@article {69848, title = {A Bayesian model to count the number of two-state emitters in a diffraction limited spot.}, journal = {Nano Lett}, year = {2025}, month = {2025 Apr 04}, abstract = {

We address the problem of inferring the number of independently blinking fluorescent light emitters, when only their combined intensity contributions can be observed. This problem occurs regularly in light microscopy of objects smaller than the diffraction limit, where one wishes to count the number of fluorescently labeled subunits. Our proposed solution directly models the photophysics of the system, as well as the blinking kinetics of the fluorescent emitters as a fully differentiable hidden Markov model, estimating a posterior distribution of the total number of emitters. We show that our model is more accurate and increases the range of countable subunits by a factor of 2 compared to current state-of-the-art methods. Furthermore, we demonstrate that our model can be used to investigate the effect of blinking kinetics on counting ability and therefore can inform optimal experimental conditions.

}, issn = {1530-6992}, doi = {10.1021/acs.nanolett.4c06304}, url = {https://doi.org/10.1021/acs.nanolett.4c06304}, author = {Hillsley, Alexander and Stein, Johannes and Tillberg, Paul W and Stern, David L and Funke, Jan} } @article {70146, title = {Chromosome scale genomes of two invasive Adelges species enable virtual screening for selective adelgicides.}, journal = {G3 (Bethesda)}, volume = {16}, year = {2025}, month = {2026 Jan 01}, abstract = {

Two invasive adelgids are associated with widespread damage to several North American conifer species. Adelges tsugae, hemlock woolly adelgid, was introduced from Japan and reproduces parthenogenetically in North America, where it has rapidly decimated Tsuga canadensis and Tsuga caroliniana (eastern and Carolina hemlocks, respectively). Adelges abietis, eastern spruce gall adelgid, introduced from Europe, forms distinctive pineapple-shaped galls on several native spruce species. While not considered a major forest pest, it weakens trees and increases susceptibility to additional stressors. Broad-spectrum insecticides that are often used to control adelgid populations can have off-target impacts on beneficial insects. Whole genome sequencing was performed on both species to aid in development of targeted solutions that may minimize ecological impact. Adelges abietis was sequenced using barcoded linked-reads from 30 pooled individuals, with Hi-C scaffolding performed using data from a single individual collected from the same host plant. Adelges tsugae used long-read sequencing from pooled nymphs. The assembled A. tsugae and A. abietis genomes, pooled from several parthenogenetic females, are 220.75 Mbp and 253.16 Mbp, respectively. Each consists of eight autosomal chromosomes, as well as two sex chromosomes (X1/X2), supporting the XX-XO sex determination system. The genomes are over 96\% complete based on BUSCO assessment. Genome annotation identified 11,424 and 12,060 protein-coding genes in A. tsugae and A. abietis, respectively. Comparative analysis of proteins across 29 hemipteran species and 14 arthropod outgroups identified 31,666 putative gene families. Gene family evolution analysis with CAFE revealed lineage-specific expansions in immune-related aminopeptidases (ERAP1) and juvenile hormone binding proteins (JHBP), contractions in juvenile hormone acid methyltransferases (JHAMT), and conservation of nicotinic acetylcholine receptors (nAChR). These genes were explored as candidate families towards a long-term objective of developing adelgid-selective insecticides. Structural comparisons of proteins across seven focal species (Adelges tsugae, Adelges abietis, Adelges cooleyi, Rhopalosiphum maidis, Apis mellifera, Danaus plexippus, and Drosophila melanogaster) revealed high conservation of nAChR and ERAP1, while JHAMT exhibited species-specific structural divergence. The potential of JHAMT as a lineage-specific target for pest control was explored through virtual screening of drugs and pesticides.

bioRxiv preprint:\ https://doi.org/10.1101/2024.11.21.624573

}, issn = {2160-1836}, doi = {10.1093/g3journal/jkaf232}, url = {https://doi.org/10.1093/g3journal/jkaf232}, author = {Glendening, Adam M and Stephens, Cole and Vuruputoor, Vidya S and Chaganti, Tesko and Myles, Meghan N and Stern, David L and Abdelalim, Muhammad and Juang, Yu-Pu and Hogenhout, Saskia A and Mathers, Thomas C and Pauloski, Nicole and Cernak, Tim A and Wegrzyn, Jill L and Fetter, Karl C} } @article {69594, title = {Activity of nested neural circuits drives different courtship songs in Drosophila.}, journal = {Nat Neurosci}, year = {2024}, month = {2024 Aug 28}, abstract = {

Motor systems implement diverse motor programs to pattern behavioral sequences, yet how different motor actions are controlled on a moment-by-moment basis remains unclear. Here, we investigated the neural circuit mechanisms underlying the control of distinct courtship songs in Drosophila. Courting males rapidly alternate between two types of song: pulse and sine. By recording calcium signals in the ventral nerve cord in singing flies, we found that one neural population is active during both songs, whereas an expanded neural population, which includes neurons from the first population, is active during pulse song. Brain recordings showed that this nested activation pattern is present in two descending pathways required for singing. Connectomic analysis reveals that these two descending pathways provide structured input to ventral nerve cord neurons in a manner consistent with their activation patterns. These results suggest that nested premotor circuit activity, directed by distinct descending signals, enables rapid switching between motor actions.

}, issn = {1546-1726}, doi = {10.1038/s41593-024-01738-9}, url = {https://doi.org/10.1038/s41593-024-01738-9}, author = {Shiozaki, Hiroshi M and Wang, Kaiyu and Lillvis, Joshua L and Xu, Min and Dickson, Barry J and Stern, David L} } @article {69678, title = {Genomes of two invasive Adelges species (hemlock woolly adelgid and pineapple gall adelgid) enable characterization of nicotinic acetylcholine receptors}, journal = {bioRxiv}, year = {2024}, month = {2024 Nov 26}, abstract = {

Two invasive hemipteran adelgids cause widespread damage to North American conifers. Adelges tsugae (the hemlock woolly adelgid) has decimated Tsuga canadensis and Tsuga caroliniana (the Eastern and Carolina hemlocks, respectively). A. tsugae was introduced from East Asia and reproduces parthenogenetically in North America, where it can kill trees rapidly. A. abietis, introduced from Europe, makes pineapple galls on several North American spruce species, and weakens trees, increasing their susceptibility to other stresses. Broad-spectrum insecticides that are often used to control adelgid populations can have off-target impacts on beneficial insects and the development of more selective chemical treatments could improve control methods and minimize ecological damage. Whole genome sequencing was performed on both species to aid in development of targeted pest control solutions and improve species conservation. The assembled A. tsugae and A. abietis genomes are 231.71 Mbp and 290.39 Mbp, respectively, each consisting of nine chromosomes and both genomes are over 96\% complete based on BUSCO assessment. Genome annotation identified 11,424 and 14,118 protein-coding genes in A. tsugae and A. abietis, respectively. Comparative analysis across 29 Hemipteran species and 14 arthropod outgroups identified 31,666 putative gene families. Gene family expansions in A. abietis included ABC transporters and carboxypeptidases involved in carbohydrate metabolism, while both species showed contractions in core histone families and oxidoreductase pathways. Gene family expansions in A. tsugae highlighted families associated with the regulation of cell differentiation and development (survival motor protein, SMN; juvenile hormone acid methyltransferase JHAMT) as well as those that may be involved in the suppression of plant immunity (clip domain serine protease-D, CLIPD; Endoplasmic reticulum aminopeptidase 1, ERAP1). Among the analyzed gene families, Nicotinic acetylcholine receptors (nAChRs) maintained consistent copy numbers and structural features across species, a finding particularly relevant given their role as targets for current forestry management insecticides. Detailed phylogenetic analysis of nAChR subunits across adelgids and other ecologically important insects revealed remarkable conservation in both sequence composition and predicted structural features, providing crucial insights for the development of more selective pest control strategies.

}, doi = {10.1101/2024.11.21.624573}, url = {https://doi.org/10.1101/2024.11.21.624573}, author = {Glendening, A M and Stephens, Cole and Vuruputoor, Vidya S and Stern, David L and Hogenhout, Saskia A and Mathers, Thomas C and Chaganti, Tesko and Pauloski, Nicole and Cernak, Tim A and Wegrzyn, Jill L and Fetter, Karl C} } @article {68068, title = {Gene structure-based homology search identifies highly divergent putative effector gene family.}, journal = {Genome Biology and Evolution}, year = {2022}, month = {2022 May 09}, abstract = {

Homology of highly divergent genes often cannot be determined from sequence similarity alone. For example, we recently identified in the aphid Hormaphis cornu a family of rapidly evolving bicycle genes, which encode novel proteins implicated as plant gall effectors, and sequence similarity search methods yielded few putative bicycle homologs in other species. Coding sequence-independent features of genes, such as intron-exon boundaries, often evolve more slowly than coding sequences, however, and can provide complementary evidence for homology. We found that a linear logistic regression classifier using only structural features of bicycle genes identified many putative bicycle homologs in other species. Independent evidence from sequence features and intron locations supported homology assignments. To test the potential roles of bicycle genes in other aphids, we sequenced the genome of a second gall-forming aphid, Tetraneura nigriabdominalis, and found that many bicycle genes are strongly expressed in the salivary glands of the gall forming foundress. In addition, bicycle genes are strongly overexpressed in the salivary glands of a non-gall forming aphid, Acyrthosiphon pisum, and in the non-gall forming generations of Hormaphis cornu. These observations suggest that Bicycle proteins may be used by multiple aphid species to manipulate plants in diverse ways. Incorporation of gene structural features into sequence search algorithms may aid identification of deeply divergent homologs, especially of rapidly evolving genes involved in host-parasite interactions.

}, issn = {1759-6653}, doi = {10.1093/gbe/evac069}, author = {Stern, David L and Han, Clair} } @article {68557, title = {Neuromuscular embodiment of feedback control elements in flight.}, journal = {Science Advances}, volume = {8}, year = {2022}, month = {2022 Dec 14}, pages = {eabo7461}, abstract = {

While insects such as are flying, aerodynamic instabilities require that they make millisecond time scale adjustments to their wing motion to stay aloft and on course. These stabilization reflexes can be modeled as a proportional-integral (PI) controller; however, it is unclear how such control might be instantiated in insects at the level of muscles and neurons. Here, we show that the b1 and b2 motor units-prominent components of the fly{\textquoteright}s steering muscle system-modulate specific elements of the PI controller: the angular displacement (integral) and angular velocity (proportional), respectively. Moreover, these effects are observed only during the stabilization of pitch. Our results provide evidence for an organizational principle in which each muscle contributes to a specific functional role in flight control, a finding that highlights the power of using top-down behavioral modeling to guide bottom-up cellular manipulation studies.

}, issn = {2375-2548}, doi = {10.1126/sciadv.abo7461}, author = {Whitehead, Samuel C and Leone, Sofia and Lindsay, Theodore and Meiselman, Matthew R and Cowan, Noah J and Dickinson, Michael H and Yapici, Nilay and Stern, David L and Shirangi, Troy and Cohen, Itai} } @article {67950, title = {Regulation of Drosophila courtship behavior by the Tlx/tailless-like nuclear receptor, dissatisfaction.}, journal = {Current Biology}, volume = {32}, year = {2022}, month = {2022 Apr 25}, pages = {1703-1714}, abstract = {

Sexually dimorphic courtship behaviors in Drosophila melanogaster develop from the activity of the sexual differentiation genes, doublesex (dsx) and fruitless (fru), functioning with other regulatory factors that have received little attention. The dissatisfaction (dsf) gene encodes an orphan nuclear receptor homologous to vertebrate Tlx and Drosophila tailless that is critical for the development of several aspects of female- and male-specific sexual behaviors. Here, we report the pattern of dsf expression in the central nervous system and show that the activity of sexually dimorphic abdominal interneurons that co-express dsf and dsx is necessary and sufficient for vaginal plate opening in virgin females, ovipositor extrusion in mated females, and abdominal curling in males during courtship. We find that dsf activity results in different neuroanatomical outcomes in females and males, promoting and suppressing, respectively, female development and function of these neurons depending upon the sexual state of dsx expression. We posit that dsf and dsx interact to specify sex differences in the neural circuitry for dimorphic abdominal behaviors.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2022.02.031}, author = {Duckhorn, Julia C and Cande, Jessica and Metkus, Mary C and Song, Hyeop and Altamirano, Sofia and Stern, David L and Shirangi, Troy R} } @article {67906, title = {Transgenic tools for targeted chromosome rearrangements allow construction of balancer chromosomes in non-melanogaster Drosophila species.}, journal = {G3 (Bethesda)}, year = {2022}, month = {2022 Feb 10}, abstract = {

Perhaps the most valuable single set of resources for genetic studies of Drosophila melanogaster is the collection of multiply-inverted chromosomes commonly known as balancer chromosomes. Balancers prevent the recovery of recombination exchange products within genomic regions included in inversions and allow perpetual maintenance of deleterious alleles in living stocks and the execution of complex genetic crosses. Balancer chromosomes have been generated traditionally by exposing animals to ionizing radiation and screening for altered chromosome structure or for unusual marker segregation patterns. These approaches are tedious and unpredictable, and have failed to produce the desired products in some species. Here I describe transgenic tools that allow targeted chromosome rearrangements in Drosophila species. The key new resources are engineered reporter genes containing introns with yeast recombination sites and enhancers that drive fluorescent reporter genes in multiple body regions. These tools were used to generate a doubly-inverted chromosome 3R in D. simulans that serves as an effective balancer chromosome.

}, issn = {2160-1836}, doi = {10.1093/g3journal/jkac030}, author = {Stern, David L} } @article {67734, title = {Distinct genetic architectures underlie divergent thorax, leg, and wing pigmentation between Drosophila elegans and D. gunungcola.}, journal = {Heredity}, volume = {127}, year = {2021}, month = {2021 Nov 01}, pages = {467-74}, abstract = {

Pigmentation divergence between Drosophila species has emerged as a model trait for studying the genetic basis of phenotypic evolution, with genetic changes contributing to pigmentation differences often mapping to genes in the pigment synthesis pathway and their regulators. These studies of Drosophila pigmentation have tended to focus on pigmentation changes in one body part for a particular pair of species, but changes in pigmentation are often observed in multiple body parts between the same pair of species. The similarities and differences of genetic changes responsible for divergent pigmentation in different body parts of the same species thus remain largely unknown. Here we compare the genetic basis of pigmentation divergence between Drosophila elegans and D. gunungcola in the wing, legs, and thorax. Prior work has shown that regions of the genome containing the pigmentation genes yellow and ebony influence the size of divergent male-specific wing spots between these two species. We find that these same two regions of the genome underlie differences in leg and thorax pigmentation; however, divergent alleles in these regions show differences in allelic dominance and epistasis among the three body parts. These complex patterns of inheritance can be explained by a model of evolution involving tissue-specific changes in the expression of Yellow and Ebony between D. elegans and D. gunungcola.

}, issn = {1365-2540}, doi = {10.1038/s41437-021-00467-0}, author = {Massey, Jonathan H and Li, Jun and Stern, David L and Wittkopp, Patricia J} } @article {67181, title = {A novel family of secreted insect proteins linked to plant gall development.}, journal = {Current Biology}, volume = {31}, year = {2021}, month = {2021 May 10}, pages = {1836}, abstract = {

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2021.01.104}, author = {Korgaonkar, Aishwarya and Han, Clair and Lemire, Andrew L and Siwanowicz, Igor and Bennouna, Djawed and Kopec, Rachel E and Andolfatto, Peter and Shigenobu, Shuji and Stern, David L} } @article {65900, title = {Cis-regulatory variation in the shavenbaby gene underlies intraspecific phenotypic variation, mirroring interspecific divergence in the same trait.}, journal = {Evolution}, year = {2020}, month = {2020 Dec 14}, abstract = {

Despite considerable progress in recent decades in dissecting the genetic causes of natural morphological variation, there is limited understanding of how variation within species ultimately contributes to species differences. We have studied patterning of the non-sensory hairs, commonly known as "trichomes," on the dorsal cuticle of first-instar larvae of Drosophila. Most Drosophila species produce a dense lawn of dorsal trichomes, but a subset of these trichomes were lost in D. sechellia and D. ezoana due entirely to regulatory evolution of the shavenbaby (svb) gene. Here, we describe intraspecific variation in dorsal trichome patterns of first-instar larvae of D. virilis that is similar to the trichome pattern variation identified previously between species. We found that a single large effect QTL, which includes svb, explains most of the trichome number difference between two D. virilis strains and that svb expression correlates with the trichome difference between strains. This QTL does not explain the entire difference between strains, implying that additional loci contribute to variation in trichome numbers. Thus, the genetic architecture of intraspecific variation exhibits similarities and differences with interspecific variation that may reflect differences in long-term and short-term evolutionary processes.

}, issn = {1558-5646}, doi = {10.1111/evo.14142}, author = {Soverna, Ana Faigon and Rodriguez, Nahuel Cabrera and Korgaonkar, Aishwarya and Hasson, Esteban and Stern, David L and Frankel, Nicol{\'a}s} } @article {65585, title = {Co-evolving wing spots and mating displays are genetically separable traits in Drosophila.}, journal = {Evolution}, volume = {74}, year = {2020}, month = {2020 May 03}, pages = {1098-1111}, abstract = {

The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, we investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and D. gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), we identified a \~{}440 kb region on the X chromosome that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior. This article is protected by copyright. All rights reserved.

}, issn = {1558-5646}, doi = {10.1111/evo.13990}, author = {Massey, Jonathan H and Rice, Gavin R and Firdaus, Anggun and Chen, Chi-Yang and Yeh, Shu-Dan and Stern, David L and Wittkopp, Patricia J} } @article {65799, title = {Dense and pleiotropic regulatory information in a developmental enhancer.}, journal = {Nature}, volume = {587}, year = {2020}, month = {2020 Nov 01}, pages = {235-39}, abstract = {

Changes in gene regulation underlie much of phenotypic evolution. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

}, issn = {1476-4687}, doi = {10.1038/s41586-020-2816-5}, author = {Fuqua, Timothy and Jordan, Jeff and van Breugel, Maria Elize and Halavatyi, Aliaksandr and Tischer, Christian and Polidoro, Peter and Abe, Namiko and Tsai, Albert and Mann, Richard S and Stern, David L and Crocker, Justin} } @article {65362, title = {A large genomic insertion containing a duplicated follistatin gene is linked to the pea aphid male wing dimorphism.}, journal = {eLife}, volume = {9}, year = {2020}, month = {2020 Mar 06}, abstract = {

Wing dimorphisms have long served as models for examining the ecological and evolutionary tradeoffs associated with alternative phenotypes. Here, we investigated the genetic cause of the pea aphid () male wing dimorphism, wherein males exhibit one of two morphologies that differ in correlated traits that include the presence or absence of wings. We mapped this trait difference to a single genomic region and, using third generation, long-read sequencing, we identified a 120 kb insertion in the wingless allele. This insertion includes a duplicated gene, which is a strong candidate gene in the minimal mapped interval to cause the dimorphism. We found that both alleles were present prior to pea aphid biotype lineage diversification, we estimated that the insertion occurred millions of years ago, and we propose that both alleles have been maintained in the species, likely due to balancing selection.

}, issn = {2050-084X}, doi = {10.7554/eLife.50608}, author = {Li, Binshuang and Bickel, Ryan D and Parker, Benjamin J and Saleh Ziabari, Omid and Liu, Fangzhou and Vellichirammal, Neetha Nanoth and Simon, Jean-Christophe and Stern, David L and Brisson, Jennifer A} } @article {64958, title = {Evolution of Mechanisms that Control Mating in Drosophila Males.}, journal = {Cell Reports}, volume = {27}, year = {2019}, month = {2019 May 28}, pages = {2527-2536.e4}, abstract = {

Genetically wired neural mechanisms inhibit mating between species because even naive animals rarely mate with other species. These mechanisms can evolve through changes in expression or function of key genes in sensory pathways or central circuits. Gr32a is a gustatory chemoreceptor that, in D.\ melanogaster, is essential to inhibit interspecies courtship and sense quinine. Similar to D.\ melanogaster, we find that D.\ simulans Gr32a is expressed in foreleg tarsi, sensorimotor appendages that inhibit interspecies courtship, and it is required to sense quinine. Nevertheless, Gr32a is not required to inhibit interspecies mating by D.\ simulans males. However, and similar to its function in D.\ melanogaster, Ppk25, a member of the Pickpocket family, promotes conspecific courtship in D.\ simulans. Together, we have identified distinct evolutionary mechanisms underlying chemosensory control of taste and courtship in closely related Drosophila species.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2019.04.104}, author = {Ahmed, Osama M and Avila-Herrera, Aram and Tun, Khin May and Serpa, Paula H and Peng, Justin and Parthasarathy, Srinivas and Knapp, Jon-Michael and Stern, David L and Davis, Graeme W and Pollard, Katherine S and Shah, Nirao M} } @article {64841, title = {Neural evolution of context-dependent fly song.}, journal = {Current Biology : CB}, volume = {29}, year = {2019}, month = {2019 Mar 08}, pages = {1089-99}, abstract = {

It is unclear where in the nervous system evolutionary changes tend to occur. To localize the source of neural evolution that has generated divergent behaviors, we developed a new approach to label and functionally manipulate homologous neurons across Drosophila species. We examined homologous descending neurons that drive courtship song in two species that sing divergent song types and localized relevant evolutionary changes in circuit function downstream of the intrinsic physiology of these descending neurons. This evolutionary change causes different species to produce divergent motor patterns in similar social contexts. Artificial stimulation of these descending neurons drives multiple song types, suggesting that multifunctional properties of song circuits may facilitate rapid evolution of song types.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2019.02.019}, author = {Ding, Yun and Lillvis, Joshua L and Cande, Jessica and Berman, Gordon J and Arthur, Benjamin J and Long, Xi and Xu, Min and Dickson, Barry J and Stern, David L} } @article {65229, title = {The yellow gene influences Drosophila male mating success through sex comb melanization.}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019 Oct 15}, abstract = {

males perform a series of courtship behaviors that, when successful, result in copulation with a female. For over a century, mutations in the gene, named for its effects on pigmentation, have been known to reduce male mating success. Prior work has suggested that influences mating behavior through effects on wing extension, song, and/or courtship vigor. Here, we rule out these explanations, as well as effects on the nervous system more generally, and find instead that the effects of on male mating success are mediated by its effects on pigmentation of male-specific leg structures called sex combs. Loss of expression in these modified bristles reduces their melanization, which changes their structure and causes difficulty grasping females prior to copulation. These data illustrate why the mechanical properties of anatomy, not just neural circuitry, must be considered to fully understand the development and evolution of behavior.

}, issn = {2050-084X}, doi = {10.7554/eLife.49388}, author = {Massey, Jonathan H and Chung, Daayun and Siwanowicz, Igor and Stern, David L and Wittkopp, Patricia J} } @article {49128, title = {Accurate and sensitive quantification of protein-DNA binding affinity.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2018}, month = {2018 Apr 02}, pages = {E3692-701}, abstract = {

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

}, issn = {1091-6490}, doi = {10.1073/pnas.1714376115}, author = {Rastogi, Chaitanya and Rube, H Tomas and Kribelbauer, Judith F and Crocker, Justin and Loker, Ryan E and Martini, Gabriella D and Laptenko, Oleg and Freed-Pastor, William A and Prives, Carol and Stern, David L and Mann, Richard S and Bussemaker, Harmen J} } @article {49092, title = {Comprehensive analysis of a cis-regulatory region reveals pleiotropy in enhancer function.}, journal = {Cell Reports}, volume = {22}, year = {2018}, month = {2018 Mar 13}, pages = {3021-3031}, abstract = {

Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. Here, we have studied the activity of the enhancers of the shavenbaby gene throughout D.\ melanogaster development. We found that all seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. We explored how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer,\ the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2018.02.073}, author = {Preger-Ben Noon, Ella and Sabar{\'\i}s, Gonzalo and Ortiz, Daniela M and Sager, Jonathan and Liebowitz, Anna and Stern, David L and Frankel, Nicol{\'a}s} } @article {49478, title = {Correlated evolution of two copulatory organs via a single cis-regulatory nucleotide change.}, journal = {Current Biology : CB}, volume = {28}, year = {2018}, month = {2018 Oct 05}, pages = {3450-7}, abstract = {

Diverse traits often covary between species [1-3]. The possibility that a single mutation could contribute to the evolution of several characters between species [3] is rarely investigated as relatively few cases are dissected at the nucleotide level. Drosophila santomea has evolved additional sex comb sensory teeth on its legs and has lost two sensory bristles on its genitalia. We present evidence that a single nucleotide substitution in an enhancer of the scute gene contributes to both changes. The mutation alters a binding site for the Hox protein Abdominal-B in the developing genitalia, leading to bristle loss, and for another factor in the developing leg, leading to bristle gain. Our study suggests that morphological evolution between species can occur through a single nucleotide change affecting several sexually dimorphic traits. VIDEO ABSTRACT.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2018.08.047}, author = {Nagy, Olga and Nuez, Isabelle and Savisaar, Rosina and Peluffo, Alexandre E and Yassin, Amir and Lang, Michael and Stern, David L and Matute, Daniel R and David, Jean R and Courtier-Orgogozo, Virginie} } @article {49318, title = {Evolution of a central neural circuit underlies Drosophila mate preferences.}, journal = {Nature}, volume = {559}, year = {2018}, month = {2018 Jul 11}, pages = {564-9}, abstract = {

Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. Here we compare pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. We show that males of both species detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. Our results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution.

}, issn = {1476-4687}, doi = {10.1038/s41586-018-0322-9}, author = {Seeholzer, Laura F and Seppo, Max and Stern, David L and Ruta, Vanessa} } @article {49573, title = {Gene flow mediates the role of sex chromosome meiotic drive during complex speciation.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 Dec 13}, abstract = {

During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build-up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, we combine high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, and . Our findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, we find that a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation.

}, issn = {2050-084X}, doi = {10.7554/eLife.35468}, author = {Meiklejohn, Colin D and Landeen, Emily L and Gordon, Kathleen E and Rzatkiewicz, Thomas and Kingan, Sarah B and Geneva, Anthony J and Vedanayagam, Jeffrey P and Muirhead, Christina A and Garrigan, Daniel and Stern, David L and Presgraves, Daven C} } @article {48976, title = {Optogenetic dissection of descending behavioral control in Drosophila.}, journal = {eLife}, year = {2018}, month = {2018}, pages = {e34275}, abstract = {

In most animals, the brain makes behavioral decisions that are transmitted by descending neurons to the nerve cord circuitry that produces behaviors. In insects, only a few descending neurons have been associated with specific behaviors. To explore how descending neurons control an insect{\textquoteright}s movements, we developed a novel method to systematically assay the behavioral effects of activating individual neurons on freely behaving terrestrial\ D. melanogaster. We calculated a two-dimensional representation of the entire behavior space explored by these flies and we associated descending neurons with specific behaviors by identifying regions of this space that were visited with increased frequency during optogenetic activation. Applying this approach across a large collection of descending neurons, we found that (1) activation of most of the descending neurons drove stereotyped behaviors, (2) in many cases multiple descending neurons activated similar behaviors, and (3) optogenetically-activated behaviors were often dependent on the behavioral state prior to activation.

}, doi = {10.7554/eLife.34275}, author = {Cande, Jessica and Namiki, Shigehiro and Qiu, Jirui and Korff, Wyatt and Card, Gwyneth M and Shaevitz, Joshua W and Stern, David L and Berman, Gordon J} } @article {48961, title = {Editorial overview: Making evolutionary sense of everything.}, journal = {Current Opinion in Genetics \& Development}, volume = {47}, year = {2017}, month = {2017 Dec}, pages = {iv-vi}, issn = {1879-0380}, doi = {10.1016/j.gde.2017.11.005}, author = {Stern, David L and Haag, Eric} } @article {48765, title = {Experimental and statistical reevaluation provides no evidence for Drosophila courtship song rhythms.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Aug 29}, pages = {9978-83}, abstract = {

From 1980 to 1992, a series of influential papers reported on the discovery, genetics, and evolution of a periodic cycling of the interval between Drosophila male courtship song pulses. The molecular mechanisms underlying this periodicity were never described. To reinitiate investigation of this phenomenon, we previously performed automated segmentation of songs but failed to detect the proposed rhythm [Arthur BJ, et al. (2013) BMC Biol 11:11; Stern DL (2014) BMC Biol 12:38]. Kyriacou et al. [Kyriacou CP, et al. (2017) Proc Natl Acad Sci USA 114:1970-1975] report that we failed to detect song rhythms because (i) our flies did not sing enough and (ii) our segmenter did not identify many of the song pulses. Kyriacou et al. manually annotated a subset of our recordings and reported that two strains displayed rhythms with genotype-specific periodicity, in agreement with their original reports. We cannot replicate this finding and show that the manually annotated data, the original automatically segmented data, and a new dataset provide no evidence for either the existence of song rhythms or song periodicity differences between genotypes. Furthermore, we have reexamined our methods and analysis and find that our automated segmentation method was not biased to prevent detection of putative song periodicity. We conclude that there is no evidence for the existence of Drosophila courtship song rhythms.

}, issn = {1091-6490}, doi = {10.1073/pnas.1707471114}, author = {Stern, David L and Clemens, Jan and Coen, Philip and Calhoun, Adam J and Hogenesch, John B and Arthur, Ben J and Murthy, Mala} } @article {48157, title = {A fully synthetic transcriptional platform for a multicellular eukaryote.}, journal = {Cell Reports}, volume = {18}, year = {2017}, month = {2017 Jan 03}, pages = {287-296}, abstract = {

Regions of genomic DNA called enhancers encode binding sites for transcription factor proteins. Binding of activators and repressors increase and reduce transcription, respectively, but it is not understood how combinations of activators and repressors generate precise patterns of transcription during development. Here, we explore this problem using\ a fully synthetic transcriptional platform in Drosophila consisting of engineered transcription factor gradients and artificial enhancers. We found that binding sites for a transcription factor that makes DNA accessible are required together with binding sites for transcriptional activators to produce a functional enhancer. Only in this context can changes in the number of activator binding sites mediate quantitative control of transcription. Using an engineered transcriptional repressor gradient, we demonstrate that overlapping repressor and activator binding sites provide more robust repression and sharper expression boundaries than non-overlapping sites. This may explain why this common motif is observed in many developmental enhancers.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2016.12.025}, author = {Crocker, Justin and Tsai, Albert and Stern, David L} } @article {48700, title = {Functional regulatory evolution outside of the minimal even-skipped stripe 2 enhancer.}, journal = {Development (Cambridge, England)}, year = {2017}, month = {2017 Jul 31}, abstract = {

Transcriptional enhancers are regions of DNA that drive precise patterns of gene expression. While many studies have elucidated how individual enhancers can evolve, most of this work has focused on what are called "minimal" enhancers, the smallest DNA regions that drive expression that approximates an aspect of native gene expression. Here we explore how the Drosophila erecta even-skipped (eve) locus has evolved by testing its activity in the divergent D. melanogaster genome. We found, as has been reported previously, that the D. erecta eve stripe 2 enhancer (eveS2) fails to drive appreciable expression in D. melanogaster (1). However, we found that a large transgene carrying the entire D. erecta eve locus drives normal eve expression, including in stripe 2. We performed a functional dissection of the region upstream of the D. erecta eveS2 region and found multiple Zelda motifs that are required for normal expression. Our results illustrate how sequences outside of minimal enhancer regions can evolve functionally through mechanisms other than changes in transcription factor binding sites that drive patterning.

}, issn = {1477-9129}, doi = {10.1242/dev.149427}, author = {Crocker, Justin and Stern, David L} } @article {48319, title = {Genetic and transgenic reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea and D. virilis.}, journal = {G3 (Bethesda, Md.)}, volume = {7}, year = {2017}, month = {2017 Mar 09}, pages = {1339-47}, abstract = {

Species of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. Studies in these species have been limited, however, by a paucity of genetic and transgenic reagents. Here we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein gene expressed in the eyes and a phiC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.

}, issn = {2160-1836}, doi = {10.1534/g3.116.038885}, author = {Stern, David L and Crocker, Justin and Ding, Yun and Frankel, Nicol{\'a}s and Kappes, Gretchen and Kim, Elizabeth and Kuzmickas, Ryan and Lemire, Andrew and Mast, Joshua D and Picard, Serge} } @article {48920, title = {Nuclear microenvironments modulate transcription from low-affinity enhancers.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Nov 02}, abstract = {

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogastershavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

}, issn = {2050-084X}, doi = {10.7554/eLife.28975}, author = {Tsai, Albert and Muthusamy, Anand K and Alves, Mariana Rp and Lavis, Luke D and Singer, Robert H and Stern, David L and Crocker, Justin} } @article {49452, title = {Pleiotropy in enhancer function is encoded through diverse genetic architectures.}, journal = {bioRxiv}, year = {2017}, month = {2017 Sep 14}, abstract = {

Developmental genes can have complex cis-regulatory regions, with multiple enhancers scattered across stretches of DNA spanning tens or hundreds of kilobases. Early work revealed remarkable modularity of enhancers, where distinct regions of DNA, bound by combinations of transcription factors, drive gene expression in defined spatio-temporal domains. Nevertheless, a few reports have shown that enhancer function may be required in multiple developmental stages, implying that regulatory elements can be pleiotropic. In these cases, it is not clear whether the pleiotropic enhancers employ the same transcription factor binding sites to drive expression at multiple developmental stages or whether enhancers function as chromatin scaffolds, where independent sets of transcription factor binding sites act at different stages. In this work we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive gene expression in multiple tissues and developmental stages at varying levels of redundancy. We have explored how this pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, whereas for a second enhancer, these roles are largely encoded by distinct transcription factor binding sites. Our data suggest that enhancer pleiotropy might be a common feature of cis-regulatory regions of developmental genes and that this pleiotropy can be encoded through multiple genetic architectures.

}, doi = {10.1101/188532}, author = {Preger-Ben Noon, Ella and Sabar{\'\i}s, Gonzalo and Ortiz, Daniela M and Sager, Jonathan and Liebowitz, Anna and Stern, David L and Frankel, Nicol{\'a}s} } @article {47553, title = {Doublesex regulates the connectivity of a neural circuit controlling Drosophila male courtship song.}, journal = {Developmental Cell}, volume = {37}, year = {2016}, month = {2016 Jun 20}, pages = {533-44}, abstract = {

It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The\ Drosophila melanogaster\ male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively. We report the discovery of male-specific thoracic interneurons{\textemdash}the TN1A neurons{\textemdash}that are required specifically for sine song. The TN1A neurons can drive the activity of a sex-non-specific wing motoneuron,\ hg1, which is also required for sine song. The male-specific connection between the TN1A neurons and the\ hg1\ motoneuron is regulated by the sexual differentiation gene\ doublesex. We find that\ doublesex\ is required in the TN1A neurons during development to increase the density of the TN1A arbors that interact with dendrites of the\ hg1motoneuron. Our findings demonstrate how a sexual differentiation gene can build a sex-specific circuit motif by modulating neuronal arborization.

{\textbullet}Doublesex-expressing TN1 neurons are necessary and sufficient for the male sine song{\textbullet}A subclass of TN1 neurons, TN1A, contributes to the sine song{\textbullet}TN1A neurons are functionally coupled to a sine song motoneuron,\ hg1{\textbullet}Doublesex\ regulates the connectivity between the TN1A and\ hg1\ neurons

It is unclear how developmental regulatory genes specify sex-specific behaviors. Shirangi et\ al. demonstrate that the\ Drosophila\ sexual differentiation gene\ doublesex\ encodes a sex-specific behavior{\textemdash}male song{\textemdash}by promoting the connectivity between the male-specific TN1A neurons and the sex-non-specific\ hg1\ neurons, which are required for production of the song.

}, doi = {10.1016/j.devcel.2016.05.012}, author = {Shirangi, Troy R and Wong, Allen M and Truman, James W and Stern, David L} } @article {48043, title = {Evolved repression overcomes enhancer robustness.}, journal = {Developmental Cell}, volume = {39}, year = {2016}, month = {2016 Nov 10}, pages = {572-84}, abstract = {

Biological systems display extraordinary robustness. Robustness of transcriptional enhancers results mainly from clusters of binding sites for the same transcription factor, and it is not clear how robust enhancers can evolve loss of expression through point mutations. Here, we report the high-resolution functional dissection of a robust enhancer of the\ shavenbaby\ gene that has contributed to morphological evolution. We found that robustness is encoded by many binding sites for the transcriptional activator Arrowhead and that, during evolution, some of these activator sites were lost, weakening enhancer activity. Complete silencing of enhancer function, however, required evolution of a binding site for the spatially restricted potent repressor Abrupt. These findings illustrate that recruitment of\ repressor binding sites can overcome enhancer robustness and may minimize pleiotropic consequences of enhancer evolution. Recruitment of repression may be a general mode of evolution to break robust regulatory linkages.

}, doi = {10.1016/j.devcel.2016.10.010}, author = {Preger-Ben Noon, Ella and Davis, Fred P and Stern, David L} } @article {47766, title = {Natural courtship song variation caused by an intronic retroelement in an ion channel gene.}, journal = {Nature}, year = {2016}, month = {2016 Aug 10}, abstract = {

Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Here, using an unbiased genetic approach, we map a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, we confirm that slo is the causal locus and resolve the causal mutation to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

}, issn = {1476-4687}, doi = {10.1038/nature19093}, author = {Ding, Yun and Berrocal, Augusto and Morita, Tomoko and Longden, Kit D and Stern, David L} } @article {47287, title = {Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors.}, journal = {Nature Genetics}, year = {2016}, month = {2016 Feb 8}, abstract = {

Genes are regulated by transcription factors that bind to regions of genomic DNA called enhancers. Considerable effort is focused on identifying transcription factor binding sites, with the goal of predicting gene expression from DNA sequence. Despite this effort, general, predictive models of enhancer function are currently lacking. Here we combine quantitative models of enhancer function with manipulations using engineered transcription factors to examine the extent to which enhancer function can be controlled in a quantitatively predictable manner. Our models, which incorporate few free parameters, can accurately predict the contributions of ectopic transcription factor inputs. These models allow the predictable {\textquoteright}tuning{\textquoteright} of enhancers, providing a framework for the quantitative control of enhancers with engineered transcription factors.

}, issn = {1546-1718}, doi = {10.1038/ng.3509}, author = {Crocker, Justin and Ilsley, Garth R and Stern, David L} } @article {47264, title = {The soft touch: low-affinity transcription factor binding sites in development and evolution.}, journal = {Current Topics in Developmental Biology}, year = {2016}, month = {2016 Jan 07}, abstract = {

Transcription factor proteins regulate gene expression by binding to specific DNA regions. Most studies of transcription factor binding sites have focused on the highest affinity sites for each factor. There is abundant evidence, however, that binding sites with a range of affinities, including very low affinities, are critical to gene regulation. Here, we present the theoretical and experimental evidence for the importance of low-affinity sites in gene regulation and development. We also discuss the implications of the widespread use of low-affinity sites in eukaryotic genomes for robustness, precision, specificity, and evolution of gene regulation.

}, doi = {10.1016/bs.ctdb.2015.11.018}, author = {Crocker, Justin and Preger-Ben Noon, Ella and Stern, David L} } @article {47972, title = {An unsupervised method for quantifying the behavior of interacting individuals.}, journal = {Physical Biology}, volume = {14}, year = {2016}, month = {2017 Feb16}, pages = {1609.09345}, abstract = {

Behaviors involving the interaction of multiple individuals are complex and frequently crucial for an animal{\textquoteright}s survival. These interactions, ranging across sensory modalities, length scales, and time scales, are often subtle and difficult to characterize. Contextual effects on the frequency of behaviors become even more difficult to quantify when physical interaction between animals interferes with conventional data analysis, e.g. due to visual occlusion. We introduce a method for quantifying behavior in fruit fly interaction that combines high-throughput video acquisition and tracking of individuals with recent unsupervised methods for capturing an animal{\textquoteright}s entire behavioral repertoire. We find behavioral differences between solitary flies and those paired with an individual of the opposite sex, identifying specific behaviors that are affected by social and spatial context. Our pipeline allows for a comprehensive description of the interaction between two individuals using unsupervised machine learning methods, and will be used to answer questions about the depth of complexity and variance in fruit fly courtship.

}, doi = {10.1088/1478-3975/aa5c50}, url = {https://arxiv.org/abs/1609.09345v1}, author = {Klibaite, Ugne and Berman, Gordon J and Cande, Jessica and Stern, David L} } @article {46535, title = {Genetic architecture and functional characterization of genes underlying the rapid diversification of male external genitalia between Drosophila simulans and Drosophila mauritiana.}, journal = {Genetics}, year = {2015}, month = {2015 Mar 16}, abstract = {

Male sexual characters are often among the first traits to diverge between closely related species and identifying the genetic basis of such changes can contribute to our understanding of their evolutionary history. However, little is known about the genetic architecture or the specific genes underlying the evolution of male genitalia. The morphology of the claspers, posterior lobes and anal plates exhibit striking differences between Drosophila mauritiana and Drosophila simulans. Using QTL and introgression-based high-resolution mapping, we identified several small regions on chromosome arms 3L and 3R that contribute to differences in these traits. However, we found that the loci underlying the evolution of clasper differences between these two species are independent from those that contribute to posterior lobe and anal plate divergence. Furthermore, while most of the loci affect each trait in the same direction and act additively, we also found evidence for epistasis between loci for clasper bristle number. In addition, we conducted an RNAi screen in D. melanogaster to investigate if positional and expression candidate genes located on chromosome 3L, are also involved in genital development. We found that six of these genes, including components of Wnt signaling and male-specific lethal 3 (msl3), regulate the development of genital traits consistent with the effects of the introgressed regions where they are located and that thus represent promising candidate genes for the evolution these traits.

}, issn = {1943-2631}, doi = {10.1534/genetics.114.174045}, author = {Tanaka, Kentaro M and Hopfen, Corinna and Herbert, Matthew R and Schl{\"o}tterer, Christian and Stern, David L and Masly, John P and McGregor, Alistair P and Nunes, Maria D S} } @article {45847, title = {Low affinity binding site clusters confer Hox specificity and regulatory robustness.}, journal = {Cell}, volume = {160}, year = {2015}, month = {2015 Jan 15}, pages = {191-203}, abstract = {

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in\ vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in\ vivo, but multiple clustered sites were required for robust expression when embryos developed in\ variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2014.11.041}, author = {Crocker, Justin and Abe, Namiko and Rinaldi, Lucrezia and McGregor, Alistair P and Frankel, Nicol{\'a}s and Wang, Shu and Alsawadi, Ahmad and Valenti, Philippe and Plaza, Serge and Payre, Fran{\c c}ois and Mann, Richard S and Stern, David L} } @article {47238, title = {A major locus controls a genital shape difference involved in reproductive isolation between Drosophila yakuba and Drosophila santomea.}, journal = {G3 (Bethesda, Md.)}, volume = {5}, year = {2015}, month = {2015 Oct 28}, pages = {2893-901}, abstract = {

Rapid evolution of genitalia shape, a widespread phenomenon in animals with internal fertilization, offers the opportunity to dissect the genetic architecture of morphological evolution linked to sexual selection and speciation. Most quantitative trait loci (QTL) mapping studies of genitalia divergence have focused on Drosophila melanogaster and its three most closely related species, D. simulans, D. mauritiana, and D. sechellia, and have suggested that the genetic basis of genitalia evolution involves many loci. We report the first genetic study of male genitalia evolution between D. yakuba and D. santomea, two species of the D. melanogaster species subgroup. We focus on male ventral branches, which harm females during interspecific copulation. Using landmark-based geometric morphometrics, we characterized shape variation in parental species, F1 hybrids, and backcross progeny and show that the main axis of shape variation within the backcross population matches the interspecific variation between parental species. For genotyping, we developed a new molecular method to perform multiplexed shotgun genotyping (MSG), which allowed us to prepare genomic DNA libraries from 365 backcross individuals in a few days using little DNA. We detected only three QTL, one of which spans 2.7 Mb and exhibits a highly significant effect on shape variation that can be linked to the harmfulness of the ventral branches. We conclude that the genetic architecture of genitalia morphology divergence may not always be as complex as suggested by previous studies.

}, issn = {2160-1836}, doi = {10.1534/g3.115.023481}, author = {Peluffo, Alexandre E and Nuez, Isabelle and Debat, Vincent and Savisaar, Rosina and Stern, David L and Orgogozo, Virginie} } @article {6826, title = {Cellular and behavioral functions of fruitless isoforms in Drosophila courtship.}, journal = {Current Biology }, volume = {24}, year = {2014}, month = {2014 Feb 3}, pages = {242-51}, abstract = {

BACKGROUND: Male-specific products of the fruitless (fru) gene control the development and function of neuronal circuits that underlie male-specific behaviors in Drosophila, including courtship. Alternative splicing generates at least three distinct Fru isoforms, each containing a different zinc-finger domain. Here, we examine the expression and function of each of these isoforms. RESULTS: We show that most fru(+) cells express all three isoforms, yet each isoform has a distinct function in the elaboration of sexually dimorphic circuitry and behavior. The strongest impairment in courtship behavior is observed in fru(C) mutants, which fail to copulate, lack sine song, and do not generate courtship song in the absence of visual stimuli. Cellular dimorphisms in the fru circuit are dependent on Fru(C) rather than other single Fru isoforms. Removal of Fru(C) from the neuronal classes vAB3 or aSP4 leads to cell-autonomous feminization of arborizations and loss of courtship in the dark. CONCLUSIONS: These data map specific aspects of courtship behavior to the level of single fru isoforms and fru(+) cell types-an important step toward elucidating the chain of causality from gene to circuit to behavior.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2013.12.015}, author = {von Philipsborn, Anne C and J{\"o}rchel, Sabrina and Tirian, Laszlo and Demir, Ebru and Morita, Tomoko and Stern, David L and Dickson, Barry J} } @article {45781, title = {Evolved differences in larval social behavior mediated by novel pheromones.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Dec 12}, abstract = {

Pheromones, chemical signals that convey social information, mediate many insect social behaviors, including navigation and aggregation. Several studies have suggested that behavior during the immature larval stages of Drosophila development is influenced by pheromones, but none of these compounds or the pheromone-receptor neurons that sense them have been identified. Here we report a larval pheromone-signaling pathway. We found that larvae produce two novel long-chain fatty acids that are attractive to other larvae. We identified a single larval chemosensory neuron that detects these molecules. Two members of the pickpocket family of DEG/ENaC channel subunits (ppk23 and ppk29) are required to respond to these pheromones. This pheromone system is evolving quickly, since the larval exudates of D. simulans, the sister species of D. melanogaster, are not attractive to other larvae. Our results define a new pheromone signaling system in Drosophila that shares characteristics with pheromone systems in a wide diversity of insects.

}, issn = {2050-084X}, doi = {10.7554/eLife.04205}, author = {Mast, Joshua D and De Moraes, Consuelo M and Alborn, Hans T and Lavis, Luke D and Stern, David L} } @article {45695, title = {Identification of loci that cause phenotypic variation in diverse species with the reciprocal hemizygosity test.}, journal = {Trends in Genetics}, volume = {30}, year = {2014}, month = {2014 Dec}, pages = {547-554}, abstract = {

The reciprocal hemizygosity test is a straightforward genetic test that can positively identify genes that have evolved to contribute to a phenotypic difference between strains or between species. The test involves a comparison between hybrids that are genetically identical throughout the genome except at the test locus, which is rendered hemizygous for alternative alleles from the two parental strains. If the two reciprocal hemizygotes display different phenotypes, then the two parental alleles must have evolved. New methods for targeted mutagenesis will allow application of the reciprocal hemizygosity test in many organisms. This review discusses the principles, advantages, and limitations of the test.

}, issn = {0168-9525}, doi = {10.1016/j.tig.2014.09.006}, author = {Stern, David L} } @article {45061, title = {Looking under the lamp post: neither fruitless nor doublesex has evolved to generate divergent male courtship in Drosophila.}, journal = {Cell Reports}, volume = {8}, year = {2014}, month = {2014 Jul 24}, pages = {363-70}, abstract = {

How do evolved genetic changes alter the nervous system to produce different patterns of behavior? We address this question using Drosophila male courtship behavior, which is innate, stereotyped, and evolves rapidly between species. D. melanogaster male courtship requires the male-specific isoforms of two transcription factors, fruitless\ and doublesex. These genes underlie genetic switches between female and male behaviors, making them excellent candidate genes for courtship behavior evolution. We tested their role in courtship evolution by transferring the entire locus for each gene from divergent species to D.\ melanogaster. We found that despite differences in Fru+ and Dsx+ cell numbers in wild-type species, cross-species transgenes rescued D.\ melanogaster courtship behavior and no species-specific behaviors were conferred. Therefore, fru and dsx are not a significant source of evolutionary variation in courtship behavior.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2014.06.023}, author = {Cande, Jessica and Stern, David L and Morita, Tomoko and Prud{\textquoteright}homme, Benjamin and Gompel, Nicolas} } @article {45029, title = {Reported Drosophila courtship song rhythms are artifacts of data analysis.}, journal = {BMC Biology}, volume = {12}, year = {2014}, month = {2014 Jun 26}, pages = {38}, abstract = {

BACKGROUND: In a series of landmark papers, Kyriacou, Hall, and colleagues reported that the average inter-pulse interval of Drosophila melanogaster male courtship song varies rhythmically (KH cycles), that the period gene controls this rhythm, and that evolution of the period gene determines species differences in the rhythm{\textquoteright}s frequency. Several groups failed to recover KH cycles, but this may have resulted from differences in recording chamber size.

RESULTS: Here, using recording chambers of the same dimensions as used by Kyriacou and Hall, I found no compelling evidence for KH cycles at any frequency. By replicating the data analysis procedures employed by Kyriacou and Hall, I found that two factors--data binned into 10-second intervals and short recordings--imposed non-significant periodicity in the frequency range reported for KH cycles. Randomized data showed similar patterns.

CONCLUSIONS: All of the results related to KH cycles are likely to be artifacts of binning data from short songs. Reported genotypic differences in KH cycles cannot be explained by this artifact and may have resulted from the use of small sample sizes and/or from the exclusion of samples that did not exhibit song rhythms.

}, issn = {1741-7007}, doi = {10.1186/1741-7007-12-38}, author = {Stern, David L} } @article {45724, title = {Aphids evolved novel secreted proteins for symbiosis with bacterial endosymbiont.}, journal = {Proc Biol Sci}, volume = {280}, year = {2013}, month = {2013 Jan 7}, pages = {20121952}, abstract = {

Aphids evolved novel cells, called bacteriocytes, that differentiate specifically to harbour the obligatory mutualistic endosymbiotic bacteria Buchnera aphidicola. The genome of the host aphid Acyrthosiphon pisum contains many orphan genes that display no similarity with genes found in other sequenced organisms, prompting us to hypothesize that some of these orphan genes are related to lineage-specific traits, such as symbiosis. We conducted deep sequencing of bacteriocytes mRNA followed by whole mount in situ hybridizations of over-represented transcripts encoding aphid-specific orphan proteins. We identified a novel class of genes that encode small proteins with signal peptides, which are often cysteine-rich, that are over-represented in bacteriocytes. These genes are first expressed at a developmental time point coincident with the incorporation of symbionts strictly in the cells that contribute to the bacteriocyte and this bacteriocyte-specific expression is maintained throughout the aphid{\textquoteright}s life. The expression pattern suggests that recently evolved secretion proteins act within bacteriocytes, perhaps to mediate the symbiosis with beneficial bacterial partners, which is reminiscent of the evolution of novel cysteine-rich secreted proteins of leguminous plants that regulate nitrogen-fixing endosymbionts.

}, keywords = {Amino Acid Sequence, Animals, Aphids, Buchnera, DNA, Complementary, Evolution, Molecular, High-Throughput Nucleotide Sequencing, In Situ Hybridization, Insect Proteins, Protein Sorting Signals, RNA, Messenger, Sequence Alignment, Sequence Analysis, RNA, Symbiosis}, issn = {1471-2954}, doi = {10.1098/rspb.2012.1952}, author = {Shigenobu, Shuji and Stern, David L} } @article {6477, title = {The genetic causes of convergent evolution.}, journal = {Nature Reviews Genetics}, volume = {14}, year = {2013}, month = {2013 Oct 9}, pages = {751-4}, type = {Review}, abstract = {

The evolution of phenotypic similarities between species, known as convergence, illustrates that populations can respond predictably to ecological challenges. Convergence often results from similar genetic changes, which can emerge in two ways: the evolution of similar or identical mutations in independent lineages, which is termed parallel evolution; and the evolution in independent lineages of alleles that are shared among populations, which I call collateral genetic evolution. Evidence for parallel and collateral evolution has been found in many taxa, and an emerging hypothesis is that they result from the fact that mutations in some genetic targets minimize pleiotropic effects while simultaneously maximizing adaptation. If this proves correct, then the molecular changes underlying adaptation might be more predictable than has been appreciated previously.

}, issn = {1471-0064}, doi = {10.1038/nrg3483advance online publication}, author = {Stern, David L} } @article {6671, title = {Motor control of Drosophila courtship song.}, journal = {Cell Reports}, volume = {5}, year = {2013}, month = {2013 Nov 14}, pages = {678-86}, abstract = {

Many animals utilize acoustic signals-or songs-to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce trains of pulses and extended tone, called pulse and sine song, respectively. Courtship songs in the genus Drosophila are exceedingly diverse, and different song features appear to have evolved independently of each other. How the nervous system allows such diversity to evolve is not understood. Here, we identify a wing muscle in D.\ melanogaster (hg1) that is uniquely male-enlarged. The hg1 motoneuron and the sexually dimorphic development of the hg1 muscle are required specifically for the sine component of the male song. In contrast, the motoneuron innervating a sexually monomorphic wing muscle, ps1, is required specifically for a feature of pulse song. Thus, individual wing motor pathways can control separate aspects of courtship song and may provide a "modular" anatomical substrate for the evolution of diverse songs.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2013.09.039}, author = {Shirangi, Troy R and Stern, David L and Truman, James W} } @article {6090, title = {Multi-channel acoustic recording and automated analysis of Drosophila courtship songs.}, journal = {BMC Biology}, volume = {11}, year = {2013}, month = {2013 Jan 31}, pages = {11}, abstract = {

Drosophila melanogaster has served as a powerful model system for genetic studies of courtship songs. To accelerate research on the genetic and neural mechanisms underlying courtship song, we have developed a sensitive recording system to simultaneously capture the acoustic signals from 32 separate pairs of courting flies as well as software for automated segmentation of songs.

}, keywords = {Animal, Animal Communication, Animals, Drosophila melanogaster, Genetic Variation, Sexual Behavior, Videotape Recording}, issn = {1741-7007}, doi = {10.1186/1741-7007-11-11}, author = {Arthur, Benjamin J and Sunayama-Morita, Tomoko and Coen, Philip and Murthy, Mala and Stern, David L} } @article {45715, title = {The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development.}, journal = {Evodevo}, volume = {4}, year = {2013}, month = {2013}, pages = {10}, abstract = {

BACKGROUND: In most species of aphid, female nymphs develop into either sexual or asexual adults depending on the length of the photoperiod to which their mothers were exposed. The progeny of these sexual and asexual females, in turn, develop in dramatically different ways. The fertilized oocytes of sexual females begin embryogenesis after being deposited on leaves (oviparous development) while the oocytes of asexual females complete embryogenesis within the mother (viviparous development). Compared with oviparous development, viviparous development involves a smaller transient oocyte surrounded by fewer somatic epithelial cells and a smaller early embryo that comprises fewer cells. To investigate whether patterning mechanisms differ between the earliest stages of the oviparous and viviparous modes of pea aphid development, we examined the expression of pea aphid orthologs of genes known to specify embryonic termini in other insects.

RESULTS: Here we show that pea aphid oviparous ovaries express torso-like in somatic posterior follicle cells and activate ERK MAP kinase at the posterior of the oocyte. In addition to suggesting that some posterior features of the terminal system are evolutionarily conserved, our detection of activated ERK in the oocyte, rather than in the embryo, suggests that pea aphids may transduce the terminal signal using a mechanism distinct from the one used in Drosophila. In contrast with oviparous development, the pea aphid version of the terminal system does not appear to be used during viviparous development, since we did not detect expression of torso-like in the somatic epithelial cells that surround either the oocyte or the blastoderm embryo and we did not observe restricted activated ERK in the oocyte.

CONCLUSIONS: We suggest that while oviparous oocytes and embryos may specify posterior fate through an aphid terminal system, viviparous oocytes and embryos employ a different mechanism, perhaps one that does not rely on an interaction between the oocyte and surrounding somatic cells. Together, these observations provide a striking example of a difference in the fundamental events of early development that is both environmentally induced and encoded by the same genome.

}, issn = {2041-9139}, doi = {10.1186/2041-9139-4-10}, author = {Bickel, Ryan D and Cleveland, Hillary C and Barkas, Joanna and Jeschke, Caitlin C and Raz, Amelie A and Stern, David L and Davis, Gregory K} } @article {45716, title = {Pupariation site preference within and between Drosophila sibling species.}, journal = {Evolution}, volume = {67}, year = {2013}, month = {2013 Sep}, pages = {2714-27}, abstract = {

Holometabolous insects pass through a sedentary pupal stage and often choose a location for pupation that is different from the site of larval feeding. We have characterized a difference in pupariation site choice within and between sibling species of Drosophila. We found that, in nature, Drosophila sechellia pupariate within their host fruit, Morinda citrifolia, and that they perform this behavior in laboratory assays. In contrast, in the laboratory, geographically diverse strains of Drosophila simulans vary in their pupariation site preference; D. simulans lines from the ancestral range in southeast Africa pupariate on fruit, or a fruit substitute, whereas populations from Europe or the New World select sites off of fruit. We explored the genetic basis for the evolved preference in puariation site preference by performing quantitative trait locus mapping within and between species. We found that the interspecific difference is controlled largely by loci on chromosomes X and II. In contrast, variation between two strains of D. simulans appears to be highly polygenic, with the majority of phenotypic effects due to loci on chromosome III. These data address the genetic basis of how new traits arise as species diverge and populations disperse.

}, keywords = {Africa, Animal Distribution, Animals, Chromosomes, Insect, Drosophila, Europe, Evolution, Molecular, Genetic Speciation, Genetic Variation, Morinda, Phylogeography, Pupa, Quantitative Trait Loci, X Chromosome}, issn = {1558-5646}, doi = {10.1111/evo.12146}, author = {Erezyilmaz, Deniz F and Stern, David L} } @article {6673, title = {The structure and evolution of cis-regulatory regions: the shavenbaby story.}, journal = {Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences}, volume = {368}, year = {2013}, month = {2013 Dec 19}, pages = {20130028}, abstract = {

In this paper, we provide a historical account of the contribution of a single line of research to our current understanding of the structure of cis-regulatory regions and the genetic basis for morphological evolution. We revisit the experiments that shed light on the evolution of larval cuticular patterns within the genus Drosophila and the evolution and structure of the shavenbaby gene. We describe the experiments that led to the discovery that multiple genetic changes in the cis-regulatory region of shavenbaby caused the loss of dorsal cuticular hairs (quaternary trichomes) in first instar larvae of Drosophila sechellia. We also discuss the experiments that showed that the convergent loss of quaternary trichomes in D. sechellia and Drosophila ezoana was generated by parallel genetic changes in orthologous enhancers of shavenbaby. We discuss the observation that multiple shavenbaby enhancers drive overlapping patterns of expression in the embryo and that these apparently redundant enhancers ensure robust shavenbaby expression and trichome morphogenesis under stressful conditions. All together, these data, collected over 13 years, provide a fundamental case study in the fields of gene regulation and morphological evolution, and highlight the importance of prolonged, detailed studies of single genes.

}, issn = {1471-2970}, doi = {10.1098/rstb.2013.0028}, author = {Stern, David L and Frankel, Nicol{\'a}s} } @article {6089, title = {TALE-mediated modulation of transcriptional enhancers in vivo.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013 Jun 30}, pages = {762-7}, abstract = {

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2543}, author = {Crocker, Justin and Stern, David L} } @article {45725, title = {Conserved regulatory architecture underlies parallel genetic changes and convergent phenotypic evolution.}, journal = {Proc Natl Acad Sci U S A}, volume = {109}, year = {2012}, month = {2012 Dec 18}, pages = {20975-9}, abstract = {

Similar morphological, physiological, and behavioral features have evolved independently in different species, a pattern known as convergence. It is known that morphological convergence can occur through changes in orthologous genes. In some cases of convergence, cis-regulatory changes generate parallel modifications in the expression patterns of orthologous genes. Our understanding of how changes in cis-regulatory regions contribute to convergence is hampered, usually, by a limited understanding of the global cis-regulatory structure of the evolving genes. Here we examine the genetic causes of a case of precise phenotypic convergence between Drosophila sechellia and Drosophila ezoana, species that diverged ~40 Mya. Previous studies revealed that changes in multiple transcriptional enhancers of shavenbaby (svb, a transcript of the ovo locus) caused phenotypic evolution in the D. sechellia lineage. It has also been shown that the convergent phenotype of D. ezoana was likely caused by cis-regulatory evolution of svb. Here we show that the large-scale cis-regulatory architecture of svb is conserved between these Drosophila species. Furthermore, we show that the D. ezoana orthologs of the evolved D. sechellia enhancers have also evolved expression patterns that correlate precisely with the changes in the phenotype. Our results suggest that phenotypic convergence resulted from multiple noncoding changes that occurred in parallel in the D. sechellia and D. ezoana lineages.

}, keywords = {Animals, Developmental Biology, Drosophila, Drosophila melanogaster, Enhancer Elements, Genetic, Evolution, Molecular, Gene Expression Regulation, Genes, Reporter, Genetic Vectors, Models, Genetic, Molecular Sequence Data, Phenotype, Species Specificity, Transcription, Genetic}, issn = {1091-6490}, doi = {10.1073/pnas.1207715109}, author = {Frankel, Nicol{\'a}s and Wang, Shu and Stern, David L} } @article {45726, title = {Evolution of multiple additive loci caused divergence between Drosophila yakuba and D. santomea in wing rowing during male courtship.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e43888}, abstract = {

In Drosophila, male flies perform innate, stereotyped courtship behavior. This innate behavior evolves rapidly between fly species, and is likely to have contributed to reproductive isolation and species divergence. We currently understand little about the neurobiological and genetic mechanisms that contributed to the evolution of courtship behavior. Here we describe a novel behavioral difference between the two closely related species D. yakuba and D. santomea: the frequency of wing rowing during courtship. During courtship, D. santomea males repeatedly rotate their wing blades to face forward and then back (rowing), while D. yakuba males rarely row their wings. We found little intraspecific variation in the frequency of wing rowing for both species. We exploited multiplexed shotgun genotyping (MSG) to genotype two backcross populations with a single lane of Illumina sequencing. We performed quantitative trait locus (QTL) mapping using the ancestry information estimated by MSG and found that the species difference in wing rowing mapped to four or five genetically separable regions. We found no evidence that these loci display epistasis. The identified loci all act in the same direction and can account for most of the species difference.

}, keywords = {Animals, Chromosome Mapping, Courtship, Drosophila, Evolution, Molecular, Female, Genetic Markers, Genotyping Techniques, Hybridization, Genetic, Male, Movement, Quantitative Trait Loci, Sexual Behavior, Animal, Species Specificity, Wing}, issn = {1932-6203}, doi = {10.1371/journal.pone.0043888}, author = {Cande, Jessica and Andolfatto, Peter and Prud{\textquoteright}homme, Benjamin and Stern, David L and Gompel, Nicolas} } @article {45730, title = {Preparation and mounting of adult Drosophila structures in Canada balsam.}, journal = {Cold Spring Harb Protoc}, volume = {2012}, year = {2012}, month = {2012 Mar}, pages = {373-5}, abstract = {

The Drosophila cuticle carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. To prepare fine "museum-quality," permanent slides, it is best to mount specimens in Canada Balsam. It is difficult to give precise recipes for Canada Balsam, because every user seems to prefer a slightly different viscosity. Dilute solutions spread easily and do not dry too rapidly while mounting specimens. The disadvantage is that there is actually less Balsam in a "drop" of the solution, and when dried, it can contract from the sides of the coverslip, sometimes disturbing the specimen. Unfortunately, there is no substitute for experience when using Canada Balsam. This protocol describes a procedure for mounting adult cuticles in Canada Balsam.

}, keywords = {Animals, Balsams, Drosophila, Microscopy, Preservation, Biological, Specimen Handling}, issn = {1559-6095}, doi = {10.1101/pdb.prot067389}, author = {Stern, David L and Sucena, Elio} } @article {45731, title = {Rapid mounting of adult Drosophila structures in Hoyer{\textquoteright}s medium.}, journal = {Cold Spring Harb Protoc}, volume = {2012}, year = {2012}, month = {2012 Jan}, pages = {107-9}, abstract = {

The Drosophila cuticle carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. This protocol describes a procedure for mounting adult cuticles in Hoyer{\textquoteright}s medium, a useful mountant for both larval and adult cuticles. The medium digests soft tissues rapidly, leaving the cuticle cleared for observation. In addition, samples can be transferred directly from water to Hoyer{\textquoteright}s medium. However, specimens mounted in Hoyer{\textquoteright}s medium degrade over time. For example, the fine denticles on the larval dorsum are best observed soon after mounting; they begin to fade after 1 week, and can disappear completely after several months. More robust features, such as the ventral denticle belts, will persist for a longer period of time. Because adults cannot profitably be mounted whole in Hoyer{\textquoteright}s medium, some dissection is necessary.

}, keywords = {Animals, Drosophila, Integumentary System, Specimen Handling}, issn = {1559-6095}, doi = {10.1101/pdb.prot067371}, author = {Stern, David L and Sucena, Elio} } @article {4650, title = {Morphological evolution caused by many subtle-effect substitutions in regulatory DNA.}, journal = {Nature}, volume = {474}, year = {2011}, month = {2011 Jun 30}, pages = {598-603}, abstract = {

Morphology evolves often through changes in developmental genes, but the causal mutations, and their effects, remain largely unknown. The evolution of naked cuticle on larvae of Drosophila sechellia resulted from changes in five transcriptional enhancers of shavenbaby (svb), a transcript of the ovo locus that encodes a transcription factor that governs morphogenesis of microtrichiae, hereafter called {\textquoteright}trichomes{\textquoteright}. Here we show that the function of one of these enhancers evolved through multiple single-nucleotide substitutions that altered both the timing and level of svb expression. The consequences of these nucleotide substitutions on larval morphology were quantified with a novel functional assay. We found that each substitution had a relatively small phenotypic effect, and that many nucleotide changes account for this large morphological difference. In addition, we observed that the substitutions had non-additive effects. These data provide unprecedented resolution of the phenotypic effects of substitutions and show how individual nucleotide changes in a transcriptional enhancer have caused morphological evolution.

}, keywords = {Animals, Base Sequence, Biological Evolution, Developmental, DNA-Binding Proteins, Drosophila, Drosophila melanogaster, Drosophila Proteins, Embryo, Enhancer Elements, Female, Gene Expression Regulation, Genetic, Larva, Male, Nonmammalian, Nucleic Acid, Phenotype, Sequence Alignment, Sequence Homology, Transcription Factors}, issn = {1476-4687}, doi = {10.1038/nature10200}, author = {Frankel, Nicol{\'a}s and Erezyilmaz, Deniz F and McGregor, Alistair P and Wang, Shu and Payre, Fran\c cois and Stern, David L} } @article {3565, title = {Multiplexed shotgun genotyping for rapid and efficient genetic mapping.}, journal = {Genome Research}, volume = {21}, year = {2011}, month = {2011 Apr}, pages = {610-7}, abstract = {

We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10 ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kb of its true position using 96 F1-backcross individuals genotyped in a single lane on an Illumina Genome Analyzer. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38-kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.

}, keywords = {Animals, Chromosome Breakpoints, Chromosome Mapping, Computational Biology, DNA, Dominant, Drosophila, Female, Genes, Genetic, Genetic Markers, Genotype, Male, Molecular Typing, Phenotype, Quantitative Trait Loci, Recombination, Research Design, Sequence Analysis}, issn = {1549-5469}, doi = {10.1101/gr.115402.110}, author = {Andolfatto, Peter and Davison, Dan and Erezyilmaz, Deniz and Hu, Tina T and Mast, Joshua and Sunayama-Morita, Tomoko and Stern, David L} } @article {45732, title = {Preparation of cuticles from feeding Drosophila larvae.}, journal = {Cold Spring Harb Protoc}, volume = {2011}, year = {2011}, month = {2011 Nov}, pages = {1394-8}, abstract = {

The Drosophila cuticle carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. Studies of the Drosophila cuticle have focused mainly on first-instar larvae and adult cuticular morphology. Although the cuticles of second- and third-instar larvae are strikingly different from those of the first instar, these differences have been poorly studied. This protocol describes three methods for preparing cuticles from fed larvae. One commonly used procedure involves manually pricking the larvae. A simpler method for preparing larval cuticles is to burst the larvae once they have been mounted. This method is used for first- and second-instar larvae and does not require pricking; it removes the gut contents by "popping" the rear of the embryo using pressure from the coverslip. If just the right amount of medium is used, the coverslip will be pulled toward the slide, applying pressure on the samples. The larvae usually burst from their posterior ends. Also presented is an alternative procedure designed specifically for the use with third-instar larvae, although the "pricking" method can be used at this stage.

}, keywords = {Animals, Dissection, Drosophila, Entomology, Female, Integumentary System, Larva}, issn = {1559-6095}, doi = {10.1101/pdb.prot066498}, author = {Stern, David L and Sucena, Elio} } @article {45733, title = {Preparation of cuticles from unhatched first-instar Drosophila larvae.}, journal = {Cold Spring Harb Protoc}, volume = {2011}, year = {2011}, month = {2011 Sep}, abstract = {

The finely sculpted cuticle of Drosophila carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. Studies of the Drosophila cuticle have focused mainly on first-instar larvae and adult cuticular morphology. This protocol describes the preparation of cuticles from larvae that have not yet hatched from the egg. It is designed for sampling all eggs laid by one or more females. This can be particularly useful, for example, when a mutation produces embryos that are unable to hatch from the egg.

}, keywords = {Animals, Drosophila, Entomology, Female, Integumentary System, Larva}, issn = {1559-6095}, doi = {10.1101/pdb.prot065532}, author = {Stern, David L and Sucena, Elio} } @book {45796, title = {Evolution, Development, and the Predictable Genome}, year = {2010}, publisher = {Roberts and Company Publishers}, organization = {Roberts and Company Publishers}, address = {Austin, TX}, abstract = {

For too long, efforts to synthesize evolution and development have failed to build a united view of the origins and evolution of biological diversity. In this groundbreaking book, David Stern sets out to draw evolutionary biology and developmental biology together by cutting through the differences that divide the disciplines and by revealing their deeper similarities. He draws upon the insights of generations of evolutionary biologists and scores of developmental biologists to build a solid foundation for future investigation of the genetic and developmental causes of diversity. Along the way, and in plain English, he explicates many of the guiding principles of evolution, population genetics, and developmental biology. Each chapter offers a clear review of fundamental principles, together with thoughtprovoking ideas that will be tested only with data emerging from current and future studies. With the basic principles established, he then offers a new way of thinking about development{\textemdash}backwards{\textemdash}to clarify precisely how the mechanisms of development influence evolution. In the same spirit, he takes a fresh look at evolution in populations, arguing that population history influences precisely how developmental mechanisms evolve. Both Stern{\textquoteright}s new perspective on development and his reassessment of the role of populations leads to the surprising conclusion that the evolution of genomes appears to be predictable. Stern argues that developmental biology and evolutionary biology are intertwined: it is impossible to understand one of them fully without understanding the other. This book provides a clear and wide-ranging introduction to evolution and development for the basic reader; graduate students will be introduced to the cutting-edge of research in evolutionary developmental biology; and experts in evolution or development will receive both an uncomplicated introduction to the other discipline and an abundance of new, provocative ideas.

Stern, David L. Evolution, Development, and the Predictable Genome. Austin, TX: Roberts and Company Publishers, 2010.

}, isbn = {9781936221011}, url = {http://www.roberts-publishers.com/evolution-development-and-the-predictable-genome-physical.html}, author = {Stern, David L} } @article {4406, title = {Michael Akam and the rise of evolutionary developmental biology.}, journal = {The International Journal of Developmental Biology}, volume = {54}, year = {2010}, month = {2010}, pages = {561-5}, abstract = {

Michael Akam has been awarded the 2007 Kowalevsky medal for his many research accomplishments in the area of evolutionary developmental biology. We highlight three tributaries of Michaels contribution to evolutionary developmental biology. First, he has made major contributions to our understanding of development of the fruit fly, Drosophila melanogaster. Second, he has maintained a consistent focus on several key problems in evolutionary developmental biology, including the evolving role of Hox genes in arthropods and, more recently, the evolution of segmentation mechanisms. Third, Michael has written a series of influential reviews that have integrated progress in developmental biology into an evolutionary perspective. Michael has also made a large impact on the field through his effective mentorship style, his selfless promotion of younger colleagues, and his leadership of the University Museum of Zoology at Cambridge and the European community of evolutionary developmental biologists.

}, keywords = {20th Century, 21st Century, Animals, Arthropods, Awards and Prizes, Biological Evolution, Developmental Biology, Drosophila melanogaster, England, Genes, History, Homeobox, Research}, issn = {1696-3547}, doi = {10.1387/ijdb.092908ds}, author = {Stern, David L and Dawes-Hoang, Rachel E} } @article {4401, title = {Phenotypic robustness conferred by apparently redundant transcriptional enhancers.}, journal = {Nature}, volume = {466}, year = {2010}, month = {2010 Jul 22}, pages = {490-3}, abstract = {

Genes include cis-regulatory regions that contain transcriptional enhancers. Recent reports have shown that developmental genes often possess multiple discrete enhancer modules that drive transcription in similar spatio-temporal patterns: primary enhancers located near the basal promoter and secondary, or {\textquoteright}shadow{\textquoteright}, enhancers located at more remote positions. It has been proposed that the seemingly redundant activity of primary and secondary enhancers contributes to phenotypic robustness. We tested this hypothesis by generating a deficiency that removes two newly discovered enhancers of shavenbaby (svb, a transcript of the ovo locus), a gene encoding a transcription factor that directs development of Drosophila larval trichomes. At optimal temperatures for embryonic development, this deficiency causes minor defects in trichome patterning. In embryos that develop at both low and high extreme temperatures, however, absence of these secondary enhancers leads to extensive loss of trichomes. These temperature-dependent defects can be rescued by a transgene carrying a secondary enhancer driving transcription of the svb cDNA. Finally, removal of one copy of wingless, a gene required for normal trichome patterning, causes a similar loss of trichomes only in flies lacking the secondary enhancers. These results support the hypothesis that secondary enhancers contribute to phenotypic robustness in the face of environmental and genetic variability.

}, keywords = {Animal Structures, Animals, Developmental, DNA-Binding Proteins, Drosophila melanogaster, Drosophila Proteins, Enhancer Elements, Gene Expression Regulation, Genetic, Larva, Models, Phenotype, Temperature, Transcription, Transcription Factors, Transgenes}, issn = {1476-4687}, doi = {10.1038/nature09158}, author = {Frankel, Nicol{\'a}s and Davis, Gregory K and Vargas, Diego and Wang, Shu and Payre, Fran{\c c}ois and Stern, David L} } @article {4411, title = {Evolution of the tan locus contributed to pigment loss in Drosophila santomea: a response to Matute et al.}, journal = {Cell}, volume = {139}, year = {2009}, month = {2009 Dec 11}, pages = {1189-96}, abstract = {

We have shown previously that the loss of abdominal pigmentation in D. santomea relative to its sister species D. yakuba resulted, in part, from cis-regulatory mutations at the tan locus. Matute et al. claim, based solely upon extrapolation from genetic crosses of D. santomea and D. melanogaster, a much more divergent species, that at least four X chromosome regions but not tan are responsible for pigmentation differences. Here, we provide additional evidence from introgressions of D. yakuba genes into D. santomea that support a causative role for tan in the loss of pigmentation and present analyses that contradict Matute et al.{\textquoteright}s claims. We discuss how the choice of parental species and other factors affect the ability to identify loci responsible for species divergence, and we affirm that all of our previously reported results and conclusions stand.

}, keywords = {Animals, Chimera, Chromosomal Proteins, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Evolution, Molecular, Non-Histone, Pigmentation, Species Specificity, X Chromosome}, issn = {1097-4172}, doi = {10.1016/j.cell.2009.11.004}, author = {Rebeiz, Mark and Ramos-Womack, Margarita and Jeong, Sangyun and Andolfatto, Peter and Werner, Thomas and True, John and Stern, David L and Carroll, Sean B} } @article {3566, title = {Is genetic evolution predictable?}, journal = {Science}, volume = {323}, year = {2009}, month = {2009 Feb 6}, pages = {746-51}, abstract = {

Ever since the integration of Mendelian genetics into evolutionary biology in the early 20th century, evolutionary geneticists have for the most part treated genes and mutations as generic entities. However, recent observations indicate that all genes are not equal in the eyes of evolution. Evolutionarily relevant mutations tend to accumulate in hotspot genes and at specific positions within genes. Genetic evolution is constrained by gene function, the structure of genetic networks, and population biology. The genetic basis of evolution may be predictable to some extent, and further understanding of this predictability requires incorporation of the specific functions and characteristics of genes into evolutionary theory.

}, keywords = {Animals, Arabidopsis, Biological Evolution, Drosophila, Epistasis, Evolution, Gene Regulatory Networks, Genes, Genetic, Genetic Speciation, Genetic Variation, Molecular, Mutation, Phenotype, Plants, Population Dynamics, Selection}, issn = {1095-9203}, doi = {10.1126/science.1158997}, author = {Stern, David L and Orgogozo, Virginie} } @article {4409, title = {How different are recently diverged species?: more than 150 phenotypic differences have been reported for the D. melanogaster species subgroup.}, journal = {Fly}, volume = {3}, year = {2009}, month = {2009 Apr-Jun}, pages = {117}, keywords = {Animals, Drosophila melanogaster, Genetic Speciation, Phenotype, Species Specificity}, issn = {1933-6942}, author = {Orgogozo, Virginie and Stern, David L} } @article {4410, title = {Similar patterns of linkage disequilibrium and nucleotide diversity in native and introduced populations of the pea aphid, Acyrthosiphon pisum.}, journal = {BMC Genetics}, volume = {10}, year = {2009}, month = {2009 May 26}, pages = {22}, abstract = {

The pea aphid, Acyrthosiphon pisum, is an emerging genomic model system for studies of polyphenisms, bacterial symbioses, host-plant specialization, and the vectoring of plant viruses. Here we provide estimates of nucleotide diversity and linkage disequilibrium (LD) in native (European) and introduced (United States) populations of the pea aphid. Because introductions can cause population bottlenecks, we hypothesized that U.S. populations harbor lower levels of nucleotide diversity and higher levels of LD than native populations.

}, keywords = {Animals, Aphids, Europe, Genetic Variation, Genetics, INDEL Mutation, Linkage Disequilibrium, North America, Nucleotides, Population}, issn = {1471-2156}, doi = {10.1186/1471-2156-10-22}, author = {Brisson, Jennifer A and Nuzhdin, Sergey V and Stern, David L} } @article {45737, title = {Aphids.}, journal = {Curr Biol}, volume = {18}, year = {2008}, month = {2008 Jun 24}, pages = {R504-5}, abstract = {

Mammalian herbivores profoundly influence plant-dwelling insects [1]. Most studies have focused on the indirect effect of herbivory on insect populations via damage to the host plant [2,3]. Many insects, however, are in danger of being inadvertently ingested during herbivore feeding. Here, we report that pea aphids (Acyrthosiphon pisum) are able to sense the elevated heat and humidity of the breath of an approaching herbivore and thus salvage most of the colony by simultaneously dropping off the plant in large numbers immediately before the plant is eaten. Dropping entails the risk of losing the host plant and becoming desiccated or preyed upon on the ground [4,5], yet pea aphids may sporadically drop when threatened by insect enemies [6]. The immediate mass dropping, however, is an adaptation to the potential destructive impact of mammalian herbivory on the entire aphid colony. The combination of heat and humidity serves as a reliable cue to impending mammalian herbivory, enabling the aphids to avoid unnecessary dropping. No defensive behavior against incidental predation by herbivores has ever been demonstrated. The pea aphids{\textquoteright} highly adaptive escape behavior uniquely demonstrates the strength of the selective pressure large mammalian herbivores impose on insect herbivores.

}, keywords = {Animals, Aphids, Female, Genome, Insect, Insect Vectors, Male, Peas, Plant Diseases, Plant Tumors, Styrax}, issn = {0960-9822}, doi = {10.1016/j.cub.2008.03.034}, author = {Stern, David L} } @article {4648, title = {The loci of evolution: how predictable is genetic evolution?}, journal = {Evolution}, volume = {62}, year = {2008}, month = {2008 Sep}, pages = {2155-77}, abstract = {

Is genetic evolution predictable? Evolutionary developmental biologists have argued that, at least for morphological traits, the answer is a resounding yes. Most mutations causing morphological variation are expected to reside in the cis-regulatory, rather than the coding, regions of developmental genes. This "cis-regulatory hypothesis" has recently come under attack. In this review, we first describe and critique the arguments that have been proposed in support of the cis-regulatory hypothesis. We then test the empirical support for the cis-regulatory hypothesis with a comprehensive survey of mutations responsible for phenotypic evolution in multicellular organisms. Cis-regulatory mutations currently represent approximately 22\% of 331 identified genetic changes although the number of cis-regulatory changes published annually is rapidly increasing. Above the species level, cis-regulatory mutations altering morphology are more common than coding changes. Also, above the species level cis-regulatory mutations predominate for genes not involved in terminal differentiation. These patterns imply that the simple question "Do coding or cis-regulatory mutations cause more phenotypic evolution?" hides more interesting phenomena. Evolution in different kinds of populations and over different durations may result in selection of different kinds of mutations. Predicting the genetic basis of evolution requires a comprehensive synthesis of molecular developmental biology and population genetics.

}, keywords = {Animals, Evolution, Humans, Molecular, Phenotype, Regulatory Elements, Transcriptional}, issn = {0014-3820}, doi = {10.1111/j.1558-5646.2008.00450.x}, author = {Stern, David L and Orgogozo, Virginie} } @article {45736, title = {Tinker where the tinkering{\textquoteright}s good.}, journal = {Trends Genet}, volume = {24}, year = {2008}, month = {2008 Jul}, pages = {317-9}, abstract = {

Do general principles govern the genetic causes of phenotypic evolution? One promising idea is that mutations in cis-regulatory regions play a predominant role in phenotypic evolution because they can alter gene activity without causing pleiotropic effects. Recent evidence that revealed the genetic basis of pigmentation pattern evolution in Drosophila santomea supports this notion. Multiple mutations that disrupt an abdominal enhancer of the pleiotropic gene tan partly explain the reduced pigmentation observed in this species.

}, keywords = {Abdomen, Animals, Biological Evolution, Drosophila melanogaster, Genes, Insect, Mutation, Phenotype, Pigmentation}, issn = {0168-9525}, doi = {10.1016/j.tig.2008.04.003}, author = {Rockman, Matthew V and Stern, David L} } @article {45739, title = {Common genome-wide patterns of transcript accumulation underlying the wing polyphenism and polymorphism in the pea aphid (Acyrthosiphon pisum).}, journal = {Evol Dev}, volume = {9}, year = {2007}, month = {2007 Jul-Aug}, pages = {338-46}, abstract = {

The pea aphid, Acyrthosiphon pisum, exhibits several environmentally cued polyphenisms, in which discrete, alternative phenotypes are produced. At low-density, parthenogenetic females produce unwinged female progeny, but at high-density females produce progeny that develop with wings. These alternative phenotypes represent a solution to the competing demands of dispersal and reproduction. Males also develop as either winged or unwinged, but these alternatives are determined by a genetic polymorphism. Winged and unwinged males are morphologically less distinct from each other than winged and unwinged females, possibly because males experience fewer trade-offs between dispersal and reproduction. To assess whether shared physiological differences mirror the shared morphological differences that characterize the wing polyphenism and polymorphism, we used a cDNA microarray representing an estimated 10\% of the coding genome (1734 genes) to examine differential transcript accumulation between winged and unwinged females and males. We identified several transcripts that differentially accumulate between winged and unwinged morphs in both sexes, the majority of which are involved in energy production. Unexpectedly, the extent of differential transcript accumulation between winged and unwinged morphs was greater for adult males than for adult females. Together, these results suggest not only that similar physiological differences underlie the polyphenism and polymorphism, but that male morphs, like females, are subject to trade-offs between reproduction and dispersal that are reflected in levels of transcript accumulation and possibly genome-wide patterns of gene regulation. These data also provide a baseline for future studies of the molecular and physiological basis of life-history trade-offs.

}, keywords = {Animals, Aphids, Female, Genome, Male, Phenotype, Polymorphism, Genetic, RNA, Sex Characteristics, Wing}, issn = {1520-541X}, doi = {10.1111/j.1525-142X.2007.00170.x}, author = {Brisson, Jennifer A and Davis, Gregory K and Stern, David L} } @article {45744, title = {The developmental genetics of microevolution.}, journal = {Novartis Found Symp}, volume = {284}, year = {2007}, month = {2007}, pages = {191-200; discussion 200-6}, abstract = {

What is the relationship between variation that segregates within natural populations and the differences that distinguish species? Many studies over the past century have demonstrated that most of the genetic variation within natural populations that contributes to quantitative traits causes relatively small phenotypic effects. In contrast, the genetic causes of quantitative differences between species are at least sometimes caused by few loci of relatively large effect. In addition, most of the results from evolutionary developmental biology are often discussed as though changes at just a few important {\textquoteright}molecular toolbox{\textquoteright} genes provide the key clues to morphological evolution. On the face of it, these divergent results seem incompatible and call into question the neo-Darwinian view that differences between species emerge from precisely the same kinds of variants that segregate much of the time in natural populations. One prediction from the classical model is that many different genes can evolve to generate similar phenotypes. I discuss our studies that demonstrate that similar phenotypes have evolved in multiple lineages of Drosophila by evolution of the same gene, shavenbaby/ovo. This evidence for parallel evolution suggests that svb occupies a privileged position in the developmental network patterning larval trichomes that makes it a favourable target of evolutionary change.

}, keywords = {Animals, Biological Evolution, Developmental Biology, Drosophila, Evolution, Molecular, Gene Expression Regulation, Developmental, Genes, Insect, Genetic Variation, Models, Biological, Models, Genetic, Models, Molecular, Phenotype, Phylogeny}, issn = {1528-2511}, author = {Stern, David L} } @article {45741, title = {Evolution. An embarrassment of switches.}, journal = {Science}, volume = {317}, year = {2007}, month = {2007 Aug 10}, pages = {758-9}, keywords = {Animals, Binding Sites, Chromatin Immunoprecipitation, Evolution, Molecular, Gene Expression Regulation, Humans, Mutation, Oligonucleotide Array Sequence Analysis, Phenotype, Regulatory Sequences, Nucleic Acid, Species Specificity, Transcription Factors, Yeasts}, issn = {1095-9203}, doi = {10.1126/science.1146921}, author = {Kruglyak, Leonid and Stern, David L} } @article {45740, title = {The function and regulation of Ultrabithorax in the legs of Drosophila melanogaster.}, journal = {Dev Biol}, volume = {308}, year = {2007}, month = {2007 Aug 15}, pages = {621-31}, abstract = {

Alterations in Hox gene expression patterns have been implicated in both large and small-scale morphological evolution. An improved understanding of these changes requires a detailed understanding of Hox gene cis-regulatory function and evolution. cis-regulatory evolution of the Hox gene Ultrabithorax (Ubx) has been shown to contribute to evolution of trichome patterns on the posterior second femur (T2p) of Drosophila species. As a step toward determining how this function of Ubx has evolved, we performed a series of experiments to clarify the role of Ubx in patterning femurs and to identify the cis-regulatory regions of Ubx that drive expression in T2p. We first performed clonal analysis to further define Ubx function in patterning bristle and trichome patterns in the legs. We found that low levels of Ubx expression are sufficient to repress an eighth bristle row on the posterior second and third femurs, whereas higher levels of expression are required to promote the development and migration of other bristles on the third femur and to repress trichomes. We then tested the hypothesis that the evolutionary difference in T2p trichome patterns due to Ubx was caused by a change in the global cis-regulation of Ubx expression. We found no evidence to support this view, suggesting that the evolved difference in Ubx function reflects evolution of a leg-specific enhancer. We then searched for the regulatory regions of the Ubx locus that drive expression in the second and third femur by assaying all existing regulatory mutations of the Ubx locus and new deficiencies in the large intron of Ubx that we generated by P-element-induced male recombination. We found that two enhancer regions previously known to regulate Ubx expression in the legs, abx and pbx, are required for Ubx expression in the third femur, but that they do not contribute to pupal expression of Ubx in the second femur. This analysis allowed us to rule out at least 100 kb of DNA in and around the Ubx locus as containing a T2p-specific enhancer. We then surveyed an additional approximately 30 kb using enhancer constructs. None of these enhancer constructs produced an expression pattern similar to Ubx expression in T2p. Thus, after surveying over 95\% of the Ubx locus, we have not been able to localize a T2p-specific enhancer. While the enhancer could reside within the small regions we have not surveyed, it is also possible that the enhancer is structurally complex and/or acts only within its native genomic context.

}, keywords = {Animals, Base Sequence, Body Patterning, DNA, Drosophila, Drosophila melanogaster, Drosophila Proteins, Enhancer Elements, Genetic, Evolution, Molecular, Extremities, Female, Gene Expression Regulation, Developmental, Genes, Homeobox, Genes, Insect, Genetic Complementation Test, Homeodomain Proteins, Male, Mutation, Recombination, Genetic, Species Specificity, Transcription Factors}, issn = {0012-1606}, doi = {10.1016/j.ydbio.2007.06.002}, author = {Davis, Gregory K and Srinivasan, Dayalan G and Wittkopp, Patricia J and Stern, David L} } @article {45738, title = {Internal and external constraints in the evolution of morphological allometries in a butterfly.}, journal = {Evolution}, volume = {61}, year = {2007}, month = {2007 Dec}, pages = {2958-70}, abstract = {

Much diversity in animal morphology results from variation in the relative size of morphological traits. The scaling relationships, or allometries, that describe relative trait size can vary greatly in both intercept and slope among species or other animal groups. Yet within such groups, individuals typically exhibit low variation in relative trait size. This pattern of high intra- and low intergroup variation may result from natural selection for particular allometries, from developmental constraints restricting differential growth among traits, or both. Here we explore the relative roles of short-term developmental constraints and natural selection in the evolution of the intercept of the allometry between the forewing and hindwing of a butterfly. First, despite a strong genetic correlation between these two traits, we show that artificial selection perpendicular to the forewing-hindwing scaling relationship results in rapid evolution of the allometry intercept. This demonstrates an absence of developmental constraints limiting intercept evolution for this scaling relationship. Mating experiments in a natural environment revealed strong stabilizing selection favoring males with the wild-type allometry intercept over those with derived intercepts. Our results demonstrate that evolution of this component of the forewing-hindwing allometry is not limited by developmental constraints in the short term and that natural selection on allometry intercepts can be powerful.

}, keywords = {Animals, Biological Evolution, Body Size, Butterflies, Genetic Variation, Phenotype, Selection, Genetic, Wing}, issn = {0014-3820}, doi = {10.1111/j.1558-5646.2007.00249.x}, author = {Frankino, W Anthony and Zwaan, Bas J and Stern, David L and Brakefield, Paul M} } @article {4649, title = {Morphological evolution through multiple cis-regulatory mutations at a single gene.}, journal = {Nature}, volume = {448}, year = {2007}, month = {2007 Aug 2}, pages = {587-90}, abstract = {

One central, and yet unsolved, question in evolutionary biology is the relationship between the genetic variants segregating within species and the causes of morphological differences between species. The classic neo-darwinian view postulates that species differences result from the accumulation of small-effect changes at multiple loci. However, many examples support the possible role of larger abrupt changes in the expression of developmental genes in morphological evolution. Although this evidence might be considered a challenge to a neo-darwinian micromutationist view of evolution, there are currently few examples of the actual genes causing morphological differences between species. Here we examine the genetic basis of a trichome pattern difference between Drosophila species, previously shown to result from the evolution of a single gene, shavenbaby (svb), probably through cis-regulatory changes. We first identified three distinct svb enhancers from D. melanogaster driving reporter gene expression in partly overlapping patterns that together recapitulate endogenous svb expression. All three homologous enhancers from D. sechellia drive expression in modified patterns, in a direction consistent with the evolved svb expression pattern. To test the influence of these enhancers on the actual phenotypic difference, we conducted interspecific genetic mapping at a resolution sufficient to recover multiple intragenic recombinants. This functional analysis revealed that independent genetic regions upstream of svb that overlap the three identified enhancers are collectively required to generate the D. sechellia trichome pattern. Our results demonstrate that the accumulation of multiple small-effect changes at a single locus underlies the evolution of a morphological difference between species. These data support the view that alleles of large effect that distinguish species may sometimes reflect the accumulation of multiple mutations of small effect at select genes.

}, keywords = {Alleles, Animals, Biological, Biological Evolution, Developmental, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Enhancer Elements, Evolution, Gene Expression Regulation, Genes, Genetic, Genetic Speciation, Models, Molecular, Mutation, Recombination, Reporter, Transcription Factors}, issn = {1476-4687}, doi = {10.1038/nature05988}, author = {McGregor, Alistair P and Orgogozo, Virginie and Delon, Isabelle and Zanet, Jennifer and Srinivasan, Dayalan G and Payre, Fran\c cois and Stern, David L} } @article {45745, title = {Sex and death in the male pea aphid, Acyrthosiphon pisum: The life-history effects of a wing dimorphism.}, journal = {J Insect Sci}, volume = {7}, year = {2007}, month = {2007}, pages = {1-9}, abstract = {

Insect dispersal dimorphisms, in which both flight-capable and flightless individuals occur in the same species, are thought to reflect a balance between the benefits and costs of dispersal. Fitness costs and benefits associated with wing dimorphism were investigated in the male pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae). In one-on-one mating competitions in small arenas between winged and wingless males, the winged aphids obtained most of the matings with virgin females. In contrast, during competition experiments in larger cages with multiple individuals of each morph, the winged males no longer had a clear mating advantage over wingless males. In the absence of competition, wingless males had marginally higher lifetime reproductive success than winged males, probably because mating winged males tended to die faster than wingless males. In the absence of females, winged males survived longer than wingless males and this difference disappeared under starvation conditions. Mating males of both morphs died significantly faster than males without access to females. There does not appear to be a direct tradeoff of dispersal ability with life history characteristics in pea aphid males, suggesting that the advantages of producing winged males may result from outbreeding.

}, keywords = {Animals, Aphids, Female, Fertility, Longevity, Male, Sex Characteristics, Sexual Behavior, Animal, Wing}, issn = {1536-2442}, doi = {10.1673/031.007.4501}, author = {Sack, Coralynn and Stern, David L} } @article {45742, title = {Variation in fiber number of a male-specific muscle between Drosophila species: a genetic and developmental analysis.}, journal = {Evol Dev}, volume = {9}, year = {2007}, month = {2007 Jul-Aug}, pages = {368-77}, abstract = {

We characterize a newly discovered morphological difference between species of the Drosophila melanogaster subgroup. The muscle of Lawrence (MOL) contains about four to five fibers in D. melanogaster and Drosophila simulans and six to seven fibers in Drosophila mauritiana and Drosophila sechellia. The same number of nuclei per fiber is present in these species but their total number of MOL nuclei differs. This suggests that the number of muscle precursor cells has changed during evolution. Our comparison of MOL development indicates that the species difference appears during metamorphosis. We mapped the quantitative trait loci responsible for the change in muscle fiber number between D. sechellia and D. simulans to two genomic regions on chromosome 2. Our data eliminate the possibility of evolving mutations in the fruitless gene and suggest that a change in the twist might be partly responsible for this evolutionary change.

}, keywords = {Animals, Drosophila, Genetic Variation, Male, Muscle Fibers, Skeletal, Quantitative Trait Loci}, issn = {1520-541X}, doi = {10.1111/j.1525-142X.2007.00174.x}, author = {Orgogozo, Virginie and Muro, Noelle M and Stern, David L} } @article {45746, title = {Developmental biology. Morphing into shape.}, journal = {Science}, volume = {313}, year = {2006}, month = {2006 Jul 7}, pages = {50-1}, keywords = {Animals, Biological Evolution, Body Size, Cell Proliferation, Drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Developmental, Genes, Insect, Homeodomain Proteins, Morphogenesis, Organ Size, Protein-Serine-Threonine Kinases, Receptors, Cell Surface, Signal Transduction, Transcription Factors, Wing}, issn = {1095-9203}, doi = {10.1126/science.1130785}, author = {Stern, David L} } @article {45750, title = {A dual-genome microarray for the pea aphid, Acyrthosiphon pisum, and its obligate bacterial symbiont, Buchnera aphidicola.}, journal = {BMC Genomics}, volume = {7}, year = {2006}, month = {Mar 2006}, pages = {50}, abstract = {

BACKGROUND: The best studied insect-symbiont system is that of aphids and their primary bacterial endosymbiont Buchnera aphidicola. Buchnera inhabits specialized host cells called bacteriocytes, provides nutrients to the aphid and has co-speciated with its aphid hosts for the past 150 million years. We have used a single microarray to examine gene expression in the pea aphid, Acyrthosiphon pisum, and its resident Buchnera. Very little is known of gene expression in aphids, few studies have examined gene expression in Buchnera, and no study has examined simultaneously the expression profiles of a host and its symbiont. Expression profiling of aphids, in studies such as this, will be critical for assigning newly discovered A. pisum genes to functional roles. In particular, because aphids possess many genes that are absent from Drosophila and other holometabolous insect taxa, aphid genome annotation efforts cannot rely entirely on homology to the best-studied insect systems. Development of this dual-genome array represents a first attempt to characterize gene expression in this emerging model system.

RESULTS: We chose to examine heat shock response because it has been well characterized both in Buchnera and in other insect species. Our results from the Buchnera of A. pisum show responses for the same gene set as an earlier study of heat shock response in Buchnera for the host aphid Schizaphis graminum. Additionally, analyses of aphid transcripts showed the expected response for homologs of known heat shock genes as well as responses for several genes with unknown functional roles.

CONCLUSION: We examined gene expression under heat shock of an insect and its bacterial symbiont in a single assay using a dual-genome microarray. Further, our results indicate that microarrays are a useful tool for inferring functional roles of genes in A. pisum and other insects and suggest that the pea aphid genome may contain many gene paralogs that are differentially regulated.

}, keywords = {Animals, Aphids, Buchnera, Expressed Sequence Tags, Gene Expression Profiling, Gene Expression Regulation, Genome, Insect, Genomics, Heat-Shock Response, Oligonucleotide Array Sequence Analysis, Symbiosis}, issn = {1471-2164}, doi = {10.1186/1471-2164-7-50}, author = {Wilson, Alex C C and Dunbar, Helen E and Davis, Gregory K and Hunter, Wayne B and Stern, David L and Moran, Nancy A} } @article {45749, title = {High-resolution quantitative trait locus mapping reveals sign epistasis controlling ovariole number between two Drosophila species.}, journal = {Genetics}, volume = {173}, year = {2006}, month = {2006 May}, pages = {197-205}, abstract = {

Identifying the genes underlying genetically complex traits is of fundamental importance for medicine, agriculture, and evolutionary biology. However, the level of resolution offered by traditional quantitative trait locus (QTL) mapping is usually coarse. We analyze here a trait closely related to fitness, ovariole number. Our initial interspecific mapping between Drosophila sechellia (8 ovarioles/ovary) and D. simulans (15 ovarioles/ovary) identified a major QTL on chromosome 3 and a minor QTL on chromosome 2. To refine the position of the major QTL, we selected 1038 additional recombinants in the region of interest using flanking morphological markers (selective phenotyping). This effort generated approximately one recombination event per gene and increased the mapping resolution by approximately seven times. Our study thus shows that using visible markers to select for recombinants can efficiently increase the resolution of QTL mapping. We resolved the major QTL into two epistatic QTL, QTL3a and QTL3b. QTL3a shows sign epistasis: it has opposite effects in two different genetic backgrounds, the presence vs. the absence of the QTL3b D. sechellia allele. This property of QTL3a allows us to reconstruct the probable order of fixation of the QTL alleles during evolution.

}, keywords = {Animals, Chromosome Mapping, Chromosomes, Crosses, Genetic, Drosophila, Epistasis, Genetic, Evolution, Molecular, Female, Ovary, Quantitative Trait Loci}, issn = {0016-6731}, doi = {10.1534/genetics.105.054098}, author = {Orgogozo, Virginie and Broman, Karl W and Stern, David L} } @article {45751, title = {Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera).}, journal = {Genome Biol}, volume = {7}, year = {2006}, month = {2006}, pages = {R21}, abstract = {

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A. pisum. An in silico profiling analysis characterized 135 transcripts specific to pea-aphid tissues (relating to bacteriocytes and parthenogenetic embryos). This project is the first to address the genetics of the Hemiptera and of a hemimetabolous insect.

}, keywords = {Animals, Aphids, Base Composition, Base Sequence, DNA, DNA, Complementary, Expressed Sequence Tags, Gene Library, Microsatellite Repeats, Peas, Phylogeny, Plant Diseases, Population Density, Transcription, Genetic}, issn = {1465-6914}, doi = {10.1186/gb-2006-7-3-r21}, author = {Sabater-Mu{\~n}oz, Beatriz and Legeai, Fabrice and Rispe, Claude and Bonhomme, Jo{\"e}l and Dearden, Peter and Dossat, Carole and Duclert, Aymeric and Gauthier, Jean-Pierre and Ducray, Dani{\`e}le Giblot and Hunter, Wayne and Dang, Phat and Kambhampati, Srini and Martinez-Torres, David and Cortes, Teresa and Moya, Andr{\`e}s and Nakabachi, Atsushi and Philippe, Cathy and Prunier-Leterme, Nathalie and Rahb{\'e}, Yvan and Simon, Jean-Christophe and Stern, David L and Wincker, Patrick and Tagu, Denis} } @article {45748, title = {The pea aphid, Acyrthosiphon pisum: an emerging genomic model system for ecological, developmental and evolutionary studies.}, journal = {Bioessays}, volume = {28}, year = {2006}, month = {2006 Jul}, pages = {747-55}, abstract = {

Aphids display an abundance of adaptations that are not easily studied in existing model systems. Here we review the biology of a new genomic model system, the pea aphid, Acyrthosiphon pisum. We then discuss several phenomena that are particularly accessible to study in the pea aphid: the developmental genetic basis of polyphenisms, aphid-bacterial symbioses, the genetics of adaptation and mechanisms of virus transmission. The pea aphid can be maintained in the laboratory and natural populations can be studied in the field. These properties allow controlled experiments to be performed on problems of direct relevance to natural aphid populations. Combined with new genomic approaches, the pea aphid is poised to become an important model system for understanding the molecular and developmental basis of many ecologically and evolutionarily relevant problems.

}, keywords = {Adaptation, Biological, Animals, Aphids, Biological Evolution, Ecology, Female, Genome, Insect, Life Cycle Stages, Male, Models, Genetic, Peas, Plant Viruses, Polymorphism, Genetic, Symbiosis}, issn = {0265-9247}, doi = {10.1002/bies.20436}, author = {Brisson, Jennifer A and Stern, David L} } @article {45756, title = {Genetic variation for an aphid wing polyphenism is genetically linked to a naturally occurring wing polymorphism.}, journal = {Proc Biol Sci}, volume = {272}, year = {2005}, month = {2005 Mar 22}, pages = {657-64}, abstract = {

Many polyphenisms are examples of adaptive phenotypic plasticity where a single genotype produces distinct phenotypes in response to environmental cues. Such alternative phenotypes occur as winged and wingless parthenogenetic females in the pea aphid (Acyrthosiphon pisum). However, the proportion of winged females produced in response to a given environmental cue varies between clonal genotypes. Winged and wingless phenotypes also occur in males of the sexual generation. In contrast to parthenogenetic females, wing production in males is environmentally insensitive and controlled by the sex-linked, biallelic locus, aphicarus (api). Hence, environmental or genetic cues induce development of winged and wingless phenotypes at different stages of the pea aphid life cycle. We have tested whether allelic variation at the api locus explains genetic variation in the propensity to produce winged females. We assayed clones from an F2 cross that were heterozygous or homozygous for alternative api alleles for their propensity to produce winged offspring. We found that clones with different api genotypes differed in their propensity to produce winged offspring. The results indicate genetic linkage of factors controlling the female wing polyphenism and male wing polymorphism. This finding is consistent with the hypothesis that genotype by environment interaction at the api locus explains genetic variation in the environmentally cued wing polyphenism.

}, keywords = {Alleles, Analysis of Variance, Animals, Aphids, Crosses, Genetic, Crowding, Female, Food Deprivation, Genes, Insect, Genetic Linkage, Genetic Variation, Genotype, Male, Phenotype, Wing}, issn = {0962-8452}, doi = {10.1098/rspb.2004.2995}, author = {Braendle, Christian and Friebe, Ilvy and Caillaud, Marina C and Stern, David L} } @article {45757, title = {Natural selection and developmental constraints in the evolution of allometries.}, journal = {Science}, volume = {307}, year = {2005}, month = {2005 Feb 4}, pages = {718-20}, abstract = {

In animals, scaling relationships between appendages and body size exhibit high interspecific variation but low intraspecific variation. This pattern could result from natural selection for specific allometries or from developmental constraints on patterns of differential growth. We performed artificial selection on the allometry between forewing area and body size in a butterfly to test for developmental constraints, and then used the resultant increased range of phenotypic variation to quantify natural selection on the scaling relationship. Our results show that the short-term evolution of allometries is not limited by developmental constraints. Instead, scaling relationships are shaped by strong natural selection.

}, keywords = {Alleles, Animals, Biological Evolution, Body Size, Butterflies, Crosses, Genetic, Female, Flight, Animal, Genetic Variation, Male, Phenotype, Reproduction, Selection, Genetic, Wing}, issn = {1095-9203}, doi = {10.1126/science.1105409}, author = {Frankino, W Anthony and Zwaan, Bas J and Stern, David L and Brakefield, Paul M} } @article {45755, title = {The origin of a mutualism: a morphological trait promoting the evolution of ant-aphid mutualisms.}, journal = {Evolution}, volume = {59}, year = {2005}, month = {2005 Apr}, pages = {921-6}, abstract = {

Mutualisms are mutually beneficial interactions between species and are fundamentally important at all levels of biological organization. It is not clear, however, why one species participates in a particular mutualism whereas another does not. Here we show that pre-existing traits can dispose particular species to evolve a mutualistic interaction. Combining morphological, ecological, and behavioral data in a comparative analysis, we show that resource use in Chaitophorus aphids (Hemiptera: Aphididae) modulates the origin of their mutualism with ants. We demonstrate that aphid species that feed on deeper phloem elements have longer mouthparts, that this inhibits their ability to withdraw their mouthparts and escape predators and that, consequently, this increases their need for protection by mutualist ants.

}, keywords = {Animals, Ants, Aphids, Biological Evolution, Body Weights and Measures, Feeding Behavior, Great Britain, Mouth, Symbiosis}, issn = {0014-3820}, author = {Shingleton, Alexander W and Stern, David L and Foster, William A} } @article {45753, title = {Pleiotropic functions of a conserved insect-specific Hox peptide motif.}, journal = {Development}, volume = {132}, year = {2005}, month = {2005 Dec}, pages = {5261-70}, abstract = {

The proteins that regulate developmental processes in animals have generally been well conserved during evolution. A few cases are known where protein activities have functionally evolved. These rare examples raise the issue of how highly conserved regulatory proteins with many roles evolve new functions while maintaining old functions. We have investigated this by analyzing the function of the ;QA{\textquoteright} peptide motif of the Hox protein Ultrabithorax (Ubx), a motif that has been conserved throughout insect evolution since its establishment early in the lineage. We precisely deleted the QA motif at the endogenous locus via allelic replacement in Drosophila melanogaster. Although the QA motif was originally characterized as involved in the repression of limb formation, we have found that it is highly pleiotropic. Curiously, deleting the QA motif had strong effects in some tissues while barely affecting others, suggesting that QA function is preferentially required for a subset of Ubx target genes. QA deletion homozygotes had a normal complement of limbs, but, at reduced doses of Ubx and the abdominal-A (abd-A) Hox gene, ectopic limb primordia and adult abdominal limbs formed when the QA motif was absent. These results show that redundancy and the additive contributions of activity-regulating peptide motifs play important roles in moderating the phenotypic consequences of Hox protein evolution, and that pleiotropic peptide motifs that contribute quantitatively to several functions are subject to intense purifying selection.

}, keywords = {Amino Acid Motifs, Animals, Conserved Sequence, Drosophila melanogaster, Drosophila Proteins, Extremities, Genes, Homeobox, Homeodomain Proteins, Phenotype, Sequence Deletion, Transcription Factors}, issn = {0950-1991}, doi = {10.1242/dev.02146}, author = {Hittinger, Chris Todd and Stern, David L and Carroll, Sean B} } @article {45754, title = {The temporal requirements for insulin signaling during development in Drosophila.}, journal = {PLoS Biol}, volume = {3}, year = {2005}, month = {2005 Sep}, pages = {e289}, abstract = {

Recent studies have indicated that the insulin-signaling pathway controls body and organ size in Drosophila, and most metazoans, by signaling nutritional conditions to the growing organs. The temporal requirements for insulin signaling during development are, however, unknown. Using a temperature-sensitive insulin receptor (Inr) mutation in Drosophila, we show that the developmental requirements for Inr activity are organ specific and vary in time. Early in development, before larvae reach the "critical size" (the size at which they commit to metamorphosis and can complete development without further feeding), Inr activity influences total development time but not final body and organ size. After critical size, Inr activity no longer affects total development time but does influence final body and organ size. Final body size is affected by Inr activity from critical size until pupariation, whereas final organ size is sensitive to Inr activity from critical size until early pupal development. In addition, different organs show different sensitivities to changes in Inr activity for different periods of development, implicating the insulin pathway in the control of organ allometry. The reduction in Inr activity is accompanied by a two-fold increase in free-sugar levels, similar to the effect of reduced insulin signaling in mammals. Finally, we find that varying the magnitude of Inr activity has different effects on cell size and cell number in the fly wing, providing a potential linkage between the mode of action of insulin signaling and the distinct downstream controls of cell size and number. We present a model that incorporates the effects of the insulin-signaling pathway into the Drosophila life cycle. We hypothesize that the insulin-signaling pathway controls such diverse effects as total developmental time, total body size and organ size through its effects on the rate of cell growth, and proliferation in different organs.

}, keywords = {Animals, Drosophila, Drosophila Proteins, Insect Hormones, Insulin, Insulin Receptor Substrate Proteins, Intracellular Signaling Peptides and Proteins, Larva, Mutation, Receptor Protein-Tyrosine Kinases, Signal Transduction, Time Factors}, issn = {1545-7885}, doi = {10.1371/journal.pbio.0030289}, author = {Shingleton, Alexander W and Das, Jayatri and Vinicius, Lucio and Stern, David L} } @article {45760, title = {Comparison of diverse protein sequences of the nuclear-encoded subunits of cytochrome C oxidase suggests conservation of structure underlies evolving functional sites.}, journal = {Mol Biol Evol}, volume = {21}, year = {2004}, month = {2004 Aug}, pages = {1572-82}, abstract = {

Interspecific comparisons of protein sequences can reveal regions of evolutionary conservation that are under purifying selection because of functional constraints. Interpreting these constraints requires combining evolutionary information with structural, biochemical, and physiological data to understand the biological function of conserved regions. We take this integrative approach to investigate the evolution and function of the nuclear-encoded subunits of cytochrome c oxidase (COX). We find that the nuclear-encoded subunits evolved subsequent to the origin of mitochondria and the subunit composition of the holoenzyme varies across diverse taxa that include animals, yeasts, and plants. By mapping conserved amino acids onto the crystal structure of bovine COX, we show that conserved residues are structurally organized into functional domains. These domains correspond to some known functional sites as well as to other uncharacterized regions. We find that amino acids that are important for structural stability are conserved at frequencies higher than expected within each taxon, and groups of conserved residues cluster together at distances of less than 5 A more frequently than do randomly selected residues. We, therefore, suggest that selection is acting to maintain the structural foundation of COX across taxa, whereas active sites vary or coevolve within lineages.

}, keywords = {Animals, Catalytic Domain, Cattle, Electron Transport Complex IV, Mitochondria, Models, Molecular, Plants, Yeasts}, issn = {0737-4038}, doi = {10.1093/molbev/msh161}, author = {Das, Jayatri and Miller, Stephen T and Stern, David L} } @article {45766, title = {A comparison of parthenogenetic and sexual embryogenesis of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea).}, journal = {J Exp Zool B Mol Dev Evol}, volume = {295}, year = {2003}, month = {2003 Feb 15}, pages = {59-81}, abstract = {

Aphids exhibit divergent modes of embryogenesis during the sexual and asexual phases of the life cycle. To explore how a single genome can give rise to these alternative developmental modes, we have initiated embryological studies of the pea aphid, Acyrthosiphon pisum. Here we present a detailed description of parthenogenetic, viviparous embryonic development in the pea aphid. We compare and contrast development of the parthenogenetic embryo with that of the embryo resulting from sexual reproduction. The primary difference between the embryos is the scale on which development occurs: early parthenogenetic development occurs in a volume approximately three orders of magnitude smaller than the sexual egg, largely because of the apparent absence of yolk in the parthenogenetic egg. This results in a drastically different duration of syncytial energid cleavage and, presumably, patterning processes in the two embryos must act at scales that differ by orders of magnitude. The eggs also develop on time scales that differ approximately by an order of magnitude and the timing of the embryonic movements, collectively called blastokinesis, have temporally shifted relative to growth of the embryo. In addition, the endosymbiotic bacteria are transferred from mother to embryo in different ways in the two embryos. Finally, the function of the serosa has diverged greatly in the two embryos: in the sexual egg the serosa deposits a thick cuticle that protects the egg, whereas the serosa of the parthenogenetic embryo is greatly reduced and its function is unclear. The pea aphid is a useful model system for examining how a single genome has evolved to allow divergent modes of development.

}, keywords = {Animals, Aphids, Embryo, Nonmammalian, Female, Germ Cells, Male, Morphogenesis, Ovum, Parthenogenesis, Reproduction}, issn = {1552-5007}, doi = {10.1002/jez.b.3}, author = {Miura, Toru and Braendle, Christian and Shingleton, Alexander and Sisk, Geoffroy and Kambhampati, Srinivas and Stern, David L} } @article {45761, title = {Developmental origin and evolution of bacteriocytes in the aphid-Buchnera symbiosis.}, journal = {PLoS Biol}, volume = {1}, year = {2003}, month = {2003 Oct}, pages = {E21}, abstract = {

Symbiotic relationships between bacteria and insect hosts are common. Although the bacterial endosymbionts have been subjected to intense investigation, little is known of the host cells in which they reside, the bacteriocytes. We have studied the development and evolution of aphid bacteriocytes, the host cells that contain the endosymbiotic bacteria Buchnera aphidicola. We show that bacteriocytes of Acyrthosiphon pisum express several gene products (or their paralogues): Distal-less, Ultrabithorax/Abdominal-A, and Engrailed. Using these markers, we find that a subpopulation of the bacteriocytes is specified prior to the transmission of maternal bacteria to the embryo. In addition, we discovered that a second population of cells is recruited to the bacteriocyte fate later in development. We experimentally demonstrate that bacteriocyte induction and proliferation occur independently of B. aphidicola. Major features of bacteriocyte development, including the two-step recruitment of bacteriocytes, have been conserved in aphids for 80-150 million years. Furthermore, we have investigated two cases of evolutionary loss of bacterial symbionts: in one case, where novel extracellular, eukaryotic symbionts replaced the bacteria, the bacteriocyte is maintained; in another case, where symbionts are absent, the bacteriocytes are initiated but not maintained. The bacteriocyte represents an evolutionarily novel cell fate, which is developmentally determined independently of the bacteria. Three of five transcription factors we examined show novel expression patterns in bacteriocytes, suggesting that bacteriocytes may have evolved to express many additional transcription factors. The evolutionary transition to a symbiosis in which bacteria and an aphid cell form a functional unit, similar to the origin of plastids, has apparently involved extensive molecular adaptations on the part of the host cell.

}, keywords = {Animals, Aphids, Buchnera, Cell Lineage, Drosophila Proteins, Evolution, Molecular, Female, Gene Expression Regulation, Bacterial, Homeodomain Proteins, Male, Microscopy, Fluorescence, Models, Biological, Parthenogenesis, Species Specificity, Symbiosis, Time Factors, Transcription Factors}, issn = {1545-7885}, doi = {10.1371/journal.pbio.0000021}, author = {Braendle, Christian and Miura, Toru and Bickel, Ryan and Shingleton, Alexander W and Kambhampati, Srinivas and Stern, David L} } @article {45763, title = {Diapause in the pea aphid (Acyrthosiphon pisum) is a slowing but not a cessation of development.}, journal = {BMC Dev Biol}, volume = {3}, year = {2003}, month = {2003 Aug 8}, pages = {7}, abstract = {

BACKGROUND: Many insects undergo a period of arrested development, called diapause, to avoid seasonally recurring adverse conditions. Whilst the phenology and endocrinology of insect diapause have been well studied, there has been comparatively little research into the developmental details of diapause. We investigated developmental aspects of diapause in sexually-produced embryos of the pea aphid, Acyrthosiphon pisum.

RESULTS: We found that early stages of embryogenesis progressed at a temperature-independent rate, characteristic of diapause, whereas later stages of embryogenesis progressed at a temperature-dependent rate. However, embryos maintained at very high temperatures during the temperature-independent stage showed severe developmental abnormalities. Under no temperature regime did embryos display a distinct resting stage. Rather, morphological development progressed slowly but continuously throughout embryogenesis.

CONCLUSION: Diapause in the pea aphid, and perhaps in many other insects, is a temperature-independent slowing but not a cessation of morphological development. This suggests that the mechanisms limiting developmental rate during diapause may be the same as those controlling developmental rate at other stages of growth.

}, keywords = {Animals, Aphids, Female, Life Cycle Stages, Male, Peas, Temperature}, issn = {1471-213X}, doi = {10.1186/1471-213X-3-7}, author = {Shingleton, Alexander W and Sisk, Geoffroy C and Stern, David L} } @article {45764, title = {The Hox gene Ultrabithorax modulates the shape and size of the third leg of Drosophila by influencing diverse mechanisms.}, journal = {Dev Biol}, volume = {256}, year = {2003}, month = {2003 Apr 15}, pages = {355-66}, abstract = {

The developmental mechanisms that regulate the relative size and shape of organs have remained obscure despite almost a century of interest in the problem and the fact that changes in relative size represent the dominant mode of evolutionary change. Here, I investigate how the Hox gene Ultrabithorax (Ubx) instructs the legs on the third thoracic segment of Drosophila melanogaster to develop with a different size and shape from the legs on the second thoracic segment. Through loss-of-function and gain-of-function experiments, I demonstrate that different segments of the leg, the femur and the first tarsal segment, and even different regions of the femur, regulate their size in response to Ubx expression through qualitatively different mechanisms. In some regions, Ubx acts autonomously to specify shape and size, whereas in other regions, Ubx influences size through nonautonomous mechanisms. Loss of Ubx autonomously reduces cell size in the T3 femur, but this reduction seems to be partially compensated by an increase in cell numbers, so that it is unclear what effect cell size and number directly have on femur size. Loss of Ubx has both autonomous and nonautonomous effects on cell number in different regions of the basitarsus, but again there is not a strong correlation between cell size or number and organ size. Total organ size appears to be regulated through mechanisms that operate at the level of the entire leg segment (femur or basitarsus) relatively independently of the behavior of individual subpopulations of cells within the segment.

}, keywords = {Animals, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Extremities, Gene Expression Regulation, Developmental, Homeodomain Proteins, Pupa, Transcription Factors}, issn = {0012-1606}, author = {Stern, David L} } @article {45767, title = {Molecular phylogenetic evidence for multiple gains or losses of ant mutualism within the aphid genus Chaitophorus.}, journal = {Mol Phylogenet Evol}, volume = {26}, year = {2003}, month = {2003 Jan}, pages = {26-35}, abstract = {

Mutualism with ants is suspected to be a highly labile trait within homopteran evolution. We used molecular phylogenetic evidence to test whether the mutualism has multiple origins within a single aphid genus. We constructed a molecular phylogeny of 15 Chaitophorus Koch (Hemiptera: Aphidoidea) species, using mitochondrial cytochrome oxidase I and II sequences. Ant tending evolved, or was lost, at least five times during Chaitophorus evolution. Parametric bootstrapping rejected the hypothesis of a single origin of ant tending in this genus. Further, the Chaitophorus made at least two host genus switches from poplars (Populus) to willow (Salix), and four switches in feeding position, from leaf feeding to stem feeding or vice versa. This is the first phylogenetic confirmation that ant tending is an evolutionarily labile trait in aphids.

}, keywords = {Animals, Ants, Aphids, Behavior, Animal, Evolution, Molecular, Phylogeny, Symbiosis}, issn = {1055-7903}, author = {Shingleton, Alexander W and Stern, David L} } @article {45762, title = {Regulatory evolution of shavenbaby/ovo underlies multiple cases of morphological parallelism.}, journal = {Nature}, volume = {424}, year = {2003}, month = {2003 Aug 21}, pages = {935-8}, abstract = {

Cases of convergent evolution that involve changes in the same developmental pathway, called parallelism, provide evidence that a limited number of developmental changes are available to evolve a particular phenotype. To our knowledge, in no case are the genetic changes underlying morphological convergence understood. However, morphological convergence is not generally assumed to imply developmental parallelism. Here we investigate a case of convergence of larval morphology in insects and show that the loss of particular trichomes, observed in one species of the Drosophila melanogaster species group, has independently evolved multiple times in the distantly related D. virilis species group. We present genetic and gene expression data showing that regulatory changes of the shavenbaby/ovo (svb/ovo) gene underlie all independent cases of this morphological convergence. Our results indicate that some developmental regulators might preferentially accumulate evolutionary changes and that morphological parallelism might therefore be more common than previously appreciated.

}, keywords = {Animals, Biological Evolution, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Gene Expression Regulation, Developmental, Genes, Insect, Larva, Molecular Sequence Data, Morphogenesis, Phylogeny, RNA, Messenger, Transcription Factors}, issn = {1476-4687}, doi = {10.1038/nature01768}, author = {Sucena, Elio and Delon, Isabelle and Jones, Isaac and Payre, Fran{\c c}ois and Stern, David L} }