The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Cuticle Maturation in Apis mellifera: Cuticular Hydrocarbons Profiles, Expression and Evolution of Desaturases and Elongases.

Os hidrocarbonetos cuticulares têm importante papel no processo de reconhecimento dos membros da colônia de insetos sociais. Muitos estudos têm mostrado variações qualitativas e quantitativas nestes compostos entre os insetos adultos. Contudo, abordagens referentes à modulação do perfil destes compostos durante a formação da cutícula são escassas, e se restringem aos estágios larval de holometábolos e de ninfas de hemimetábolos. O principal objetivo dessa pesquisa foi caracterizar o perfil de hidrocarbonetos cuticulares e a expressão de genes potencialmente relacionados à sua biossíntese durante o processo de formação e maturação da cutícula adulta. Os perfis de hidrocarbonetos foram caracterizados por meio de GC/MS e mostraram diferenças quantitativas marcantes que significativamente discriminaram as cutículas pupal, adulta-farata e adulta. Em paralelo, sequências de enzimas que catalisam a desaturação (desaturases) ou elongação (elongases) de lipídeos, disponíveis no banco de dados do NCBI, foram utilizadas para o desenho de primers e estudo da expressão gênica por meio de RT-qPCR. Cinco genes de desaturases, e oito genes de elongases mostraram variação de expressão estatisticamente significante no tegumento de abelhas adultas em comparação com pupas e adultas-faratas. Testes de correlação entre os perfis de expressão gênica e de hidrocarbonetos cuticulares evidenciaram os genes potencialmente envolvidos com a biossíntese destes compostos para a formação e maturação da cutícula. Estes resultados corroboram a hipótese de que nos insetos sociais, a cutícula só amadurece completamente por ocasião do início da atividade de forrageamento. Associando estes dados a análises de evolução molecular das desaturases e elongases, pudemos sugerir as etapas da via de síntese de hidrocarbonetos catalisadas por estas enzimas, e assim eleger genes candidatos a futuro silenciamento mediado por RNA de interferência para pesquisa de função.Cuticular hydrocarbons are important for recognition of nestmates in social insect colonies. Many studies have shown qualitative and quantitative variations in the cuticular hydrocarbons between adult insects. However, approaches on developmental profiles of these compounds during cuticle formation and differentiation are scarce, and restricted to larval stages of holometabolous and nymphs of hemimetabolous. The main objective of this work was to characterize the cuticular hydrocarbons profiles and the expression of genes potentially involved in the biosynthesis of these compounds during the synthesis and differentiation of the adult cuticle in the honeybee. The hydrocarbons profiles were characterized using GC/MS and showed remarkable quantitative differences, thus discriminating the pupal, pharate-adult and adult cuticles from each other. In parallel, we used annotated sequences of enzymes catalyzing lipid desaturation (desaturases) or elongation (elongases), available in NCBI data bank, for primers design and gene expression analysis using RT-qPCR. Five desaturase genes and eight elongase genes showed statistically significant expression changes in the integument of adult bees in comparison to pupae and pharate-adults. Correlation tests supported roles of some of the desaturase and elongase genes in hydrocarbons biosynthesis for incorporation into adult cuticle. In addition, these results go along with the hypothesis that in social insects the cuticle is just completed when the insect starts forager activity. Taken together, these data and an analysis on the molecular evolution of desaturases and elongases allowed suggesting the steps in the pathway of cuticular hydrocarbons biosynthesis that are catalyzed by these enzymes, and also allowed to elect candidate genes for further functional studies using gene silencing mediated by RNAi

Machine Learning Supports Long Noncoding RNAs as Expression Markers for Endometrial Carcinoma

Uterine corpus endometrial carcinoma (UCEC) is the second most common type of gynecological tumor. Several research studies have recently shown the potential of different ncRNAs as biomarkers for prognostics and diagnosis in different types of cancers, including UCEC. Thus, we hypothesized that long noncoding RNAs (lncRNAs) could serve as efficient factors to discriminate solid primary (TP) and normal adjacent (NT) tissues in UCEC with high accuracy. We performed an in silico differential expression analysis comparing TP and NT from a set of samples downloaded from the Cancer Genome Atlas (TCGA) database, targeting highly differentially expressed lncRNAs that could potentially serve as gene expression markers. All analyses were performed in R software. The receiver operator characteristics (ROC) analyses and both supervised and unsupervised machine learning indicated a set of 14 lncRNAs that may serve as biomarkers for UCEC. Functions and putative pathways were assessed through a coexpression network and target enrichment analysis

Microsporidia with Vertical Transmission Were Likely Shaped by Nonadaptive Processes

Microsporidia have the leanest genomes among eukaryotes, and their physiological and genomic simplicity has been attributed to their intracellular, obligate parasitic life-style. However, not all microsporidia genomes are small or lean, with the largest dwarfing the smallest ones by at least an order of magnitude. To better understand the evolutionary mechanisms behind this genomic diversification, we explore here two clades of microsporidia with distinct life histories, Ordospora and Hamiltosporidium, parasitizing the same host species, Daphnia magna. Based on seven newly assembled genomes, we show that mixed-mode transmission (the combination of horizontal and vertical transmission), which occurs in Hamiltosporidium, is found to be associated with larger and AT-biased genomes, more genes, and longer intergenic regions, as compared with the exclusively horizontally transmitted Ordospora. Furthermore, the Hamiltosporidium genome assemblies contain a variety of repetitive elements and long segmental duplications. We show that there is an excess of nonsynonymous substitutions in the microsporidia with mixed-mode transmission, which cannot be solely attributed to the lack of recombination, suggesting that bursts of genome size in these microsporidia result primarily from genetic drift. Overall, these findings suggest that the switch from a horizontal-only to a mixed mode of transmission likely produces population bottlenecks in Hamiltosporidium species, therefore reducing the effectiveness of natural selection, and allowing their genomic features to be largely shaped by nonadaptive processes

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, <i>Apis mellifera</i>

<div><p>Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, <i>Apis mellifera</i>. RNAi-mediated knockdown of <i>AmBurs α</i> and <i>AmBurs β</i> bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of <i>AmBurs α</i>, <i>AmBurs β</i> and <i>AmRk</i> is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of <i>AmBurs α</i>, <i>AmBurs β</i> and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.</p></div

Expression of <i>AmBurs α and AmBurs β</i> in the brain, and expression of <i>AmRk</i> in the integument during adult cuticle formation.

<p><b>(A)</b> The main molting events (pupal ecdysis, apolysis, synthesis and differentiation of the adult cuticle, adult ecdysis) are indicated in relation to the ecdysteroid titer variation and adult cuticle phenotype. Pupa (Pw), pharate-adults (Pp, Pdp, Pb, Pbl, Pbm and Pbd) and newly-emerged adults (NE) are the successive phases of the honeybee development. Relative quantification of <b>(B)</b> <i>AmBurs α</i> and <b>(C)</b> <i>AmBurs β</i> mRNAs in the brain of pupae (Pw), pharate adults (Pb, Pbl) and newly emerged-adults (NE). <b>(D)</b> Relative quantification of bursicon receptor (<i>AmRk</i>) transcripts in the integument of the same developmental phases. Transcript levels determined through RT-qPCR. Bars represent mean ± standard error (se) of three samples, each prepared with seven brains or seven thoracic/abdominal integuments. The asterisk indicates statistically significant increase of transcripts in pharate adults in relation to the pupal (Pw) phase (Student's t-test, p<0.05). <b>(E-G)</b> mRNA levels (FPKM+1) in the brain <b>(E, F)</b> and in the integument <b>(G)</b> of pharate adults (Pbm phase) and newly-ecdysed adults (NE phase) determined through RNA-seq. Levels of <i>AmBurs α</i> and <i>AmBurs β</i> were identified in two brain libraries, each prepared with five pooled brains extracted from the Pbm or NE phases. Levels of <i>AmRk</i> were identified in six integument libraries (three from Pbm and three from NE phases), each prepared with five integuments. The asterisk in <b>G</b> indicates a statistically significant difference (adjusted p value = 0.001).</p

The expression of genes encoding structural cuticular proteins was up- or downregulated by <i>AmBurs α</i> and <i>AmBurs β</i> knockdown.

<p><b>(A)</b> Pupae (Pw) were injected in the brain with ds<i>AmBurs α</i> or ds<i>AmBurs β</i>, and integument samples were collected when bees reached the Pbd and NE phases. Transcript levels of <b>(B)</b> <i>AmCPR14</i>, <b>(C)</b> <i>AmCPR3</i>, <b>(D)</b> <i>AmTwdl1</i>, <b>(E)</b> <i>AmTwdl2</i>, <b>(F)</b> <i>Amapd2</i> and <b>(G)</b> <i>Amapd3</i> were quantified in the integument through RT-qPCR using <i>AmRP49</i> as reference gene. Bars represent mean ± se of four samples, each prepared with a single integument. Different letters on the bars indicate statistically significant differences (Student's t-test, p<0,05). RNA-seq using integument samples support the presence of <b>(H)</b> <i>AmCPR14</i>, <b>(I)</b> <i>AmCPR3</i>, <b>(J)</b> <i>AmTwdl1</i>, <b>(K)</b> <i>AmTwdl2</i>, <b>(L)</b> <i>Amapd2</i> and <b>(M)</b> <i>Amapd3</i> transcripts during adult cuticle formation in pharate-adults (Pbm phase) and show that, except for <i>Amapd2</i> <b>(L)</b> (adjusted p-value = 0.226), there is a significant decrease in transcript levels after ecdysis (NE phase) (adjusted p-values ≤ 0.001).</p

Effect of <i>AmBurs α</i> and <i>AmBurs β</i> knockdown on the expression of genes encoding enzymes with roles in the melanization/sclerotization pathway, and on the expression of the bursicon receptor.

<p><b>(A)</b> Pupae (Pw) were injected in the brain with ds<i>AmBurs α</i> or ds<i>AmBurs β</i>, and integument samples were collected when bees reached the Pbl pharate-adult phase and after ecdysis (NE phase). Transcript levels of <b>(B)</b> <i>Amddc</i>, <b>(C)</b> <i>Amth</i>, <b>(D)</b> <i>Ampxd</i> and <b>(E</b>) <i>AmRk</i> were quantified in the integument through RT-qPCR using <i>AmRP49</i> as reference gene. Bars represent mean ± se of four samples, each prepared with the integument of a single bee. Different letters on the bars indicate statistically significant differences (Student's t-test, p<0,05). RNA-seq using integument samples support the presence of <b>(F)</b> <i>Amddc</i>, <b>(G)</b> <i>Amth</i>, <b>(H)</b> <i>Ampxd</i> and <b>(I)</b> <i>AmRk</i> transcripts during adult cuticle formation in pharate-adults (Pbm phase) and show that, except for <i>Amth</i> <b>(B)</b>, which was up-regulated, there is a significant decrease in transcript levels after ecdysis (NE phase) (adjusted p-values ≤ 0.001).</p

20E-dependent expression of <i>AmBurs α</i>, <i>AmBurs β</i> and <i>AmRk</i>.

<p><b>(A)</b> Representation of the moment of 20E injection (Pw phase) and collection of brain and integument samples at the Pb and Pbd pharate adult phases. External aspect of the developing honeybees injected with 20E and (C) controls. <i>AmBurs α</i> transcript levels in the brain of <b>(B)</b> Pb and <b>(C)</b> Pbd phases. <i>AmBurs β</i> transcript levels in the brain of <b>(D)</b> Pb and <b>(E)</b> Pbd phases. <i>AmRk</i> transcript levels in the integument of <b>(F)</b> Pb and <b>(G)</b> Pbd phases. Transcript levels quantified through RT-qPCR. Bars represent mean ± se of three samples, each prepared with two brains or two integuments. The asterisks indicate statistically significant differences (Student's t-test, p<0,05).</p