Journal of Experimental Zoology Part B: Molecular and Developmental Evolution

The model zebrafish (Danio rerio) belongs to the Danioninae subfamily with a range of informative phenotypes. However, the craniofacial diversity across the subfamily is not fully described. To better understand craniofacial phenotypes across Danioninae we used microCT and 3D geometric morphometrics to capture skull shapes from nine species. The Danio species examined showed largely similar skull shapes, although D. aesculapii, the sister species to D. rerio showed a unique morphology. Two non-Danio species examined, Chela dadiburjori and Devario aequipinnatus showed distinct skull morphologies unique from those of other species examined. Thyroid hormone regulates skeletal development and remodeling, and we asked if changes in developmental thyroid hormone metabolism could underlie some of the craniofacial diversity across Danioninae. We reared two Danio species under altered thyroid profiles, finding that hypothyroid individuals from both species showed corresponding morphological shifts in skull shape. Hypothyroid Danios showed skull morphologies closer to that of Chela and unlike any of the examined wild-type Danio species. We provide an examination of the evolved craniofacial diversity across Danioninae, and demonstrate that alterations to thyroid hormone have the capacity to create unique skull phenotypes.

Most eukaryotic genes have been vertically transmitted to the present from distant ancestors. However, variable gene number across species indicates that gene gain and loss also occurs. While new genes typically originate as products of duplications and rearrangements of pre-existing genes, putative de novo genes - genes born out of previously non-genic sequence - have been identified. Previous studies of de novo genes in Drosophila have provided evidence that expression in male reproductive tissues is common. However, no studies have focused on female reproductive tissues. Here we begin addressing this gap in the literature by analyzing the transcriptomes of three female reproductive tract organs (spermatheca, seminal receptacle, and parovaria) in three species - our focal species, D. melanogaster - and two closely related species, D. simulans and D. yakuba, with the goal of identifying putative D. melanogaster-specific de novo genes expressed in these tissues. We discovered several candidate genes, which, consistent with the literature, tend to be short, simple, and lowly expressed. We also find evidence that some of these genes are expressed in other D. melanogaster tissues and both sexes. The relatively small number of candidate genes discovered here is similar to that observed in the accessory gland, but substantially fewer than that observed in the testis.

The repeated evolution of reduced skeletal armour in threespine stickleback provides an excellent model for understanding the genetic basis of morphological change. Here, we investigated the genetic and developmental mechanisms underlying the loss of the second dorsal spine in a freshwater stickleback population from North Uist, Scotland. Crosses between spineless freshwater and fully spined marine individuals confirmed a genetic basis for the trait, with inheritance patterns inconsistent with simple Mendelian expectations. A bulk segregant analysis of F3 hybrids revealed a strong genomic signal on chromosome VI, overlapping the hoxdb cluster, echoing previous findings in another spine-reduced stickleback population. Developmental series of the Scottish freshwater and marine populations using skeletal stainings and transcriptomic analyses of dorsal tissue showed delayed and incomplete cartilage formation and altered gene expression during critical stages of dorsal skeletal development in spineless fish, notably of several hoxdb genes. Our findings indicate that while the loss of dorsal spines has evolved repeatedly, it may involve distinct alleles across populations, with the same locus possibly playing a central role in this case.

The lake sturgeon (Acipenser fulvescens) is an ancient, octoploid fish faced with conservation challenges across its range in North America but a lack of genomic resources has hindered molecular research in the species. To support such research we aimed to provide a transcriptomic database from 13 tissues: brain, esophagus, gill, head kidney, heart, white muscle, liver, glandular stomach, muscular stomach, anterior intestine, pyloric cecum, spiral valve, and rectum. The transcriptomes for each tissue were sequenced and assembled individually from a mean of 98.3 million ({+/-}38.9 million std. dev.) reads each. In addition, an overall transcriptome was assembled and annotated with all data used for each tissue-specific transcriptome. All assembled transcriptomes and their annotations were made publicly available as a scientific resource. The non-gut transcriptomes provide important resources for many research avenues, however, the gut represents a compartmentalized organ system with compartmentalized functions and the sequenced gut tissues were from each of these portions. Therefore, we focused our analysis on mRNA transcribed in different tissues of the gut and explored evidence of microbiome regulation. Gene set enrichment analyses were used to reveal the presence of photoperiod and circadian-related transcripts in the pyloric caecum, which may support periodicity in lake sturgeon digestion. Similar analyses were used to identify different types of innate immune regulation across the gut, while analyses of unique transcripts annotated to microbes revealed heterogeneous genera and genes among different gut tissues. The present results provide a scientific resource and information about the mechanisms of compartmentalized function across gut tissues in a phylogenetically ancient vertebrate.

The development of animal model systems is dependent on the standardization of husbandry protocols that increase fecundity and reduce generation time. The blind Mexican tetra, Astyanax mexicanus, is an emerging genetic vertebrate model for evolution and biomedical research. Surface and cave populations of A. mexicanus have independently evolved, providing a model system for studying the genetic basis of divergent biological traits. While a rapid increase in the use of A. mexicanus has led to the generation of genetic tools including gene-editing and transgenesis, a slow and inconsistent growth rate remains a major limitation to the expanded application of A. mexicanus. The optimization of husbandry protocols that maximizes high-nutrient feed, smaller tank densities and larger tank sizes across development, would facilitate faster growth and expand the use of this model. Here, we describe standardized husbandry practices that optimize growth through a high protein diet, increased feeding, growth sorting of larvae and juveniles, and tank size transitions based on standard length. These changes to husbandry had a significant effect on growth rates and decreased the age of sexual maturity in comparison to our previous protocols. To determine whether our nutritional change and increased feeding impacted behavior, we tested fish in exploration and schooling assays. We found that a change in diet had no effect on the behaviors we tested, suggesting that increased feeding and rapid growth will not impact the natural variation in behavioral traits. Taken together, this standardized husbandry protocol will accelerate the development of A. mexicanus as a genetic model.

Developmental systems may preferentially produce certain types of variation and, thereby, bias phenotypic evolution. This is a central issue in evolutionary developmental biology, albeit somewhat understudied. Here we focus on the shape of the first upper molar which shows a clear, repeated tendency for anterior elongation at different scales from within mouse populations to between species of the Mus genus. In contrast, the lower molar displays more evolutionary stability. We compared upper and lower molar development of mouse strains representative of this fine variation (DUHi: elongated molars and FVB: short molars). Using a novel quantitative approach to examine small-scale developmental variation, we identified temporal, spatial and functional differences in tooth signaling centers between the two strains, likely due to different tuning of the activation-inhibition mechanisms ruling signaling center patterning. Based on the spatio-temporal dynamics of signaling centers and their lineage tracing, we show an intrinsic difference in the fate of signaling centers between lower and upper jaw of both strains. This can explain why variations in activation-inhibition parameters between strains are turned into anterior elongation in the upper molar only. Finally, although the \"elongated\" DUHi strain was inbred, first molar elongation was variable in adults, and we found high levels of intra-strain developmental variation in upper molar development. This is consistent with the inherent developmental instability of the upper molar system enabling the morphological variability of the tooth phenotype.\n\nIn conclusion, we have uncovered developmental properties that underlie the molars capacity for repeated phenotypic change, or said differently, that underlie a \"line of least resistance\". By focusing on the developmental basis of fine phenotypic variation, our study also challenges some common assumptions and practices in developmental and evolutionary developmental biology.

Sex has a major effect on the metabolome. However, we do not yet understand the degree to which these quantitative sex differences in metabolism are associated with anatomical dimorphism and modulated by sex-specific tissues. In the fruit fly, Drosophila melanogaster, knocking out the doublesex (dsx) gene gives rise to adults with intermediate sex characteristics. Here we sought to determine the degree to which this key node in sexual development leads to sex differences in the fly metabolome. We measured 91 metabolites across head, thorax and abdomen in Drosophila, comparing the differences between distinctly sex-dimorphic flies with those of reduced sexual dimorphism: dsx null flies. Notably, in the reduced dimorphism flies, we observed a sex difference in only 1 of 91 metabolites, kynurenate, whereas 51% of metabolites (46/91) were significantly different between wildtype XX and XY flies in at least one tissue, suggesting that dsx plays a major role in sex differences in fly metabolism. Kynurenate was consistently higher in XX flies in both the presence and absence of functioning dsx. We observed tissue-specific consequences of knocking out dsx. Metabolites affected by sex were significantly enriched in branched chain amino acid metabolism and the mTOR pathway. This highlights the importance of considering variation in genes that cause anatomical sexual dimorphism when analyzing sex differences in metabolic profiles and interpreting their biological significance.

Body shape diversity in vertebrates reflects a complex interplay between functional demands, environmental constraints, and internal developmental mechanisms. Various environments have promoted diverse morphological adaptations not only under natural but also domesticated conditions. One of the most remarkable examples of artificially induced morphology is found in the domesticated ornamental goldfish (Carassius auratus), which has diversified into numerous strains with strikingly different body shapes through prolonged human selection. In this study, we compared the body shapes of representative goldfish strains: the single-tail common goldfish (wild-type), Ryukin, Oranda, Pearl scale, and Ranchu. Our analysis revealed that the Ryukin and Pearl scale strains exhibit significantly greater body circularity in dorsal view compared to the other strains. Further anatomical and histological analyses showed that Pearl scale goldfish possess a thicker lateral body wall along with increased adipose tissue accumulation and reduced muscle fiber density, unlike Ryukin goldfish. These findings suggest that similar globular body shapes in different goldfish strains have arisen through distinct developmental pathways, exemplifying morphological convergence accompanied by histological divergence. We further discuss adipose accumulation in Pearl scale goldfish in relation to natural examples, providing insight into how function, morphology, and tissue organization may be interlinked in the evolution of globular body shapes.

Synovial joints, characterized by reciprocally congruent and lubricated articular surfaces separated by a cavity, are hypothesized to have evolved from continuous cartilaginous joints for increased mobility and improved load bearing. To test the evolutionary origins of synovial joints, we examine the morphology, genetic, and molecular mechanisms required for the development and function of the joints in elasmobranchs and cyclostomes. We find the presence of cavitated and articulated joints in elasmobranchs, such as the little skate (Leucoraja erinacea) and bamboo shark (Chiloscyllium plagiosum), and the expression of lubrication-related proteoglycans such as aggrecan and glycoproteins such as hyaluronic acid receptor (CD44) at the articular surfaces in little skates. Sea lampreys (Petromyozon marinus), a representative of cyclostomes, are devoid of articular cavities but express proteoglycan-linking proteins throughout their cartilaginous skeleton, suggesting that the expression of proteoglycans is primitively not limited to the articular cartilage. Analysis of the development of joints in the little skate reveals the expression of growth differentiation factor-5 (Gdf5) and {beta}-catenin at the joint interzone before the process of cavitation, indicating the involvement of BMP and Wnt-signaling pathway, and reliance on muscle contraction for the process of joint cavitation, similar to tetrapods. In conclusion, our results show that synovial joints are present in elasmobranchs but not cyclostomes, and therefore, synovial joints originated in the common ancestor of extant gnathostomes. A review of fossils from the extinct clades along the gnathostome stem further shows that synovial joints likely arose in the common ancestor of gnathostomes. Our results have implications for understanding how the evolution of synovial joints around 400 mya in our vertebrate ancestors unlocked motor behaviors such as feeding and locomotion. Author summaryWe owe our mobility and agility to synovial joints, characterized by a lubricated joint cavity between the bony elements. Due to the cavity, synovial joints function by bones sliding relative to each other, allowing an extensive range of motion and heightened stability compared to fused or cartilaginous joints that function by bending. Using histological and protein expression analysis, we show that reciprocally articulated, cavitated, and lubricated joints are present in elasmobranchs such as skates and sharks but not in cyclostomes such as the sea lamprey. Furthermore, the development of the little skate joints relies on genetic regulatory mechanisms such as BMP and Wnt-signalling, similar to tetrapods. Thus, our results show that synovial joints are present in elasmobranchs but not in cyclostomes. In conclusion, synovial joints originated in the common ancestor of jawed vertebrates. Furthermore, a review of fossil taxa along the gnathostome stem shows that cavitated joints that function by relative sliding of articulating surfaces originated at the common ancestor of all gnathostomes. Our results have consequences for understanding how the evolution of cavitated and lubricated joints in ancient vertebrates impacted behaviors like feeding and locomotion 400 million years ago.

Specific character traits of mineralized endoskeletal tissues need to be clearly defined and comprehensively examined among extant chondrichthyans (elasmobranchs, such as sharks and skates, and holocephalans, such as chimaeras) to understand their evolution. For example, tiles of mineralized polygonal structures called tesserae occur at cartilage surfaces in chondrichthyans, but recent studies showing trabecular mineralization at elasmobranch cartilage surfaces suggest that tesserae are not as common as previously thought. Also, while areolar mineralized tissue in elasmobranchs is generally considered a unique, shared chondrichthyan feature, some chondrichthyan species demonstrate bone-like tissues in both a specific region of tesserae termed the cap zone and continuous (not tiled) mineralized neural arches. To clarify the distribution of specific endoskeletal features among extant chondrichthyans, adult skeletal tissues in a holocephalan chimaera (spotted ratfish) and two elasmobranchs (small-spotted catshark and little skate) were characterized using synchrotron radiation and desktop micro-CT imaging, and histological and immunofluorescent assays. Endoskeletal mineralization in the ratfish, catshark, and little skate varied both quantitively in tissue mineral density (TMD), and qualitatively in the morphology and localization of mineralized structures and tissues. For example, TMD of several skeletal elements was significantly lower in ratfish, compared to catshark and little skate. Trabecular and areolar mineralization were shared among these extant chondrichthyan species, but tesserae and bone-like tissues were not. Interestingly, three separate analyses argued that the adult chimaera endoskeleton has features of the embryonic little skate endoskeleton. Generally, this study proposes specific terminology for character states of the extant chondrichthyan endoskeleton and infers those states in ancestral chondrichthyans with reference to fossil data.

Chicken domestication began at least 3,500 years ago for purposes of divination, cockfighting, and food. Prior to industrial scale chicken production, domestication selected larger birds with increased egg production. In the mid-20th century companies began intensive selection with the broiler (meat) industry focusing on improved feed conversion, rapid growth, and breast muscle yield. Here we present proteomic analysis comparing the Ross 708 modern broiler line with the UIUC legacy line. Comparing the breast muscle proteome between modern broilers and legacy lines not selected for these growth traits identifies cellular processes that have responded to human directed evolution. Mass spectrometry was used to identify differences in protein levels in the breast muscle of 6-day old chicks from Modern and Legacy lines. The results highlighted elevated levels of stress proteins, ribosomal proteins, and proteins that participate in the innate immune pathway in the Modern chickens. Furthermore, the comparative analyses indicated differences in the levels of proteins involved in multiple biochemical pathways. In particular, the Modern line had elevated levels of proteins affecting the pentose phosphate pathway, TCA cycle, and fatty acid oxidation and reduced protein levels of the first phase of glycolysis. These analyses provide hypotheses linking the morphometric changes driven by human directed selection to biochemical pathways. The results also have implications for the onset of Wooden Breast disease that arose due to selection for rapid breast muscle growth and is a major problem in the poultry industry.

Dietary restriction is a putative key to a healthier and longer life, but these benefits may come at a trade-off with reproductive fitness and may affect the following generation(s). The potential inter- and transgenerational effects of starvation are particularly poorly understood in vertebrates when they originate from the paternal line. We utilised the externally fertilising zebrafish amenable to a split-egg clutch design to explore the male-specific effects of starvation on fertility and fitness of offspring independently of maternal contribution. Eighteen days of fasting resulted in reduced fertility in exposed males. While average offspring survival was not affected, we detected higher larval growth in offspring from starved males and increased malformation rates at 24 hours post fertilisation in the F2 embryos produced by the offspring of the starved males. The transcriptome analysis of embryos from starved and fed fathers revealed robust and reproducible induction of muscle composition genes and a contrasting repressive effect on lipid metabolism and lysosome genes. A large proportion of these genes showed enrichment in the yolk syncytial layer suggesting gene regulatory responses associated with metabolism of nutrients through paternal impact on extra embryonic tissues which are loaded with maternally deposited factors. We compared the embryo transcriptome to adult transcriptome datasets and demonstrated comparable repressive effects on metabolism-associated genes. These similarities suggest a physiologically relevant, directed and potentially adaptive response transmitted by the father, independently from the offsprings nutritional state, which was defined by the mother.

Optimization of reproduction under dietary adversity is an important aspect of diet-dependent adaptation. Yet, little is known about the mechanism of such adaptive evolution. Here, we investigated a set of experimentally evolved populations of Drosophila melanogaster where early-life fecundity evolved as an adaptation to chronic protein restriction. We tested the role of resource acquisition and macronutrient storage, and changes in ovarian function that could have allowed such reproductive adaptation. We show that higher early-life fecundity was associated with the increased larval feeding rate, aiding in accumulation of higher protein content at the time of eclosion. Further evidence also suggests increase in reproductively allocated lipid content. Evolved females were found to have larger ovaries that also had a higher number of mature, post-vitellogenic oocytes that seem to readily allow the attainment of higher fecundity. Our results further show the evolution of plasticity in ovariole count (depending on mating status) and total egg-chamber count in females. These results are important in understanding the putative role of genetic variance and covariances that aid or limit the evolution of reproductive optimization, especially under nutritional adversity.

Using a cross-fostering experiment, we provide evidence for the contribution of both genetic differentiation and phenotypic plasticity to troglomorphic character development in the recently discovered cave form of Barbatula barbatula, an evolutionarily young lineage and first cavefish described in Europe, the northernmost record. We established reproducing populations of cave- and surface-dwelling loaches to produce cave, surface, and hybrid offspring and reared the F1 fish in a common garden setting in total darkness (DD) to simulate cave conditions as well as under the natural photoperiod (DL). We observed significant differences in the occurrence and extent of typical troglomorphic target characters among the offspring groups. Regardless of rearing conditions, cave fish exhibited smaller eyes, lighter body coloration, longer barbels, and larger olfactory epithelium than seen in surface fish. Hybrids in both rearing conditions generally showed an intermediate level of these traits. Surface and hybrid DD fish differed from the DL groups, resembling the cave fish phenotype in several traits, including eye size and body pigmentation. In contrast, cave and hybrid DL fish groups resembled surface fish phenotypes. Results confirmed that troglomorphic traits arise from heritable genetic differentiation of cave from surface forms and that phenotypic plasticity contributes to the process of adaptation to novel light conditions.

The elemental composition of organisms relates to a suite of functional traits that change during development in response to environmental conditions. It may be a part of a phenomenon known as developmental programming, which hypothetically creates phenotypes that are better adapted to their environments. However, associations between developmental speed and elemental body composition are not well understood. We compared body mass, elemental body composition, food uptake and fat metabolism of Drosophila melanogaster Oregon-R male fruit flies across the time gradient of their larval development. The results showed that flies with intermediate and rapid developmental speeds were heavier than slowly developing flies. Slowly developing flies had higher body carbon concentration than rapidly developing and intermediate flies. Rapidly developing flies had the highest body nitrogen concentration, while slowly developing flies had higher body nitrogen levels than flies with intermediate speed of development. The carbon-to-nitrogen ratio was therefore lower in rapidly developing flies than in slow and intermediate flies. Feeding rates were lowest in the slowly developing flies. The amount of storage fats was highest in the intermediate group. This means that the growth of rapidly developing flies is not suppressed by stress and they actively convert the food they consume into growth with less emphasis on storage build-up, suggesting bet-hedging in the larval development. In contrast, flies in the intermediate developmental group had the greatest fat reserves which optimize fitness under many climatic conditions. Low food intake may slow down development and the accumulation of body fat reserves in slowly developing flies. However, at the cost of slower growth, their phenotype conceivably facilitates survival under higher stochasticity of their ephemeral environments spoiled by metabolic waste due to high density of conspecifics. Overall, this study suggests that bet-hedging may be a common developmental strategy in fruit flies to cope with environmental uncertainty.

Predator-prey dynamics provide critical insight into overall coral reef health. It has been shown that predator-prey relationships link the relative brain size of predators to their prey. Predation pressure forces prey to use decision-making skills that require higher cognition by inspecting and identifying predators and then adjusting their behavior to achieve the highest chance for survival. However, the predation pressure that prey face outweighs the pressure predators face to find prey, resulting in prey having larger relative brain sizes than their predators. There is little data on the relative brain size of fishes with few natural predators such as Pterois volitans. This study compared the brain mass to body mass ratio of Pterois volitans, which have very few natural predators and thus very little predation pressure, to the brain mass to body mass ratio of their prey, possible predators, competitors, and taxonomically similar fish. Lionfish had a significantly smaller relative brain size than their predators, prey, and competitors, but was not significantly smaller than taxonomically similar fish. These results demonstrate that the morphological anti-predator adaptation of venomous spines causes little predation pressure. Thus, lionfish do not use the same cognitive skills as other prey or predators and, in turn, have smaller relative brain sizes.

Environmental change, such as increased rates of urbanization, can induce shifts in phenotypic plasticity with some individuals adapting to city life while others are displaced. A key trait that can facilitate adaptation is the degree at which animals respond to stress. This stress response has a heritable component and exhibits intra- and inter-individual variation. However, the mechanisms behind this variability and whether they might be responsible for adaptation to different environments are not known. Variation in DNA methylation can be a potential mechanism that mediates environmental effects on the stress response. We used an inter- and intra-environmental cross-foster experiment to analyze the contribution of DNA methylation to early-life phenotypic variation. We found that at hatching, urban house wren (Troglodytes aedon) offspring had increased methylation as compared to their rural counterparts, and observed plasticity in methylation as offspring aged, indicating developmental effects of the rearing environment on methylation. Differential methylation analyses showed that cellular respiration genes were differentially expressed at hatching and behavioral and metabolism genes were differentially expressed at fledgling. Lastly, hyper-methylation of a single gene (CNTNAP2) is associated with increased glucocorticoid levels. These differential methylation patterns linked to a specific physiological phenotype suggest that DNA methylation may be a mechanism by which individuals adapt to novel environments. Characterizing genetic and environmental influences on methylation is critical for understanding the role of epigenetic mechanisms in evolutionary adaptation.

Cilia and flagella were one of the characteristic traits of the last eukaryotic common ancestor and as such, are highly conserved among eukaryotes. Their proteomic makeup is consequently remarkably similar throughout all eukaryotic lineages. Recently, one subgroup of ciliary transport proteins in mammalian cells, the Bardet-Biedl Syndrome (BBS) proteins, was shown to have the ability to traverse the nuclear envelope, and to engage in protein-protein-interactions that modulate gene expression, signalling cascades, and cell homeostasis. Insects have been critically understudied in cilia biology because of their highly specialised cilia being localised on only a small subset of cell types. In this study, we present evidence that the BBSome, a hetero-octameric ciliary transport complex of BBS proteins, is largely conserved in multiple insect lineages. Using the honeybee Apis mellifera as a study system to explore BBS-associated gene expression, our analyses suggest that not all BBSome-associated genes are expressed equally, indicating possible non-ciliary functions. We also demonstrate that the expression of individual BBS proteins varies significantly between the tissues of queens and males in A. mellifera, especially in neuronal tissue. This result raises the question of what role BBS proteins play in these tissues and whether they are involved in gene regulation in insects. The potential gene regulatory function of BBS proteins should be explored in other eukaryotes due to their high degree of conservation.

BackgroundMarine organisms with sessile adults commonly possess motile larval stages that make settlement decisions based on integrating environmental sensory cues. Phototaxis, the movement toward or away from light, is a common behavioral characteristic of aquatic and marine metazoan larvae, and of algae, protists, and fungi. In cnidarians, behavioral genomic investigations of motile planulae larvae have been conducted in anthozoans (corals and sea anemones) and scyphozoans (true jellyfish), but such studies are presently lacking in hydrozoans. Here, we examined the behavioral genomics of phototaxis in planulae of the hydrozoan Hydractinia symbiolongicarpus. ResultsA behavioral phototaxis study of day 3 planulae indicated preferential phototaxis to green (523 nm) and blue (470 nm) wavelengths of light, but not red (625 nm) wavelengths. A developmental transcriptome study where planula larvae were collected from four developmental time points for RNA-seq revealed that many genes critical to the physiology and development of ciliary photosensory systems are dynamically expressed in planula development and correspond to the expression of phototactic behavior. Microscopical investigations using immunohistochemistry and in situ hybridization demonstrated that several transcripts with predicted function in photoreceptors, including cnidops class opsin, CNG ion channel, and CRX-like transcription factor, localize to ciliated bipolar sensory neurons of the aboral sensory neural plexus, which is associated with the direction of phototaxis and the site of settlement. ConclusionsThe phototactic preference displayed by planulae is consistent with the shallow sandy marine habitats they experience in nature. Our genomic investigations add further evidence of similarities between cnidops-mediated photoreceptors of hydrozoans and other cnidarians and ciliary photoreceptors as found in the eyes of humans and other bilaterians, suggesting aspects of their shared evolutionary history.

During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have found network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. Here, we performed RNA-seq in whole Drosophila melanogaster terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the terminalia is highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. Our results are housed online in a searchable database (flyterminalia.pitt.edu) where they can serve as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.\n\nSummaryWe performed RNA-seq in whole Drosophila melanogaster terminalia (genitalia and analia) followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the pupal terminalia is highly patterned with specific transcription factors marking separate structures and substructures. Our results are housed online in a searchable database (flyterminalia.pitt.edu) where they can serve as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.