Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster.

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. Here, we characterize the Drosophila tuSz1 mutant strain, which mounts an aberrant immune response against its own fat body. We demonstrate that this autoimmunity is the result of two mutations: 1) a mutation in the GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Our data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals.

Maternal Priming of Offspring Immune System in Drosophila.

Immune priming occurs when a past infection experience leads to a more effective immune response upon a secondary exposure to the infection or pathogen. In some instances, parents are able to transmit immune priming to their offspring, creating a subsequent generation with a superior immune capability, through processes that are not yet fully understood. Using a parasitoid wasp, which infects larval stages of Drosophila melanogaster, we describe an example of an intergenerational inheritance of immune priming. This phenomenon is anticipatory in nature and does not rely on parental infection, but rather, when adult fruit flies are cohabitated with a parasitic wasp, they produce offspring that are more capable of mounting a successful immune response against a parasitic macro-infection. This increase in offspring survival correlates with a more rapid induction of lamellocytes, a specialized immune cell. RNA-sequencing of the female germline identifies several differentially expressed genes following wasp exposure, including the peptiodoglycan recognition protein-LB (PGRP-LB). We find that genetic manipulation of maternal PGRP-LB identifies this gene as a key element in this intergenerational phenotype.

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens.

BACKGROUND: The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. RESULTS: Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. CONCLUSION: We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

Neural circuitry of social learning in Drosophila requires multiple inputs to facilitate inter-species communication.

Drosophila species communicate the threat of parasitoid wasps to naïve individuals. Communication of the threat between closely related species is efficient, while more distantly related species exhibit a dampened, partial communication. Partial communication between D. melanogaster and D. ananassae about wasp presence is enhanced following a period of cohabitation, suggesting that species-specific natural variations in communication 'dialects' can be learned through socialization. In this study, we identify six regions of the Drosophila brain essential for dialect training. We pinpoint subgroups of neurons in these regions, including motion detecting neurons in the optic lobe, layer 5 of the fan-shaped body, the D glomerulus in the antennal lobe, and the odorant receptor Or69a, where activation of each component is necessary for dialect learning. These results reveal functional neural circuits that underlie complex Drosophila social behaviors, and these circuits are required for integration several cue inputs involving multiple regions of the Drosophila brain.

Transgeneratonal inheritance of ethanol preference is caused by maternal NPF repression.

Rapid or even anticipatory adaptation to environmental conditions can provide a decisive fitness advantage to an organism. The memory of recurring conditions could also benefit future generations; however, neuronally-encoded behavior isn't thought to be inherited across generations. We tested the possibility that environmentally triggered modifications could allow 'memory' of parental experiences to be inherited. In Drosophila melanogaster, exposure to predatory wasps leads to inheritance of a predisposition for ethanol-rich food for five generations. Inhibition of Neuropeptide-F (NPF) activates germline caspases required for transgenerational ethanol preference. Further, inheritance of low NPF expression in specific regions of F1 brains is required for the transmission of this food preference: a maternally derived NPF locus is necessary for this phenomenon, implicating a maternal epigenetic mechanism of NPF-repression. Given the conserved signaling functions of NPF and its mammalian NPY homolog in drug and alcohol disorders, these observations raise the intriguing possibility of NPY-related transgenerational effects in humans.

New Drosophila Long-Term Memory Genes Revealed by Assessing Computational Function Prediction Methods.

A major bottleneck to our understanding of the genetic and molecular foundation of life lies in the ability to assign function to a gene and, subsequently, a protein. Traditional molecular and genetic experiments can provide the most reliable forms of identification, but are generally low-throughput, making such discovery and assignment a daunting task. The bottleneck has led to an increasing role for computational approaches. The Critical Assessment of Functional Annotation (CAFA) effort seeks to measure the performance of computational methods. In CAFA3, we performed selected screens, including an effort focused on long-term memory. We used homology and previous CAFA predictions to identify 29 key Drosophila genes, which we tested via a long-term memory screen. We identify 11 novel genes that are involved in long-term memory formation and show a high level of connectivity with previously identified learning and memory genes. Our study provides first higher-order behavioral assay and organism screen used for CAFA assessments and revealed previously uncharacterized roles of multiple genes as possible regulators of neuronal plasticity at the boundary of information acquisition and memory formation.

Correction: Drosophila species learn dialects through communal living.

[This corrects the article DOI: 10.1371/journal.pgen.1007430.].

Drosophila species learn dialects through communal living.

Many species are able to share information about their environment by communicating through auditory, visual, and olfactory cues. In Drosophila melanogaster, exposure to parasitoid wasps leads to a decline in egg laying, and exposed females communicate this threat to naïve flies, which also depress egg laying. We find that species across the genus Drosophila respond to wasps by egg laying reduction, activate cleaved caspase in oocytes, and communicate the presence of wasps to naïve individuals. Communication within a species and between closely related species is efficient, while more distantly related species exhibit partial communication. Remarkably, partial communication between some species is enhanced after a cohabitation period that requires exchange of visual and olfactory signals. This interspecies "dialect learning" requires neuronal cAMP signaling in the mushroom body, suggesting neuronal plasticity facilitates dialect learning and memory. These observations establish Drosophila as genetic models for interspecies social communication and evolution of dialects.

Nematocytes: Discovery and characterization of a novel anculeate hemocyte in Drosophila falleni and Drosophila phalerata.

Immune challenges, such as parasitism, can be so pervasive and deleterious that they constitute an existential threat to a species' survival. In response to these ecological pressures, organisms have developed a wide array of novel behavioral, cellular, and molecular adaptations. Research into these immune defenses in model systems has resulted in a revolutionary understanding of evolution and functional biology. As the field has expanded beyond the limited number of model organisms our appreciation of evolutionary innovation and unique biology has widened as well. With this in mind, we have surveyed the hemolymph of several non-model species of Drosophila. Here we identify and describe a novel hemocyte, type-II nematocytes, found in larval stages of numerous Drosophila species. Examined in detail in Drosophila falleni and Drosophila phalerata, we find that these remarkable cells are distinct from previously described hemocytes due to their anucleate state (lacking a nucleus) and unusual morphology. Type-II nematocytes are long, narrow cells with spindle-like projections extending from a cell body with high densities of mitochondria and microtubules, and exhibit the ability to synthesize proteins. These properties are unexpected for enucleated cells, and together with our additional characterization, we demonstrate that these type-II nematocytes represent a biological novelty. Surprisingly, despite the absence of a nucleus, we observe through live cell imaging that these cells remain motile with a highly dynamic cellular shape. Furthermore, these cells demonstrate the ability to form multicellular structures, which we suggest may be a component of the innate immune response to macro-parasites. In addition, live cell imaging points to a large nucleated hemocyte, type-I nematocyte, as the progenitor cell, leading to enucleation through a budding or asymmetrical division process rather than nuclear ejection: This study is the first to report such a process of enucleation. Here we describe these cells in detail for the first time and examine their evolutionary history in Drosophila.

A systems level approach to temporal expression dynamics in Drosophila reveals clusters of long term memory genes.

The ability to integrate experiential information and recall it in the form of memory is observed in a wide range of taxa, and is a hallmark of highly derived nervous systems. Storage of past experiences is critical for adaptive behaviors that anticipate both adverse and positive environmental factors. The process of memory formation and consolidation involve many synchronized biological events including gene transcription, protein modification, and intracellular trafficking: However, many of these molecular mechanisms remain illusive. With Drosophila as a model system we use a nonassociative memory paradigm and a systems level approach to uncover novel transcriptional patterns. RNA sequencing of Drosophila heads during and after memory formation identified a number of novel memory genes. Tracking the dynamic expression of these genes over time revealed complex gene networks involved in long term memory. In particular, this study focuses on two functional gene clusters of signal peptides and proteases. Bioinformatics network analysis and prediction in combination with high-throughput RNA sequencing identified previously unknown memory genes, which when genetically knocked down resulted in behaviorally validated memory defects.

Machine Learning Analysis Identifies Drosophila Grunge/Atrophin as an Important Learning and Memory Gene Required for Memory Retention and Social Learning.

High-throughput experiments are becoming increasingly common, and scientists must balance hypothesis-driven experiments with genome-wide data acquisition. We sought to predict novel genes involved in Drosophila learning and long-term memory from existing public high-throughput data. We performed an analysis using PILGRM, which analyzes public gene expression compendia using machine learning. We evaluated the top prediction alongside genes involved in learning and memory in IMP, an interface for functional relationship networks. We identified Grunge/Atrophin (Gug/Atro), a transcriptional repressor, histone deacetylase, as our top candidate. We find, through multiple, distinct assays, that Gug has an active role as a modulator of memory retention in the fly and its function is required in the adult mushroom body. Depletion of Gug specifically in neurons of the adult mushroom body, after cell division and neuronal development is complete, suggests that Gug function is important for memory retention through regulation of neuronal activity, and not by altering neurodevelopment. Our study provides a previously uncharacterized role for Gug as a possible regulator of neuronal plasticity at the interface of memory retention and memory extinction.

Genomic characterization of patient-derived xenograft models established from fine needle aspirate biopsies of a primary pancreatic ductal adenocarcinoma and from patient-matched metastatic sites.

N-of-1 trials target actionable mutations, yet such approaches do not test genomically-informed therapies in patient tumor models prior to patient treatment. To address this, we developed patient-derived xenograft (PDX) models from fine needle aspiration (FNA) biopsies (FNA-PDX) obtained from primary pancreatic ductal adenocarcinoma (PDAC) at the time of diagnosis. Here, we characterize PDX models established from one primary and two metastatic sites of one patient. We identified an activating KRAS G12R mutation among other mutations in these models. In explant cells derived from these PDX tumor models with a KRAS G12R mutation, treatment with inhibitors of CDKs (including CDK9) reduced phosphorylation of a marker of CDK9 activity (phospho-RNAPII CTD Ser2/5) and reduced viability/growth of explant cells derived from PDAC PDX models. Similarly, a CDK inhibitor reduced phospho-RNAPII CTD Ser2/5, increased apoptosis, and inhibited tumor growth in FNA-PDX and patient-matched metastatic-PDX models. In summary, PDX models can be constructed from FNA biopsies of PDAC which in turn can enable genomic characterization and identification of potential therapies.

Social communication of predator-induced changes in Drosophila behavior and germ line physiology.

Behavioral adaptation to environmental threats and subsequent social transmission of adaptive behavior has evolutionary implications. In Drosophila, exposure to parasitoid wasps leads to a sharp decline in oviposition. We show that exposure to predator elicits both an acute and learned oviposition depression, mediated through the visual system. However, long-term persistence of oviposition depression after predator removal requires neuronal signaling functions, a functional mushroom body, and neurally driven apoptosis of oocytes through effector caspases. Strikingly, wasp-exposed flies (teachers) can transmit egg-retention behavior and trigger ovarian apoptosis in naive, unexposed flies (students). Acquisition and behavioral execution of this socially learned behavior by naive flies requires all of the factors needed for primary learning. The ability to teach does not require ovarian apoptosis. This work provides new insight into genetic and physiological mechanisms that underlie an ecologically relevant form of learning and mechanisms for its social transmission.

A novel paradigm for nonassociative long-term memory in Drosophila: predator-induced changes in oviposition behavior.

Learning processes in Drosophila have been studied through the use of Pavlovian associative memory tests, and these paradigms have been extremely useful in identifying both genetic factors and neuroanatomical structures that are essential to memory formation. Whether these same genes and brain compartments also contribute to memory formed from nonassociative experiences is not well understood. Exposures to environmental stressors such as predators are known to induce innate behavioral responses and can lead to new memory formation that allows a predator response to persist for days after the predator threat has been removed. Here, we utilize a unique form of nonassociative behavior in Drosophila where female flies detect the presence of endoparasitoid predatory wasps and alter their oviposition behavior to lay eggs in food containing high levels of alcohol. The predator-induced change in fly oviposition preference is maintained for days after wasps are removed, and this persistence in behavior requires a minimum continuous exposure time of 14 hr. Maintenance of this behavior is dependent on multiple long-term memory genes, including orb2, dunce, rutabaga, amnesiac, and Fmr1. Maintenance of the behavior also requires intact synaptic transmission of the mushroom body. Surprisingly, synaptic output from the mushroom body (MB) or the functions of any of these learning and memory genes are not required for the change in behavior when female flies are in constant contact with wasps. This suggests that perception of this predator that leads to an acute change in oviposition behavior is not dependent on the MB or dependent on learning and memory gene functions. Because wasp-induced oviposition behavior can last for days and its maintenance requires a functional MB and the wild-type products of several known learning and memory genes, we suggest that this constitutes a paradigm for a bona fide form of nonassociative long-term memory that is not dependent on associated experiences.

A role for nematocytes in the cellular immune response of the drosophilid Zaprionus indianus.

The melanotic encapsulation response mounted by Drosophila melanogaster against macroparasites, which is based on haemocyte binding to foreign objects, is poorly characterized relative to its humoral immune response against microbes, and appears to be variable across insect lineages. The genus Zaprionus is a diverse clade of flies embedded within the genus Drosophila. Here we characterize the immune response of Zaprionus indianus against endoparasitoid wasp eggs, which elicit the melanotic encapsulation response in D. melanogaster. We find that Z. indianus is highly resistant to diverse wasp species. Although Z. indianus mounts the canonical melanotic encapsulation response against some wasps, it can also potentially fight off wasp infection using two other mechanisms: encapsulation without melanization and a non-cellular form of wasp killing. Zaprionus indianus produces a large number of haemocytes including nematocytes, which are large fusiform haemocytes absent in D. melanogaster, but which we found in several other species in the subgenus Drosophila. Several lines of evidence suggest these nematocytes are involved in anti-wasp immunity in Z. indianus and in particular in the encapsulation of wasp eggs. Altogether, our data show that the canonical anti-wasp immune response and haemocyte make-up of the model organism D. melanogaster vary across the genus Drosophila.

Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity.

Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.

Fruit flies medicate offspring after seeing parasites.

Hosts have numerous defenses against parasites, of which behavioral immune responses are an important but underappreciated component. Here we describe a behavioral immune response that Drosophila melanogaster uses against endoparasitoid wasps. We found that when flies see wasps, they switch to laying eggs in alcohol-laden food sources that protect hatched larvae from infection. This change in oviposition behavior, mediated by neuropeptide F, is retained long after wasps are removed. Flies respond to diverse female larval endoparasitoids but not to males or pupal endoparasitoids, showing that they maintain specific wasp search images. Furthermore, the response evolved multiple times across the genus Drosophila. Our data reveal a behavioral immune response based on anticipatory medication of offspring and outline a nonassociative memory paradigm based on innate parasite recognition by the host.

Mgat1-dependent N-glycosylation of membrane components primes Drosophila melanogaster blood cells for the cellular encapsulation response.

In nature, larvae of the fruitfly Drosophila melanogaster are commonly infected by parasitoid wasps, and so have evolved a robust immune response to counter wasp infection. In this response, fly immune cells form a multilayered capsule surrounding the wasp egg, leading to death of the parasite. Many of the molecular mechanisms underlying this encapsulation response are conserved with human immune responses. Our findings suggest that protein N-glycosylation, a common protein post-translational modification of human immune proteins, may be one such conserved mechanism. We found that membrane proteins on Drosophila immune cells are N-glycosylated in a temporally specific manner following wasp infection. Furthermore we have identified mutations in eight genes encoding enzymes of the N-glycosylation pathway that decrease fly resistance to wasp infection. More specifically, loss of protein N-glycosylation in immune cells following wasp infection led to the formation of defective capsules, which disintegrated over time and were thereby unsuccessful at preventing wasp development. Interestingly, we also found that one species of Drosophila parasitoid wasp, Leptopilina victoriae, targets protein N-glycosylation as part of its virulence mechanism, and that overexpression of an N-glycosylation enzyme could confer resistance against this wasp species to otherwise susceptible flies. Taken together, these findings demonstrate that protein N-glycosylation is a key player in Drosophila cellular encapsulation and suggest that this response may provide a novel model to study conserved roles of protein glycosylation in immunity.

High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster.

Among the most common parasites of Drosophila in nature are parasitoid wasps, which lay their eggs in fly larvae and pupae. D. melanogaster larvae can mount a cellular immune response against wasp eggs, but female wasps inject venom along with their eggs to block this immune response. Genetic variation in flies for immune resistance against wasps and genetic variation in wasps for virulence against flies largely determines the outcome of any fly-wasp interaction. Interestingly, up to 90% of the variation in fly resistance against wasp parasitism has been linked to a very simple mechanism: flies with increased constitutive blood cell (hemocyte) production are more resistant. However, this relationship has not been tested for Drosophila hosts outside of the melanogaster subgroup, nor has it been tested across a diversity of parasitoid wasp species and strains. We compared hemocyte levels in two fly species from different subgroups, D. melanogaster and D. suzukii, and found that D. suzukii constitutively produces up to five times more hemocytes than D. melanogaster. Using a panel of 24 parasitoid wasp strains representing fifteen species, four families, and multiple virulence strategies, we found that D. suzukii was significantly more resistant to wasp parasitism than D. melanogaster. Thus, our data suggest that the relationship between hemocyte production and wasp resistance is general. However, at least one sympatric wasp species was a highly successful infector of D. suzukii, suggesting specialists can overcome the general resistance afforded to hosts by excessive hemocyte production. Given that D. suzukii is an emerging agricultural pest, identification of the few parasitoid wasps that successfully infect D. suzukii may have value for biocontrol.

Alcohol consumption as self-medication against blood-borne parasites in the fruit fly.

Plants and fungi often produce toxic secondary metabolites that limit their consumption, but herbivores and fungivores that evolve resistance gain access to these resources and can also gain protection against nonresistant predators and parasites. Given that Drosophila melanogaster fruit fly larvae consume yeasts growing on rotting fruit and have evolved resistance to fermentation products, we decided to test whether alcohol protects flies from one of their common natural parasites, endoparasitoid wasps. Here, we show that exposure to ethanol reduces wasp oviposition into fruit fly larvae. Furthermore, if infected, ethanol consumption by fruit fly larvae causes increased death of wasp larvae growing in the hemocoel and increased fly survival without need of the stereotypical antiwasp immune response. This multifaceted protection afforded to fly larvae by ethanol is significantly more effective against a generalist wasp than a wasp that specializes on D. melanogaster. Finally, fly larvae seek out ethanol-containing food when infected, indicating that they use alcohol as an antiwasp medicine. Although the high resistance of D. melanogaster may make it uniquely suited to exploit curative properties of alcohol, it is possible that alcohol consumption may have similar protective effects in other organisms.