heat-tolerance-assays-using-drosophila-activity-monitor-system-guide

Heat Tolerance Assays Using the <i>Drosophila</i> Activity Monitor System: A Guide to an Executable Application for Data Analysis

The study of heat tolerance in&#8239;Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to ...

University of Nebraska at Kearney

Human immunodeficiency virus type 1 (HIV-1) persists as a pandemic even though new information about the virus is being discovered on a daily basis. If the brain becomes infected, HIV-1 encephalitis or HIV-1-associated dementia may develop. There is much to be learned about the modes of action and mechanisms of genes and proteins, and their interactions that underlie HIV-1 infection. Drosophila melanogaster has been used successfully to study genes and proteins related to HIV-1 infection, including but not limited to the disturbance of antimicrobial responses by viral protein U and the identification of D. melanogaster analogs to the serine palmitoyltransferase 5 and 6 proteins that play a role in activation of transcription by the HIV-1 Tat protein in human cells. We believe that utilizing D. melanogaster as a complementary system for the study of genes and proteins related to HIV-1 infection will provide useful information that will lead to new studies designed to enhance our understanding of the mechanistic roles of these molecules. In the present study, we focus on the utilization of D. melanogaster as a complementary system for studying HIV-1 related genes and proteins, why this research should be extended, and why this complementary system is an important method for enhancing our understanding of the genetics involved in HIV-1 infection.

Drosophila melanogaster as a complementary system for studying HIV-1-related genes and proteins.

OTK18 is a human transcriptional suppressor implicated in the regulation of human immunodeficiency virus type-one infection of mononuclear phagocytes. It is ubiquitously expressed in all normal tissues, but its normal homeostatic function is yet to be characterized. One hypothesis is that OTK18 aids in the regulation of the innate immune system. To test this hypothesis, cDNA microarray analysis was performed on the total RNA extracted from Drosophila melanogaster embryonic Schneider 2 (S2) cells transfected with either pEGFP-OTK18 (enhanced green fluorescent protein) or empty vector controls (pEGFP-N3) for 6, 12 and 24 h. cDNA microarray analysis revealed differential expression of genes known to be important in regulation of Drosophila innate immunity. The expression levels of two genes, Metchnikowin and CG16708 were verified by quantitative real-time reverse transcription PCR. These results suggest a role for OTK18 in innate immunity.

Characterization of gene expression regulated by human OTK18 using Drosophila melanogaster as a model system for innate immunity.

Drosophila melanogaster is an ideal model organism for various types of aging studies. They are easy to maintain, relatively inexpensive, have short life cycles, provide large sample sizes, and can be genetically manipulated via various methods for testing. The 49(th) Annual Drosophila Research Conference, held in San Diego, CA (April 2-6, 2008), had over 30 poster presentations and eight platform talks devoted to physiology and aging, and seven presentations in a longevity and functional senescence workshop. The data presented via these avenues included life span manipulation, physiological related genes, candidate aging genes, gene expression, signaling, and using D. melanogaster as a model for age related disease, to name a few. This report provides highlights of some of the information presented in the poster, platform and workshop presentations.

Advances in age-old questions.

The intent of this study was to characterize the effect OTK18 upregulation in monocytic cells had on neuronal survival. The human monocytic cell line, U937, was differentiated into macrophages or left as an undifferentiated monocyte. These cells were transfected with a plasmid containing the enhanced green fluorescent protein and OTK18 (pEGFP-OTK18) or an empty control vector (pEGFP-N3). The supernatants from the transfected U937 cells were used to culture rat neuronal cells (PC12). A live/dead assay was performed to determine the effect of culturing on cell survival. The protein levels of the neurotoxin, tumor necrosis factor alpha (TNF-alpha), and the neurotrophin, neurotrophin three (NT3), were determined by enzyme linked immunosorbent assay. The results of the live/dead assay showed differential cell survival between conditions with pEGFP-OTK18 when compared to the control empty vector. Quantitative real-time polymerase chain reaction assays demonstrated that OTK18 had an increased expression level when compared to the control. Lastly, NT3 protein levels were upregulated in treated cells with increased OTK18 expression, suggesting that OTK18 may play a role in neurotrophin production and consequently support neuronal survival.

The effect of OTK18 upregulation in U937 cells on neuronal survival.

Nora virus is a recently discovered RNA picorna-like virus that produces a persistent infection in Drosophila melanogaster, but the antiviral pathway or change in gene expression is unknown. We performed cDNA microarray analysis comparing the gene expression profiles of Nora virus infected and uninfected wild-type D. melanogaster. This analysis yielded 58 genes exhibiting a 1.5-fold change or greater and p-value less than 0.01. Of these genes, 46 were up-regulated and 12 down-regulated in response to infection. To validate the microarray results, qRT-PCR was performed with probes for Chorion protein 16 and Troponin C isoform 4, which show good correspondence with cDNA microarray results. Differential regulation of genes associated with Toll and immune-deficient pathways, cytoskeletal development, Janus Kinase-Signal Transducer and Activator of Transcription interactions, and a potential gut-specific innate immune response were found. This genome-wide expression profile of Nora virus infection of D. melanogaster can pinpoint genes of interest for further investigation of antiviral pathways employed, genetic mechanisms, sites of replication, viral persistence, and developmental effects.

Differential gene expression related to Nora virus infection of Drosophila melanogaster.

Aging is a complex process characterized by a steady decline in an organism's ability to perform life-sustaining tasks. In the present study, two cages of approximately 12,000 mated Drosophila melanogaster females were used as a source of RNA from individuals sampled frequently as a function of age. A linear model for microarray data method was used for the microarray analysis to adjust for the box effect; it identified 1,581 candidate aging genes. Cluster analyses using a self-organizing map algorithm on the 1,581 significant genes identified gene expression patterns across different ages. Genes involved in immune system function and regulation, chorion assembly and function, and metabolism were all significantly differentially expressed as a function of age. The temporal pattern of data indicated that gene expression related to aging is affected relatively early in life span. In addition, the temporal variance in gene expression in immune function genes was compared to a random set of genes. There was an increase in the variance of gene expression within each cohort, which was not observed in the set of random genes. This observation is compatible with the hypothesis that D. melanogaster immune function genes lose control of gene expression as flies age.

Genome-Wide Gene Expression in relation to Age in Large Laboratory Cohorts of Drosophila melanogaster.

A Drosophila melanogaster genome-wide transcriptome dataset is available for studies on temporal patterns of gene expression. Gene expression was measured using two-dye color oligonucleotide arrays derived from Version 2 of the Drosophila Genomics Resource Center. A total of 15,158 oligonucleotide probes corresponded to a high proportion of the coding genes in the genome. The source of the flies was a highly genetically heterogeneous population maintained in an overlapping generation population regime. This regime was designed to maintain life history traits so that they were similar to those found in natural populations. Flies collected for the cohorts were obtained in a short period of time in a carefully controlled manner before virgin females and males were allowed to mate. Mated females were introduced into two large population cages in unusually high numbers (approximately 12,000 per cage) for a Drosophila laboratory longevity study. Samples were taken weekly from each cohort for 11 weeks; only a small proportion of surviving flies were present at the last two collection time points and thus they were exceptionally old compared to those collected in early-to-midlife samples. The data set is useful for studies of temporal patterns of gene expression as flies age. The very large size of each cohort, and relatively frequent incidence of temporal samples, allows for a fine-scale study of gene expression from young to very old flies.

A dataset for assessing temporal changes in gene expression during the aging process of adult Drosophila melanogaster.

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

 depends upon the innate immune system to regulate and combat viral infection. This is a complex, yet widely conserved process that involves a number of immune pathways and gene interactions. In addition, expression of genes involved in immunity are differentially regulated as the organism ages. This is particularly true for viruses that demonstrate chronic infection, as is seen with Nora virus. Nora virus is a persistent non-pathogenic virus that replicates in a horizontal manner in . The genes involved in the regulation of the immune response to Nora virus infection are largely unknown. In addition, the temporal response of immune response genes as a result of infection has not been examined. In this study,  either infected with Nora virus or left uninfected were aged for 2, 10, 20 and 30 days. The RNA from these samples was analyzed by next generation sequencing (NGS) and the resulting immune-related genes evaluated by utilizing both the PANTHER and DAVID databases, as well as comparison to lists of immune related genes and FlyBase. The data demonstrate that Nora virus infected  exhibit an increase in immune related gene expression over time. In addition, at day 30, the data demonstrate that a persistent immune response may occur leading to an upregulation of specific immune response genes. These results demonstrate the utility of NGS in determining the potential immune system genes involved in Nora virus replication, chronic infection and involvement of antiviral pathways.

Analysis of immune-related genes during Nora virus infection of  using next generation sequencing.

A reaction-based sensor (NAS-1) showed a high affinity and sensitivity to HSO3- via a nucleophilic addition reaction in the aqueous media, giving dual signals from absorption and emission spectra. NAS-1 was successfully applied in RK13 epithelial cells to detect HSO3- in a cellular environment.

Investigation of a Sensing Strategy Based on a Nucleophilic Addition Reaction for Quantitative Detection of Bisulfite (HSO).

Study of the novel RNA virus, Nora virus, which is a persistent, picorna-like virus that replicates in the gut of  offers insight into human innate immunity and other picorna-like viruses. Nora virus infection leads to a locomotor abnormality and upregulation of two candidate target proteins, Vago and Virus-induced RNA 1 (Vir-1). These proteins are uncharacterized in response to Nora virus. We hypothesize that Nora virus is circulating in the hemolymph of Nora virus-infected , allowing for migration beyond the primary site of replication in the gut. Analysis by qRT-PCR demonstrated biphasic viral load and corresponding  and  transcription levels, suggesting transcription of  and  occurs in response to viral infection. However, Vir-1 is also present in virus-free  suggesting basal expression or alternative functions. Presence of Nora virus RNA and the Viral Protein 4b (VP4b), in hemolymph of infected  supports the hypothesized circulation of Nora virus in the hemolymph. The study suggests that impaired locomotor function may be due to transport of Nora virus from the gut to the brain via the hemolymph.

Nora Virus VP4b and  Circulate in Hemolymph of Infected  with Coordinate Expression of Vago and Vir-1.

Gastrointestinal microflora is a key component in the maintenance of health and longevity across many species. In humans and mice, nonpathogenic viruses present in the gastrointestinal tract enhance the effects of the native bacterial microbiota. However, it is unclear whether nonpathogenic gastrointestinal viruses, such as Nora virus that infects , lead to similar observations. Longevity analysis of Nora virus infected (NV+) and uninfected (NV-)  in relationship to presence (B+) or absence (B-) of the native gut bacteria using four different treatment groups, NV+/B+, NV+/B-, NV-/B+, and NV-/B-, was conducted. Data from the longevity results were tested via Kaplan-Meier analysis and demonstrated that Nora virus can be detrimental to the longevity of the organism, whereas bacterial presence is beneficial. These data led to the hypothesis that gastrointestinal bacterial composition varies from NV+ to NV- flies. To test this, NV+ and NV- virgin female flies were collected and aged for 4 days. Surface sterilization followed by dissections of the fat body and the gastrointestinal tract, divided into crop (foregut), midgut, and hindgut, were performed. Ribosomal 16S DNA samples were sequenced to determine the bacterial communities that comprise the microflora in the gastrointestinal tract of NV+ and NV- . When analyzing operational taxonomic units (OTUs), the data demonstrate that the NV+ samples consist of more OTUs than NV- samples. The NV+ samples were both more rich and diverse in OTUs compared to NV-. When comparing whole body samples to specific organs and organ sections, the whole fly was more diverse in OTUs, whereas the crop was the most rich. These novel data are pertinent in describing where Nora virus infection may be occurring within the gastrointestinal tract, as well as continuing discussion between the relationship of persistent viral and bacterial interaction.

Effect of Nora virus infection on native gut bacterial communities of .

The positive influence of undergraduate research and mentoring on student success in STEM fields has been well-established. However, the role that the gender of a research mentor may play in the undergraduate research experience warrants further investigation. This is an especially critical issue to address, since the lack of female role models in STEM fields is acknowledged as an impediment to the success and progress of women pursuing STEM-careers. To evaluate how the gender of undergraduate research mentors influences the research experience of students, we collected and analyzed surveys from undergraduates and alumni who had completed undergraduate research at the University of Nebraska at Kearney. We found that even though students did not select mentors based on gender, there were differences in how students perceived their mentors, depending on the gender of their mentors. Interestingly, students with female mentors were more likely than students with male mentors to report that their research experience had prepared them for a career in science. Further, our gender-pairing analyses revealed that students who expressed that the gender of their mentor had contributed to their relationship with their mentor were more likely to have a female mentor. Our data indicate that female mentors favorably influence the undergraduate research experience of both male and female students. Finally, our study reinforces the conclusions of previous studies demonstrating that undergraduate research and mentoring are beneficial for students. Overall, our findings support that, for students to fully benefit from their undergraduate research experience, undergraduate research opportunities for students should include an equitable representation of female mentors.

Female mentors positively contribute to undergraduate STEM research experiences.

 is widely known for its resistance and virulence causing public health concerns. However, antibiotic tolerance is also a contributor to chronic and relapsing infections. Previously, it has been demonstrated that persister formation is dependent on reduced tricarboxylic acid (TCA) cycle activity. Persisters have been extensively examined in terms of antibiotic tolerance but tolerance to antimicrobial peptides (AMPs) remains largely unexplored. AMPs are a key component of both the human and  innate immune response. TCA cycle mutants were tested to determine both antibiotic and AMP tolerance. Challenging with multiple classes of antibiotics led to increased persister formation (100- to 1,000-fold). Similarly, TCA mutants exhibited AMP tolerance with a 100- to 1,000-fold increase in persister formation when challenged with LL-37 or human β-defensin 3 (hβD3). The ability of TCA cycle mutants to tolerate the innate immune system was further examined with a  model. Both males and females infected with TCA cycle mutants exhibited increased mortality and had higher bacterial burdens (1.5 log) during the course of the infection. These results suggest increasing the percentage of persister cells leads to increased tolerance to components of the innate immune system.

Interruption of the tricarboxylic acid cycle in  leads to increased tolerance to innate immunity.

 Nora virus (DmNV) is a novel picorna-like virus first characterized in 2006. Since then, Nora virus has been detected in several non- species, including insects in the Orders Hymenoptera, Lepidoptera, Coleoptera, and Orthoptera. The objective of this study was to determine if DmNV could infect individuals of other species of invertebrates besides . The presence of DmNV in native invertebrates and commercially available stocks was determined. Laboratory-reared , , , , , and  were intentionally infected with DmNV. In addition, native invertebrates were collected and  stocks were purchased and screened for DmNV presence using reverse transcription-polymerase chain reaction (RT-PCR) before being intentionally infected for study. All  species and other invertebrates, except , that were intentionally infected with DmNV ended up scoring positive for the virus via RT-PCR. DmNV infection was also detected in three native invertebrates (, , and ) and all commercially available stocks tested. These findings suggest that DmNV readily infects individuals of other species of invertebrates, while also appearing to be an endemic virus in both wild and laboratory  populations. The detection of DmNV in commercially available stocks presents a cautionary message for scientists using these stocks in studies of virology and immunology.

Characterization of Cross-Species Transmission of  Nora Virus.

To quantify heat tolerance in insects, two manual observation measures are typically implemented: the time to physiological collapse at a static noxious temperature (time to knockdown; TKD) or the temperature at which collapse occurs as temperature increases (critical thermal maximum; CT). Both assay modalities focus on physiological collapse, neglecting the prior behavioral processes. In this study, the locomotion response of Drosophila melanogaster to relatively high temperature (39 and 40.5 °C) was quantified using the TriKinetics Drosophila Activity Monitor (DAM2 system). The absence of locomotion was defined as the state of physiological collapse resulting from extended exposure to high temperature. An easy-to-use executable application that allows the user to automatically extract individual TKD from the activity data was developed. For validation, manual TKD assays were performed in parallel to automated assays across multiple factors, including sex, hardening, recovery time after hardening, and assay temperature, which gave similar results. In terms of behavioral aspects, heat hardening consistently led to reduced activity during a subsequent heat stress, irrespective of assay temperature, sex, or recovery time after hardening. Our automated heat tolerance assay utilizing the DAM2 system is one way to expand the scope of the heat tolerance phenotype to include a behavioral component in conjunction with the traditional TKD measure.

Kimberly A. Carlson

Department of Biology

University of Nebraska at Kearney

Heat Tolerance Assays Using the Drosophila Activity Monitor System: A Guide to an Executable Application for Data Analysis

Kimberly A. Carlson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

University of Nebraska at Kearney

Heat Tolerance Assays Using the Drosophila Activity Monitor System: A Guide to an Executable Application for Data Analysis

Department of Biology