RESUMEN

While undergraduate research has been shown to be a high-impact educational practice, it is logistically impossible for all undergraduate biology majors to have long-term faculty-mentored research experience. Therefore, biology educators and researchers must devise opportunities to engage more students in undergraduate research outside of working directly in their labs. Course-Based Research Experiences (CREs), structured as authentic research experiences, are one such opportunity. In this work, we describe the effects of a CRE with biomedical relevance on students' research skills, attitudes toward science, and perceptions of scientific research and scientific researchers. Results demonstrate that students gained experience in independent research skills including designing their own research project, being accountable for part of a project, and writing a research proposal. Students' perceptions of scientific research and researchers, assessed by the Draw-A-Researcher Task, did not show changes among the whole group, but individual analysis yielded meaningful results related to students' personal changes in how they perceived research and researchers, including their perception of themselves as researchers. This work demonstrates the substantial impact of CREs on upper-level biology undergraduate and graduate students.

RESUMEN

Innate immune responses that allow hosts to survive infection depend on the action of multiple conserved signaling pathways. Pathogens and parasites in turn have evolved virulence factors to target these immune signaling pathways in an attempt to overcome host immunity. Consequently, the interactions between host immune molecules and pathogen virulence factors play an important role in determining the outcome of an infection. The immune responses of Drosophila melanogaster provide a valuable model to understand immune signaling and host-pathogen interactions. Flies are commonly infected by parasitoid wasps and mount a coordinated cellular immune response following infection. This response is characterized by the production of specialized blood cells called lamellocytes that form a tight capsule around wasp eggs in the host hemocoel. The conserved JAK-STAT signaling pathway has been implicated in lamellocyte proliferation and is required for successful encapsulation of wasp eggs. Here we show that activity of Stat92E, the D. melanogaster STAT ortholog, is induced in immune tissues following parasitoid infection. Virulent wasp species are able to suppress Stat92E activity during infection, suggesting they target JAK-STAT pathway activation as a virulence strategy. Furthermore, two wasp species (Leptopilina guineaensis and Ganaspis xanthopoda) suppress phenotypes associated with a gain-of-function mutation in hopscotch, the D. melanogaster JAK ortholog, indicating that they inhibit the activity of the core signaling components of the JAK-STAT pathway. Our data suggest that parasitoid wasp virulence factors block JAK-STAT signaling to overcome fly immune defenses.

Asunto(s)

Proteínas de Drosophila , Drosophila melanogaster , Interacciones Huésped-Parásitos , Quinasas Janus , Factores de Transcripción STAT , Transducción de Señal , Avispas , Animales , Drosophila melanogaster/parasitología , Factores de Transcripción STAT/metabolismo , Quinasas Janus/metabolismo , Virulencia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Inmunidad Innata

RESUMEN

The ability to overcome stressful environments is critical for pathogen survival in the host. One challenge for bacteria is the exposure to reactive chlorine species (RCS), which are generated by innate immune cells as a critical part of the oxidative burst. Hypochlorous acid (HOCl) is the most potent antimicrobial RCS and is associated with extensive macromolecular damage in the phagocytized pathogen. However, bacteria have evolved defense strategies to alleviate the effects of HOCl-mediated damage. Among these are RCS-sensing transcriptional regulators that control the expression of HOCl-protective genes under non-stress and HOCl stress. Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils during pathogenesis; however, their responses to and defenses from HOCl are still completely unexplored. Here, we present evidence that UPEC strains tolerate higher levels of HOCl and are better protected from neutrophil-mediated killing compared with other E. coli. Transcriptomic analysis of HOCl-stressed UPEC revealed the upregulation of an operon consisting of three genes, one of which encodes the transcriptional regulator RcrR. We identified RcrR as a HOCl-responsive transcriptional repressor, which, under non-stress conditions, is bound to the operator and represses the expression of its target genes. During HOCl exposure, however, the repressor forms reversible intermolecular disulfide bonds and dissociates from the DNA resulting in the derepression of the operon. Deletion of one of the target genes renders UPEC significantly more susceptible to HOCl and phagocytosis indicating that the HOCl-mediated induction of the regulon plays a major role for UPEC's HOCl resistance. IMPORTANCE How do pathogens deal with antimicrobial oxidants produced by the innate immune system during infection? Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils and, therefore, must counter elevated levels of the antimicrobial oxidant HOCl to establish infection. Our study provides fundamentally new insights into a defense mechanism that enables UPEC to fend off the toxic effects of HOCl stress. Intriguingly, the defense system is predominantly found in UPEC and absent in noninvasive enteropathogenic E. coli. Our data suggest expression of the target gene rcrB is exclusively responsible for UPEC's increased HOCl tolerance in culture and contributes to UPEC's survival during phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Asunto(s)

Infecciones por Escherichia coli , Proteínas de Escherichia coli , Infecciones Urinarias , Escherichia coli Uropatógena , Humanos , Escherichia coli Uropatógena/metabolismo , Cloro/farmacología , Cloro/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Hipocloroso/farmacología , Escherichia , Infecciones Urinarias/microbiología , Infecciones por Escherichia coli/microbiología , Oxidación-Reducción , Antibacterianos/farmacología , Oxidantes/farmacología , Disulfuros/metabolismo

RESUMEN

Organisms are commonly infected by a diverse array of pathogens and mount functionally distinct responses to each of these varied immune challenges. Host immune responses are characterized by the induction of gene expression, however, the extent to which expression changes are shared among responses to distinct pathogens is largely unknown. To examine this, we performed meta-analysis of gene expression data collected from Drosophila melanogaster following infection with a wide array of pathogens. We identified 62 genes that are significantly induced by infection. While many of these infection-induced genes encode known immune response factors, we also identified 21 genes that have not been previously associated with host immunity. Examination of the upstream flanking sequences of the infection-induced genes lead to the identification of two conserved enhancer sites. These sites correspond to conserved binding sites for GATA and nuclear factor κB (NFκB) family transcription factors and are associated with higher levels of transcript induction. We further identified 31 genes with predicted functions in metabolism and organismal development that are significantly downregulated following infection by diverse pathogens. Our study identifies conserved gene expression changes in Drosophila melanogaster following infection with varied pathogens, and transcription factor families that may regulate this immune induction.

RESUMEN

Avian eggshell pigmentation may provide information about a female's physiological condition, in particular her state of oxidative balance. Previously we found that female house wrens (Troglodytes aedon Vieillot, 1809) with lighter, less-maculated, and redder ground-colored shells were older and produced heavier offspring than females laying darker, browner eggs. The strong pro-oxidant protoporphyrin is responsible for this species' eggshell pigmentation, so differences in pigmentary coloration may be related to eggshell protoporphyrin content and reflect female oxidative balance and condition during egg-formation. Therefore, we tested the assumption that egg-surface coloration is related to the amount of protoporphyrin in the shell matrix. We analyzed digital photographs of eggs to determine maculation coverage as a measure of the overall ground coloration of the egg and its red-, green-, and blue-channel pixel values. Pigments were then extracted from these same eggs and analyzed using high-performance liquid chromatography. There was a strong, positive relationship between eggshell redness and protoporphyrin content of eggshells, but no relationship between percent maculation and protoporphyrin content. Thus, when older, larger females deposit more protoporphyrin in their eggshells, this may reflect a tolerance for high levels of circulating protoporphyrin or an effective mechanism for off-loading protoporphyrin into the eggshell matrix.

RESUMEN

As organisms age, they often accumulate protein aggregates that are thought to be toxic, potentially leading to age-related diseases. This accumulation of protein aggregates is partially attributed to a failure to maintain protein homeostasis. A variety of genetic factors have been linked to longevity, but how these factors also contribute to protein homeostasis is not completely understood. In order to understand the relationship between aging and protein aggregation, we tested how a gene that regulates lifespan and age-dependent locomotor behaviors, p38 MAPK (p38Kb), influences protein homeostasis as an organism ages. We find that p38Kb regulates age-dependent protein aggregation through an interaction with starvin, a regulator of muscle protein homeostasis. Furthermore, we have identified Lamin as an age-dependent target of p38Kb and starvin.

Asunto(s)

Envejecimiento/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Longevidad/genética , Sistema de Señalización de MAP Quinasas/genética , Proteostasis/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Envejecimiento/genética , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Eliminación de Gen , Laminas/metabolismo , Locomoción/genética , Macroautofagia/genética , Músculos/metabolismo , Estrés Oxidativo/genética , Fenotipo , Proteolisis , Interferencia de ARN , Proteínas Quinasas p38 Activadas por Mitógenos/genética

RESUMEN

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.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila melanogaster/inmunología , Proteínas de la Matriz Extracelular/metabolismo , Tolerancia Inmunológica/inmunología , Quinasas Janus/metabolismo , Mutación , Autotolerancia , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas de la Matriz Extracelular/genética , Glicosilación , Hemocitos , Quinasas Janus/genética

RESUMEN

In nature, Drosophila melanogaster larvae are infected by parasitoid wasps and mount a cellular immune response to this infection. Several conserved signaling pathways have been implicated in coordinating this response, however our understanding of the integration and regulation of these pathways is incomplete. Members of the S1A serine protease family have been previously linked to immune functions, and our findings suggest roles for two S1A family members, CG10764 and CG4793 in the cellular immune response to parasitoid infection.

RESUMEN

The interactions between Drosophila melanogaster and the parasitoid wasps that infect Drosophila species provide an important model for understanding host-parasite relationships. Following parasitoid infection, D. melanogaster larvae mount a response in which immune cells (hemocytes) form a capsule around the wasp egg, which then melanizes, leading to death of the parasitoid. Previous studies have found that host hemocyte load; the number of hemocytes available for the encapsulation response; and the production of lamellocytes, an infection induced hemocyte type, are major determinants of host resistance. Parasitoids have evolved various virulence mechanisms to overcome the immune response of the D. melanogaster host, including both active immune suppression by venom proteins and passive immune evasive mechanisms. We identified a previously undescribed parasitoid species, Asobara sp. AsDen, which utilizes an active virulence mechanism to infect D. melanogaster hosts. Asobara sp. AsDen infection inhibits host hemocyte expression of msn, a member of the JNK signaling pathway, which plays a role in lamellocyte production. Asobara sp. AsDen infection restricts the production of lamellocytes as assayed by hemocyte cell morphology and altered msn expression. Our findings suggest that Asobara sp. AsDen infection alters host signaling to suppress immunity.

RESUMEN

Scientists are sequencing new genomes at an increasing rate with the goal of associating genome contents with phenotypic traits. After a new genome is sequenced and assembled, structural gene annotation is often the first step in analysis. Despite advances in computational gene prediction algorithms, most eukaryotic genomes still benefit from manual gene annotation. This requires access to good genome browsers to enable annotators to visualize and evaluate multiple lines of evidence (e.g., sequence similarity, RNA sequencing [RNA-Seq] results, gene predictions, repeats) and necessitates many volunteers to participate in the work. To address the technical barriers to creating genome browsers, the Genomics Education Partnership (GEP; https://gep.wustl.edu/) has partnered with the Galaxy Project (https://galaxyproject.org) to develop G-OnRamp (http://g-onramp.org), a web-based platform for creating UCSC Genome Browser Assembly Hubs and JBrowse genome browsers. G-OnRamp also converts a JBrowse instance into an Apollo instance for collaborative genome annotations in research and educational settings. The genome browsers produced can be transferred to the CyVerse Data Store for long-term access. G-OnRamp enables researchers to easily visualize their experimental results, educators to create Course-based Undergraduate Research Experiences (CUREs) centered on genome annotation, and students to participate in genomics research. In the process, students learn about genes/genomes and about how to utilize large datasets. Development of G-OnRamp was guided by extensive user feedback. Sixty-five researchers/educators from >40 institutions participated through in-person workshops, which produced >20 genome browsers now available for research and education. Genome browsers generated for four parasitoid wasp species have been used in a CURE engaging students at 15 colleges and universities. Our assessment results in the classroom demonstrate that the genome browsers produced by G-OnRamp are effective tools for engaging undergraduates in research and in enabling their contributions to the scientific literature in genomics. Expansion of such genomics research/education partnerships will be beneficial to researchers, faculty, and students alike.

Asunto(s)

Biología Computacional/educación , Biología Computacional/métodos , Genoma , Genómica/educación , Genómica/métodos , Anotación de Secuencia Molecular , Programas Informáticos , Algoritmos , Animales , Secuencia de Bases , Gráficos por Computador , Bases de Datos Genéticas , Drosophila melanogaster , Humanos , Análisis de Secuencia de ARN , Estudiantes , Interfaz Usuario-Computador

RESUMEN

As organisms are constantly exposed to the damaging effects of oxidative stress through both environmental exposure and internal metabolic processes, they have evolved a variety of mechanisms to cope with this stress. One such mechanism is the highly conserved p38 MAPK (p38K) pathway, which is known to be post-translationally activated in response to oxidative stress, resulting in the activation of downstream antioxidant targets. However, little is known about the role of p38K transcriptional regulation in response to oxidative stress. Therefore, we analyzed the p38K gene family across the genus Drosophila to identify conserved regulatory elements. We found that oxidative stress exposure results in increased p38K protein levels in multiple Drosophila species and is associated with increased oxidative stress resistance. We also found that the p38Kb genomic locus includes conserved AP-1 and lola-PT transcription factor consensus binding sites. Accordingly, over-expression of these transcription factors in D. melanogaster is sufficient to induce transcription of p38Kb and enhances resistance to oxidative stress. We further found that the presence of a putative lola-PT binding site in the p38Kb locus of a given species is predictive of the species' survival in response to oxidative stress. Through our comparative genomics approach, we have identified biologically relevant putative transcription factor binding sites that regulate the expression of p38Kb and are associated with resistance to oxidative stress. These findings reveal a novel mode of regulation for p38K genes and suggest that transcription may play as important a role in p38K-mediated stress responses as post-translational modifications.

Asunto(s)

Proteínas de Drosophila , Drosophila , Estrés Oxidativo , Factores de Transcripción , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética

RESUMEN

Differences in avian eggshell pigmentation could be an honest signal of female quality that males use to inform their nestling provisioning effort. We investigated whether among-individual variation in protoporphyrin-based eggshell pigmentation in house wrens (Troglodytes aedon) reflects female fitness-associated traits and whether males use that information. Females laying lighter clutches were older and larger than females laying darker clutches. Nestlings hatching from lighter clutches had greater size-corrected mass on post-hatch day 11, a measure that strongly predicts survival and recruitment to the breeding population. To test whether male provisioning effort responds to clutch pigmentation, we used a reciprocal clutch cross-fostering design, swapping dark with light clutches and light with dark; in controls, we swapped light with light clutches and dark with dark. Shortly before hatching, clutches were returned to their original nest to avoid confounding effects of nestling quality on male provisioning. Contrary to the sexual selection hypothesis, clutch pigmentation had no effect on male provisioning. Males were probably able to observe eggshell pigmentation and thus had information about female quality, but they did not use this information to modulate their nestling provisioning. This may be because of constraints on species-specific reproductive opportunities, or because variation in eggshell protoporphyrin serves other functions.

RESUMEN

Hymenopteran parasitoid wasps are a diverse collection of species that infect arthropod hosts and use factors found in their venoms to manipulate host immune responses, physiology, and behaviour. Whole parasitoid venoms have been profiled using proteomic approaches, and here we present a bioinformatic characterization of the venom protein content from Ganaspis sp. 1, a parasitoid that infects flies of the genus Drosophila. We find evidence that diverse evolutionary processes including multifunctionalization, co-option, gene duplication, and horizontal gene transfer may be acting in concert to drive venom gene evolution in Ganaspis sp.1. One major role of parasitoid wasp venom is host immune evasion. We previously demonstrated that Ganaspis sp. 1 venom inhibits immune cell activation in infected Drosophila melanogaster hosts, and our current analysis has uncovered additional predicted virulence functions. Overall, this analysis represents an important step towards understanding the composition and activity of parasitoid wasp venoms.

Asunto(s)

Venenos de Artrópodos/genética , Evolución Molecular , Avispas/genética , Animales , Venenos de Artrópodos/metabolismo , Drosophila melanogaster/inmunología , Drosophila melanogaster/parasitología , Duplicación de Gen , Transferencia de Gen Horizontal , Evasión Inmune , Proteoma/genética , Proteoma/metabolismo , Avispas/patogenicidad

RESUMEN

The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (∼5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in D. ananassae To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes (e.g., larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae Compared to D. melanogaster, the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5' ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains.

Asunto(s)

Cromosomas/genética , Drosophila/genética , Retroelementos/genética , Animales , Composición de Base/genética , Secuencia de Bases , Codón/genética , Femenino , Perfilación de la Expresión Génica , Genes de Insecto , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Wolbachia/genética

RESUMEN

The Caenorhabditis elegans gene sup-26 encodes a well-conserved RNA-recognition motif-containing RNA-binding protein (RBP) that functions in dendrite morphogenesis of the PVD sensory neuron. The Drosophila ortholog of sup-26, alan shepard (shep), is expressed throughout the nervous system and has been shown to regulate neuronal remodeling during metamorphosis. Here, we extend these studies to show that sup-26 and shep are required for the development of diverse cell types within the nematode and fly nervous systems during embryonic and larval stages. We ascribe roles for sup-26 in regulating dendrite number and the expression of genes involved in mechanosensation within the nematode peripheral nervous system. We also find that in Drosophila, shep regulates dendrite length and branch order of nociceptive neurons, regulates the organization of neuronal clusters of the peripheral nervous system and the organization of axons within the ventral nerve cord. Taken together, our results suggest that shep/sup-26 orthologs play diverse roles in neural development across animal species. Moreover, we discuss potential roles for shep/sup-26 orthologs in the human nervous system.

Asunto(s)

Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/genética , Embrión no Mamífero/metabolismo , Sistema Nervioso/metabolismo , Proteínas de Unión al ARN/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/metabolismo , Dendritas/genética , Dendritas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Metamorfosis Biológica/genética , Microscopía Confocal , Morfogénesis/genética , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo

RESUMEN

The regulation of dendritic branching is critical for sensory reception, cell-cell communication within the nervous system, learning, memory, and behavior. Defects in dendrite morphology are associated with several neurologic disorders; thus, an understanding of the molecular mechanisms that govern dendrite morphogenesis is important. Recent investigations of dendrite morphogenesis have highlighted the importance of gene regulation at the posttranscriptional level. Because RNA-binding proteins mediate many posttranscriptional mechanisms, we decided to investigate the extent to which conserved RNA-binding proteins contribute to dendrite morphogenesis across phyla. Here we identify a core set of RNA-binding proteins that are important for dendrite morphogenesis in the PVD multidendritic sensory neuron in Caenorhabditis elegans. Homologs of each of these genes were previously identified as important in the Drosophila melanogaster dendritic arborization sensory neurons. Our results suggest that RNA processing, mRNA localization, mRNA stability, and translational control are all important mechanisms that contribute to dendrite morphogenesis, and we present a conserved set of RNA-binding proteins that regulate these processes in diverse animal species. Furthermore, homologs of these genes are expressed in the human brain, suggesting that these RNA-binding proteins are candidate regulators of dendrite development in humans.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Morfogénesis/fisiología , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética

RESUMEN

The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways.

Asunto(s)

Ritmo Circadiano/fisiología , Drosophila melanogaster/fisiología , Neuronas/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Animales Modificados Genéticamente , Relojes Circadianos/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Factores de Transcripción MEF2/fisiología , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Mutantes/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética

RESUMEN

In nature, larvae of the fruit fly Drosophila melanogaster are commonly infected by parasitoid wasps. Following infection, flies mount an immune response termed cellular encapsulation in which fly immune cells form a multilayered capsule that covers and kills the wasp egg. Parasitoids have thus evolved virulence factors to suppress cellular encapsulation. To uncover the molecular mechanisms underlying the antiwasp response, we and others have begun identifying and functionally characterizing these virulence factors. Our recent work on the Drosophila parasitoid Ganaspis sp.1 has demonstrated that a virulence factor encoding a SERCA-type calcium pump plays an important role in Ganaspis sp.1 virulence. This venom SERCA antagonizes fly immune cell calcium signaling and thereby prevents the activation of the encapsulation response. In this way, the study of wasp virulence factors has revealed a novel aspect of fly immunity, namely a role for calcium signaling in fly immune cell activation, which is conserved with human immunity, again illustrating the marked conservation between fly and mammalian immune responses. Our findings demonstrate that the cellular encapsulation response can serve as a model of immune cell function and can also provide valuable insight into basic cell biological processes.

Asunto(s)

Drosophila melanogaster/parasitología , Interacciones Huésped-Parásitos , Avispas/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Drosophila melanogaster/inmunología , Femenino , Inmunidad Celular , Virulencia , Venenos de Avispas/farmacología , Avispas/patogenicidad

RESUMEN

The fruit fly Drosophila melanogaster and its endoparasitoid wasps are a developing model system for interactions between host immune responses and parasite virulence mechanisms. In this system, wasps use diverse venom cocktails to suppress the conserved fly cellular encapsulation response. Although numerous genetic tools allow detailed characterization of fly immune genes, lack of wasp genomic information has hindered characterization of the parasite side of the interaction. Here, we use high-throughput nucleic acid and amino acid sequencing methods to describe the venoms of two related Drosophila endoparasitoids with distinct infection strategies, Leptopilina boulardi and L. heterotoma. Using RNA-seq, we assembled and quantified libraries of transcript sequences from female wasp abdomens. Next, we used mass spectrometry to sequence peptides derived from dissected venom gland lumens. We then mapped the peptide spectral data against the abdomen transcriptomes to identify a set of putative venom genes for each wasp species. Our approach captured the three venom genes previously characterized in L. boulardi by traditional cDNA cloning methods as well as numerous new venom genes that were subsequently validated by a combination of RT-PCR, blast comparisons, and secretion signal sequence search. Overall, 129 proteins were found to comprise L. boulardi venom and 176 proteins were found to comprise L. heterotoma venom. We found significant overlap in L. boulardi and L. heterotoma venom composition but also distinct differences that may underlie their unique infection strategies. Our joint transcriptomic-proteomic approach for endoparasitoid wasp venoms is generally applicable to identification of functional protein subsets from any non-genome sequenced organism.

Asunto(s)

Drosophila melanogaster/parasitología , Interacciones Huésped-Parásitos/inmunología , Venenos de Avispas/genética , Avispas/genética , Animales , Antioxidantes/metabolismo , Drosophila melanogaster/inmunología , Femenino , Glucólisis , Evasión Inmune , Inmunidad Innata , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Masculino , Anotación de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcriptoma , Venenos de Avispas/metabolismo , Avispas/fisiología

RESUMEN

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.

Asunto(s)

Calcio/metabolismo , Drosophila melanogaster/inmunología , Drosophila melanogaster/parasitología , Inmunidad Celular/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/farmacología , Venenos de Avispas/enzimología , Avispas/química , Animales , Secuencia de Bases , Western Blotting , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Hemocitos/inmunología , Hemocitos/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/análisis , Análisis de Secuencia de ADN , Factores de Virulencia/farmacología , Avispas/genética , Avispas/patogenicidad