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Genomic dissection of behavioral maturation in the honey bee.

Honey bees undergo an age-related, socially regulated transition from working in the hive to foraging that has been previously associated with changes in the expression of thousands of genes in the brain. To understand the meaning of these changes, we conducted microarray analyses to examine the following: (i) the ontogeny of gene expression preceding the onset of foraging, (ii) the effects of physiological and genetic factors that influence this behavioral transition, and (iii) the effects of foraging experience. Although >85% of approximately 5,500 genes showed brain differences, principal component analysis revealed discrete influences of age, behavior, genotype, environment, and experience. Young bees not yet competent to forage showed extensive, age-related expression changes, essentially complete by 8 days of age, coinciding with previously described structural brain changes. Subsequent changes were not age-related but were largely related to effects of juvenile hormone (JH), suggesting that the increase in JH that influences the hive bee-forager transition may cause many of these changes. Other treatments that also influence the onset age of foraging induced many changes but with little overlap, suggesting that multiple pathways affect behavioral maturation. Subspecies differences in onset age of foraging were correlated with differences in JH and JH-target gene expression, suggesting that this endocrine system mediates the genetic differences. We also used this multifactorial approach to identify candidate genes for behavioral maturation. This successful dissection of gene expression indicates that, for social behavior, gene expression in the brain can provide a robust indicator of the interaction between hereditary and environmental information.

Aging↗

Genome scan for cis-regulatory DNA motifs associated with social behavior in honey bees.

Honey bees (Apis mellifera) undergo an age-related, socially regulated transition from working in the hive to foraging, which is associated with changes in the expression of thousands of genes in the brain. To begin to study the cis-regulatory code underlying this massive social regulation of gene expression, we used the newly sequenced honey bee genome to scan the promoter regions of eight sets of behaviorally related genes differentially expressed in the brain in the context of division of labor among worker bees, for 41 cis-regulatory motifs previously characterized in Drosophila melanogaster. Binding sites for the transcription factors Hairy, GAGA, Adf1, Cf1, Snail, and Dri, known to function in nervous system development, olfactory learning, or hormone binding in Drosophila, were significantly associated with one or more gene sets. The presence of some binding sites also predicted expression patterns for as many as 71% of the genes in some gene sets. These results suggest that there is a robust relationship between cis and social regulation of brain gene expression, especially considering that we studied <15% of all known transcription factors. These results also suggest that transcriptional networks involved in the regulation of development in Drosophila are used to regulate behavioral development in adult honey bees. However, differences in gene regulation between these two processes are suggested by the finding that the promoter regions for the behaviorally related bee genes differed in both motif occurrence and G/C content relative to their Drosophila orthologs.

Animals↗

Identification of a Drosophila muscle development gene with structural homology to mammalian early growth response transcription factors.

In Drosophila, stripe (sr) gene function is required for normal muscle development. Some mutations disrupt embryonic muscle development and are lethal. Other mutations cause total loss of only a single muscle in the adult. Molecular analysis shows that sr encodes a predicted protein containing a zinc finger motif. This motif is homologous to the DNA binding domains encoded by members of the early growth response (egr) gene family. In mammals, expression of egr genes is induced by intercellular signals, and there is evidence for their role in many developmental events. The identification of sr as an egr gene and its pattern of expression suggest that it functions in muscle development via intercellular communication.

Amino Acid Sequence↗

Identification and characterization of a Drosophila nuclear receptor with the ability to inhibit the ecdysone response.

In a search for retinoid X receptor-like molecules in Drosophila, we have identified an additional member of the nuclear receptor superfamily, XR78E/F. In the DNA-binding domain, XR78E/F is closely related to the mammalian receptor TR2, as well as to the nuclear receptors Coup-TF and Seven-up. We demonstrate that XR78E/F binds as a homodimer to direct repeats of the sequence AGGTCA. In transient transfection assays, XR78E/F represses ecdysone signaling in a DNA-binding-dependent fashion. XR78E/F has its highest expression in third-instar larvae and prepupae. These experiments suggest that XR78E/F may play a regulatory role in the transcriptional cascade triggered by the hormone ecdysone in Drosophila.

Amino Acid Sequence↗

Evolutionary changes in the expression pattern of a developmentally essential gene in three Drosophila species.

The hypothesis that morphological evolution may largely result from changes in gene regulation rather than gene structure has been difficult to test. Morphological differences among insects are often apparent in the cuticle structures produced. The dopa decarboxylase (Ddc) and alpha-methyldopa hypersensitive (amd) genes arose from an ancient gene duplication. In Drosophila, they have evolved nonoverlapping functions, including the production of distinct types of cuticle, and for Ddc, the production of the neurotransmitters, dopamine and serotonin. The amd gene is particularly active in the production of specialized flexible cuticles in the developing embryo. We have compared the pattern of amd expression in three Drosophila species. Several regions of expression conserved in all three species but, surprisingly, a unique domain of expression is found in Drosophila simulans that does occur in the closely related (2-5 million years) Drosophila melanogaster or in the more remote (40-50 million years) Drosophila virilis. The "sudden" appearance of a completely new and robust domain of expression provides a glimpse of evolutionary variation resulting from changes in regulation of structural gene expression.

Animals↗

Evolution of sexual isolation during secondary contact: genotypic versus phenotypic changes in laboratory populations.

We monitored the phenotypic and genotypic changes that occur when two behavioral races come into contact in laboratory populations. Drosophila melanogaster from Zimbabwe and nearby regions (Z type) show strong but asymmetric sexual isolation from their cosmopolitan counterparts (M type). Crosses of Z females and M males do not take place readily when other choices are available. At least 15 loci are known to control Z-type mating preferences and performance. By thoroughly mixing the genomes of the two types in laboratory populations, we artificially created maximum secondary contact. Despite the strength of sexual selection favoring Z-type male characters, Z-type behavior is eliminated or greatly diminished in all 12 hybrid populations after only 60 generations. This trend is consistent with the spread of the M-type behavior throughout the world as well as a detailed analysis of fitness components. Surprisingly, in contrast with the phenotypic convergence toward the M-type, genotypic samples broadly covering the genomic regions of mapped behavioral loci show no such trend. The genome appears to be "fine grained," with adjacent loci having different evolutionary dynamics and genealogical histories.

Alleles↗

Horka, a dominant mutation of Drosophila, induces nondisjunction and, through paternal effect, chromosome loss and genetic mosaics.

Fs(3)Horka (Horka) was described as a dominant female-sterile mutation of Drosophila melanogaster. Genetic and cytological data show that Horka induces mostly equational nondisjunction during spermatogenesis but not chromosome loss and possesses a dominant paternal effect: the X, second, third and the fourth chromosomes, but not the Y, are rendered unstable while undergoing spermatogenesis and may be lost in the descending zygotes. The frequency of Horka-induced chromosome loss is usually 2-4% but varies with the genetic background and can be over 20%. The X chromosome loss occurs during the gonomeric and the initial cleavage divisions. Loss of the X and fourth chromosomes shows no correlation. We propose, based on similarities in the mutant phenotypes with the chromosome destabilizing mutations nonclaret disjunctional and paternal loss, that the normal Horka+ product is required for function of the centromeres and/or nearby regions. Horka is a convenient tool for the generation of gynandromorphs, autosome mosaics and for the study of gene expression in mosaics.

Animals↗

Beaded of Goldschmidt, an antimorphic allele of Serrate, encodes a protein lacking transmembrane and intracellular domains.

Serrate (Ser) is an essential gene in Drosophila melanogaster best known for the Ser dominant (SerD) allele and its effects on wing development. Animals heterozygous or homozygous for SerD are viable and exhibit loss of wing margin tissue and associated bristles and hairs. The Beaded of Goldschmidt (BdG) allele of Ser, when heterozygous to wild type, will also produce animals exhibiting loss of wing margin material. However, animals homozygous for BdG exhibit a larval lethal phenotype comparable to animals homozygous for loss-of-function Ser alleles. BdG is a partial duplication of the Ser locus with a single 5' Ser-homologous region and two distinct 3' regions. Meiotic recombination between BdG and a wild-type Ser chromosome demonstrated that only one DNA lesion, caused by the insertion of a transposable roo element into the coding regions of the Ser transcript, appears capable of generating BdG phenotypes. Due to the roo insertion, the protein product is predicted to be prematurely truncated and lack an extracellular cysteine-rich region along with the transmembrane and intracellular domains found within the normal SERRATE (SER) protein. The loss of these protein domains apparently contributes to the antimorphic nature of this mutation.

Alleles↗

Intragenomic rDNA ITS2 variation in the neotropical Anopheles (Nyssorhynchus) albitarsis complex (Diptera: Culicidae).

We cloned and sequenced the rDNA internal transcribed spacer 2 (ITS2) of 4 species belonging to the neotropical Anopheles (Nyssorhynchus) albitarsis complex, that is, A. albitarsis; A. albitarsis B; Anopheles marajoara, a proven malaria vector; and Anopheles deaneorum, a suspected vector. Even though the ITS2 sequences of these species were very similar (< or =1.17% divergence), we found differences suitable for species identification and intragenomic variation of possible consequence in phylogenetic reconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existence of partially correlated subsets of clones in A. albitarsis is hypothesized either to be separate rDNA loci or to be semi-independently evolving portions of a single rDNA locus. No differences were found between males and females, suggesting that similar rDNA arrays exist on both the X and Y chromosomes. In addition, highly variant clones, possibly pseudogenes, were found in A. marajoara from Venezuela.

Animals↗

Molecular distinction between populations of Gonatocerus morrilli, egg parasitoids of the glassy-winged sharpshooter from Texas and California: do cryptic species exist?

Two molecular methods were utilized to distinguish geographic populations of Gonatocerus morrilli (Howard) from Texas and California and to test the possibility that this species could exist as a species-complex. Inter-Simple Sequence Repeat-Polymerase Chain Reactions (ISSR-PCR) were performed with a 5'-anchored ISSR primer. Twenty-five markers were generated with four populations (40 individuals) of G. morrilli. Twenty-three were polymorphic and the percentage of polymorphic loci was 92%. Most markers could be considered diagnostic since there was no band sharing between the Texas and California populations. Such differences typically are not found unless the populations are reproductively isolated. Exact tests for population differentiation indicated significant differences in marker frequencies among the populations. Comparison of other genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated excellent agreement between GST and theta values, 0.92 and 0.94, respectively, indicating that about 92 to 94% of the variance was distributed among populations. The average genetic divergence (D), as measured by genetic distance, was extremely high (Nei = 0.82 and Reynolds = 2.79). A dendrogram based on Nei's genetic distance separated the Texas and California populations into two clusters, respectively. Amplification of the Internal Transcribed Spacer-1 (ITS-1) region showed no size differences, whereas the ITS-2 DNA fragment varied in size between the two geographic populations. The ITS-2 fragment sizes were about 865 and 1099 base pairs for the California and Texas populations, respectively. The present study using the two molecular methods provides novel data critical to the glassy-winged sharpshooter/Pierce's disease biological control program in California.

Animals↗

The Fire Ant Social Chromosome Exerts a Major Influence on Genome Regulation.

Supergenes underlying complex trait polymorphisms ensure that sets of coadapted alleles remain genetically linked. Despite their prevalence in nature, the mechanisms of supergene effects on genome regulation are poorly understood. In the fire ant Solenopsis invicta, a supergene containing over 500 individual genes influences trait variation in multiple castes to collectively underpin a colony level social polymorphism. Here, we present results of an integrative investigation of supergene effects on gene regulation. We present analyses of ATAC-seq data to investigate variation in chromatin accessibility by supergene genotype and STARR-seq data to characterize enhancer activity by supergene haplotype. Integration with gene co-expression analyses, newly mapped intact transposable elements (TEs), and previously identified copy number variants (CNVs) collectively reveals widespread effects of the supergene on chromatin structure, gene transcription, and regulatory element activity, with a genome-wide bias for open chromatin and increased expression in the presence of the derived supergene haplotype, particularly in regions that harbor intact TEs. Integrated consideration of CNVs and regulatory element divergence suggests each evolved in concert to shape the expression of supergene encoded factors, including several transcription factors that may directly contribute to the trans-regulatory footprint of a heteromorphic social chromosome. Overall, we show how genome structure in the form of a supergene has wide-reaching effects on gene regulation and gene expression.

Animals↗

Comparative genomics of accessory gland protein genes in Drosophila melanogaster and D. pseudoobscura.

Male accessory gland protein genes (Acps) evolve rapidly in the melanogaster species subgroup of Drosophila. However, conservation of Acps in more diverged lineages is poorly understood. We used comparisons of the D. melanogaster and D. pseudoobscura genome sequences, along with empirical investigation of D. pseudoobscura transcription, to assay the D. pseudoobscura genome for orthologs of 13 D. melanogaster Acps (Acp26Aa, Acp26Ab, Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp70A, Acp76A, Acp95EF, and Acp98AB). We find that Acp26Aa, Acp26Ab, Acp32CD, and Acp53Ea are present at the expected microsyntenic locations of D. pseudoobscura. Acp62F and Acp70A are also present, although they are located in nonsyntenic regions. For six of the remaining seven Acps, computational and molecular biological evidence suggests they are D. melanogaster orphans. The weighted average of interspecific amino acid identity for alignable residues across the six orthologous Acps is 35.6%. Population genetic data for D. pseudoobscura Acp26Aa show that this gene has been evolving under directional selection, as it has been in D. melanogaster/D. simulans. All four D. melanogaster Acps we analyze from chromosome arm 3L are absent from the homologous D. pseudoobscura XR chromosome arm, which was autosomal before an X chromosome-autosome fusion event in the D. pseudoobscura lineage. This observation is consistent with the hypothesis that male-advantage genes on the Drosophila X chromosome are disfavored by natural selection.

Animals↗

Differences in genome size between closely related species: the Drosophila melanogaster species subgroup.

Genome size varies considerably among organisms due to differences in the amplification, deletion, and divergence of various kinds of repetitive sequences, including the transposable elements, which constitute a large fraction of the genome. However, while the changes in genome size observed at a wide taxonomic level have been thoroughly investigated, we still know little about the process involved in closely related species. We estimated genome sizes and the reverse transcriptase-related sequence (RTRS) content in the nine species of the Drosophila melanogaster species subgroup. We showed that the species differ with regard to their genome size and that the RTRS content is correlated with genome size for all species except Drosophila orena. The genome of D. orena, which is 1.6-fold as big as that of D. melanogaster, has in fact not undergone any major increase in its RTRS content.

Animals↗

Intron length evolution in Drosophila.

I present data on the evolution of intron lengths among 3 closely related Drosophila species, D. melanogaster, Drosophila simulans, and Drosophila yakuba. Using D. yakuba as an outgroup, I mapped insertion and deletion mutations in 148 introns (spanning approximately 30 kb) to the D. melanogaster and D. simulans lineages. Intron length evolution in the 2 sister species has been different: in D. melanogaster, X-linked introns have increased slightly in size, whereas autosomal ones have decreased slightly in size; in D. simulans, both X-linked and autosomal introns have decreased in size. To understand the possible evolutionary causes of these lineage- and chromosome-specific patterns of intron evolution, I studied insertion-deletion (indel) polymorphism and divergence in D. melanogaster. Small insertion mutations segregate at elevated frequencies and enjoy elevated probabilities of fixation, particularly on the X chromosome. In contrast, there is no detectable X chromosome effect on fixations in D. simulans. These findings suggest X chromosome-specific selection or biased gene conversion-gap repair favoring insertions in D. melanogaster but not in D. simulans. These chromosome- and lineage-specific patterns of indel substitution are not easily explained by existing general population genetic models of intron length evolution. Genomic data from D. melanogaster further suggest that the forces described here affect introns and intergenic regions similarly.

Animals↗

Heterogeneity in regional GC content and differential usage of codons and amino acids in GC-poor and GC-rich regions of the genome of Apis mellifera.

The honeybee (Apis mellifera) has a genome with a wide variation in GC content showing 2 clear modal GC values, in some ways reminiscent of an isochore-like structure. To gain insight into causes and consequences of this pattern, we used a comparative approach to study the genome-wide alignment of primarily coding sequence of A. mellifera with Drosophila melanogaster and Anopheles gambiae. The latter 2 species show a higher average GC content than A. mellifera and no indications of bimodality, suggesting that the GC-poor mode is a derived condition in honeybee. In A. mellifera, synonymous sites of genes generally adopt the GC content of the region in which they reside. A large proportion of genes in GC-poor regions have not been assigned to the honeybee assembly because of the low sequence complexity of their genome neighborhood. The synonymous substitution rate between A. mellifera and the other species is very close to saturation, but analyses of nonsynonymous substitutions as well as amino acid substitutions indicate that the GC-poor regions are not evolving faster than the GC-rich regions. We describe the codon usage and amino acid usage and show that they are remarkably heterogeneous within the honeybee genome between the 2 different GC regions. Specifically, the genes located in GC-poor regions show a much larger deviation in both codon usage bias and amino acid usage from the Dipterans than the genes located in the GC-rich regions.

Amino Acids↗

Drosophila RpS3a, a novel Minute gene situated between the segment polarity genescubitus interruptus and dTCF.

Genetic analysis of the small chromosome 4 of Drosophila has been hampered by the virtual lack of recombination. The segment polarity gene cubitus interruptus (ci) maps to the most intensively studied locus on this chromosome. Up to four complementation groups have been found to be associated with ci. We and others have recently characterized a second segment polarity gene, dTCF or pan, 12 kb upstream of ci, in a head-to-head configuration. During the course of these studies we identified a transcription unit in the intergenic region. We report here the cloning of cDNAs from this transcription unit, which encode the Drosophila homologue of the human ribosomal protein S3a (RpS3a). The RpS3a gene is expressed ubiquitously and throughout development. A Minute allele, M(4)101, linked tightly to ci, was found to harbour an integration of a Doc retroposon in the promotor region of RpS3a. Thus, like other Minute loci, M(4)101 encodes a component of the protein synthesis machinery. These data further unravel the complex genetics surrounding the ci and dTCF loci.

Amino Acid Sequence↗

FlyRNAi: the Drosophila RNAi screening center database.

RNA interference (RNAi) has become a powerful tool for genetic screening in Drosophila. At the Drosophila RNAi Screening Center (DRSC), we are using a library of over 21,000 double-stranded RNAs targeting known and predicted genes in Drosophila. This library is available for the use of visiting scientists wishing to perform full-genome RNAi screens. The data generated from these screens are collected in the DRSC database (http://flyRNAi.org/cgi-bin/RNAi_screens.pl) in a flexible format for the convenience of the scientist and for archiving data. The long-term goal of this database is to provide annotations for as many of the uncharacterized genes in Drosophila as possible. Data from published screens are available to the public through a highly configurable interface that allows detailed examination of the data and provides access to a number of other databases and bioinformatics tools.

Animals↗

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc.

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in our study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification.

ATP-Binding Cassette Transporters↗