Periodic Addition of Glucose Suppressed Cyanobacterial Abundance in Additive Lake Water Samples during the Entire Bloom Season.

Previously, we showed that prophylactic addition of glucose to Harsha Lake water samples could inhibit cyanobacteria growth, at least for a short period of time. The current study tested cyanobacterial control with glucose for the entire Harsha Lake bloom season. Water samples (1000 ml) were collected weekly from Harsha Lake during the algal-bloom season starting June 9 and lasting until August 24, 2022. To each of two 7-liter polypropylene containers, 500 ml of Harsha Lake water was added, and the containers were placed in a controlled environment chamber. To one container labeled "Treated," 0.15 g of glucose was added, and nothing was added to the container labeled "Control." After that, three 25 ml samples from each container were collected and used for 16S rRNA gene sequencing each week. Then 1000 ml of Harsha Lake water was newly collected each week, with 500 ml added to each container, along with the addition of 0.15 g glucose to the "Treated" container. Sequencing data were used to examine differences in the composition of bacterial communities between Treated and Control containers. Treatment with glucose altered the microbial communities by 1) reducing taxonomic diversity, 2) largely eliminating cyanobacterial taxa, and 3) increasing the relative abundance of subsets of non-cyanobacterial taxa (such as Proteobacteria and Actinobacteriota). These effects were observed across time despite weekly inputs derived directly from Lake water. The addition of glucose to a container receiving weekly additions of Lake water suppressed the cyanobacterial populations during the entire summer bloom season. The glucose appears to stimulate the diversity of certain bacterial taxa at the expense of the cyanobacteria.

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese.

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland-a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

Metagenomic mapping of cyanobacteria and potential cyanotoxin producing taxa in large rivers of the United States.

Cyanobacteria and cyanotoxin producing cyanobacterial blooms are a trending focus of current research. Many studies focus on bloom events in lentic environments such as lakes or ponds. Comparatively few studies have explored lotic environments and fewer still have examined the cyanobacterial communities and potential cyanotoxin producers during ambient, non-bloom conditions. Here we used a metagenomics-based approach to profile non-bloom microbial communities and cyanobacteria in 12 major U.S. rivers at multiple time points during the summer months of 2019. Our data show that U.S. rivers possess microbial communities that are taxonomically rich, yet largely consistent across geographic location and time. Within these communities, cyanobacteria often comprise significant portions and frequently include multiple species with known cyanotoxin producing strains. We further characterized these potential cyanotoxin producing taxa by deep sequencing amplicons of the microcystin E (mcyE) gene. We found that rivers containing the highest levels of potential cyanotoxin producing cyanobacteria consistently possess taxa with the genetic potential for cyanotoxin production and that, among these taxa, the predominant genus of origin for the mcyE gene is Microcystis. Combined, these data provide a unique perspective on cyanobacteria and potential cyanotoxin producing taxa that exist in large rivers across the U.S. and can be used to better understand the ambient conditions that may precede bloom events in lotic freshwater ecosystems.

Transcriptomic exploration of the Coleopteran wings reveals insight into the evolution of novel structures associated with the beetle elytron.

The acquisition of novel traits is central to organismal evolution, yet the molecular mechanisms underlying this process are elusive. The beetle forewings (elytra) are evolutionarily modified to serve as a protective shield, providing a unique opportunity to study these mechanisms. In the past, the orthologs of genes within the wing gene network from Drosophila studies served as the starting point when studying the evolution of elytra (candidate genes). Although effective, candidate gene lists are finite and only explore genes conserved across species. To go beyond candidate genes, we used RNA sequencing and explored the wing transcriptomes of two Coleopteran species, the red flour beetle (Tribolium castaneum) and the Japanese stag beetle (Dorcus hopei). Our analysis revealed sets of genes enriched in Tribolium elytra (57 genes) and genes unique to the hindwings, which possess more "typical" insect wing morphologies (29 genes). Over a third of the hindwing-enriched genes were "candidate genes" whose functions were previously analyzed in Tribolium, demonstrating the robustness of our sequencing. Although the overlap was limited, transcriptomic comparison between the beetle species found a common set of genes, including key wing genes, enriched in either elytra or hindwings. Our RNA interference analysis for elytron-enriched genes in Tribolium uncovered novel genes with roles in forming various aspects of morphology that are unique to elytra, such as pigmentation, hardening, sensory development, and vein formation. Our analyses deepen our understanding of how gene network evolution facilitated the emergence of the elytron, a unique structure critical to the evolutionary success of beetles.

Prophylactic Addition of Glucose Suppresses Cyanobacterial Abundance in Lake Water.

To mitigate harmful cyanobacterial blooms (HCBs), toxic algicides have been used, but alternative methods of HCB prevention are needed. Our goal was to test the prophylactic addition of glucose to inhibit HCB development, using Microcystis and the toxin microcystin as the HCB model. Water samples were collected weekly, from 4 June to 2 July, from Harsha Lake in southwestern Ohio during the 2021 algal bloom season. From each weekly sample, a 25 mL aliquot was frozen for a 16S rRNA gene sequencing analysis. Then, 200 mL of Harsha Lake water was added to each of the three culture flasks, and glucose was added to create concentrations of 0 mM (control), 1.39 mM, or 13.9 mM glucose, respectively. The microcystin concentration in each flask was measured after 1 and 2 weeks of incubation. The results showed an 80 to 90% reduction in microcystin concentrations in glucose-treated water compared to the control. At the end of the second week of incubation, a 25 mL sample was also obtained from each of the culture flasks for molecular analysis, including a 16S rRNA gene sequencing and qPCR-based quantification of Microcystis target genes. Based on these analyses, the glucose-treated water contained significantly lower Microcystis and microcystin producing gene (mcy) copy numbers than the control. The 16S rRNA sequencing analysis also revealed that Cyanobacteria and Proteobacteria were initially the most abundant bacterial phyla in the Harsha Lake water, but as the summer progressed, Cyanobacteria became the dominant phyla. However, in the glucose-treated water, the Cyanobacteria decreased and the Proteobacteria increased in weekly abundance compared to the control. This glucose-induced proteobacterial increase in abundance was driven primarily by increases in two distinct families of Proteobacteria: Devosiaceae and Rhizobiaceae. In conclusion, the prophylactic addition of glucose to Harsha Lake water samples reduced Cyanobacteria's relative abundance, Microcystis numbers and microcystin concentrations and increased the relative abundance of Proteobacteria compared to the control.

Doublesex mediates species-, sex-, environment- and trait-specific exaggeration of size and shape.

Context-dependent trait exaggeration is a major contributor to phenotypic diversity. However, the genetic modifiers instructing development across multiple contexts remain largely unknown. We use the arthropod tibia, a hotspot for segmental differentiation, as a paradigm to assess the developmental mechanisms underlying the context-dependent structural exaggeration of size and shape through nutritional plasticity, sexual dimorphism and segmental differentiation. Using an RNAseq approach in the sexually dimorphic and male-polyphenic dung beetle Digitonthophagus gazella, we find that only a small portion (3.7%) of all transcripts covary positively in expression level with trait size across contexts. However, RNAi-mediated knockdown of the conserved sex-determination gene doublesex suggests that it functions as a context-dependent master mediator of trait exaggeration in D. gazella as well as the closely related dung beetle Onthophagus taurus. Taken together, our findings suggest (i) that the gene networks associated with trait exaggeration are highly dependent on the precise developmental context, (ii) that doublesex differentially shapes morphological exaggeration depending on developmental contexts and (iii) that this context-specificity of dsx-mediated trait exaggeration may diversify rapidly. This mechanism may contribute to the resolution of conflict arising from environment-dependent antagonistic selection among sexes and divergent developmental contexts in a wide range of animals.

From descent with modification to the origins of novelty.

Descent with modification is the foundational framework of all of evolution. Yet evolutionary novelties are defined as lacking affinities to structures that already existed in the ancestral state, i.e. to somehow emerge in the absence of homology. We posit that reconciling both perspectives necessitates the existence of a type of innovation gradient that allows descent with modification to seed the initiation of a novel trait, which once in existence can then diversify into its variant forms. Recent work on diverse, textbook examples of morphological novelties illustrate the value of the innovation gradient concept. Innovations as profound and diverse as insect wings, beetle horns, and treehopper helmets derive from homologous source tissues instructed in their development by homologous gene regulatory networks. Yet rather than rendering these traits no longer novel, we posit that discoveries such as these call for a reassessment of the usefulness of defining evolutionary novelty as necessitating the absence of homology. Instead, we need to redirect our attention to how ancestral homologies scaffold and bias the innovation gradient to facilitate hotspots of innovation in some places, and deep conservation elsewhere.

A Case of Toxicity from Cannabidiol Gummy Ingestion.

A 56-year-old male with no known history of substance abuse and no known prior medical conditions presented via ambulance to the emergency department after being found by coworkers with bizarre behavior, vomiting, and slurred speech. He had legally purchased cannabidiol (CBD) gummies marketed for pain and anxiety relief at a gas station several hours prior. Vitals upon arrival were temperature 36.8 Celsius, heart rate (HR) 79, respiratory rate (RR) 12, blood pressure (BP) 113/60, and oxygen saturation (O2) of 84% on room air that improved upon arousal. Physical exam showed an obese man in no acute distress with a depressed level of consciousness but who awoke to painful stimuli. Neuro exam was significant for dysarthric, hypophonic speech. Labs were significant for a primary respiratory acidosis with concomitant mild lactic acid elevation, normal bicarbonate, and normal anion gap. A comprehensive urine toxicology screen including cannabis was negative. Vital signs three hours after presentation deteriorated, showing: HR 47, RR 8-12, BP 88/52, O2 78%. Electrocardiogram (EKG) revealed sinus bradycardia. The patient progressively became more obtunded and required constant stimuli in order to maintain a patent airway. Non-invasive positive pressure ventilation was not administered due to persistent emesis. The patient underwent supportive care with intravenous fluids, oxygen, anti-emetics, continuous stimulation, and close neurologic monitoring with full recovery by the following morning. Further, patient history revealed that he had consumed two packages of CBD gummies, totaling 370 mg total of CBD (serving size on the package was 30 mg). He felt the products were healthy and safe based on packaging and therefore did not believe they would have any adverse effects. CBD is one of many cannabinoids found in marijuana and marijuana-derived products. It is generally considered safe unlike its more psychoactive counterpart, tetrahydrocannabinol (THC), which has been linked to seizures, respiratory depression, and cardiovascular complications. CBD has surged in popularity recently, being marketed in oils, capsules, and candies as a health supplement, claiming to treat a wide variety of medical conditions such as glaucoma, pain, and even having beneficial effects on cancer prevention. Most currently available studies do not look at isolated CBD nor their synthetic equivalents, and purity is not guaranteed, thus leading to unforeseen side effects and toxicities. Moreover, these compounds do not show on traditional toxicology screens, posing a diagnostic dilemma for physicians. This case of respiratory depression and cardiovascular compromise in a relatively healthy man is just one example of the importance of considering synthetic CBD toxicity in the differential diagnosis, as there is little data available for recognizing and treating this condition.

Integrating evolutionarily novel horns within the deeply conserved insect head.

BACKGROUND: How novel traits integrate within ancient trait complexes without compromising ancestral functions is a foundational challenge in evo-devo. The insect head represents an ancient body region patterned by a deeply conserved developmental genetic network, yet at the same time constitutes a hot spot for morphological innovation. However, the mechanisms that facilitate the repeated emergence, integration, and diversification of morphological novelties within this body region are virtually unknown. Using horned Onthophagus beetles, we investigated the mechanisms that instruct the development of the dorsal adult head and the formation and integration of head horns, one of the most elaborate classes of secondary sexual weapons in the animal kingdom. RESULTS: Using region-specific RNAseq and gene knockdowns, we (i) show that the head is compartmentalized along multiple axes, (ii) identify striking parallels between morphological and transcriptional complexity across regions, yet (iii) fail to identify a horn-forming gene module. Instead, (iv) our results support that sex-biased regulation of a shared transcriptional repertoire underpins the formation of horned and hornless heads. Furthermore, (v) we show that embryonic head patterning genes frequently maintain expression within the dorsal head well into late post-embryonic development, thereby possibly facilitating the repurposing of such genes within novel developmental contexts. Lastly, (vi) we identify novel functions for several genes including three embryonic head patterning genes in the integration of both posterior and anterior head horns. CONCLUSIONS: Our results illuminate how the adult insect head is patterned and suggest mechanisms capable of integrating novel traits within ancient trait complexes in a sex- and species-specific manner. More generally, our work underscores how significant morphological innovation in developmental evolution need not require the recruitment of new genes, pathways, or gene networks but instead may be scaffolded by pre-existing developmental machinery.

Developmental bias in horned dung beetles and its contributions to innovation, adaptation, and resilience.

Developmental processes transduce diverse influences during phenotype formation, thereby biasing and structuring amount and type of phenotypic variation available for evolutionary processes to act on. The causes, extent, and consequences of this bias are subject to significant debate. Here we explore the role of developmental bias in contributing to organisms' ability to innovate, to adapt to novel or stressful conditions, and to generate well integrated, resilient phenotypes in the face of perturbations. We focus our inquiry on one taxon, the horned dung beetle genus Onthophagus, and review the role developmental bias might play across several levels of biological organization: (a) gene regulatory networks that pattern specific body regions; (b) plastic developmental mechanisms that coordinate body wide responses to changing environments and; (c) developmental symbioses and niche construction that enable organisms to build teams and to actively modify their own selective environments. We posit that across all these levels developmental bias shapes the way living systems innovate, adapt, and withstand stress, in ways that can alternately limit, bias, or facilitate developmental evolution. We conclude that the structuring contribution of developmental bias in evolution deserves further study to better understand why and how developmental evolution unfolds the way it does.

Beetle horns evolved from wing serial homologs.

Understanding how novel complex traits originate is a foundational challenge in evolutionary biology. We investigated the origin of prothoracic horns in scarabaeine beetles, one of the most pronounced examples of secondary sexual traits in the animal kingdom. We show that prothoracic horns derive from bilateral source tissues; that diverse wing genes are functionally required for instructing this process; and that, in the absence of Hox input, prothoracic horn primordia transform to contribute to ectopic wings. Once induced, however, the transcriptional profile of prothoracic horns diverges markedly from that of wings and other wing serial homologs. Our results substantiate the serial homology between prothoracic horns and insects wings and suggest that other insect innovations may derive similarly from wing serial homologs and the concomitant establishment of structure-specific transcriptional landscapes.

The origins of novelty from within the confines of homology: the developmental evolution of the digging tibia of dung beetles.

Understanding the origin of novel complex traits is among the most fundamental goals in evolutionary biology. The most widely used definition of novelty in evolution assumes the absence of homology, yet where homology ends and novelty begins is increasingly difficult to parse as evo devo continuously revises our understanding of what constitutes homology. Here, we executed a case study to explore the earliest stages of innovation by examining the tibial teeth of tunnelling dung beetles. Tibial teeth are a morphologically modest innovation, composed of relatively simple body wall projections and contained fully within the fore tibia, a leg segment whose own homology status is unambiguous. We first demonstrate that tibial teeth aid in multiple digging behaviours. We then show that the developmental evolution of tibial teeth was dominated by the redeployment of locally pre-existing gene networks. At the same time, we find that even at this very early stage of innovation, at least two genes that ancestrally function in embryonic patterning and thus entirely outside the spatial and temporal context of leg formation, have already become recruited to help shape the formation of tibial teeth. Our results suggest a testable model for how developmental evolution scaffolds innovation.

Teaching to Teach: An Effective and Feasible Teaching Curriculum for Internal Medicine Interns.

OBJECTIVES: New competency requirements from the Accreditation Council for Graduate Medical Education have prompted greater emphasis on developing residents' teaching skills. Many residents make their first foray into teaching during internship, making it an important yet underrecognized opportunity to develop basic teaching skills. In addition, in the current graduate medical education climate, residents' tasks are compressed into an even shorter time, which has caused teaching opportunities and expectations to be balanced with the need for efficiency. After performing needs assessment surveys of medicine interns and medical students, we developed an interns-as-teachers curriculum to equip internal medicine interns with skills specific to their unique role as medical student teachers. METHODS: We conducted a workshop focused around four specific skills: role modeling, using teachable moments (ie, teaching on the fly), thinking out loud, and coaching. We evaluated the curriculum by comparing pre- and postcurricular teaching knowledge, attitudes, and self-reported teaching behaviors among 51 interns in the intervention group with 20 interns in the comparison group from the previous year's class. RESULTS: Sixty-one interns participated in the curriculum, and 51 (84%) completed both surveys. Knowledge and several self-reported teaching behaviors improved significantly among the intervention group, but not in the comparison group. CONCLUSIONS: Interns participating in a half-day interns-as-teachers workshop aimed at preparing them to teach medical students in clinical settings achieved significant improvement in teaching knowledge and in several core, self-reported teaching behaviors.

Dual evolutionary origin of insect wings supported by an investigation of the abdominal wing serial homologs in Tribolium.

The origin of insect wings is still a highly debated mystery in biology, despite the importance of this evolutionary innovation. There are currently two prominent, but contrasting wing origin hypotheses (the tergal origin hypothesis and the pleural origin hypothesis). Through studies in the Tribolium beetle, we have previously obtained functional evidence supporting a third hypothesis, the dual origin hypothesis. Although this hypothesis can potentially unify the two competing hypotheses, it requires further testing from various fields. Here, we investigated the genetic regulation of the tissues serially homologous to wings in the abdomen, outside of the appendage-bearing segments, in Tribolium We found that the formation of ectopic wings in the abdomen upon homeotic transformation relies not only on the previously identified abdominal wing serial homolog (gin-trap), but also on a secondary tissue in the pleural location. Using an enhancer trap line of nubbin (a wing lineage marker), we were able to visualize both of these two tissues (of tergal and pleural nature) contributing to form a complete wing. These results support the idea that the presence of two distinct sets of wing serial homologs per segment represents an ancestral state of the wing serial homologs, and can therefore further support a dual evolutionary origin of insect wings. Our analyses also uncovered detailed Hox regulation of abdominal wing serial homologs, which can be used as a foundation to elucidate the molecular mechanisms that have facilitated the evolution of bona fide insect wings, as well as the diversification of other wing serial homologs.

Functional value of elytra under various stresses in the red flour beetle, Tribolium castaneum.

Coleoptera (beetles) is a massively successful order of insects, distinguished by their evolutionarily modified forewings called elytra. These structures are often presumed to have been a major driving force for the successful radiation of this taxon, by providing beetles with protection against a variety of harsh environmental factors. However, few studies have directly demonstrated the functional significance of the elytra against diverse environmental challenges. Here, we sought to empirically test the function of the elytra using Tribolium castaneum (the red flour beetle) as a model. We tested four categories of stress on the beetles: physical damage to hindwings, predation, desiccation, and cold shock. We found that, in all categories, the presence of elytra conferred a significant advantage compared to those beetles with their elytra experimentally removed. This work provides compelling quantitative evidence supporting the importance of beetle forewings in tolerating a variety of environmental stresses, and gives insight into how the evolution of elytra have facilitated the remarkable success of beetle radiation.

Neofunctionalization of embryonic head patterning genes facilitates the positioning of novel traits on the dorsal head of adult beetles.

The origin and integration of novel traits are fundamental processes during the developmental evolution of complex organisms. Yet how novel traits integrate into pre-existing contexts remains poorly understood. Beetle horns represent a spectacular evolutionary novelty integrated within the context of the adult dorsal head, a highly conserved trait complex present since the origin of insects. We investigated whether otd1/2 and six3, members of a highly conserved gene network that instructs the formation of the anterior end of most bilaterians, also play roles in patterning more recently evolved traits. Using ablation-based fate-mapping, comparative larval RNA interference (RNAi) and transcript sequencing, we found that otd1/2, but not six3, play a fundamental role in the post-embryonic formation of the adult dorsal head and head horns of Onthophagus beetles. By contrast, neither gene appears to pattern the adult head of Tribolium flour beetles even though all are expressed in the dorsal head epidermis of both Onthophagus and Tribolium We propose that, at least in beetles, the roles of otd genes during post-embryonic development are decoupled from their embryonic functions, and that potentially non-functional post-embryonic expression in the dorsal head facilitated their co-option into a novel horn-patterning network during Onthophagus evolution.

RNAi screening of developmental toolkit genes: a search for novel wing genes in the red flour beetle, Tribolium castaneum.

The amazing array of diversity among insect wings offers a powerful opportunity to study the mechanisms guiding morphological evolution. Studies in Drosophila (the fruit fly) have identified dozens of genes important for wing development. These genes are often called candidate genes, serving as an ideal starting point to study wing development in other insects. However, we also need to explore beyond the candidate genes to gain a more comprehensive view of insect wing evolution. As a first step away from the traditional candidate genes, we utilized Tribolium (the red flour beetle) as a model and assessed the potential involvement of a group of developmental toolkit genes (embryonic patterning genes) in beetle wing development. We hypothesized that the highly pleiotropic nature of these developmental genes would increase the likelihood of finding novel wing genes in Tribolium. Through the RNA interference screening, we found that Tc-cactus has a less characterized (but potentially evolutionarily conserved) role in wing development. We also found that the odd-skipped family genes are essential for the formation of the thoracic pleural plates, including the recently discovered wing serial homologs in Tribolium. In addition, we obtained several novel insights into the function of these developmental genes, such as the involvement of mille-pattes and Tc-odd-paired in metamorphosis. Despite these findings, no gene we examined was found to have novel wing-related roles unique in Tribolium. These results suggest a relatively conserved nature of developmental toolkit genes and highlight the limited degree to which these genes are co-opted during insect wing evolution.

Larval RNA interference in the red flour beetle, Tribolium castaneum.

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle's body cavity. In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Insights into insect wing origin provided by functional analysis of vestigial in the red flour beetle, Tribolium castaneum.

Despite accumulating efforts to unveil the origin of insect wings, it remains one of the principal mysteries in evolution. Currently, there are two prominent models regarding insect wing origin: one connecting the origin to the paranotal lobe and the other to the proximodorsal leg branch (exite). However, neither hypothesis has been able to surpass the other. To approach this conundrum, we focused our analysis on vestigial (vg), a critical wing gene initially identified in Drosophila. Our investigation in Tribolium (Coleoptera) has revealed that, despite the well-accepted view of vg as an essential wing gene, there are two groups of vg-dependent tissues in the "wingless" first thoracic segment (T1). We show that one of these tissues, the carinated margin, also depends on other factors essential for wing development (such as Wingless signal and apterous), and has nubbin enhancer activity. In addition, our homeotic mutant analysis shows that wing transformation in T1 originates from both the carinated margin and the other vg-dependent tissue, the pleural structures (trochantin and epimeron). Intriguingly, these two tissues may actually be homologous to the two proposed wing origins (paranotal lobes and exite bearing proximal leg segments). Therefore, our findings suggest that the vg-dependent tissues in T1 could be wing serial homologs present in a more ancestral state, thus providing compelling functional evidence for the dual origin of insect wings.