We address the problem of inferring the number of independently blinking fluorescent light emitters, when only their combined intensity contributions can be observed at each timepoint. This problem occurs regularly in light microscopy of objects that are smaller than the diffraction limit, where one wishes to count the number of fluorescently labelled subunits. Our proposed solution directly models the photo-physics of the system, as well as the blinking kinetics of the fluorescent emitters as a fully differentiable hidden Markov model. Given a trace of intensity over time, our model jointly estimates the parameters of the intensity distribution per emitter, their blinking rates, as well as a posterior distribution of the total number of fluorescent emitters. We show that our model is consistently more accurate and increases the range of countable subunits by a factor of two compared to current state-of-the-art methods, which count based on autocorrelation and blinking frequency, Further-more, we demonstrate that our model can be used to investigate the effect of blinking kinetics on counting ability, and therefore can inform experimental conditions that will maximize counting accuracy.

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.

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.

Although different animal species often exhibit extensive variation in many behaviors, typically scientists examine one or a small number of behaviors in any single study. Here, we propose a new framework to simultaneously study the evolution of many behaviors. We measured the behavioral repertoire of individuals from six species of fruit flies using unsupervised techniques and identified all stereotyped movements exhibited by each species. We then fit a Generalized Linear Mixed Model to estimate the intra- and inter-species behavioral covariances, and, by using the known phylogenetic relationships among species, we estimated the (unobserved) behaviors exhibited by ancestral species. We found that much of intra-specific behavioral variation has a similar covariance structure to previously described long-time scale variation in an individual's behavior, suggesting that much of the measured variation between individuals of a single species in our assay reflects differences in the status of neural networks, rather than genetic or developmental differences between individuals. We then propose a method to identify groups of behaviors that appear to have evolved in a correlated manner, illustrating how sets of behaviors, rather than individual behaviors, likely evolved. Our approach provides a new framework for identifying co-evolving behaviors and may provide new opportunities to study the mechanistic basis of behavioral evolution.

Large collections of full-length cDNAs are important resources for genome annotation and functional genomics. We report the creation of a collection of 50 599 full-length cDNA clones from the pea aphid, Acyrthosiphon pisum. Sequencing from 5’ and 3’ ends of the clones generated 97 828 high-quality expressed sequence tags, representing approximately 9000 genes. These sequences were imported to AphidBase and are shown to play crucial roles in both automatic gene prediction and manual annotation. Our detailed analyses demonstrated that the full-length cDNAs can further improve gene models and can even identify novel genes that are not included in the current version of the official gene set. This full-length cDNA collection can be utilized for a wide variety of functional studies, serving as a community resource for the study of the functional genomics of the pea aphid.

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.

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.

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.

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.

The pea aphid, Acyrthosiphon pisum, exhibits several environmentally cued, discrete, alternate phenotypes (polyphenisms) during its life cycle. In the case of the reproductive polyphenism, differences in day length determine whether mothers will produce daughters that reproduce either sexually by laying fertilized eggs (oviparous sexual reproduction), or asexually by allowing oocytes to complete embryogenesis within the mother without fertilization (viviparous parthenogenesis). Oocytes and embryos that are produced asexually and develop within the mother develop more rapidly, are yolk-free, and much smaller than oocytes and embryos that are produced sexually. These overt differences suggest that there may be underlying differences in the molecular mechanisms of pattern formation. Indeed, our preliminary comparative gene expression work suggests that there are important differences in the terminal patterning system, involving the Torso pathway, between viviparous and oviparous development. We have so far examined the expression of homologs of torso-like and capicua, members of the Drosophila Torso pathway. We have detected clear differential expression of torso-like and possible differential expression of capicua. Establishing such differences in the expression of patterning genes between these developmental modes is a first step toward understanding how a single genome manages to direct patterning events in such different embryological contexts.