Identification of genome-wide genetic and epigenetic changes due to cannibalism in Drosophila melanogaster.

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A.TITLE OF PROPOSED PROJECT:

Identification of genome-wide genetic and epigenetic changes due to cannibalism in Drosophila melanogaster.

C1.RESEARCH AREA

Basic life sciences; proteomics and genomics

D.PROJECT DIGEST. Describe the proposed research geared to the non-specialist reader (max. 250 words).

The physical attributes of an organism are not determined solely by its DNA, unlike the notion held by earlier scientists. Recent work has indicated that the environment and its interaction with the organism’s genetic makeup together determine its physical characteristics. In particular, the effect of food on such characteristics is widely researched and recognized. A specialized subgroup of such an interaction is the cannibalistic behavior displayed by some organisms. The effect of cannibalism on the genetic makeup of organisms and its effect on the proteins expressed has not been studied. This study aims at filling this gap in our knowledge, and help us understand better how the food an organism ingests impacts not only its own life but also its progeny. 

Project DEtails

1. PROJECT SUMMARY

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Describe the proposed research. (max. 100 words)

The project aims at looking at the impact of a cannibalistic diet on the inner workings of a cell. Differences in the protein expression profiles and the rates of mutations/epimutations in fruit flies (Drosophila melanogaster) reared on normal food sources versus those reared on a cannibalistic diet will help shed light on some of these molecular interactions.

2.PROPOSED GOALS/OBJECTIVES

HYPOTHESIS/BASIS OF RESEARCH

Dietary factors have been shown to effect phenotypes of organisms by mechanisms that include, but might not be limited to, the modulation of the epigenome of the organism. The most striking example for this epigenetic regulation was provided by Jirtle and Waterland (Waterland and Jirtle 2003) who demonstrated that the absence or presence of supplements such as vitamin B12 and folic acid in the diets of pregnant mice had profound effect on the phenotype of the progeny, with a lack of these supplements leading to the birth of yellow, obese and disease-prone mice. It was shown that this drastic phenotypic variation was caused by the activation or suppression of the Agouti gene. Apart from modulating gene expression levels, environmental stresses have also been shown to change the rate of genetic (Belfield, Gan et al. 2012) and epigenetic (Jiang, Mithani et al. 2014) changes in organisms. Thus, it can be hypothesized that a change from a purely herbivorous diet to a completely carnivorous (cannibalistic diet) will affect the proteomic and/or genomic/epigenomic makeup of the flies.

RESEARCH OBJECTIVES

  1. Identify changes in protein expression patterns between cannibalistic and normal flies by microarray analysis
  1. Determine the rates of genomic changes in normal and cannibalistic flies by next generation sequencing
  1. Determine the rates of epigenomic changes in normal and cannibalistic flies by next generation sequencing after bisulphite treatment.
  1. Analysis of the data obtained

3.INTRODUCTION

The introduction should consist of three paragraphs; the first paragraph should indicate the scientific hypothesis/commercial basis on which the project is based. The second paragraph should introduce the precise nature of the project, and the final paragraph should indicate the proposed objectives in the light of the first two paragraphs and explain clearly what the reader will see in the main body of the proposal.

The complexity seen in multicellular organisms has long baffled humans and hence been a source of scientific intrigue. Another layer of complexity is added to this study with the identification of phenomenon that indicated an interaction between the organism and its environment, not just at the macro level, but at the molecular level. Studies have documented the effect of the dietary variation on the phenotypic variation within a species with many changes being stably heritable and predisposing the organism to various diseases (Jirtle and Skinner 2007). This implies that these environmental factors are able to influence the proteomics, genetics or epigentics of the organism. Stresses, whether biotic or abiotic, can influence the rate of genetic and epigenetic changes that an organism accumulates over time. Flies reared on a diet very different from their usual herbivorous diets can thus be expected to have altered patterns of protein expression and/or have genetic or epigenetic changes.

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As an adaptive mechanism under stress, Drosophila melanogaster is known to turn cannibalistic (Vijendravarma, Narasimha et al. 2012). This project aims at identifying the changes such a drastic behavioral response might have on the proteome, genome and epigenome of the flies. Flies raised exclusively on a cannibalistic diet for 15 generations will be compared to flies raised on normal lab media by looking at differences in their transcriptome by microarray analysis and by looking for rates of change in DNA bases and their associated methyl marks by Next Generation Sequencing and bisulphite treatment followed by Next Generation Sequencing. The data obtained from these experiments would then be analyzed to identify any differences that might arise due to the cannibalistic behavior of the flies.

The objectives of the project include rearing flies successfully on a cannibalistic diet for 15 generations, performing microarray on the cDNA obtained from normally raised and the experimental flies, using Next Generation Sequencing for detecting rates of changes in the genome of normally raised and the experimental flies and using Next Generation Sequencing coupled with bisulphite treatment for detecting rates of changes in the genome of normally raised and the experimental flies.

4A.BACKGROUND OF THE RESEARCH PROBLEMS TO BE ADDRESSED (Not to exceed two pages)

A comprehensive and up-to-date literature survey clearly highlighting the existing gaps and what new information will be added to the existing pool of knowledge.

Drosophila melanogaster or fruit fly, as the name suggests, is predominantly herbivorous in its feeding patterns, at the larval and adult stages, relieving on rotting vegetative matter and the microbes therein to provide it with nutrition for growth and development (Carson and Hartt 1971). However, various normally herbivorous insects, including locusts and crickets, engage in carnivorous feeding in conditions of over-population or nutritional stress (Richardson, Mitchell et al. 2010). Reports of carnivorous/cannibalistic behaviors exhibited by other Drosophila species, such as Drosophila hydei, led to the speculation that D. melanogaster might also show aberrant feeding patterns under stress conditions with limited nutrient supply. Upon investigation it was revealed that such deviations do exist, at least under lab conditions, with 1st and 2nd instar larvae attacking and feeding on larger 3rd instar larvae (Vijendravarma, Narasimha et al. 2013). However, studies to date have implicated only larval cannibalism. Unpublished data from our lab has shown that contrary to previous reports, larvae exhibit both larval and egg cannibalism and that adults also feed on the damaged carcasses of other flies and small insects (unpublished, Ahmad et al. 2014). In light of the establishment of a protocol to rear flies exclusively on cannibalistic diet in our lab we propose to study the prolonged effects of the change in dietary patterns on the cell’s inner working in Drosophila melanogaster. Such exclusive rearing has not been in reported elsewhere and hence no research on its effects has been carried out.

4B.RESEARCH PLAN/ METHODOLOGY: SCHEDULE/PHASING

Phase 1

Raise CantonS (wild type) flies on normal and cannibalistic diet for 15 generations at 25ºC. In each generation keep 100+ flies to account for any fluctuations in fly number due to death. Each generation would take 10-15 days to hatch from eggs into adult flies, depending on the lab conditions. From the parental stocks samples would be prepared for microarray, NGS and bisulphite sequencing.

Phase 2

After rearing the flies for 15 generations (F15) samples would be taken for microarray, NGS and bisulphite sequencing from both the control and experimental groups. All samples would be in triplicates.

Phase 3

Performing microarray, NGS, bisulphite sequencing on the collected samples.

Phase 4

Analysis of the data.

4C.REFERENCES

Ahmad, M., Tariq, M., and Afzal, A. J., (2014). “New insights in dynamic dietary patterns of Drosophila melanogaster larvae and adults”.

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Belfield, E. J., X. Gan, A. Mithani, C. Brown, C. Jiang, K. Franklin, E. Alvey, A. Wibowo, M. Jung and K. Bailey (2012). "Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana." Genome research 22(7): 1306-1315.

Carson, H. L. and C. E. Hartt (1971). The ecology of Drosophila breeding sites, University of Hawaii Foundation Lyon Arboretum Fund.

Jiang, C., A. Mithani, E. J. Belfield, R. Mott, L. D. Hurst and N. P. Harberd (2014). "Environmentally responsive genome-wide accumulation of de novo Arabidopsis thaliana mutations and epimutations." Genome research 24(11): 1821-1829.

Jirtle, R. L. and M. K. Skinner (2007). "Environmental epigenomics and disease susceptibility." Nature reviews genetics 8(4): 253-262.

Richardson, M. L., R. F. Mitchell, P. F. Reagel and L. M. Hanks (2010). "Causes and consequences of cannibalism in noncarnivorous insects." Annual review of entomology 55: 39-53.

Vijendravarma, R. K., S. Narasimha and T. J. Kawecki (2012). "Evolution of foraging behaviour in response to chronic malnutrition in Drosophila melanogaster." Proceedings of the Royal Society B: Biological Sciences: rspb20120966.

Vijendravarma, R. K., S. Narasimha and T. J. Kawecki (2013). "Predatory cannibalism in Drosophila melanogaster larvae." Nature communications 4: 1789.

Waterland, R. A. and R. L. Jirtle (2003). "Transposable elements: targets for early nutritional effects on epigenetic gene regulation." Molecular and cellular biology 23(15): 5293-5300.

5. IMPACT (of proposed research on teaching/training of manpower, institutional capability building and on local industry)

The impact of the study would be solely in filling up the knowledge gap in the effect of cannibalism on an organism’s genetic and epigenetic makeup. Findings from this study would shed some light on and help us understand what the evolutionary switch between cannibalism and carnivorous behavior is.

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