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Lupita Sanchez, Genomics Analysis—BIOL310, May 17th2018
Functional Analysis of Drosophila sechellia fed on L-DOPA shows effects on oogenesis, response to stress, and extracellular region
Abstract:
Drosophila Sechellia has specialized on the ripe fruit of its host, Morinda citrifolia, despite the toxicity that results from the presence of high levels of carboxylic acids, primarily octanoic acid (OA). D. sechellia has evolved a resistance for OA, allowing it to take advantage of other important components of the fruit, such as L-DOPA (Lavista-Llanos, 2014). Research studies identified molecular polymorphisms in the catsup gene resulting in elevated levels of dopamine in D. sechellia and associated this alteration with infertility caused by maternal arrest of oogenesis (Lavista-Llanos, 2014). It was proposed that the presence of L-DOPA is enough to reverse the effects, thus making it necessary for the successful reproduction in D. sechellia (Lavista-Llanos, 2014). Here, we identify genes responsive to L-DOPA by performing differential gene expression analysis using RNA-sequencing (RNA-seq) on control and L-DOPA exposed D. sechellia and found 642 significantly differentiated expressed genes. Gene Ontology (GO) term enrichment showed significant enrichment for genes involved in oogenesis in upregulated genes, supporting earlier work conducted on differential gene expression on flies exposed to L-DOPA. In addition, GO term enrichment showed significant enrichment for genes responsive to stress and genes involved in the extracellular region for downregulated genes, providing a valuable new focus of research for further experiments.
Introduction:
Many insects have evolved a preference for a single plant, leading to a small number of insects to become specialists (Huang & Erezyilmaz, 2015). When such shifts occur, insects are obligated to evolve new physiological, morphological, and behavioral adaptations in order to survive their new environments (Dworkin, 2009). Although we know much about the ecological changes that led to these adaptations, the genetic mechanisms by which they occurred are still unclear (Lanno, 2017). The endemic species from the Seychelles Islands, Drosophila sechellia, provides a great model organism for studying these specializations, as it has adapted to feed and oviposit on the ripe fruit of M. citrifolia (Huang & Erezyilmaz, 2015). The toxicity of this fruit is due to high levels of octanoic acid (OA), to which D. sechellia has evolved a resistance for while OA has shown to be toxic to all other drosophilids within its clade (Huang & Erezyilmaz, 2015).
Besides the presence of OA, M. citrifolia contains other key chemicals that may be important role in the specialization of D. sechellia (Lavista-Llanos, 2014). Unlike its sibling generalist species, D. sechellia is known to produce lower levels of L-DOPA, and molecular polymorphisms in the protein catsup have been proposed to cause this deficit (Stensmyr, 2016) (Lavista-Llanos, 2014). Catsup is a pleiotropic quantitative trait and serves as a negative regulator of tyrosine hydroxylase (TH) (Carbone, 2006). It is the fourth locus (ple) found in the Ddc gene cluster that functions in the catecholamine metabolism (Stathakis, 1999). Dopamine is one type of catecholamine, and the biosynthesis of dopamine begins with the hydroxylation of L-DOPA, a reaction that is catalyzed by TH (Stathakis, 1999). Mutant proteins in Drosophila can lead to high levels of dopamine that give rise to abnormalities, including small ovaries, poor egg production, cell apoptosis, which overall lead to female sterility and may even cause lethality (Stathakis, 1999) (Lavista-Llanos, 2014).
In various insects, dopamine serves as a precursor in the process of cuticle sclerotization and melanization (Riemensperger, 2011). Sclerotization forms the exoskeleton, which protects the bodies of insects by preventing foreign organisms from invading (Verlinden, 2018). Similarly, melanin protects the exoskeleton from harmful radiation, as well as it aids in the defense against pathogens by helping in wound healing (Verlinden, 2018). In Drosophila, dopamine controls egg production, making this hormone a critical contributor to the specialization of D. sechellia on M. citrifolia.
Earlier work has shown that L-DOPA is enough to increase the expression of genes involved in oogenesis as well as reverse other various effects caused by mutant catsup gene (Lavista-Llanos, 2014). In this study, we exposed adult female D. sechellia flies to control food and food containing L-DOPA and identified genes that were differentially expressed among the two environments. Our results showed that the presence of L-DOPA is beneficial in the reproductive success of D. sechellia. Furthermore, we reveal the negative effects of L-DOPA, proposing a new area of research for further analysis.
Methods:
Adult female flies were exposed to control or L-DOPA (10mg/ml) containing food. The RNA was extracted from both groups of flies and libraries were made from the mRNA. These libraries were sent to the University of Michigan Sequencing Core Facility where sequencing was performed on an Illumina Hiseq 4000. The sequencing yielded six sequencing reads that were analyzed using the RNA-sequencing (RNA-seq) pipeline (Figure 1).
The RNA-seq pipeline was carried out in Galaxy, where FASTQC, a program used to perform simple quality control checks on high throughout sequence data, was used to ensure that the raw data was suitable for further statistical analysis. FASTQC outputs were divided into 12 sections, including a category for overrepresented sequences that were analyzed using NCBI blast. The sequence reads were then aligned to the reference D. sechellia genome using Bowtie 2. The alignment output file was put into Cuffdiff along with the D. sechellia gff3 file, resulting in a set of genes that showed differential expression. From here, the data was analyzed using R, a programming language used to perform statistical analysis and generate visual graphics. A second set of genes of D. melanogaster was imported onto R, and the D. sechellia genes that resulted from Cuffdiff were merged with their respected orthologs. This resulted in 642 differentially expressed genes that were analyzed using GO term enrichment, a tool used to show enriched terms for biological processes, molecular function, and cellular components that are important to the response of D. sechellia to L-DOPA.
Figure 1: RNA-seq pipeline

Figure 1 Legend: RNA-seq pipeline used to perform differential gene expression analysis.
Results:
Overrepresented Sequences
A normal high throughput sequences data contains a diverse set of sequences without a single sequence making up a large fraction of the whole. Overrepresented sequences can arise as a result of contamination or due to their biological importance to the life of the organism. For all three samples of Drosophila fed on L-DOPA, there were overrepresented sequences that resulted in both a warning and an error, which occurs when any sequence is found to be present more than 0.01% or 1% of the total, respectively. When inputted into NCBI BLAST, which reports the source of overrepresented sequences, all three samples resulted in several overrepresented sequences that produced a significant alignment to D. ananassae GF27906, ncRNA. Sample 2 yielded distinct results than the other two samples, as one of the overrepresented sequences aligned to D. melanogaster 18S ribosomal RNA. A second overrepresented sequence from sample 2 aligned to D. melanogaster 28S ribosomal RNA, and D. erecta 28S ribosomal RNA. A third overrepresented sequences resulted in an alignment to D. melanogaster 18S ribosomal RNA too, as well as to the 18S ribosomal RNA of various Drosophila species. These results suggest that the 18S ribosomal RNA in Drosophila has been well conserved throughout evolution, indicating the importance of this sequence to the development of the organism.

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FASTQC
In order to identify differentially expressed genes in D. sechellia when exposed to control food and food containing L-DOPA, we performed RNA-seq on three control samples and three L-DOPA samples. This produced six sequencing libraries (Table 1 and 2). 945 genes were differentially expressed (Graph 1), which were then merged with D. melanogaster orthologs, resulting in 642 significantly expressed genes. Out of these genes, 371 genes were upregulated and 271 of them were downregulated in response to L-DOPA (Table 3).

Table 1: FASTQC results for control samples
Control Rep 1
(76332) Control Rep 2
(76333) Control Rep 3
(76334)
19222060 20704811 17696868
Table 1 Legend: The second row represents the total number of mapped reads for each sequencing library for the control samples.
Table 2: FASTC results for L-DOPA Samples
L-DOPA Rep 1
(76335) L-DOPA Rep 2
(76336) L-DOPA Rep 3
(76337)
19576162 14508205 17432600
Table 2 Legend: The second column represents total number of mapped reads for each sequencing library for the L-DOPA samples.

Table 3: Differentially expressed genes
Differentially expressed genes (after merging with D. melanogaster) Upregulated
(Out of 642) Downregulated
(Out of 642)
945(642) 371 271
Table 3 Legend: The second column represents the number of differentially expressed genes and the corresponding number of upregulated vs. downregulated genes.
Graph 1: Gene Expression in Response to L-DOPA
Graph 1 Legend: The red dots in this graph represent the genes that were significantly expressed (p-value<0.05). The black dots represent the genes that were not significantly expressed. The genes represented here are all the genes that were differentially expressed in D. sechellia (945). After merging with a set of D. melanogaster genes, 303 genes were lost, which resulted in 642 differentially expressed genes that were used for the remainder of this analysis.
GO Term Enrichment Analysis
The 642 differentially expressed genes were analyzed using GO term enrichment, resulting in many terms enriched for various biological processes, molecular functions, and cellular components. Upregulated genes showed significant enrichment for biological processes devoted to oogenesis (Appendix, Table 5), while downregulated genes showed significant enrichment for biological processes involved in response to stress and cellular components involving the extracellular region (Table 4).
Table 4: Significantly differentiated expressed genes responsive to stress and involved in extracellular region
Gene (D. melanogaster ortholog) Expression in Control
(RPKM) Expression in L-DOPA
(RPKM)
GM20557 (def) 3500.27 1129.23
GM21465 (AttC) 646.053 186.403
GM25706 (Edin ) 317.049 85.0071
GM12960( Peritrophin-15a) 6265.22 3043.23
GM13371 (CG9672) 49.9246 19.1902
GM21866 (IM2) 2620.72 690.724
Table 4 Legend: All these genes showed significant enrichment for the cellular component of the extracellular region. The ones highlighted in green were significantly enriched for response to stress.
Graph 2:

Graph 2 Legend: This is a graphical representation of the differential expression in control vs L-DOPA. All six genes were downregulated when exposed to L-DOPA.
Discussion:
In this study, we identify genes important in the specialization of D. sechellia on M. citrifolia. Forty-six genes involved in oogenesis, which is the process of formation of a female gamete from a female germ cell, were upregulated when exposed to L-DOPA, confirming with previous work that L-DOPA is enough to increase oogenesis in this organism. Since D. sechellia lacks the ability to produce L-DOPA at similar levels as its sibling species, it must have a secondary source of L-DOPA in order to reproduce. The need for higher levels of L-DOPA to increase oogenesis is a possible explanation for the organism’s specialization on its host (Lavista-Llanos, 2014).
GO term enrichment showed that while L-DOPA increases the expression of genes involved in oogenesis, it results in the downregulation of genes responsive to stress and genes involved in the extracellular region. Response to stress refers to any exposure of the cell to an environment that puts it at risk and reduces cell viability (Nadal, 2011). This suggests that D. sechellia fed on L-DOPA leads to a decrease in the ability of the organism to respond to rapid changes in the environment. Stressors, such as changes in temperature, pH, or any other alteration to the environment, require immediate cellular responses to maximize survival of the organism (Nadal, 2011). The downregulation of these genes indicates that exposure to L-DOPA could be lethal to D. sechellia. Additionally, genes downregulated in the extracellular region, the space external to the outermost structure of the cell, is another negative effect of L-DOPA. The composition of the extracellular region could be affected as a result of the decrease in expression of these genes, and in turn affect the structural support it provides for the tissue as well as intercellular communication. Interestingly, these genes showed a similar pattern of expression when exposed to OA. A recent study showed that when D. sechellia was fed on food containing OA, these genes were significantly enriched for immune and defense response, concluding that exposure to OA leads to a weakened immune system and may result in lethality (Lanno, 2017). This overlap could be significant, and further research is necessary to reach more concrete conclusions revolving this observation.
Appendix:
Table 5: Significantly differentiated expressed genes in oogenesis
Gene
(D. sechellia) Expression in control
(RPKM) Expression in L-DOPA
(RPKM)
GM19220 512.609 143.217
GM22256 11.2179 19.7122
GM26235 27.1157 45.5546
GM21605 109.583 46.9668
GM12699 42.5382 136.816
GM13834 22.6783 6.98147
GM16408 31.1858 53.704
GM17351 33.0623 13.8737
GM17674 37.8408 162.272
GM17981 225.752 90.6404
GM17984 410.549 83.8142
GM21594 103.236 387.561
GM22735 92.2632 227.137
GM14198 23.2574 50.0911
GM11593 17.4912 71.9849
GM22263 15.8141 27.3131
GM13710 38.1086 78.1845
GM20762 18.3633 38.005
GM24530 29.6117 52.6892
GM13910 11.0321 3.68314
GM20349 69.2606 121.661
GM16821 2.76023 14.8965
GM17444 10.9907 19.6873
GM17532 23.8664 67.0682
GM25591 23.209 43.9896
GM10162 44.5685 88.1826
GM20140 38.2971 85.0295
GM15515 31.91 54.1341
GM16021 22.2072 39.9559
GM12700 42.7749 126.563
GM10156 18.2079 37.0461
GM21616 229.913 90.6178
GM20076 21.7678 43.0133
GM21940 73.0111 40.3122
GM18917 8.05944 15.8219
GM18584 3821.66 888.546
GM19827 47.3151 85.0085
GM26622 20.8608 44.3437
GM23619 7.34802 13.1964
GM17983 9444.02 2742.91
GM12060 26.1375 13.7096
GM19914 55.8632 105.999
GM25580 21.8755 38.0901
GM17128 32.1977 57.8682
GM14107 10.8778 21.0398
GM18798 30.3472 52.2032
Table 5 Legend: The complete lists of genes enriched for oogenesis. Out of the 46 genes, 34 of them were upregulated in response to L-DOPA. The other 12 were downregulated. The second and third column represent the expression in control vs. L-DOPA containing food, respectively.
References:
Carbone, Mary A. “Phenotypic Variation and Natural Selection at Catsup, a Pleiotropic Quantitative Trait Gene in Drosophila.” Cell, W.M. Keck Center for Behavioral Biology, 9 May 2006.

Dworkin, Ian, and Corbin D. Jones. “Genetic Changes Accompanying the Evolution of Host Specialization in Drosophila Sechellia.” Advances in Pediatrics., U.S. National Library of Medicine, Feb. 2009.

Huang, Yan, and Deniz Erezyilmaz. “The Genetics of Resistance to Morinda Fruit Toxin During the Postembryonic Stages in Drosophila Sechellia.” G3: Genes | Genomes | Genetics, G3: Genes, Genomes, Genetics, 1 Oct. 2015.
Lanno, Stephen M., et al. “Transcriptomic Analysis of Octanoic Acid Response in Drosophila Sechellia Using RNA-Sequencing.” G3: Genes | Genomes | Genetics, G3: Genes, Genomes, Genetics, 1 Dec. 2017.

Lavista-Llanos, Sofía, et al. “Dopamine Drives Drosophila Sechellia Adaptation to Its Toxic Host.” Advances in Pediatrics., U.S. National Library of Medicine, 9 Dec. 2014.

Nadal, Eulàlia de, et al. “Controlling Gene Expression in Response to Stress.” Nature News, Nature Publishing Group, 3 Nov. 2011.

Riemensperger, Thomas, et al. “Behavioral Consequences of Dopamine Deficiency in the Drosophila Central Nervous System.” PNAS, National Academy of Sciences, 11 Jan. 2011.
Stathakis, Dean G., et al. “The Catecholamines up (Catsup) Protein of Drosophila Melanogaster Functions as a Negative Regulator of Tyrosine Hydroxylase Activity.” Genetics, Genetics, 1 Sept. 1999.
Stensmyr, Marcus C. “Evolutionary Genetics: Smells like a Pseudo-Pseudogene.” Egyptian Journal of Medical Human Genetics, Elsevier, 19 Dec. 2016.
Verlinden, Heleen. “Dopamine Signaling in Locusts and Other Insects.” Egyptian Journal of Medical Human Genetics, Elsevier, 20 Apr. 2018, 

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