Accurate characterization of gene structure and transcript isoform expression is an essential prerequisite for studying a gene’s function and regulation. During insect embryogenesis, the single-minded (sim ; FlyBaseID:FBgn0004666 ) gene is a critical and conserved regulator of the central nervous system midline (Nambu et al., 1991; Zinzen et al., 2006; Linne et al., 2012). Sim, a bHLH-Pas domain transcription factor, heterodimerizes with the related bHLH-Pas protein Tango to bind to regulatory elements and stimulate the expression of key genes involved in the development of midline neuronal and glial cell lineages (Nambu et al., 1990; Wharton et al., 1994; Sonnenfeld et al., 1997; Watson and Crews, 2012). sim ’s structure, transcriptional profile, and regulation are all well-characterized in Drosophila melanogaster , where sim is expressed from the early blastoderm stage and remains expressed throughout embryogenesis (Thomas et al., 1988; Wheeler et al., 2006). Post-embryogenesis, sim is involved in axon guidance in the brain, development of the optic lobe and genital discs, and high expression in the follicular cells of the ovaries (Pielage et al., 2002; Freer et al., 2011; Oramas et al., 2023). Three promoters control the expression of D. melanogaster sim , giving rise to virtually-identical isoforms: an ‘early’ transcript, which is the predominant transcript from embryonic stages 5 through 9, a ‘late’ transcript, which is the predominantly expressed transcript from stage 9 onward throughout embryogenesis and in other developmental stages, and an ovarian isoform, which is expressed in the follicular cells of the developing egg, promoting maturation (Freer et al., 2011; Knapp et al., 2020). The ‘late’ embryonic promoter also produces a second nearly-identical alternatively-spliced transcript from roughly stage 9 throughout embryogenesis (Freer et al., 2011).

Recently, we have been studying how sim is regulated in the distantly-related mosquito Aedes aegypti (Schember et al., 2024; Sterling-Lentsch and Halfon, 2024). However, only two transcripts were annotated in the Ae. aegypti genome for the sim ortholog AAEL011013 , leading us to wonder if the genome annotation was complete. Although RNA-seq data exist for Ae. aegypti embryos (e.g. Biedler et al., 2012; Akbari et al., 2013), an early transcript that is expressed transiently and only in the small population of midline cells could easily be missed in RNA-Seq characterization due to low coverage depth. Here, we report on the identification of such an early sim transcript, thus demonstrating equivalency in gene structure and promoter usage between D. melanogaster and Ae. aegypti .

To test for the presence of a distinct unannotated early Ae. aegypti sim transcript, we collected Ae. aegypti embryos at 4h after egg laying (AEL) and processed them for 5’-RACE. We identified a novel transcript with a transcription start site (TSS) 10,263 bp upstream of Exon II of AAEL011013 ( Fig. 1B ) and confirmed its existence with allele-specific intron-spanning RT-PCR primers ( Fig. 1C ). This places the early sim promoter as the most proximal to the shared Exon II, with the AAEL011013-RC (ovarian) and AAEL011013-RB (late) TSSs being 58,005 and 106,578 bp upstream, respectively. These results are consistent with the sim gene structure of D. melanogaster , where the early promoter is the most proximal to the common Exon II of the three defined sim promoters. Further testing of the expression of all sim transcripts using allele-specific intron-spanning RT-PCR ( Fig. 1C ) demonstrated that the early sim promoter is active as early as 4h AEL and sharply decreases expression by 18h AEL, at which point embryos are approximately equivalent to Drosophila stage 10-12. Meanwhile, the late sim transcript ( AAEL11013-RB ) is first detectable at 10h AEL (stage 8-10) and continues to be expressed throughout embryogenesis and through all life stages with the exception of the male carcass. The third sim isoform, AAEL11013-RC , was detectable only in 48h post-bloodmeal ovaries. The onset of transcript expression in Ae. aegypti is therefore consistent with that in D. melanogaster, where expression from the early promoter tails off by stage 11 and the ovarian promoter drives expression exclusively in the ovaries (Freer et al., 2011). Detection of the early-promoter and AAEL11013-RB sim transcripts in female, but not male, carcass may be due to the presence of expression in ovarian tissue, since the assayed carcasses consisted of the entirety of the mosquito minus the head. Three in-frame ATG putative start codons are present within the early sim transcript and are conserved among four additional Aedes spp: Ae. albopictus , Ae. formosus , Ae. mascarensis , and Ae. polynesiensis ( Fig. 1A ). Alignment and conservation analysis of all five Aedes spp. shows two strong blocks of conservation at the beginning of the predicted TSS through the first 100 bp of exon I. A third block of conservation was identified spanning the predicted splice donor site ( Fig. 1A ), with conservation in the region between the promoter and the splice donor remaining ≥60%. Conceptual translation of the orthologous exon I in all Ae . spp. indicates that either of the two ATG present within exon I could be the start codon, encoding 53 or 61 predicted N-terminal residues respectively, as all species retain in-frame ORFs irrespective of start codon choice. As such, whether the putative exon I of the early sim transcript is entirely or only partially 5’UTR cannot be determined from our study. Local alignment of the D. melanogaster early - specific N-terminal residues against those predicted for the Ae. aegypti early-specific isoform suggests a moderate degree of conservation (28.6% identity; 42.9% similarity) , but whether or not these residues are translated or relevant for early Sim function remains to be determined . Our work further refines the structure of the sim locus in Ae. aeygpti, which will facilitate future studies, and underscores the need for careful manual annotation and experiments beyond bulk RNA-seq to ensure reliable description of minor transcripts .

Mosquito rearing. Ae. aegypti (strain Liverpool) were maintained at 27°C and 75% humidity under a 12:12 (L:D) photoperiod. Larval mosquitoes were fed ad libitum with a slurry of ground Tetramin fish flake (Tetra, Blacksburg, VA), and adults were provided with raisins as a sugar source and defibrinated sheep blood (Hardy Diagnostics, Santa Maria, CA) supplemented with 1 µM ATP for blood feeding.

Embryo collection and aging . Five day old adult females were provided with a bloodmeal, and four days following, 20-30 hypergravid females were collected and placed into a 50 mL conical tube containing Whatman #1 filter paper wetted with deionized water. Females were allowed to oviposit for 1h and embryos were subsequently aged on wetted Whatman #1 filter paper at 27°C for the indicated time periods of “hours after egg laying” hours AEL.

RNA extraction, 5’RACE, RT-PCR. Total RNA was collected by homogenizing ~10 mg of embryos in Trizol (ThermoFisher Scientific, Grand Island, NY) according to the manufacturer’s instructions. A total of 3 μg of RNA collected from 4h AEL embryos was used for the 5’-RACE, utilizing the 5’-RLM Generacer kit (ThermoFisher Scientific, Grand Island, NY) and first strand synthesis was conducted using oligo dT and Superscript III reverse transcriptase (ThermoFisher Scientific, Grand Island, NY). PCR products were amplified using sim -specific reverse primers and RACE PCR products were sequenced using nanopore sequencing (Plasmidsaurus, Louisville, KY). The cDNA templates for the time course RT-PCR were reverse transcribed in 20 μL reactions from 500 ng of total RNA using the RevertAid First Strand cDNA synthesis kit (ThermoFisher Scientific, Grand Island, NY) according to the manufacturer’s instructions. First strand cDNA was subsequently diluted 5-fold and 1 μL was used for each of four 15 μL RT-PCR reactions specific for: 1) intron-spanning sim early transcript, 2) intron-spanning sim late transcript, 3) intron-spanning sim ovarian transcript, and 4) rpS7 control. Five μL of each PCR product was then run out on a 1.5% agarose gel in 1xTBE and stained with ethidium bromide.

Promoter alignment. The full loci for sim for Ae. aegypti (AaegL5.0; GCA_002204515.1) , Ae. albopictus (AalbF5; GCA_035046485.1) , Ae. formosus (Aaf_Bf05_pri1.0; GCA_052575885.1) , Ae. mascarensis (Am_MascCH02_pri1.0; GCA_052575835.1) , and Ae. polynesiensis (Smith_Apoly_02182021; GCA_051529985.1) were downloaded from NCBI (ncbi.nlm.nih.gov) and aligned using MAFFT. The DNA regions matching to the predicted early sim transcript promoter from Ae. aegypti were then manually extracted and aligned to identify regions of full and partial conservation among the five mosquito species.