of mitochondrial malfunction. Hence, there is a significant focus on studying the role of mitochondria in cancer, ageing, neurodegenerative diseases, and the field of developmental biology. Mitochondria could exist as discrete organelles in the cell; however, they have the ability to fuse, resulting in the formation of interconnected reticular structures. The dynamic changes between these forms correlate with mitochondrial function and mitochondrial health, and consequently, there is a significant scientific interest in uncovering the specific molecular constituents that govern these transitions. Moreover, the specialized mitochondria display a wide array of variable morphologies in their cristae formations. These inner mitochondrial structures are closely associated with the specific functions performed by the mitochondria. In multiple cases, the presence of mitochondrial dysfunction has been linked to male sterility, as it has been observed to cause a range of abnormal spermatogenesis and sperm phenotypes in different species. This review aims to elucidate the dynamic alterations and functions of mitochondria in germ cell development during the spermatogenesis of Drosophila melanogaster.

Developmental and Housekeeping Genes: Two Types of Genetic Organization in the Drosophila Genome.

We developed a procedure for locating genes on Drosophila melanogaster polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which differed markedly in morphological and genetic properties. This was reached through the use of our original methods of molecular and genetic analysis, electron microscopy, and bioinformatics data processing. Analysis of the genome-wide distribution of these properties led us to a bioinformatics model of the Drosophila genome organization, in which the genome was divided into two groups of genes. One was constituted by 65, in which the genome was divided into two groups, 62 genes that are expressed in most cell types during life cycle and perform basic cellular functions (the so-called "housekeeping genes"). The other one was made up of 3162 genes that are expressed only at particular stages of development ("developmental genes"). These two groups of genes are so different that we may state that the genome has two types of genetic organization. Different are the timings of their expression, chromatin packaging levels, the composition of activating and deactivating proteins, the sizes of these genes, the lengths of their introns, the organization of the promoter regions of the genes, the locations of origin recognition complexes (ORCs), and DNA replication timings.

Elevated ozone disrupts mating boundaries in drosophilid flies.

Animals employ different strategies to establish mating boundaries between closely related species, with sex pheromones often playing a crucial role in identifying conspecific mates. Many of these pheromones have carbon-carbon double bonds, making them vulnerable to oxidation by certain atmospheric oxidant pollutants, including ozone. Here, we investigate whether increased ozone compromises species boundaries in drosophilid flies. We show that short-term exposure to increased levels of ozone degrades pheromones of Drosophila melanogaster, D. simulans, D. mauritiana, as well as D. sechellia, and induces hybridization between some of these species. As many of the resulting hybrids are sterile, this could result in local population declines. However, hybridization between D. simulans and D. mauritiana as well as D. simulans and D. sechellia results in fertile hybrids, of which some female hybrids are even more attractive to the males of the parental species. Our experimental findings indicate that ozone pollution could potentially induce breakdown of species boundaries in insects.

Hippo Signaling at the Hallmarks of Cancer and Drug Resistance.

Originally identified in Drosophila melanogaster in 1995, the Hippo signaling pathway plays a pivotal role in organ size control and tumor suppression by inhibiting proliferation and promoting apoptosis. Large tumor suppressors 1 and 2 (LATS1/2) directly phosphorylate the Yki orthologs YAP (yes-associated protein) and its paralog TAZ (also known as WW domain-containing transcription regulator 1 [WWTR1]), thereby inhibiting their nuclear localization and pairing with transcriptional coactivators TEAD1-4. Earnest efforts from many research laboratories have established the role of mis-regulated Hippo signaling in tumorigenesis, epithelial mesenchymal transition (EMT), oncogenic stemness, and, more recently, development of drug resistances. Hippo signaling components at the heart of oncogenic adaptations fuel the development of drug resistance in many cancers for targeted therapies including KRAS and EGFR mutants. The first U.S. food and drug administration (US FDA) approval of the imatinib tyrosine kinase inhibitor in 2001 paved the way for nearly 100 small-molecule anti-cancer drugs approved by the US FDA and the national medical products administration (NMPA). However, the low response rate and development of drug resistance have posed a major hurdle to improving the progression-free survival (PFS) and overall survival (OS) of cancer patients. Accumulating evidence has enabled scientists and clinicians to strategize the therapeutic approaches of targeting cancer cells and to navigate the development of drug resistance through the continuous monitoring of tumor evolution and oncogenic adaptations. In this review, we highlight the emerging aspects of Hippo signaling in cross-talk with other oncogenic drivers and how this information can be translated into combination therapy to target a broad range of aggressive tumors and the development of drug resistance.

Numerous Serine/Threonine Kinases Affect Blood Cell Homeostasis in Drosophila melanogaster.

Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.

Feeding Drosophila gut microbiomes from young and old flies modifies the microbiome.

It is becoming increasingly evident that the myriad of microbes in the gut, within cells and attached to body parts (or roots of plants), play crucial roles for the host. Although this has been known for decades, recent developments in molecular biology allow for expanded insight into the abundance and function of these microbes. Here we used the vinegar fly, Drosophila melanogaster, to investigate fitness measures across the lifetime of flies fed a suspension of gut microbes harvested from young or old flies, respectively. Our hypothesis was that flies constitutively enriched with a 'Young microbiome' would live longer and be more agile at old age (i.e. have increased healthspan) compared to flies enriched with an 'Old microbiome'. Three major take home messages came out of our study: (1) the gut microbiomes of young and old flies differ markedly; (2) feeding flies with Young and Old microbiomes altered the microbiome of recipient flies and (3) the two different microbial diets did not have any effect on locomotor activity nor lifespan of the recipient flies, contradicting our working hypothesis. Combined, these results provide novel insight into the interplay between hosts and their microbiomes and clearly highlight that the phenotypic effects of gut transplants and probiotics can be complex and unpredictable.

The BKCa (slo) channel regulates the cardiac function of Drosophila.

The large conductance, calcium, and voltage-active potassium channels (BKCa) were originally discovered in Drosophila melanogaster as slowpoke (slo). They are extensively characterized in fly models as ion channels for their roles in neurological and muscular function, as well as aging. BKCa is known to modulate cardiac rhythm and is localized to the mitochondria. Activation of mitochondrial BKCa causes cardioprotection from ischemia-reperfusion injury, possibly via modulating mitochondrial function in adult animal models. However, the role of BKCa in cardiac function is not well-characterized, partially due to its localization to the plasma membrane as well as intracellular membranes and the wide array of cells present in mammalian hearts. Here we demonstrate for the first time a direct role for BKCa in cardiac function and cardioprotection from IR injury using the Drosophila model system. We have also discovered that the BKCa channel plays a role in the functioning of aging hearts. Our study establishes the presence of BKCa in the fly heart and ascertains its role in aging heart function.

Developmental changes in nuclear lamina components during germ cell differentiation.

The nuclear lamina (NL) changes composition for regulation of nuclear events. We investigated changes that occur in Drosophila oogenesis, revealing switches in NL composition during germ cell differentiation. Germline stem cells (GSCs) express only LamB and predominantly emerin, whereas differentiating nurse cells predominantly express LamC and emerin2. A change in LamC-specific localization also occurs, wherein phosphorylated LamC redistributes to the nuclear interior only in the oocyte, prior to transcriptional reactivation of the meiotic genome. These changes support existing concepts that LamC promotes differentiation, a premise that was tested. Remarkably ectopic LamC production in GSCs did not promote premature differentiation. Increased LamC levels in differentiating germ cells altered internal nuclear structure, increased RNA production, and reduced female fertility due to defects in eggshell formation. These studies suggest differences between Drosophila lamins are regulatory, not functional, and reveal an unexpected robustness to level changes of a major scaffolding component of the NL.

The BTB-ZF gene Bm-mamo regulates pigmentation in silkworm caterpillars.

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor of dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermal features in some insects.

Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Endocrine Regulation of Aging in the Fruit Fly Drosophila melanogaster.

The past few decades have witnessed increasing research clarifying the role of endocrine signaling in the regulation of aging in both vertebrates and invertebrates. Studies using the model organism fruit fly Drosophila melanogaster have largely advanced our understanding of evolutionarily conserved mechanisms in the endocrinology of aging and anti-aging. Mutations in single genes involved in endocrine signaling modify lifespan, as do alterations of endocrine signaling in a tissue- or cell-specific manner, highlighting a central role of endocrine signaling in coordinating the crosstalk between tissues and cells to determine the pace of aging. Here, we review the current landscape of research in D. melanogaster that offers valuable insights into the endocrine-governed mechanisms which influence lifespan and age-related physiology.

Biological aging of two innate behaviors of Drosophila melanogaster: Escape climbing versus courtship learning and memory.

Motor and cognitive aging can severely affect life quality of elderly people and burden health care systems. In search for diagnostic behavioral biomarkers, it has been suggested that walking speed can predict forms of cognitive decline, but in humans, it remains challenging to separate the effects of biological aging and lifestyle. We examined a possible association of motor and cognitive decline in Drosophila, a genetic model organism of healthy aging. Long term courtship memory is present in young male flies but absent already during mid life (4-8 weeks). By contrast, courtship learning index and short term memory (STM) are surprisingly robust and remain stable through mid (4-8 weeks) and healthy late life (>8 weeks), until courtship performance collapses suddenly at ~4.5 days prior to death. By contrast, climbing speed declines gradually during late life (>8 weeks). The collapse of courtship performance and short term memory close to the end of life occur later and progress with a different time course than the gradual late life decline in climbing speed. Thus, during healthy aging in male Drosophila, climbing and courtship motor behaviors decline differentially. Moreover, cognitive and motor performances decline at different time courses. Differential behavioral decline during aging may indicate different underlying causes, or alternatively, a common cause but different thresholds for defects in different behaviors.

Morphogenesis: Setting the pace of embryo folding.

Tissue folding is a key process for shape generation during embryonic development. A new study reports how a fold in the Drosophila embryo forms by a propagating trigger wave.

Developmental neuroscience: Building sex-specific adult circuitry from common larval origins.

The development of sex-specific neural circuitry is critical for reproductive behaviors. A new study traces the developmental origin of female-specific neurons that underlie an adult mating behavior to larval neurons common to both sexes in Drosophila.

Dynamic encoding of temperature in the central circadian circuit coordinates physiological activities.

The circadian clock regulates animal physiological activities. How temperature reorganizes circadian-dependent physiological activities remains elusive. Here, using in-vivo two-photon imaging with the temperature control device, we investigated the response of the Drosophila central circadian circuit to temperature variation and identified that DN1as serves as the most sensitive temperature-sensing neurons. The circadian clock gate DN1a's diurnal temperature response. Trans-synaptic tracing, connectome analysis, and functional imaging data reveal that DN1as bidirectionally targets two circadian neuronal subsets: activity-related E cells and sleep-promoting DN3s. Specifically, behavioral data demonstrate that the DN1a-E cell circuit modulates the evening locomotion peak in response to cold temperature, while the DN1a-DN3 circuit controls the warm temperature-induced nocturnal sleep reduction. Our findings systematically and comprehensively illustrate how the central circadian circuit dynamically integrates temperature and light signals to effectively coordinate wakefulness and sleep at different times of the day, shedding light on the conserved neural mechanisms underlying temperature-regulated circadian physiology in animals.

Comprehensive insights into the impact of bacterial indole-3-acetic acid on sensory preferences in Drosophila melanogaster.

Several bacteria of environmental and clinical origins, including some human-associated strains secrete a cross-kingdom signaling molecule indole-3-acetic acid (IAA). IAA is a tryptophan (trp) derivative mainly known for regulating plant growth and development as a hormone. However, the nutritional sources that boost IAA secretion in bacteria and the impact of secreted IAA on non-plant eukaryotic hosts remained less explored. Here, we demonstrate significant trp-dependent IAA production in Pseudomonas juntendi NEEL19 when provided with ethanol as a carbon source in liquid cultures. IAA was further characterized to modulate the odor discrimination, motility and survivability in Drosophila melanogaster. A detailed analysis of IAA-fed fly brain proteome using high-resolution mass spectrometry showed significant (fold change, ± 2; p ≤ 0.05) alteration in the proteins governing neuromuscular features, audio-visual perception and energy metabolism as compared to IAA-unfed controls. Sex-wise variations in differentially regulated proteins were witnessed despite having similar visible changes in chemo perception and psychomotor responses in IAA-fed flies. This study not only revealed ethanol-specific enhancement in trp-dependent IAA production in P. juntendi, but also showed marked behavioral alterations in flies for which variations in an array of proteins governing odor discrimination, psychomotor responses, and energy metabolism are held responsible. Our study provided novel insights into disruptive attributes of bacterial IAA that can potentially influence the eukaryotic gut-brain axis having broad environmental and clinical implications.