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For yeast cells, tolerance to high levels of ethanol is vital both in their natural environment and in industrially relevant conditions. We recently genotyped experimentally evolved yeast strains adapted to high levels of ethanol and identified mutations linked to ethanol tolerance. In this study, by integrating genomic sequencing data with quantitative proteomics profiles from six evolved strains (data set identifier PXD006631) and construction of protein interaction networks, we elucidate exactly how the genotype and phenotype are related at the molecular level. Our multi-omics approach points to the rewiring of numerous metabolic pathways affected by genomic and proteomic level changes, from energy-producing and lipid pathways to differential regulation of transposons and proteins involved in cell cycle progression. One of the key differences is found in the energy-producing metabolism, where the ancestral yeast strain responds to ethanol by switching to respiration and employing the mitochondrial electron transport chain. In contrast, the ethanol-adapted strains appear to have returned back to energy production mainly via glycolysis and ethanol fermentation, as supported by genomic and proteomic level changes. This work is relevant for synthetic biology where systems need to function under stressful conditions, as well as for industry and in cancer biology, where it is important to understand how the genotype relates to the phenotype. ispartof: JOURNAL OF PROTEOME RESEARCH vol:20 issue:8 pages:3840-3852 ispartof: location:United States status: published

In many animal species, sex determination is accomplished by heterogamety i.e., one of the sexes produces two types of gametes, which upon fertilization will direct the development toward males or females. Both male (“XY”) and female (“ZW”) heterogamety are known to occur and can be easily distinguished when the sex-chromosomes are morphologically different. However, this approach fails in cases of homomorphic sex chromosomes, such as the sand fly Lutzomyia longipalpis s.l. (Psychodidae, Diptera), which is the main vector of visceral leishmaniosis in Brazil. In order to identify the heterogametic sex in L. longipalpis s.l., we did a whole-genome sequencing of males and females separately and used the “Y chromosome Genome Scan” (YGS) method to find sex-specific sequences. Our results, which were confirmed by PCR, show that L. longipalpis s.l. has XY system. The YGS method can be especially useful in situations in which no morphological difference is observed in the sex-chromosomes or when fresh specimens are not readily available.

The vinegar flyDrosophila melanogasteris a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4000 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the groups and genera in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred well-supported group and species trees for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

BackgroundThe Drosophilidae family is traditionally divided into two subfamilies: Drosophilinae and Steganinae. This division is based on morphological characters, and the two subfamilies have been treated as monophyletic in most of the literature, but some molecular phylogenies have suggested Steganinae to be paraphyletic. To test the paraphyletic-Steganinae hypothesis, here, we used genomic sequences of eight Drosophilidae (three Steganinae and five Drosophilinae) and two Ephydridae (outgroup) species and inferred the phylogeny for the group based on a dataset of 1,028 orthologous genes present in all species (> 1,000,000 bp). This dataset includes three genera that broke the monophyly of the subfamilies in previous works. To investigate possible biases introduced by small sample sizes and automatic gene annotation, we used the same methods to infer species trees from a set of 10 manually annotated genes that are commonly used in phylogenetics.ResultsMost of the 1,028 gene trees depicted Steganinae as paraphyletic with distinct topologies, but the most common topology depicted it as monophyletic (43.7% of the gene trees). Despite the high levels of gene tree heterogeneity observed, species tree inference in ASTRAL, in PhyloNet, and with the concatenation approach strongly supported the monophyly of both subfamilies for the 1,028-gene dataset. However, when using the concatenation approach to infer a species tree from the smaller set of 10 genes, we recovered Steganinae as a paraphyletic group. The pattern of gene tree heterogeneity was asymmetrical and thus could not be explained solely by incomplete lineage sorting (ILS).ConclusionsSteganinae was clearly a monophyletic group in the dataset that we analyzed. In addition to ILS, gene tree discordance was possibly the result of introgression, suggesting complex branching processes during the early evolution of Drosophilidae with short speciation intervals and gene flow. Our study highlights the importance of genomic data in elucidating contentious phylogenetic relationships and suggests that phylogenetic inference for drosophilids based on small molecular datasets should be performed cautiously. Finally, we suggest an approach for the correction and cleaning of BUSCO-derived genomic datasets that will be useful to other researchers planning to use this tool for phylogenomic studies.

Three North American cactophilic Drosophila species, D. mojavensis, D. arizonae, and D. navojoa, are of considerable evolutionary interest owing to the shift from breeding in Opuntia cacti to columnar species. The 3 species form the “mojavensis cluster” of Drosophila. The genome of D. mojavensis was sequenced in 2007 and the genomes of D. navojoa and D. arizonae were sequenced together in 2016 using the same technology (Illumina) and assembly software (AllPaths-LG). Yet, unfortunately, the D. navojoa genome was considerably more fragmented and incomplete than its sister species, rendering it less useful for evolutionary genetic studies. The D. navojoa read dataset does not fully meet the strict insert size required by the assembler used (AllPaths-LG) and this incompatibility might explain its assembly problems. Accordingly, when we re-assembled the genome of D. navojoa with the SPAdes assembler, which does not have the strict AllPaths-LG requirements, we obtained a substantial improvement in all quality indicators such as N50 (from 84 kb to 389 kb) and BUSCO coverage (from 77% to 97%). Here we share a new, improved reference assembly for D. navojoa genome, along with a RNAseq transcriptome. Given the basal relationship of the Opuntia breeding D. navojoa to the columnar breeding D. arizonae and D. mojavensis, the improved assembly and annotation will allow researchers to address a range of questions associated with the genomics of host shifts, chromosomal rearrangements and speciation in this group.

Y chromosomes play important roles in sex determination and male fertility. In several groups (e.g., mammals) there is strong evidence that they evolved through gene loss from a common X-Y ancestor, but in Drosophila the acquisition of new genes plays a major role. This conclusion came mostly from studies in two species. Here we report the identification of the 22 Y-linked genes in D. willistoni. They all fit the previously observed pattern of autosomal or X-linked testis-specific genes that duplicated to the Y. The ratio of gene gains to gene losses is ~25 in D. willistoni, confirming the prominent role of gene gains in the evolution of Drosophila Y chromosomes. We also found four large segmental duplications (ranging from 62 kb to 303 kb) from autosomal regions to the Y, containing ~58 genes. All but four of these duplicated genes became pseudogenes in the Y or disappeared. In the GK20609 gene the Y-linked copy remained functional, whereas its original autosomal copy degenerated, demonstrating how autosomal genes are transferred to the Y chromosome. Since the segmental duplication that carried GK20609 contained six other testis-specific genes, it seems that chance plays a significant role in the acquisition of new genes by the Drosophila Y chromosome.

Although infection with the Zika virus was first recognized in 1942, it received little attention until 2007, when a true pandemic spread throughout Africa, Asia, and the Americas. Since then, numerous forms of central nervous system involvement have been described, mainly malformations related to congenital infection. Although the neuroimaging findings in congenital Zika syndrome are not pathognomonic, many are quite suggestive of the diagnosis, and radiologists should be prepared to interpret such findings accordingly. The objective of this article is to review the computed tomography and magnetic resonance imaging findings in congenital Zika syndrome. Resumo A infecção pelo vírus Zika, apesar de conhecida desde 1942, apresentou destaque somente a partir de 2007, quando uma verdadeira pandemia se espalhou pela África, Ásia e Américas. Durante este período, numerosas formas de acometimento do sistema nervoso central têm sido descritas, principalmente as malformações relacionadas a infecção congênita. Apesar de os achados de neuroimagem na síndrome congênita pelo vírus Zika não serem patognomônicos, muitos são bastante sugestivos, devendo o radiologista estar preparado para saber interpretar e sugerir o diagnóstico. O objetivo deste artigo é revisar os achados de tomografia computadorizada e ressonância magnética da síndrome congênita pelo vírus Zika.

Background: In 1947, Zika virus (ZIKV) was first discovered in Monkeys, in Zika Forest, in Uganda, Africa. Five years later, (1952) the first human Zika infection was detected in Nigeria, Africa. After this date, only sporadic cases happened, until the first 3 epidemics occurred, all outside Africa. The first epidemic was in Yap Island in 2007, the second in French Polynesia in 2013, and the third in 2015 in the northeast of Brazil, and then the spread to the Americas in 2015 and 2016. However, it was only after the epidemic in the northeast of Brazil, in the first half of 2015, that many babies were born with microcephaly in the second semester of that same year and in 2016. Until now, every year, some babies are still born with congenital ZIKV syndrome (CZVS). Summary: The objective of this article is to describe infrequent and rarely discussed imaging (computed tomography and magnetic resonance imaging) findings of CZVS, in addition to those classically described such as a simplified gyral pattern, ventriculomegaly, corpus callosum dysgenesis, craniofacial disproportion, and redundant scalp, thus suggesting an increase in the spectrum of neurological findings related to the syndrome.

In many insects, the Y chromosome plays a key role in sexual determination and male fertility. The Chagas disease vectorTriatoma infestanshas 22 autosomal chromosomes and a pair of XY sex chromosomes. However, the knowledge on the Y chromosome of this species, its genetic content or its biological function, is very poor. Due to repetitive DNA, Y chromosome sequences are poorly assembled in genome projects, hindering structural and functional studies on Y-linked genes. Our group has developed many of the bioinformatic tools to identify Y-linked sequences in assembled genomes. Here, we describe the identification of a γ-dynein heavy chain linked to the Y-chromosome ofT. infestans. This protein is orthologous to theDrosophila melanogasterY-linked genekl-3. InD. melanogaster, dyneins of the Y chromosome are known as male fertility factors and their deletion causes male infertility. We performed knockdown of thekl-3expression to ascertain its function inT. infestans. Our results showed that injection of dsKL3 reduced, significantly, the fertility ofT. infestansmales (pkl-3maintains its functional role as essential for male fertility inT. infestans. Hence, it seems that the Y-chromosome ofT. infestanshas a key role in male fertility. This is the first report of akl-3orthologue linked to the Y chromosome of an insect species outside the diptera clade. In addition to the first report of a Y-linked gene inT. infestanswith a role for male fertility, this finding is of great relevance for the study of the evolution of Y chromosomes and further studies that could lead to novel approaches in insect control.

Genome assembly depends critically on read length. Two recent technologies, from Pacific Biosciences (PacBio) and Oxford Nanopore, produce read lengths >20 kb, which yield de novo genome assemblies with vastly greater contiguity than those based on Sanger, Illumina, or other technologies. However, the very high error rates of these two new technologies (∼15% per base) makes assembly imprecise at repeats longer than the read length and computationally expensive. Here we show that the contiguity and quality of the assembly of these noisy long reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in Illumina reads (which account for ∼95% of the distinct k-mers) are deemed sequencing errors and ignored at the seed alignment step. By focusing on the ∼5% of k-mers that are error free, read overlap sensitivity is dramatically increased. Of equal importance, the validation procedure can be extended to exclude repetitive k-mers, which prevents read miscorrection at repeats and further improves the resulting assemblies. We tested the k-mer validation procedure using one long-read technology (PacBio) and one assembler (MHAP/Celera Assembler), but it is very likely to yield analogous improvements with alternative long-read technologies and assemblers, such as Oxford Nanopore and BLASR/DALIGNER/Falcon, respectively.

The work was supported by CNPq (Science without Borders program) and CAPES. Lutzomyia longipalpis is the main vector of visceral Leishmaniasis in the Americas and is composed of a species complex. Males of this sand-fly produce acoustic signals during copulation and different patterns are observed among Brazilian populations. Such acoustic signals are commonly involved in species recognition. However, since the song is only produced during copulation it is not clear how it affects mating success or contributes to sexual isolation. Another aspect that may affect reproductive success is the presence of food. Since hematophagy is such an important aspect of L. longipalpis biology, we wanted to test if blood-feeding can influence the reproductive behaviour of this insect. We performed crossing experiments removing males' wings (silencing them) and playing back either the homo-specific or the hetero-specific song to either unfed or blood-fed females. Our results showed that both songs and blood-feeding affect insemination success, but not the frequency of copulation. In trials where females were not blood-fed song clearly affected insemination; males with wings, and males with homo-specific song playback had a higher insemination success than wingless males (no song) and trials with hetero-specific song. Blood-feeding females prior to the trials increased insemination in all groups including the control group which suggests that mating happens simultaneously with, or immediately after, the blood meal. Blood-fed females also seemed to discriminate less against the wrong song or the lack of song (wingless) one day after feeding, however trials with the correct song still had higher insemination rates. Altogether, our results show that both the male copulatory courtship songs and female blood-feeding are important for reproductive success and as such are important components of the sexual behaviour of L. longipalpis. Postprint

Two new Brazilian species of Drosophila (subgenus Drosophila) are described and illustrated: Drosophila asymmetrica sp. nov. and Drosophila peixotoi sp. nov. Both species were collected, and emerged, from inflorescences of Goeppertia monophylla (Marantaceae) in the urban Forest Reserve of the Instituto de Biociências da Universidade de São Paulo and their types will be deposited in the Museu de Zoologia da USP. The former species, which could not be assigned to any known group, has a conspicuously asymmetric aedeagus and a narrow oviscapt valve. The latter species belongs to the guarani group and is closely related to D. guaru, D. ornatifrons and D. subbadia, from which it can be distinguished by the presence of just one conspicuous large black spine at inner lower tip of cercus instead of two spines. Keywords: Breeding site, Caeté, Drosophilinae, Feeding site, Taxonomy

Background Anopheles (Kerteszia) cruzii is the primary vector of human and simian malarias in Brazilian regions covered by the Atlantic Rainforest. Previous studies found that An. cruzii presents high levels of behavioural, chromosomal and molecular polymorphisms, which led to the hypothesis that it may be a complex of cryptic species. Here, An. cruzii specimens were collected in five sites in South-East Brazil located at different altitudes on the inner and coastal slopes of two mountain ranges covered by Atlantic Rainforest, known as Serra do Mar and Serra da Mantiqueria. Partial sequences for two genes (Clock and cpr) were generated and compared with previously published sequences from Florianópolis (southern Brazil). Genetic diversity was analysed with estimates of population structure (FST) and haplotype phylogenetic trees in order to understand how many species of the complex may occur in this biome and how populations across the species distribution are related. Results The sequences from specimens collected at sites located on the lower coastal slopes of Serra do Mar (Guapimirim, Tinguá and Sana) clustered together in the phylogenetic analysis, while the major haplotypes from sites located on higher altitude and at the continental side of the same mountains (Bocaina) clustered with those from Serra da Mantiqueira (Itatiaia), an inner mountain range. These two An. cruzii lineages showed statistically significant genetic differentiation and fixed characters, and have high FST values typical of between species comparisons. Finally, in Bocaina, where the two lineages occur in sympatry, we found deviations from Hardy-Weinberg equilibrium due to a deficit of heterozygotes, indicating partial reproductive isolation. These results strongly suggest that at least two distinct lineages of An. cruzii (provisorily named “Group 1” and “Group 2”) occur in the mountains of South-East Brazil. Conclusions At least two genetically distinct An. cruzii lineages occur in the Atlantic Forest covered mountains of South-East Brazil. The co-occurrence of distinct lineages of An. cruzii (possibly incipient species) in those mountains is an interesting biological phenomenon and may have important implications for malaria prevalence, Plasmodium transmission dynamics and control. Electronic supplementary material The online version of this article (10.1186/s13071-018-2615-0) contains supplementary material, which is available to authorized users.

In December 2006, 495 drosophilids belonging to 19 species were aspirated from inflorescences of Calathea monophylla (Vellozo) Körn at a forest reserve in the city of Séo Paulo, state of Séo Paulo, and 42 specimens belonging to three species of Drosophila were aspirated from those of Calathea cylindrica (Roscoe) Karl Schumann in an urban forest in the city of Rio de Janeiro, state of Rio de Janeiro, Brazil. In addition, 20 inflorescences of C. monophylla and 14 inflorescences of C. cylindrica were collected and observed for the emergence of flies in the laboratories. In total, 137 drosophilids belonging to Zygothrica dispar (Wiedemann, 1830) plus five species of Drosophila (Drosophila griseolineata Duda and four undescribed species) emerged from C. monophylla inflorescences, and a total of 22 specimens, all belonging to two undescribed species of Drosophila, emerged from those of C. cylindrica. Drosophila calatheae sp. nov., ungrouped but related to both the xanthopallescens Pipkin and the bromeliae Patterson & Stone species groups, is described based on both aspirated and emerged flies from C. monophylla inflorescences from Séo Paulo city. This new species was also reared in the laboratory with a new medium recipe, thus providing larvae for chromosomal studies. Fil: Vaz, S. C.. Universidade de Sao Paulo; Brasil Fil: Vilela, C. R.. Universidade de Sao Paulo; Brasil Fil: Krsticevic, Flavia Jorgelina. Universidade Federal do Rio de Janeiro; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Carvalho, A. B.. Universidade Federal do Rio de Janeiro; Brasil

Journal of Neuroradiology - In Press.Proof corrected by the author Available online since samedi 30 decembre 2017

Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes.

The Y chromosome of Drosophila melanogaster has

Contrary to the pattern seen in mammalian sex chromosomes, where most Y-linked genes have X-linked homologs, the Drosophila X and Y chromosomes appear to be unrelated. Most of the Y-linked genes have autosomal paralogs, so autosome-to-Y transposition must be the main source of Drosophila Y-linked genes. Here we show how these genes were acquired. We found a previously unidentified gene (flagrante delicto Y, FDY) that originated from a recent duplication of the autosomal gene vig2 to the Y chromosome of Drosophila melanogaster. Four contiguous genes were duplicated along with vig2, but they became pseudogenes through the accumulation of deletions and transposable element insertions, whereas FDY remained functional, acquired testis-specific expression, and now accounts for ∼20% of the vig2-like mRNA in testis. FDY is absent in the closest relatives of D. melanogaster, and DNA sequence divergence indicates that the duplication to the Y chromosome occurred ∼2 million years ago. Thus, FDY provides a snapshot of the early stages of the establishment of a Y-linked gene and demonstrates how the Drosophila Y has been accumulating autosomal genes.

The autosomal gene Mst77F of Drosophila melanogaster is essential for male fertility. In 2010, Krsticevic et al. (Genetics 184: 2952307) found 18 Y-linked copies of Mst77F ("Mst77Y"), which collectively account for 20% of the functional Mst77F-like mRNA. The Mst77Y genes were severely misassembled in the then-available genome assembly and were identified by cloning and sequencing polymerase chain reaction products. The genomic structure of the Mst77Y region and the possible existence of additional copies remained unknown. The recent publication of two long-read assemblies of D. melanogaster prompted us to reinvestigate this challenging region of the Y chromosome. We found that the Illumina Synthetic Long Reads assembly failed in the Mst77Y region, most likely because of its tandem duplication structure. The PacBio MHAP assembly of the Mst77Y region seems to be very accurate, as revealed by comparisons with the previously found Mst77Y genes, a bacterial artificial chromosome sequence, and Illumina reads of the same strain. We found that the Mst77Y region spans 96 kb and originated from a 3.4-kb transposition from chromosome 3L to the Y chromosome, followed by tandem duplications inside the Y chromosome and invasion of transposable elements, which account for 48% of its length. Twelve of the 18 Mst77Y genes found in 2010 were confirmed in the PacBio assembly, the remaining six being polymerase chain reaction2induced artifacts. There are several identical copies of some Mst77Y genes, coincidentally bringing the total copy number to 18. Besides providing a detailed picture of the Mst77Y region, our results highlight the utility of PacBio technology in assembling difficult genomic regions such as tandemly repeated genes. Fil: Krsticevic, Flavia Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas. Universidad Nacional de Rosario. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina Fil: Schrago, Carlos G.. Universidade Federal do Rio de Janeiro; Brasil Fil: Carvalho, A. Bernardo. Universidade Federal do Rio de Janeiro; Brasil

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads., This work was supported by NIH grants P50 HG00750 (G.M.R.), R01 HG00747 (G.H.K.), and R01 HG002673 (S.E.C.) and performed under U.S. Department of Energy Contracts DE-AC0376SF00098 and DE-AC02-05CH11231, University of California. I.F.Z. was supported by grant 13-04-40137 from the Russian Federation; E.N.A. was supported by grant 12-04-00874-a from the Russian Federation; P.D. was supported by a grant from the Instituto Pasteur-Fondazione Cenci Bolognetti; A.V. was supported by Ministerio de Economía y Competitividad grant BFU2011-30295-C02-01; and A.B.C. was supported by NIH grant R01 GM064590 and grants from Fundaçao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immunedeficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods., La lista completa de autores que integran el documento puede consultarse en el archivo, Este documento tiene una corrección (ver documento relacionado)., Centro Regional de Estudios Genómicos, Instituto de Investigaciones Bioquímicas de La Plata

Whole genome shotgun assemblies have proven remarkably successful in reconstructing the bulk of euchromatic genes, with the only limit appearing to be determined by the sequencing depth. For genes imbedded in heterochromatin, however, the low cloning efficiency of repetitive sequences, combined with the computational challenges, demand that additional clues be used to annotate the sequences. One approach that has proven very successful in identifying protein coding genes in Y-linked heterochromatin of Drosophila melanogaster has been to make a BLASTable database of the small, unmapped contigs and fragments leftover at the end of a shotgun assembly, and to attempt to capture these by blasting with an appropriate query sequence. This approach often yields a staggered alignment of contigs from the unmapped set to the query sequence, as though the disjoint contigs represent small portions of the gene. Further inspection frequently shows that the contigs are broken by very large, heterochromatic introns. Methods of this sort are being expanded to make best use of all available clues to determine which unmapped contigs are associated with genes. These include use of EST libraries, and, in the case of the Y chromosome, testing of male specific genes and reduced shotgun depth of relevant contigs. It appears much more hopeful than anyone would have imagined that whole genome shotgun assemblies can recover the great bulk of even heterochromatic genes.

Three recent findings are making a deep impact on our understanding of the Drosophila Y. First, the sequencing of the Drosophila genome and the development of proper computational methods increased the number of known single-copy Y-linked genes from 1 to 16, and revealed a chromosome packed with genes acquired from the autosomes. Second, this, coupled with the finding that B-chromosomes are able to show very regular segregation from the X chromosome, reinforce the hypothesis that the Drosophila Y is a specialized B-chromosome, instead of a degenerated homologue of the X. Third and finally, Y chromosomes seem to have a strong effect on male fitness.

Y chromosomes, with their reduced effective population size, lack of recombination, and male-limited transmission, present a unique collection of constraints for the operation of natural selection. Male-limited transmission may greatly increase the efficacy of selection for male-beneficial mutations, but the reduced effective size also inflates the role of random genetic drift. Together, these defining features of the Y chromosome are expected to influence rates and patterns of molecular evolution on the Y as compared with X-linked or autosomal loci. Here, we use sequence data from 11 genes in 9 Drosophila species to gain insight into the efficacy of natural selection on the Drosophila Y relative to the rest of the genome. Drosophila is an ideal system for assessing the consequences of Y-linkage for molecular evolution in part because the gene content of Drosophila Y chromosomes is highly dynamic, with orthologous genes being Y-linked in some species whereas autosomal in others. Our results confirm the expectation that the efficacy of natural selection at weakly selected sites is reduced on the Y chromosome. In contrast, purifying selection on the Y chromosome for strongly deleterious mutations does not appear to be compromised. Finally, we find evidence of recurrent positive selection for 4 of the 11 genes studied here. Our results thus highlight the variable nature of the mode and impact of natural selection on the Drosophila Y chromosome.

The molecular identity and function of the Drosophila melanogaster Y-linked fertility factors have long eluded researchers. Although the D. melanogaster genome sequence was recently completed, the fertility factors still were not identified, in part because of low cloning efficiency of heterochromatic Y sequences. Here we report a method for iterative blast searching to assemble heterochromatic genes from shotgun assemblies, and we successfully identify kl-2 and kl-3 as 1β- and γ-dynein heavy chains, respectively. Our conclusions are supported by formal genetics with X-Y translocation lines. Reverse transcription–PCR was successful in linking together unmapped sequence fragments from the whole-genome shotgun assembly, although some sequences were missing altogether from the shotgun effort and had to be generated de novo . We also found a previously undescribed Y gene, polycystine-related ( PRY ). The closest paralogs of kl-2 , kl-3 , and PRY (and also of kl-5 ) are autosomal and not X-linked, suggesting that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes arising de novo from the autosomes.

SR chromosomes are the best-known case of sex chromosome meiotic drive. These X chromosomes cause the production of female-biased progenies in several Drosophila species. Due to their meiotic drive advantage, they are expected to spread and become fixed, resulting in population extinction due to the lack of males. However, this apparently does not occur: SR chromosomes are maintained in balanced polymorphisms, resulting from the equilibrium between their meiotic drive advantage and deleterious fitness effects. In this paper we review the current explanations for their deleterious effects and we argue that it is highly improbable that all newly emerged SR are sufficiently deleterious to avoid fixation. Unbalanced SR almost certainly arise and go unnoticed because of three possible outcomes: (i) fixation followed by extinction of the population or species; (ii) fixation followed by the emergence and fixation of drive suppressors, restoring the normal 1:1 sexual proportion; or (iii) transformation into balanced SR due to partial suppression. If these outcomes really occur, then extant cases of sex-chromosome meiotic drive such as SR, causing small deviations on the population sexual proportion, are only the tip of the iceberg and strong sexual proportion shifts (possibly followed by extinction) are a more common feature of species evolution than is usually assumed.

Chromosomal organization is sufficiently evolutionarily stable that large syntenic blocks of genes can be recognized even between species as distantly related as mammals and puffer fish (450 million years (Myr) of divergence). In Diptera, the gene content of the X chromosome and the autosomes is well conserved: in Drosophila more than 95% of the genes have remained on the same chromosome arm in the 12 sequenced species (63 Myr of divergence, traversing 400 Myr of evolution), and the same linkage groups are clearly recognizable in mosquito genomes (260 Myr of divergence). Here we investigate the conservation of Y-linked gene content among the 12 sequenced Drosophila species. We found that only a quarter of the Drosophila melanogaster Y-linked genes (3 out of 12) are Y-linked in all sequenced species, and that most of them (7 out of 12) were acquired less than 63 Myr ago. Hence, whereas the organization of other Drosophila chromosomes traces back to the common ancestor with mosquitoes, the gene content of the D. melanogaster Y chromosome is much younger. Gene losses are known to have an important role in the evolution of Y chromosomes, and we indeed found two such cases. However, the rate of gene gain in the Drosophila Y chromosomes investigated is 10.9 times higher than the rate of gene loss (95% confidence interval: 2.3-52.5), indicating a clear tendency of the Y chromosomes to increase in gene content. In contrast with the mammalian Y chromosome, gene gains have a prominent role in the evolution of the Drosophila Y chromosome.

The Y chromosome and other heterochromatic regions present special challenges for genome sequencing and for the annotation of genes. Here we describe two new genes (ARY and WDY) on the Drosophila melanogaster Y, bringing its number of known single-copy genes to 12. WDY may correspond to the fertility factor kl-1.

Most sexually reproducing species have sexual proportions around 1:1. This major biological phenomenon remained unexplained until 1930, when Fisher proposed that it results from a mechanism of natural selection. Here we report the first experimental test of his model that obeys all its assumptions. We used a naturally occurring X-Y meiotic drive system—the sex-ratio trait of Drosophila mediopunctata—to generate female-biased experimental populations. As predicted by Fisher, these populations evolved toward equal sex proportions due to natural selection, by accumulation of autosomal alleles that direct the parental reproductive effort toward the rare sex. Classical Fisherian evolution is a rather slow mechanism: despite a very large amount of genetic variability, the experimental populations evolved from 16% of males to 32% of males in 49 generations and would take 330 generations (29 years) to reach 49%. This slowness has important implications for species potentially endangered by skewed sexual proportions, such as reptiles with temperature sex determination.

Heritability of sexual proportion in experimental sex-ratio populations of Drosophila mediopunctata

Classically, Y chromosomes are thought to originate from X chromosomes through a process of degeneration and gene loss. Now, the availability of 12 Drosophila genomes provides an opportunity to study the origin and evolution of Y chromosomes in an informative phylogenetic context. Surprisingly, the majority of Drosophila Y-linked genes are recent acquisitions from autosomes and Y chromosome gene gains are more frequent than gene losses. Moreover, the Drosophila pseudoobscura Y chromosome lacks homology with the Y of most Drosophila species. Thus, the Drosophila Y has a different evolutionary history from canonical Y chromosomes (such as the mammalian Y) and it also might have a different origin.

Affiliations des auteurs : cf page 216 de l'article; International audience; Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

We report a genome-wide search of Y-linked genes in Drosophila pseudoobscura . All six identifiable orthologs of the D. melanogaster Y-linked genes have autosomal inheritance in D. pseudoobscura . Four orthologs were investigated in detail and proved to be Y-linked in D. guanche and D. bifasciata , which shows that less than 18 million years ago the ancestral Drosophila Y chromosome was translocated to an autosome in the D. pseudoobscura lineage. We found 15 genes and pseudogenes in the current Y of D. pseudoobscura , and none are shared with the D. melanogaster Y. Hence, the Y chromosome in the D. pseudoobscura lineage appears to have arisen de novo and is not homologous to the D. melanogaster Y.

The sex-ratio trait is the production of female-biased progenies due to X-linked meiotic drive in males of several Drosophila species. The driving X chromosome (called SR) is not fixed due to at least two stabilizing factors: natural selection (favoring ST, the nondriving standard X) and drive suppression by either Y-linked or autosomal genes. The evolution of autosomal suppression is explained by Fisher's principle, a mechanism of natural selection that leads to equal proportion of males and females in a sexually reproducing population. In fact, sex-ratio expression is partially suppressed by autosomal genes in at least three Drosophila species. The population genetics of this system is not completely understood. In this article we develop a mathematical model for the evolution of autosomal suppressors of SR (sup alleles) and show that: (i) an autosomal suppressor cannot invade when SR is very deleterious in males (c < ⅓, where c is the fitness of SR/Y males); (ii) “SR/ST, sup/+” polymorphisms occur when SR is partially deleterious (∼0.3 < c < 1); while (iii) SR neutrality (c = 1) results in sup fixation and thus in total abolishment of drive. So, surprisingly, as long as there is any selection against SR/Y males, neutral autosomal suppressors will not be fixed. In that case, when a polymorphic equilibrium exists, the average female proportion in SR/Y males' progeny is given approximately by (ac+1−a+a2(c+1)2+1−4ac)∕4ac, where a is the fitness of SR/ST females.

The heterochromatic state of the Drosophila Y chromosome has made the cloning and identification of Y-linked genes a challenging process. Here, we report application of a procedure to identify Y-linked gene fragments from the unmapped residue of the whole genome sequencing effort. Previously identified Y-linked genes appear in sequenced scaffolds as individual exons, apparently because many introns have become heterochromatic, growing to enormous size and becoming virtually unclonable. A TBLASTN search using all known proteins as query sequences, tested against a blastable database of the unmapped fragments, produced a number of matches consistent with this scenario. Reverse transcription–PCR and genetic methods were used to confirm those that are expressed, Y-linked genes. The five genes reported here include three protein phosphatases ( Pp1-Y1 , Pp1-Y2 , and PPr-Y ), an occludin-related gene ( ORY ), and a coiled-coils gene ( CCY ). This brings the total to nine protein-coding genes identified on the Drosophila Y chromosome. ORY and CCY may correspond, respectively, to the fertility factors ks-1 and ks-2 , whereas the three protein phosphatases represent novel genes. There remains a strong functional coherence to male function among the genes on the Drosophila Y chromosome.

Intron sizes vary widely among different genes and among homologous genes of different species. The distribution of intron sizes may be maintained in a steady state, reflecting the processes of insertion and deletion of gene sequences, or it may be that the distribution is constrained by natural selection1,2,3. If intron size is governed by natural selection, there should be a statistical association between this size and the rate of recombination per map unit of the genome, assuming that natural selection is less effective in genomic regions of low recombination4,5,6. Here we show that larger introns of Drosophila melanogaster occur preferentially in regions of low recombination, which is consistent with large introns having a deleterious effect. The association is significant (P=40.001, linear regression), despite the fact that no effort was made to stratify the data by other factors that affect intron size, such as the size of the associated coding region7 or the presence of regulatory sequences inside the intron.

In several Drosophila species there is a trait known as “sex-ratio”: males carrying certain X chromosomes (called “SR”) produce female biased progenies due to X-Y meiotic drive. In Drosophila mediopunctata this trait has a variable expression due to Y-linked suppressors of sex-ratio expression, among other factors. There are two types of Y chromosomes (suppressor and nonsuppressor) and two types of SR chromosomes (suppressible and unsuppressible). Sex-ratio expression is suppressed in males with the SRsuppressible/Ysuppressor genotype, whereas the remaining three genotypes produce female biased progenies. Now we have found that ∼10–20% of the Y chromosomes from two natural populations 1500 km apart are suppressors of sex-ratio expression. Preliminary estimates indicate that Ysuppressor has a meiotic drive advantage of 6% over Ynonsuppressor. This Y polymorphism for a nonneutral trait is unexpected under current population genetics theoly. We propose that this polymorphism is stabilized by an equilibrium between meiotic drive and natural selection, resulting from interactions in the population dynamics of X and Y alleles. Numerical simulations showed that this mechanism may stabilize nonneutral Y polymorphisms such as we have found in D. mediopunctata.

Many organisms have sexual recombination, yet its advantages are not fully understood. A new study of the fruit fly Drosophila miranda shows that recombination is essential for removing deleterious mutations from the genome and for allowing beneficial mutations to be incorporated.

Background: Most eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly. Results: WGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm. Conclusions: Whole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes.

Background Due to an abundance of repetitive DNA, the annotation of heterochromatic regions of the genome such as the Y chromosome is problematic. The Y chromosome is involved in key biological functions such as male-fertility and sex-determination and hence, accurate identification of its sequences is vital. The hemipteran insect Rhodnius prolixus is an important vector of Chagas disease, a trypanosomiasis affecting 6–7 million people worldwide. Here we report the identification of the first Y-linked genes of this species. Results The R. prolixus genome was recently sequenced using separate libraries for each sex and the sequences assembled only with male reads are candidates for Y linkage. We found 766 such candidates and PCR tests with the ten largest ones, confirmed Y-linkage for all of them, suggesting that "separate libraries" is a reliable method for the identification of Y-linked sequences. BLAST analyses of the 766 candidate scaffolds revealed that 568 scaffolds contained genes or part of putative genes. We tested Y-linkage for 36 candidates and found that nine of them are Y-linked (the PCR results for the other 25 genes were inconclusive or revealed autosomal/X-linkage). Hence, we describe in this study, for the first time, Y-linked genes in the R. prolixus genome: two zinc finger proteins (Znf-Y1 and Znf-Y2), one metalloproteinase (Met-Y), one aconitase/iron regulatory protein (Aco-Y) and five genes devoid of matches in any database (Rpr-Y1 to Rpr-Y5). Expression profile studies revealed that eight genes are expressed mainly in adult testis (some of which presented a weak expression in the initial developmental stages), while Aco-Y has a gut-restricted expression. Conclusions In this study we showed that the approach used for the R. prolixus genome project (separate sequencing of male and female DNA) is key to easy and fast identification of sex-specific (e.g. Y chromosome sequences). The nine new R. prolixus Y-linked genes reported here provide unique markers for population and phylogenetic analysis and further functional studies of these genes may answer some questions about sex determination, male fertility and Y chromosome evolution in this important species. Fundo Devido à abundância de DNA repetitivo, a anotação de regiões heterocromáticas do genoma, como o cromossomo Y, é problemática. O cromossomo Y está envolvido em funções biológicas importantes, como fertilidade masculina e determinação do sexo e, portanto, a identificação precisa de suas sequências é vital. O inseto hemíptero Rhodnius prolixus é um importante vetor da doença de Chagas, uma tripanossomíase que afeta 6 a 7 milhões de pessoas em todo o mundo. Aqui relatamos a identificação dos primeiros genes ligados ao Y desta espécie. Resultados O genoma de R. prolixus foi recentemente sequenciado usando bibliotecas separadas para cada sexo e as sequências montadas apenas com reads masculinos são candidatas à ligação Y. Encontramos 766 desses candidatos e testes de PCR com os dez maiores, confirmaram a ligação Y para todos eles, sugerindo que "bibliotecas separadas" é um método confiável para a identificação de sequências ligadas ao Y. Análises BLAST dos 766 scaffolds candidatos revelaram que 568 scaffolds continham genes ou parte de genes putativos. Testamos a ligação Y para 36 candidatos e descobrimos que nove deles estão ligados ao Y (os resultados da PCR para os outros 25 genes foram inconclusivos ou revelaram ligação autossômica/X). Assim, descrevemos neste estudo, pela primeira vez, genes ligados ao Y no genoma de R. prolixus: duas proteínas dedo de zinco (Znf-Y1 e Znf-Y2), uma metaloproteinase (Met-Y), uma aconitase/ferro proteína reguladora (Aco-Y) e cinco genes desprovidos de correspondências em qualquer banco de dados (Rpr-Y1 a Rpr-Y5). Estudos de perfil de expressão revelaram que oito genes são expressos principalmente em testículos adultos (alguns dos quais apresentaram uma expressão fraca nos estágios iniciais de desenvolvimento), enquanto o Aco-Y tem uma expressão restrita ao intestino. Conclusões Neste estudo, mostramos que a abordagem usada para o projeto genoma de R. prolixus (sequenciamento separado de DNA masculino e feminino) é a chave para a identificação fácil e rápida de sexo específico (por exemplo, sequências do cromossomo Y). Os nove novos genes ligados ao Y de R. prolixus relatados aqui fornecem marcadores únicos para análise populacional e filogenética e estudos funcionais adicionais desses genes podem responder a algumas perguntas sobre determinação do sexo, fertilidade masculina e evolução do cromossomo Y nesta importante espécie.