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○ Females on average had more crystal cells present than males. ○ The genes Ncc69 and Cbp53E were investigated because of the varying amounts of crystal cell based on gender. ○ The gene Unc-89 was also investigated to determine if it had a role in the varying amounts of crystal cells throughout all the genotypes ● ● Banerjee, Utpal, et al. “Drosophila as a Genetic Model for Hematopoiesis.” Genetics, vol. 211, no. 2, 1 Feb. 2019, pp. 367–417., doi:10.1534/genetics.118.300223. ● FlyBase. FlyBase Gene Report: Dmel\Unc-89, flybase.org/reports/FBgn0053519. ● Katzemich, Anja, et al. “Binding Partners of the Kinase Domains in Drosophila Obscurin and Their Effect on the Structure of the Flight Muscle.” Journal of Cell Science, The Company of Biologists Ltd, 15 Sept. 2015, jcs.biologists.org/content/128/18/3386. ● “The Hippo Pathway Regulates Hematopoiesis in Drosophila Melanogaster.” Current Biology, Cell Press, 30 Oct. 2014, www.sciencedirect.com/science/article/pii/S0960982214013402. ● Cattenoz, Pierre B et al. “Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila.” Genetics vol. 202,1 (2016): 191-219. doi:10.1534/genetics.115.182154 ● Makhijani, Kalpana, and Katja Brückner. “Of blood cells and the nervous system: hematopoiesis in the Drosophila larva.” Fly vol. 6,4 (2012): 254-60. doi:10.4161/fly.22267 ● Hagel KR, Beriont J, Tessier CR (2015) Drosophila Cbp53E Regulates Axon Growth at the Neuromuscular Junction. PLoS ONE 10(7): e0132636. https://doi.org/10.1371/journal.pone.0132636 ● Michele Markstein, “Drosophila workers unite!” Crystal Cell Formation ○ The average number of crystal cells melanized were similar between each gender, except for in line 28179. ○ The sex of the larva had an effect on line 28179. The average number of melanized crystal cells in female larva was more than males within the same genome. This could be due to a large variation in one of the genders. Genes ● Unc-89 ○ The gene Unc-89 encodes a protein located at the midline of the muscle thick filaments that is needed for assembly of a symmetrical sarcomere. It is involved in myogenesis and the Hippo signalling pathway. (Katzemich, et al. 2015) ○ The hippo pathway was found to regulate Hematopoiesis. The hippo pathway turns on the protein lozenge and the protein lozenge causes blood stem cells to differentiate into crystal cells. So, if there was more of the protein lozenge being produced then there could be an increase in the number of crystal cells, and vise versa.(Milton, et al. 2014) Gene Differences Between Sexes ● Cpb53E ○ The gene Cbp53E, which is involved in the negative regulation of response to wounding and neuronal growth, was more likely to have a single nucleotide polymorphism(SNP) in females than males. ○ This gene might affect crystal cell melanization because of its role in the negative regulation of response to wound healing since crystal cells facilitate wound healing by melanizing. ○ Cbp53Es’ role in neuronal growth may also influence blood cell development because hemocytes rely on the peripheral nervous system to localize and proliferate.(Brückner, et al. 2012; Hagel, et al. 2015) ○ Furthermore the SNP on this gene might have an affect on crystal cell melanization because it is a minor allele in the genotype 28137, which has the highest average number of crystal cells, with females having more crystal cells on average than males. ● Ncc69 ○ Ncc69 has alleles that manifest in reticular associated glial cells. Microglial, a type of glial cell, acts as the primary defense to immune responses of the nervous system. When considering crystal cell developmental factors the alleles associated with glial cells in the Ncc69 genome may be the reason for line 28179 having a drastically high crystal cell difference and line 28247 having a slightly higher crystal cell difference in females. (Cattenoz, 2016) ○ In addition, GCM is likely to trigger the expression of target genes being that it is known to control the gene loco in late glial cell differentiation. Therefore, if GCM plays a large role in glial cell development then Ncc69 could be another one of its possible target genes. This may be the reason for higher crystal cell amounts in females due to the microglial correlation with immune responses. (Cattenoz, 2016) ○ Treat the larva with methoprene and compare the number of crystal cells that melanize. ○ Investigate further to find if the genes are specifically associated with crystal cell melanization. ○ Overexpress GMC in females and males that both have the Ncc69 gene. A ○ Heat Shocking: The third instar larvae are being heated at 70 degrees celsius for 10 minutes in the DNA Thermal Cycler. Then the crystal cells melonized. ○ Scoring: The number of crystal cells were manually-counted on the dorsal, ventral, and lateral sides using a clicker. ○ Drosophila have almost as many genes as humans do, 14,000 compared 21,000 genes in humans. They also have a homolog of over 65% of genes associated with human diseases.(Markstein ) ○ Drosophila have a 6-10 day life cycle from an embryo to an adult. ○ There are three different types of blood cell types that are found in Drosophila which are cystall cells, plasmatocytes, and lamellocytes.(Banerjee, et al. 2019) ○ Crystal cells facilitate wound healing, innate immunity, and the hypoxic response. Under stress conditions crystal cells melanize.(Banerjee, et al. 2019) ○ Blood cells develop in the larval hematopoietic organ (aka Lymph Gland)and the Dorsal vessel.(Banerjee, et al. 2019) ○ A genome-wide association study(GWAS) scans markers across complete sets of genomes to find genetic variations associated with a disease. A GWAS of drosophila can be used to find if certain genes influence the number of crystal cells in drosophila of different genotypes. Natural Variations in Blood Composition of Drosophila Natalie Santana, Suzaul Sobhan, Anwar Benmallouk, and Rebecca Spokony Department of Natural Sciences Baruch College, CUNY, 17 Lexington Avenue, New York, NY 10010 ACKNOWLEDGMENTS We would like to thank Areeba Choudhury, Jessica Sparacio, Marium Sarder for training and supervising us throughout our research. FUTURE DIRECTIONS ○ Further visual analysis of larvae reveals that the average number of crystal cells differ in different genotypes. With genotype 28137 having the highest number of crystal cells on average and genotype 28223 having the lowest number of crystal cells on average. RESULTS INTRODUCTION METHODS REFERENCES DISCUSSION ○ The average number of crystal cells differ between the males and females of each genotype. Females from genotype 28179 on average had higher amounts of crystal cells compared to the males. Despite the variation between the males and females, the genotype 28137 has the highest average number of crystal cells in both sexes. Number Of Crystal Cells In A Variety Of Drosophila Genotypes Genotypes 25174 28196 28137 28223 28179 28247 55707 Male Female OVERVIEW Figure 3: Heat shocking Process Figure 2: Hematopoietic Organ Mean Number of Crystal Cells Varies Between Different Drosophila Genotypes Mean Number of Crystal Cells in Males and Females in Different Drosophila Genotypes Figure 1: Life Cycle of Drosophila Melanogaster
