Genetically Encoded CRISPR Components Yield Efficient Gene Editing in the Invasive Pest Drosophila suzukii.

Originally from Asia, Drosophila suzukii Matsumura is a global pest of economically important soft-skinned fruits. Also commonly known as spotted wing drosophila, it is largely controlled through repeated applications of broad-spectrum insecticides by which resistance has been observed in the field. There is a pressing need for a better understanding of D. suzukii biology and for developing alternative environmentally friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here, we describe genetically modified strains that encode three different terminators and four different promoters to express Cas9 robustly in both the soma and/or germline of D. suzukii. The Cas9 strains were rigorously evaluated through genetic crossing to transgenic strains that encode single-guide RNAs targeting the conserved X-linked yellow body and white eye genes. We find that several Cas9/gRNA strains display remarkably high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in D. suzukii and may even provide tools useful for the development of new genetic strategies for control of this invasive species.

Evidence for a cryptic parasitoid species reveals its suitability as a biological control agent.

Uncertainty about the taxonomic status and the specificity of a species commonly prevent its consideration as a candidate for biological control of pest organisms. Here we use a combination of molecular analysis and crossing experiments to gather evidence that the parasitoid wasp Ganaspis brasiliensis, a candidate for biological control of the invasive spotted wing drosophila Drosophila suzukii, is a complex of at least two cryptic species. Complementary experiments demonstrate that individuals from one genetic group readily parasitize several drosophila species regardless of their food source while individuals from the other one are almost exclusively specific to larvae feeding in ripening fruits. Because only D. suzukii attacks ripening fruits in its area of invasion, parasitoids from this second group appear to be well suited as a biological control agent. Our study demonstrates the need for a combination of biosystematics with biological and ecological investigations for the development of safe and efficient biological control programs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes' large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Texture features explain the susceptibility of grapevine cultivars to Drosophila suzukii (Diptera: Drosophilidae) infestation in ripening and drying grapes.

Grapevine is a well-known host plant of the invasive pest Drosophila suzukii, but its susceptibility to pest oviposition and development greatly depends on the cultivar. To address environmental sustainability during viticultural zoning planning, new vineyard plantation and Integrated Pest Management programmes, it is essential to take pest pressure and cultivar susceptibility into account. To determine the different grapevine cultivars susceptibility to D. suzukii, we tested twelve widely spread cultivars during the ripening period. We also tested three cultivars during the drying period for raisin wine production. The infestation and emergence rates were consequently related to chemical and texture features of the berries to explain the role of skin and pulp characteristics in determining the nature of the susceptibility. Our results showed that susceptibility to D. suzukii infestation varies across cultivars. On ripening grapes, infestation is primarily influenced by skin and pulp firmness, elasticity and consistency. Suitability for egg development resulted mainly related to skin and pulp deformation. In a drying loft, infestation may also occur in relation to skin and pulp consistency. Lastly, we discuss the practical implication of the underestimated role of berry texture in D. suzukii oviposition and emergence success, in both ripening and drying grapes.

Measuring Crop Motility and Food Passaging in Drosophila.

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

The Role of Lozenge in Drosophila Hematopoiesis.

Drosophila hematopoiesis is comparable to mammalian differentiation of myeloid lineages, and therefore, has been a useful model organism in illustrating the molecular and genetic basis for hematopoiesis. Multiple novel regulators and signals have been uncovered using the tools of Drosophila genetics. A Runt domain protein, lozenge, is one of the first players recognized and closely studied in the hematopoietic lineage specification. Here, we explore the role of lozenge in determination of prohemocytes into a special class of hemocyte, namely the crystal cell, and discuss molecules and signals controlling the lozenge function and its implication in immunity and stress response. Given the highly conserved nature of Runt domain in both invertebrates and vertebrates, studies in Drosophila will enlighten our perspectives on Runx-mediated development and pathologies.

Exploring cold quarantine to mango fruit against fruit fly using artificial ripening.

Mango quarantine is based mainly on heat treatment, with a possible deterioration of fruit quality. We studied the effects of cold quarantine (19 days storage at 2 °C) on fruit quality of commercial mango cvs. Keitt and Shelly for three consecutive years. Chilling injury (CI) occurs in mango fruit stored at temperatures lower than 12 °C. By reanalysing our previous transcriptome, we found that under sub-optimal temperature storage (5 °C), the fruit increases its ethylene biosynthesis and osmolarity by activating sugar metabolism, thereby probably reducing its freezing point. Similarly, ripe fruit with higher sugar concentration should be more resistant to cold-storage stress. Here, mango fruit was artificially ripened with 150 ppm ethylene. The control group, stored at 2 °C, suffered from severe CI, whereas the combined treatment of artificial ripening, modified atmosphere (fruit were enclosed in perforated bags) and subsequent low-temperature conditioning resulted in a significant reduction in CI to satisfactory levels for consumer acceptance (taste, aroma and texture). The combined treatment reduced lipid peroxidation and maintained flavour, leading to a novel cold-quarantine treatment for mango fruit. Thus, by reversing the supply chain and storing ripe and ready-to-eat fruit, cold quarantine was enabled for mango, and possibly other chilling-susceptible fruits.

Excess active P13K rescues huntingtin-mediated neuronal cell death but has no effect on axonal transport defects.

High levels of oxidative stress is detected in neurons affected by many neurodegenerative diseases, including huntington's disease. Many of these diseases also show neuronal cell death and axonal transport defects. While nuclear inclusions/accumulations likely cause cell death, we previously showed that cytoplasmic axonal accumulations can also contribute to neuronal death. However, the cellular mechanisms responsible for activating cell death is unclear. One possibility is that perturbations in normal axonal transport alter the function of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-pathway, a signal transduction pathway that promotes survival/growth in response to extracellular signals. To test this proposal in vivo, we expressed active PI3K in the context of pathogenic huntingtin (HTT-138Q) in Drosophila larval nerves, which show axonal transport defects and neuronal cell death. We found that excess expression of active P13K significantly suppressed HTT-138Q-mediated neuronal cell death, but had no effect on HTT-138Q-mediated axonal transport defects. Expression of active PI3K also rescued Paraquat-mediated cell death. Further, increased levels of pSer9 (inactive) glycogen synthase kinase 3ß was seen in HTT-138Q-mediated larval brains, and in dynein loss of function mutants, indicating the modulation of the pro-survival pathway. Intriguingly, proteins in the PI3K/AKT-pathway showed functional interactions with motor proteins. Taken together our observations suggest that proper axonal transport is likely essential for the normal function of the pro-survival PI3K/AKT-signaling pathway and for neuronal survival in vivo. These results have important implications for targeting therapeutics to early insults during neurodegeneration and death.

Accurate elimination of superfluous attachment cells is critical for the construction of functional multicellular proprioceptors in Drosophila.

Here, we show for the first time that developmental cell death plays a critical role in the morphogenesis of multicellular proprioceptors in Drosophila. The most prominent multicellular proprioceptive organ in the fly larva, the pentascolopidial (LCh5) organ, consists of a cluster of five stretch-responsive sensory organs that are anchored to the cuticle via specialized attachment cells. Stable attachment of the organ to the cuticle is critical for its ability to perceive mechanical stimuli arising from muscle contractions and the resulting displacement of its attachment sites. We now show that five attachment cells are born within the LCh5 lineage, but three of them are rapidly eliminated, normally, by apoptosis. Strong genetic evidence attests to the existence of an autophagic gene-dependent safeguard mechanism that guarantees elimination of the unwanted cells upon perturbation of the apoptotic pathway prior to caspase liberation. The removal of the three superfluous cells guarantees the right ratio between the number of sensory organs and the number of attachment cells that anchor them to the cuticle. This accurate matching seems imperative for the attachment of cell growth and functionality and is thus vital for normal morphogenesis and functionality of the sensory organ.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Yeast red pigment modifies cloned human α-synuclein pathogenesis in Parkinson disease models in Saccharomyces cerevisiae and Drosophila melanogaster.

Recently, we identified the yeast red pigment (RP), a polymer of 1-(5'-Phosphoribosyl)-5-aminoimidazole, as a novel potential anti-amyloid agent for the therapy of neurodegenerative diseases. The purpose of this study was to further validate RP for treatment of Parkinson's disease (PD) and to clarify molecular mechanisms involved in the reduction of amyloid cytotoxicity. We investigated RP effects in vivo using Saccharomyces cerevisiae and Drosophila melanogaster PD models. Western blot analysis revealed reduction in the levels of insoluble α-synuclein in both models, while soluble α-synuclein decreased only in Drosophila. In both models RP significantly reduced α-synuclein cytotoxicity, as was revealed by immunohistochemistry in Drosophila (p < 0.001, n = 27 flies per genotype/assay) and by flow cytometry in yeast (p < 0.05). Data obtained from the yeast PD model suggests that RP antitoxic effects are associated with a drop in ROS accumulation, and slower cellular transition from the early to late apoptotic stage. Using Drosophila brain tissue sections, we have demonstrated that RP helps to compensate for an α-synuclein-mediated reduction in the number of dopaminergic neurons and leads to better performance in animal climbing tests (p < 0.001, n = 120-150 flies per genotype/assay). Taken together, these results demonstrate the potential of RP for the treatment of PD, at least in model systems.

Phosphatidylserine Externalization Results from and Causes Neurite Degeneration in Drosophila.

Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an "eat-me" signal allowing recognition of degenerating neurites by phagocytes. Here we show that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. Our findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron - dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

MicroRNAs mir-184 and let-7 alter Drosophila metabolism and longevity.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression associated with many complex biological processes. By comparing miRNA expression between long-lived cohorts of Drosophila melanogaster that were fed a low-nutrient diet with normal-lived control animals fed a high-nutrient diet, we identified miR-184, let-7, miR-125, and miR-100 as candidate miRNAs involved in modulating aging. We found that ubiquitous, adult-specific overexpression of these individual miRNAs led to significant changes in fat metabolism and/or lifespan. Most impressively, adult-specific overexpression of let-7 in female nervous tissue increased median fly lifespan by ~22%. We provide evidence that this lifespan extension is not due to alterations in nutrient intake or to decreased insulin signaling.

A Floral Fragrance, Methyl Benzoate, is An Efficient Green Pesticide.

Over-reliance on synthetic pesticides in insect pest control has caused widespread public and scientific concerns for human health and the environment, especially since many insect pests have already developed resistances to conventional pesticides and Bt products. For this reason, there is a considerable interest in development of alternative control methods for insect pest management. Based on laboratory studies, we report that methyl benzoate (MB), a naturally-occurring compound in many plants, may possess toxicity against various stages of a variety of insect pests, including the brown marmorated stinkbug, Halyomorpha halys, diamondback moth, Plutella xylostella, and tobacco hornworm, Manduca sexta, as well as the spotted wing drosophila, Drosophila suzukii. Based on our laboratory toxicity data, MB was at least 5 to 20 times more toxic than the conventional pyrethroid (ß-cyfluthrin), sulfur &pyrethrin mixture, and some organic commercial products available on the market against H. halys, P. xylostella, and M. sexta, eggs. Because MB is considered an environment-friendly, it has great potential to be used as an alternative tool to synthetic pesticide for insect pest management in crop production, thereby, reducing threats to natural ecosystems and human health caused by over-application of conventional synthetic pesticides.

The Wide Potential Trophic Niche of the Asiatic Fruit Fly Drosophila suzukii: The Key of Its Invasion Success in Temperate Europe?

The Asiatic fruit fly Drosophila suzukii has recently invaded Europe and North and South America, causing severe damage to fruit production systems. Although agronomic host plants of that fly are now well documented, little is known about the suitability of wild and ornamental hosts in its exotic area. In order to study the potential trophic niche of D. suzukii with relation to fruit characteristics, fleshy fruits from 67 plant species were sampled in natural and anthropic ecosystems (forests, hedgerows, grasslands, coastal areas, gardens and urban areas) of the north of France and submitted to experimental infestations. A set of fruit traits (structure, colour, shape, skin texture, diameter and weight, phenology) potentially interacting with oviposition choices and development success of D. suzukii was measured. Almost half of the tested plant species belonging to 17 plant families allowed the full development of D. suzukii. This suggests that the extreme polyphagy of the fly and the very large reservoir of hosts producing fruits all year round ensure temporal continuity in resource availability and contribute to the persistence and the exceptional invasion success of D. suzukii in natural habitats and neighbouring cultivated systems. Nevertheless, this very plastic trophic niche is not systematically beneficial to the fly. Some of the tested plants attractive to D. suzukii gravid females stimulate oviposition but do not allow full larval development. Planted near sensitive crops, these "trap plants" may attract and lure D. suzukii, therefore contributing to the control of the invasive fly.

Genetic changes accompanying the evolution of host specialization in Drosophila sechellia.

Changes in host specialization contribute to the diversification of phytophagous insects. When shifting to a new host, insects evolve new physiological, morphological, and behavioral adaptations. Our understanding of the genetic changes responsible for these adaptations is limited. For instance, we do not know how often host shifts involve gain-of-function vs. loss-of-function alleles. Recent work suggests that some genes involved in odor recognition are lost in specialists. Here we show that genes involved in detoxification and metabolism, as well as those affecting olfaction, have reduced gene expression in Drosophila sechellia-a specialist on the fruit of Morinda citrifolia. We screened for genes that differ in expression between D. sechellia and its generalist sister species, D. simulans. We also screened for genes that are differentially expressed in D. sechellia when these flies chose their preferred host vs. when they were forced onto other food. D. sechellia increases expression of genes involved with oogenesis and fatty acid metabolism when on its host. The majority of differentially expressed genes, however, appear downregulated in D. sechellia. For several functionally related genes, this decrease in expression is associated with apparent loss-of-function alleles. For example, the D. sechellia allele of Odorant binding protein 56e (Obp56e) harbors a premature stop codon. We show that knockdown of Obp56e activity significantly reduces the avoidance response of D. melanogaster toward M. citrifolia. We argue that apparent loss-of-function alleles like Obp56e potentially contributed to the initial adaptation of D. sechellia to its host. Our results suggest that a subset of genes reduce or lose function as a consequence of host specialization, which may explain why, in general, specialist insects tend to shift to chemically similar hosts.