Gene model for the ortholog of Ilp3 in Drosophila eugracilis

Gene model for the ortholog of Insulin-like peptide 3 (Ilp3) in the D. eugracilis Apr. 2013 (BCM- HGSC/Deug_2.0) (DeugGB2) Genome Assembly of D. eugracilis (GenBank Accession: GCA_000236325.2) of Drosophila eugracilis. This ortholog was characterized as part of a dataset to study the evolution of the Insulin/ insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

This article reports a predicted gene model generated by undergraduate work using a structured gene model annotation protocol defined by the Genomics Education Partnership (GEP; thegep.org) for Course-based Undergraduate Research Experience (CURE). The following information in quotes may be repeated in other articles submitted by participants using the same GEP CURE protocol for annotating Drosophila species orthologs of Drosophila melanogaster genes in the insulin signaling pathway. “Computational gene predictions in non-model organisms often can be improved by careful manual annotation and curation, allowing for more accurate analyses of gene and genome evolution (Mudge and Harrow 2016; Tello-Ruiz et al., 2019). The Genomics Education Partnership (thegep.org) uses web-based tools to allow undergraduates to participate in course- based research by generating manual annotations of genes in non-model species (Rele et al., 2023). These models of orthologous genes across species, such as the one presented here, then provide a reliable basis for further evolutionary genomic analyses when made available to the scientific community. The particular gene ortholog described here, Insulin-like peptide 3 (Ilp3) in D. eugracilis, was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila.” (Myers et al., 2024). “The IIS pathway is a highly conserved signaling pathway in animals and is central to mediating organismal responses to nutrients (Hietakangas and Cohen 2009; Grewal 2009)” (Myers et al., 2024). “Invertebrate insulins function similarly to metazoan insulin-like growth factors and play a role in cell and organ growth (Chan 2000). In Drosophila, seven insulin-like peptides (Ilp1-Ilp7) have a two-chain structure similar to vertebrate insulin and interact with the sole insulin-like receptor, InR, to initiate the insulin signaling cascade (Brogiolo et al., 2001; Nässel and Broeck 2016). Like the Ilp2 and Ilp5 genes, the Ilp3 gene is expressed in median neurosecretory cells (MNCs) in the brain (Ikeya et al., 2002). While the seven Ilps act redundantly with respect to .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 20, 2025. ; https://doi.org/10.1101/2025.08.16.670678doi: bioRxiv preprint 2 promoting growth, they also have unique expression patterns and functions (Ikeya et al., 2002; Grönke et al., 2010). Ilp3 may act with the transcription factor dFOXO in a positive feedback loop to regulate Ilp2 and Ilp5 secretion from MNCs (Grönke et al., 2010). In female Drosophila, ablation of MNCs or knockout of Ilp3 have been shown to reduce fecundity and remating rates (Grönke et al., 2010; Wigby et al., 2011). Knockout of Ilp3 also results in sleep defects (Yamaguchi et al., 2022).” (Gruys et al., 2024). “D. eugracilis (NCBI taxon ID 29029) is part of the melanogaster species group within the subgenus Sophophora of the genus Drosophila (Pélandakis and Solignac, 1993). It was first described as Tanygastrella gracilis by Duda (1924) and revised to Drosophila eugracilis by Bock and Wheeler (1972). D. eugracilis is found in humid tropical and subtropical forests across southeast Asia (https://www.taxodros.uzh.ch, accessed 1 Feb 2023).” (Morgan et al., 2022). We propose a gene model for the D. eugracilis ortholog of the D. melanogaster Insulin-like peptide 3 (Ilp3) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_017080989.1 (Locus ID LOC108114482) in the D. eugracilis Apr. 2013 (BCM- HGSC/Deug_2.0) (DeugGB2) Genome Assembly of D. eugracilis (GCA_000236325.2 - Chen et al., 2014). This model is based on RNA-Seq data from D. eugracilis (PRJNA63469) and Ilp3 in D. melanogaster using FlyBase release FB2024_02 (GCA_000001215.4; Gramates et al., 2022; Jenkins et al., 2022; Larkin et al., 2021). Synteny The target gene, Ilp3, occurs on chromosome 3L in D. melanogaster and is nested by CG32052 alongside Insulin-like peptide 4 (Ilp4) (upstream) and Insulin-like peptide 2 (Ilp2) (downstream). Ilp3 is flanked further upstream by Inhibitor-2 (I-2) and CG43897, which nests Insulin-like peptide 5 (Ilp5) and downstream by Insulin-like peptide 1 (Ilp1) and Z band alternatively spliced PDZ-motif protein 67 (Zasp67). The tblastn search of D. melanogaster Ilp3-PA (query) against the D. eugracilis Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly of D. eugracilis (GCA_000236325.2 - Chen et al., 2014) placed the putative ortholog of Ilp3 within scaffold scf7180000409711 (KB465257.1) at locus LOC108114482 (XP_017080989.1)— with an E-value of 7e-16 and a percent identity of 46.43%. Furthermore, the putative ortholog is nested by LOC108114479 (XP_017080986.1) alongside LOC108114483 (XP_017080990.1) upstream, and LOC108113894 (XP_017080088.1) and LOC108114481 (XP_017080988.1) downstream (E-value: 0.0, 1e-38, 1e-68 and 3e-53; identity: 91.26%, 60.00%, 57.40% and 59.71%, respectively, as determined by blastp; Figure 1A, Altschul et al., 1990). The putative ortholog is flanked further upstream by LOC108114226 (XP_017080582.1) and LOC108114224 (XP_017080566.1), which nests LOC108114228 (XP_017080583.1); that correspond to I-2, CG43897 and Ilp5 in D. melanogaster (E-value: 1e-100, 0.0 and 2e-38; identity: 85.37%, 80.83% and 56.25%, respectively, as determined by blastp). The putative ortholog of Ilp3 is flanked downstream by LOC108114480 (XP_017080987.1) and LOC108114478 (XP_017080982.1), which correspond to Ilp1 and Zasp67 in D. melanogaster (E-value: 3e-59 and 0.0; identity: 63.64% and 82.66%, respectively, as determined by blastp). The putative ortholog assignment for Ilp3 in D. eugracilis is supported by the following evidence: The genes surrounding the Ilp3 ortholog are orthologous to the genes at the same locus in D. melanogaster, aside from the insertion of CG33483. Local synteny is completely conserved, supported by results generated from blastp, so we conclude that LOC108114482 is the correct ortholog of Ilp3 in D. eugracilis (Figure 1A). .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 20, 2025. ; https://doi.org/10.1101/2025.08.16.670678doi: bioRxiv preprint 3 Protein Model Ilp3 in D. eugracilis has two CDSs within the genome sequence. The unique protein sequence (Ilp3-PA) is translated from one mRNA isoform (Ilp3-RA; Figure 1B). Relative to the ortholog in D. melanogaster, the CDS number and protein isoform count are conserved. The sequence of Ilp3-PA in D. eugracilis has 68.63% identity (E-value: 2e-48) with the protein-coding isoform Ilp3-PA in D. melanogaster, as determined by blastp (Figure 1C). Regions which lack conservation are highlighted in purple in the dot plot and protein alignment (I, II, III and IV in Figure 1C and Figure 1D, respectively). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession BK059552). This gene model can also be seen within the target genome at this TrackHub.

“Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2023). Briefly, students use the GEP instance of the UCSC Genome Browser v.435 (https://gander.wustl.edu; Kent WJ et al., 2002; Navarro Gonzalez et al., 2021) to examine the genomic neighborhood of their reference IIS gene in the D. melanogaster genome assembly (Aug. 2014; BDGP Release 6 + ISO1 MT/dm6). Students then retrieve the protein sequence for the D. melanogaster reference gene for a given isoform and run it using tblastn against their target Drosophila species genome assembly on the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al., 1990) to identify potential orthologs. To validate the potential ortholog, students compare the local genomic neighborhood of their potential ortholog with the genomic neighborhood of their reference gene in D. melanogaster. This local synteny analysis includes at minimum the two upstream and downstream genes relative to their putative ortholog. They also explore other sets of genomic evidence using multiple alignment tracks in the Genome Browser, including BLAT alignments of RefSeq Genes, Spaln alignment of D. melanogaster proteins, multiple gene prediction tracks (e.g., GeMoMa, Geneid, Augustus), and modENCODE RNA-Seq from the target species. Detailed explanation of how these lines of genomic evidenced are leveraged by students in gene model development are described in Rele et al. (2023). Genomic structure information (e.g., CDSs, intron-exon number and boundaries, number of isoforms) for the D. melanogaster

gene is retrieved through the Gene Record Finder (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html; Rele et al., 2023). Approximate splice sites within the target gene are determined using tblastn using the CDSs from the D. melanogaster reference gene. Coordinates of CDSs are then refined by examining aligned modENCODE RNA-Seq data, and by applying paradigms of molecular biology such as identifying canonical splice site sequences and ensuring the maintenance of an open reading frame across hypothesized splice sites. Students then confirm the biological validity of their target gene model using the Gene Model Checker (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html; Rele et al., 2023), which compares the structure and translated sequence from their hypothesized target gene model against the D. melanogaster reference gene model. At least two independent models for a gene are generated by students under mentorship of their faculty course instructors. Those models are then reconciled by a third independent researcher mentored by the project leaders to produce the final model. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol.” (Gruys et al., 2025) .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 20, 2025. ; https://doi.org/10.1101/2025.08.16.670678doi: bioRxiv preprint 4 Figure 1: Ilp3 gene model comparison between Drosophila eugracilis and Drosophila melanogaster orthologs (A) Synteny comparison of the genomic neighborhoods for Ilp3 in Drosophila melanogaster and D. eugracilis. Thin underlying arrows indicate the DNA strand within which the target gene –Ilp3–is located in D. melanogaster (top) and D. eugracilis (bottom). Thin arrows pointing to the left indicate that Ilp3 is on the negative (-) strand in D. eugracilis and D. melanogaster. The wide gene arrows pointing in the same direction as Ilp3 are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Ilp3 are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. eugracilis indicate orthology to the corresponding gene in D. melanogaster, while black gene arrows indicate non-orthology . Gene symbols given in the D. eugracilis gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. eugracilis. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al., 2014). The coding-regions of Ilp3 in D. eugracilis are displayed in the User Supplied Track (black); coding CDSs are depicted by .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 20, 2025. ; https://doi.org/10.1101/2025.08.16.670678doi: bioRxiv preprint 5 thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref -Seq genes for D. eugracilis), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA -Seq from Adult Females, Adult Males and Mixed Embryos (red, light blue and purple, respectively; alignment of Illumina RNA-Seq reads from D. eugracilis), and Splice Junctions Predicted by regtools using D. eugracilis RNA-Seq (PRJNA63469). The splice junction shown in green (JUNC00079281) has a read- depth of 50. (C) Dot Plot of Ilp3-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. eugracilis (y-axis). Amino acid number is indicated along the left and bottom; CDS number is indicated along the top and right, and CDSs are also highlighted with alternating colors. Line breaks in the dot plot indicate mismatching amino acids at the specified location between species. Regions that lack conservation are highlighted in purple (Box Ia, IIa, IIIa and IVa, respectively). (D) Protein alignment between D. melanogaster Ilp3-PA and its putative ortholog in D. eugracilis. The alternating colored rectangles represent adjacent exons. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. Areas highlighted in purple (Ib, IIb, IIIb and IVb) correspond to similarly labeled regions in the dot plot (Ia, IIa, IIIa and IVa). Supplemental files: 1. Zip file containing a FASTA, PEP, GFF files for the gene model 2. Figure 1 in high resolution Metadata: Bioinformatics, Genomics, Drosophila, Genotype Data, New Finding

We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model, Madeline L. Gruys for retrofitting this model and Laura K. Reed for overseeing the project. Thank you to FlyBase for providing the definitive database for Drosophila melanogaster gene models. Further, we would like to thank the editors and developers at the journal microPublication: Biology for assistance in developing the template for these single gene ortholog publications. Funding This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.

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