Arpita Kulkarni,Ben Ewen-Campen,Kanta Terao,Yukihisa Matsumoto,Yaolong Li,Takayuki Watanabe,Jonchee A. Kao,Swapnil S. Parhad,Guillem Ylla,Makoto Mizunami,Cassandra G. Extavour
oskar acts with the transcription factor Creb to regulate long-term memory in crickets
co-option,neuroblast,mushroom body,Kenyon cells,orphan genes
Significance When new genes evolve, why are they not immediately eliminated from the genome, given that the organism did not previously need them for survival? Here, we examine the hypothesis that novel genes can survive by evolving regulatory interactions with preexisting genes that control essential fitness traits. We provide experimental evidence that the unique insect-specific oskar gene plays a role in long-term memory through expression in adult neural stem cells. We show that oskar is expressed and required in stem cells of the adult cricket brain for long-term memory. Further, we provide evidence that oskar’s role in long-term memory involves regulation by the transcription factor Creb, a conserved player in long-term memory that predates the origin of oskar in animals.
oskar acts with the transcription factor Creb to regulate long-term memory in crickets

When new genes evolve, why are they not immediately eliminated from the genome, given that the organism did not previously need them for survival? Here, we examine the hypothesis that novel genes can survive by evolving regulatory interactions with preexisting genes that control essential fitness traits. We provide experimental evidence that the unique insect-specific oskar gene plays a role in long-term memory through expression in adult neural stem cells. We show that oskar is expressed and required in stem cells of the adult cricket brain for long-term memory. Further, we provide evidence that oskar’s role in long-term memory involves regulation by the transcription factor Creb, a conserved player in long-term memory that predates the origin of oskar in animals.

oskar (osk) is an insect-specific gene first discovered in Drosophila melanogaster, where it plays a critical role in germ line specification (1). osk messenger RNA is localized to the posterior of the developing D. melanogaster oocyte (2, 3). Local translation and anchoring of Oskar (Osk) protein leads to the posterior accumulation of the messenger RNA and protein products of several genes with conserved expression and function in animal germ lines, including vasa and piwi (2, 4, 5). Collectively called germ plasm, these cytoplasmic contents act as necessary and sufficient determinants to specify embryonic germ cells (2, 3). The current model of Osk function in D. melanogaster germ plasm assembly is that it serves as a scaffolding protein, facilitating the assembly of the ribonucleoprotein complexes that contain germ plasm components (2, 6, 7). Interestingly, osk and several other genes originally identified as D. melanogaster germ line genes, including vasa, pumilio, staufen, orb, and piwi-related genes including aubergine and argonaute 3, have since been shown to have a variety of roles in animal nervous systems (8–14). For example, in D. melanogaster, osk RNA interference (RNAi) in larval dendritic arborization neurons disrupts nanos mRNA localization, ultimately leading to a defect in dendrite morphogenesis and an associated defect in motor response to mechanical stimulation (12). Furthermore, osk plays a role in the embryonic nervous system, but not in the germ line, in a hemimetabolous insect, the cricket Gryllus bimaculatus, where it is important for proper neuroblast divisions and subsequent axonal patterning (15). Our recent analysis of hundreds of previously unidentified osk orthologs across insects showed that osk is expressed in at least a dozen somatic tissues in species across the insect tree (16). This suggests that a somatic function of osk may be ancestral. However, the precise roles of osk in any somatic tissue, including the nervous system, remain largely unknown. Here, we demonstrate a role for osk in the adult brain of the cricket G. bimaculatus, in a population of neural stem cells in the mushroom body that persist throughout adult life. We show that osk, as well as Piwi and Vasa, is enriched in a population of adult neuroblasts in the mushroom body, and that RNAi targeting osk or piwi in adult crickets impairs long-term, but not short-term, memory formation in an olfactory associative learning assay (17). We also provide evidence that osk and piwi function in a regulatory feedback loop with the cyclic adenosine monophosphate response element binding protein (Creb), a transcription factor with well-described conserved roles in long-term memory across metazoans (18). Our data demonstrate a somatic role for osk in the nervous system and shed light on how a novel gene may acquire critical roles by integrating with preexisting gene regulatory systems comprising older, conserved genes.

We previously showed that neuroblasts in the cricket embryo express osk, vasa, and piwi, and that osk is required for correct neuroblast division and embryonic nervous system morphology (15). Interestingly, in many insects, including crickets, a subset of embryonic neuroblasts persists in the brain throughout adulthood and continuously gives rise to new neurons called Kenyon cells that comprise the mushroom body (19–21). This contrasts with flies like D. melanogaster, in which neuroblasts die prior to adulthood (22), and in which adult brains are thus essentially devoid of neurogenesis (23) (although there are reports of potential stem cells in adult D. melanogaster brains (23, 24), which may be damage-dependent rather than homeostatic in function (25), and which remain controversial (26)). Given the role of osk in embryonic neuroblasts of crickets, we asked whether osk also plays a role in the adult mushroom body neuroblasts (MBNBs). We used in situ hybridization to examine osk expression in the adult brain and found expression in a cluster of cells with the large, round nuclei and diffuse chromatin characteristic of stem cells, at the apex of each of the two lobes of the mushroom body, consistent with descriptions of adult neuroblasts in orthopterans (Fig. 1A). EdU colocalization (Fig. 1B) confirmed the identity of these cells as neuroblasts, the only proliferative cells in the adult brain (27). We also found that MBNBs express high levels of Vasa and Piwi proteins (Fig. 1E). Previous research has shown that MBNBs play an important role in long-term olfactory memory formation in Orthoptera (28). Scotto-Lomassesse (28) found that ablation of MBNBs using irradiation led to a dramatic reduction in olfactory, but not visual, learning after 24 and 48 h, suggesting that newborn mushroom body neurons produced by those neuroblasts play a role in forming new olfactory memories. We therefore sought to test whether osk, expressed specifically in MBNBs, functions in these cells in the context of long-term memory formation. We first tested whether osk regulates the proliferation or survival of adult MBNBs. Using an established technique for systemic RNAi in the adult cricket brain (29), we injected double-stranded osk RNA (dsRNA) into the head capsule and confirmed the efficiency of osk knockdown via quantitative PCR (Fig. 2D and SI Appendix, Table S1) and small RNA profiling of oskRNAi brains (SI Appendix, Tables S2–S4 and Fig. S1). oskRNAi adult mushroom bodies showed no gross anatomical defects relative to controls (Fig. 1; and SI Appendix, Fig. S1). Moreover, neither the total number of neuroblasts (P > 0.05), nor the number of neuroblasts undergoing mitosis as revealed by EdU labeling (P > 0.05), was statistically significantly different between oskRNAi adult brains and controls (Fig. 1 C and D). We stained oskRNAi and control brains with cleaved caspase-3, a marker for apoptosis, and did not observe any evidence of cell death (SI Appendix, Fig. S2A). We noted that one described role for piwi in the Drosophila germ line is to prevent DNA damage caused by transposon mobilization (30). However, we observed no detectable increase in γH2A staining, a marker for DNA damage, in oskRNAi brains (SI Appendix, Fig. S2B). These data suggest that osk is not required for the proliferation, survival, or genomic integrity of adult neuroblasts. However, the specific expression of osk, Piwi, and Vasa in the MBNBs suggested that some or all of these genes could play a role related to memory or learning.

The mushroom body is the anatomical substrate for olfactory memory and learning in insects (27, 31, 32), and ablation of the mushroom body or of the adult MBNBs impairs these processes (28, 33, 34). Based on previous observations that MBNBs play a role in long-term olfactory memory formation in crickets (28), we hypothesized that osk might play a role in this process. To test this hypothesis, we assessed the memory of oskRNAi adult male crickets at 1 h (“short-term memory”) and 1 d (“long-term memory”) post-training using well-established cricket olfactory behavior assays as previously described (17). Briefly, the crickets were injected with double-stranded RNA against the gene of interest (osk) or a control gene (DsRed) (35), and then subjected to odor preference tests (allowed to freely visit peppermint and vanilla odor sources, quantifying the time spent at each odor source) and conditioning trials (peppermint odor was paired with a water reward, and relative preference for this rewarded odor was compared before and after the conditioning) (17). In control crickets (injected with dsRNA targeting DsRed (35)), four training sessions led to a significant (P < 0.05) short-term preference for the rewarded odor (peppermint) at 1 h after training (short-term; Fig. 2A). Trained control crickets retained this learned preference (P < 0.01) even at 1 d after training (Fig. 2A, “DsRed ”), demonstrating that long-term memory formation is intact in these controls. However, although oskRNAi crickets formed and retained memory for the rewarded odor by 1 h after training (short term; Fig. 2A osk dsRNA #1, P < 0.001), this memory was lost by 1 d after training (long term; Fig. 2A osk dsRNA #1, P > 0.05), indicating a specific impairment of long-term memory formation. These results were reproducible in a second experiment using a nonoverlapping fragment of osk dsRNA (Fig. 2A, osk dsRNA #2; P < 0.05 for short term, P > 0.05 for long term), suggesting that the impact was specific to osk knockdown. The efficacy of the knockdown was confirmed via qPCR (Fig. 2D and SI Appendix, Table S1) and small RNA sequencing (SI Appendix, Fig. S1 and Tables S1 and S2), indicating that osk is required for cricket long-term memory. Since both Piwi and Vasa were coexpressed with osk in cricket MBNBs (15) (Fig. 1E), we also assessed the role of these two genes in olfactory memory. We found that piwi (Fig. 2B, P < 0.001) but not vasa (SI Appendix, Fig. S3A, P > 0.05; SI Appendix, Fig. S3B) was also required for cricket long-term memory. qPCR analyses showed that osk RNAi led to a notable decrease in piwi transcript levels (Fig. 2D), suggesting that osk positively regulates piwi in the cricket brain. However, osk transcript levels remained unaffected in piwiRNAi animals (Fig. 2E). Consistent with the phenotype of the single gene knockdowns, oskRNAi/piwiRNAi double knockdown adults also showed a long-term memory impairment phenotype (Fig. 2C, P < 0.01). Thus, osk and piwi do not globally disrupt olfaction, learning, or short-term memory formation, but are required for consolidation of long-term memory in this species.

To understand how a novel gene like osk might have gained a role in an ancient animal function like long-term memory consolidation, we investigated the hypothesis that it might interact with conserved regulators of animal memory. To test this hypothesis, we asked whether we could detect a functional or regulatory interaction between osk and a highly conserved transcription factor with well-documented roles in long-term memory formation across animals, Creb (18). We first identified putative Creb orthologs in the G. bimaculatus genome (36) using a combination of basic local alignment search tool searches and phylogenetic analyses (Fig. 3A, and SI Appendix, Table S5). These analyses yielded two high-confidence Creb orthologs, which we called CrebA and CrebB based on their closest D. melanogaster Creb gene relative (Fig. 3A). Analysis of previously generated transcriptomes (37) showed that both genes are expressed in adult cricket brains (SI Appendix, Table S6). We performed CrebA and CrebB RNAi experiments and discovered that CrebA (but not CrebB; SI Appendix, Fig. S3C) was required for long-term memory in crickets (Fig. 3B; P < 0.003 and P < 0.001 for dsRNA#1 and dsRNA#2, respectively; SI Appendix, Fig. S3C). Using qPCR, we then asked whether transcript levels of this memory regulator were altered in osk or piwi knockdown conditions and found consistent downregulation of CrebA transcript levels in both single and double RNAi backgrounds (Fig. 2 D–F). In contrast, and consistent with the observation that vasaRNAi had no long-term memory impact (SI Appendix, Fig. S3A), qPCR revealed no reduction of CrebA transcripts in vasaRNAi conditions (SI Appendix, Fig. S3B). This suggests that the long-term memory defects observed in oskRNAi and piwi RNAi conditions (Fig. 2 D–F) are due to a downregulation of CrebA in these animals.

Creb proteins are transcription factors that bind cyclic adenosine monophosphate response element (CRE) binding sites within the regulatory regions of target genes to initiate transcription (18) (Fig. 4A). Since target gene transcription and new protein synthesis is crucial for long-term memory formation, and given the similarity in long-term memory phenotypes of oskRNAi, piwiRNAi, and CrebARNAi animals, we asked whether osk or piwi might also be Creb target genes in this cricket. qPCR revealed that transcript levels of both osk and piwi are significantly decreased in CrebARNAi conditions (Fig. 4B), suggesting that osk/piwi and CrebA may interact in a positive feedback loop to regulate each other’s transcript levels. To evaluate the possibility that osk or piwi might be direct transcriptional targets of CrebA, we examined the genomic sequences within 10 kb upstream of both loci and found two bioinformatically predicted CRE binding sites within the 6 kb upstream of the transcription start sites for osk and piwi (Fig. 4C and SI Appendix, Supplementary File 1). These predicted CRE binding sites were found twice as frequently as we would expect to find such sequences in a randomly generated sequence of this length (Materials and Methods). Electrophoretic mobility shift assays showed that protein(s) within the adult cricket brain bind specifically to the predicted CRE sites of osk (Fig. 4D and SI Appendix, Table S7). Given the current lack of species-specific CrebA reagents for this cricket species, we cannot rule out the interpretation that a protein(s) other than CrebA present in the adult cricket brain is causing the observed mobility shift by binding the predicted CRE sites of osk. However, given our functional data indicating that RNAi against osk, piwi, and CrebA all yield long-term memory defects (Figs. 2 and 3B), that osk and piwi transcript levels are reduced in CrebARNAi brains (Fig. 4B), and that osk and piwi genomic loci contain predicted CRE binding sites (Fig. 4C), the results of our gel shift assay are consistent with the hypothesis that cricket osk is a direct transcriptional target of CrebA.

We have discovered a role for oskar in the adult cricket brain (Fig. 1). We have shown that osk, Piwi, and Vasa are coexpressed in MBNBs (Fig. 1 A and B), a population of neural stem cells required for long-term olfactory memory formation (28), and that knockdown of osk and piwi disrupts olfactory long-term memory formation. The precise role that the MBNBs play in memory formation remains unknown, as does the molecular role of Osk in these cells. In D. melanogaster, where there are no adult neural stem cells in the mushroom body, olfactory long-term memory requires the ~2,500 differentiated Kenyon cell neurons of the mushroom body (38), which respond with high selectivity to a small number of stimuli, allowing the mushroom body to house an explicit representation of a large number of olfactory cues (38, 39). Specific olfactory stimuli are associated with learned behavioral responses via specific sets of neurons connecting the mushroom body to other brain regions in a protein synthesis-dependent fashion, to form long-term memories (9, 40). Thus, one possibility is that adult-born Kenyon cells in G. bimaculatus (and other insects that display adult neurogenesis in the mushroom body) are recruited into an existing circuit and allow for a constantly increasing repertoire of olfactory associations. Our results suggest that osk could play a role in this process, as osk RNAi disrupts long-term memory. We note that of the two mammalian brain regions known to undergo adult neurogenesis, one (the subventricular zone) contributes to the olfactory bulb, and neurogenesis in this region is involved in olfactory memory (41). Given that adult D. melanogaster lacks the MBNBs seen in G. bimaculatus (42), a straightforward test for a directly comparable osk function in this fruit fly is not possible. However, although D. melanogaster mushroom body stem cells are absent in adults, analogous MBNBs remain mitotically active late into pupal development (43). Thus, it will be interesting to test whether osk functions in these neuroblasts during larval and/or pupal stages. We note that an insertion of an enhancer trap transposable element over 3 kb upstream of the osk transcription start site was recovered in an insertional mutagenesis screen for long-term memory in D. melanogaster (8). However, this insertion has not been confirmed as compromising the sequence or function of the osk locus, nor has osk been tested directly to confirm a potential role in D. melanogaster learning or memory. Although D. melanogaster lacks adult MBNBs, it is possible that osk could function in fruit fly olfactory long-term memory in a neuroblast-independent manner. A recent study of the mushroom body output neurons has suggested that long-term memory involves the activity-dependent derepression of mRNAs localized to granules containing Pumilio, Staufen, and Orb (oo18 RNA-binding protein) proteins (9). Given that Osk nucleates similar granules containing these proteins in the Drosophila oocyte (44, 45), and Osk’s ability to nucleate phase-transitioned granules in D. melanogaster cells (46), it would be interesting to test whether Osk is involved in the formation and/or activity of these granules in the brain. Given our recent observation of highly similar molecular interactions of conserved molecules in animal germ lines and neural cells (14), future studies could test whether additional genes traditionally known as “germ line” genes other than vasa and piwi, for example staufen (47) and tudor (48), also function in G. bimaculatus adult neuroblasts, which would suggest that osk acts with conserved molecular partners in different cellular contexts. Because mushroom bodies derived from neuroblasts are a conserved arthropod brain structure across and beyond insects (49–52), it seems unlikely that oskar played a role in the evolution of insect mushroom body neurogenesis per se. Both germ cells and neuroblasts are stem cells that give rise to highly specialized daughter cells while remaining proliferative for long periods of time. Thus, the original role of osk in both cell types could conceivably be related to stem cell maintenance and/or asymmetric division. Indeed, many different highly conserved “germ line genes” including vasa, nanos, and piwi are found in a variety of multipotent cells in diverse animals (14, 53), raising the possibility that such genes were involved in establishing multipotency rather than specifying germ cell fate per se. In our previous examination of the distribution of osk orthologs across insects, we observed that crickets are not the only insects reported to express osk in the brain, as osk transcripts are detected in transcriptomes from the brains of cockroach, wasp, and beetle species as well (16). This is consistent with the hypothesis that osk played an ancient neural role of some kind in insects. We previously showed evidence supporting the hypothesis that evolved changes in the biophysical characteristics of Oskar protein may have driven the evolution of a novel mechanism of germ line specification in the holometabolous insects (16). A broader understanding of the putative ancestral and derived function(s) of osk thus requires additional studies of phylogenetically diverse insects, as well as further detailed biochemical analysis in the context of Drosophila germ cells and neurons. Our results provide an example of how novel genes may find stable homes in preexisting genetic regulatory circuits. In the case of osk, we hypothesize that by evolving or acquiring binding sites responsive to the conserved transcription factor Creb, osk may have gained expression in the brain, opening the door for potential participation in neural roles. An alternative hypothesis to de novo evolution of CRE sites to explain osk’s expression in the cricket brain is that osk inherited CRE binding sites from an ancestral sequence that contributed to osk’s genesis. Our previous work suggested that osk arose through a fusion of a eukaryotic LOTUS domain in the 5′ position, coding for osk’s N-terminal LOTUS domain, and a prokaryotic SGNH hydrolase-like domain in the 3′ position, coding for osk’s C-terminal OSK domain (54). In this scenario, a preexisting LOTUS domain-containing gene could have donated not just its LOTUS domain, but also some upstream 5′ regulatory sequences, including CRE sites and/or neural expression elements, to osk. This hypothesis might predict that extant LOTUS domain-containing genes might display one or both of CRE-binding sites, or expression in the brain. To test this hypothesis, we searched the G. bimaculatus genome for LOTUS domain-containing genes and identified five such genes (SI Appendix, Supplementary File 2). These were osk, Tdrd5, Tdrd7, limkain b1, and an uncharacterized gene with annotation ID GBI_15344 (SI Appendix, Supplementary File 2). In transcriptomes previously generated from the adult brain (55), we detected levels of all four non-oskar LOTUS domain-containing genes at levels at least as high as those detected for osk (SI Appendix, Fig. S4 and Table S8). Moreover, in the 10 kb upstream of the first predicted codon of these genes, we detected a putative CRE binding site (SI Appendix, Table S9). With the caveat that our transcriptomes do not provide spatial or cell-type resolution for the expression data, both of these findings are consistent with the hypothesis that whatever eukaryotic LOTUS domain-containing sequence was the ancestor of osk’s LOTUS domain, it also contributed one or both of CRE-responsive or brain-expressed upstream regulatory sequences to osk. Future studies will be needed to elucidate the molecular mechanisms of osk gene products in the cricket brain, and specifically in learning and memory. We further speculate that the biophysical properties of Osk protein that make it effective at sequestering RNAs and participating in translational control in the germ line (56–58) may have been advantageous in promoting the rapid translation needed for the synaptic plasticity that underlies learning and memory. These include Osk’s ability to form phase-transitioned condensates (46, 59), its regions of high predicted disorder (16, 46, 59), and its ability to achieve and maintain asymmetric subcellular localization, all of which are well known in the germ line and may have provided a selective advantage to Osk in the context of promoting neuronal function.

G. bimaculatus husbandry, in situ hybridization, immunostaining, olfactory learning assays, RNAi, and qPCR were performed as previously described. See SI Appendix, Extended Materials and Methods for references and detailed protocols.

Cell proliferation was assayed using the Click-iT EdU Alexa 488 kit (Life Technologies, Cat# C10637). Crickets were injected with 10 to 15 μL EdU either into the abdomen or into the head capsule through the median ocellus (both methods successfully labeled dividing neuroblasts), and brains were dissected to visualize EdU incorporation 4 h postinjection. The brains were dissected and desheathed in ice-cold 1× phosphate buffered saline. Calyces were removed with a microscalpel and incubated in 0.1M citric acid for 15 to 30 min on a poly-lysinated slide (Sigma Aldrich, Cat. No. P8920-100ML). The calyces were then spread into a monolayer by adding a Sigma cote-covered coverslip, and the entire slide was flash-frozen in liquid nitrogen. The coverslip was removed, leaving the mushroom body monolayer on the slide. Slides were air-dried and were then fixed for 15 min in 4% paraformaldehyde. EdU detection was then carried out following manufacturer’s instructions. EdU-positive cells were photographed under epifluorescence on a Zeiss AxioImager Z.1 compound microscope using Zen and manually quantified in ImageJ. For any mushroom body where the EdU-positive cluster of cells was damaged or destroyed during preparation, that sample was discarded and not included in the analysis. For tissue double stained to visualize transcripts and EdU incorporation simultaneously, in situ hybridization was conducted before the visualization of incorporated EdU. AxioImager Z.1, LSM 780, or LSM 880 confocal microscopes (Zeiss) were used for microscopy, driven by AxioVision or Zen (Zeiss).

Unmated adult male crickets within 1 week of their final molt to adulthood were injected with dsRNA as described above (see RNA interference). At 48 h after injection, brains (SI Appendix, Table S2) were dissected in ice-cold 1× phosphate buffered saline and transferred into Trizol, following which total RNA was extracted from them following manufacturer’s protocols. Next, RNA was size selected for 18 to 30 nt size range after denaturing polyacrylamide gel electrophoresis. A 2S rRNA specific oligo was used for 2S rRNA depletion. The small RNAs were ligated at the 3′ and 5′ ends by the respective adapters and purified by denaturing polyacrylamide gel electrophoresis after each ligation. PCR was performed after reverse transcription. The PCR product was gel purified from an agarose gel to obtain the final library. The libraries were sequenced using Illumina NextSeq500 1×75bp. The resulting data (SI Appendix, Table S3) were uploaded onto the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database and are publicly available under the BioProject ID PRJNA837371 (60).

Putative orthologs of Creb/ATF family members from several animal species were initially identified by basic local alignment search tool (BLAST) searches (SI Appendix, Table S5) and then downloaded from NCBI. These sequences were then used to search for putative G. bimaculatus CrebA orthologs in the G. bimaculatus genome (36). All identified sequences were then aligned with MAFFT (v 7.510) (61). A maximum likelihood tree was created in RAxML using the PROTGAMMAWAG model (62) and plotted with the FigTree package v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) (Fig. 3B).

A position frequency matrix (PFM) for the full CRE octameric palindrome was obtained from the JASPAR database (an open-source database for transcription factor binding sites (63) (SI Appendix, Supplementary File 1). In addition to CRE, PFMs for the TATA box were also obtained from the same database. We included TATA box proximity among our search criteria for putative CRE sites, since TATA boxes are often a feature of functional promoters, and functional promoter-proximal CRE sites are reported as often occupied by Creb. These raw PFM data (SI Appendix, Supplementary File 1) were then used as an input in Find Individual Motif Occurrence (FIMO) in the MEME suite [a motif-based sequence analysis tool (64)], and up to 10 Kb of the genome sequence upstream of the predicted transcription start site for each of the G. bimaculatus oskar, piwi, and vasa was scanned for the presence of the CRE and TATA motifs using the annotated G. bimaculatus genome (36), and using P < 0.0001 as the stringency criteria. For comparison, G. bimaculatus beta actin, alpha tubulin, and FGFR loci were subjected to the same analyses (also with P < 0.0001 as stringency criteria) to assess the possibility that any randomly chosen G. bimaculatus gene would be predicted to have CRE sites in the 10Kb region upstream of their transcription start site using this method (SI Appendix, Supplementary File 2). We found that for the latter three genes, there were no CRE predictions in their upstream regions (up to 10 Kb from the transcription start site). Further, we bioinformatically generated one thousand 10 Kb long DNA fragments of random sequence using the “random DNA sequence” tool in the Sequence Manipulation Suite (65) and then tested them for CRE prediction. Our results indicate that a CRE site is expected to occur in a randomly generated sequence at a frequency of ~1.8 CRE sites for every 10 Kb tested (SI Appendix, Supplementary Files 1 and 2).

We searched the annotated G. bimaculatus genome (36) for genes whose protein product was predicted to contain Pfam motif PF12872, corresponding to the LOTUS domain. This search retrieved five genes (GBI_01840 “oskar,” GBI_13502 “TDRD5/tejas”, GBI_15344 “uncharacterized,” GBI_15604 “limkain b1,” and GBI_03370 “TDRD7/tapas”). We assessed the expression of these genes in brains and gonads using previously published RNA-Seq libraries (55) available at NCBI (PRJNA564136). We analyzed the RNA-Seq data as in ref. 16, including removing adapters and reads shorter than 20 nucleotides with Cutadapt v3.4 (66) and quantifying the gene expression in transcripts per million with RSEM v1.2.29 (67), using STAR v2.7.0e1 (68) as read mapper against the G. bimaculatus genome (36) (SI Appendix, Table S8). For each of these genes, we retrieved and searched the 10Kb upstream of the first codon annotated for CRE sites as described above in “Bioinformatic Prediction of CRE Sites in osk and piwi Upstream Regulatory Region” (SI Appendix, Table S9).

Based on bioinformatic predictions of putative CRE sites, primers were designed in the upstream regulatory regions of osk (SI Appendix, Table S7 #1 and #2). Once both CRE sites were sequence confirmed by Sanger sequencing, the ~30bp fragments containing each CRE site were synthetically generated as duplexes (with 3′A overhangs) for use as Electrophoretic Mobility Shift Assay (EMSA) preprobes (SI Appendix, Table S7; CRE site in bold). The 3′A overhangs were then used to clone all EMSA preprobes into a pGEM-T easy vector following manufacturer’s instructions (Promega, catalog number A1360) using One-Shot chemically competent TOP10 E. coli cells (Thermo-Fisher, catalog number C4040-06).

Once cloned, pGEM-T easy specific duplex forward primer (5′Cy5-ACGTCGCATGCTCCCGGCCATG, reverse complement 5′Cy5-CATGGCCGGGAGCATGCGACGT) and reverse primer (5′Cy5-GTCGACCTGCAGGCGGCCGCGAATT, reverse complement 5-Cy5-AATTCGCGGCCGCCTGCAGGTCGAC) were designed with 5′Cy5 modifications to amplify inserts and generate fluorescently labeled double-stranded EMSA probes, using a two-step PCR program with the following conditions: (98 °C for 60 s (×1 cycle); 98 °C for 15 s followed by 72 °C for 30 s (×30 cycles); 72 °C for 5 min (×1 cycle) (SI Appendix, Table S7). The PCR product was loaded onto a 1% agarose gel, and the desired bands were gel eluted following IBI Scientific’s PCR purification and gel elution kit (catalog number IB47030) in 30 μL water. A second round of PCR amplification following the conditions described above was performed using the eluted DNA from previous steps to increase probe yield. All steps starting with the first round of PCR were done in the dark to protect fluorescently labeled probes. Probe concentrations were measured using a Nanodrop and diluted to a final concentration of 40 fmol/probe for use in electrophoretic mobility shift assays (69). Twenty percent native polyacrylamide gele electrophoresis gels were used to study gel shifts. Gels were imaged using an Azure Sapphire Biomolecular Imager (VWR).

Brains were dissected from unmated G. bimaculatus males within 1 wk of their final molt that were anesthetized briefly on ice prior to dissection in 1× phosphate buffered saline. Nuclear protein extracts were prepared from dissected brains following manufacturer’s instructions (Abcam Nuclear Extraction Kit, catalog number ab113474).

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