Longitudinal analysis on parasite diversity in honeybee colonies: new taxa, high frequency of mixed infections and seasonal patterns of variation

To evaluate the influence that parasites have on the losses of Apis mellifera it is essential to monitor their presence in the colonies over time. Here we analysed the occurrence of nosematids, trypanosomatids and neogregarines in five homogeneous colonies for up to 21 months until they collapsed. The study, which combined the use of several molecular markers with the application of a massive parallel sequencing technology, provided valuable insights into the epidemiology of these parasites: (I) it enabled the detection of parasite species rarely reported in honeybees (Nosema thomsoni, Crithidia bombi, Crithidia acanthocephali) and the identification of two novel taxa; (II) it revealed the existence of a high rate of co-infections (80% of the samples harboured more than one parasite species); (III) it uncovered an identical pattern of seasonal variation for nosematids and trypanosomatids, that was different from that of neogregarines; (IV) it showed that there were no significant differences in the fraction of positive samples, nor in the levels of species diversity, between interior and exterior bees; and (V) it unveiled that the variation in the number of parasite species was not directly linked with the failure of the colonies

Estimating translational selection in Eukaryotic Genomes

Natural selection on codon usage is a pervasive force that acts on a large variety of prokaryotic and eukaryotic genomes. Despite this, obtaining reliable estimates of selection on codon usage has proved complicated, perhaps due to the fact that the selection coefficients involved are very small. In this work, a population genetics model is used to measure the strength of selected codon usage bias, S, in 10 eukaryotic genomes. It is shown that the strength of selection is closely linked to expression and that reliable estimates of selection coefficients can only be obtained for genes with very similar expression levels. We compare the strength of selected codon usage for orthologous genes across all 10 genomes classified according to expression categories. Fungi genomes present the largest S values (2.24–2.56), whereas multicellular invertebrate and plant genomes present more moderate values (0.61–1.91). The large mammalian genomes (human and mouse) show low S values (0.22–0.51) for the most highly expressed genes. This might not be evidence for selection in these organisms as the technique used here to estimate S does not properly account for nucleotide composition heterogeneity along such genomes. The relationship between estimated S values and empirical estimates of population size is presented here for the first time. It is shown, as theoretically expected, that population size has an important role in the operativity of translational selection

Holistic screening of collapsing honey bee colonies in Spain: A case study

Background: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse. Results: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly. Conclusion: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified

Evidence for Pervasive Adaptive Protein Evolution in Wild Mice

The relative contributions of neutral and adaptive substitutions to molecular evolution has been one of the most controversial issues in evolutionary biology for more than 40 years. The analysis of within-species nucleotide polymorphism and between-species divergence data supports a widespread role for adaptive protein evolution in certain taxa. For example, estimates of the proportion of adaptive amino acid substitutions (alpha) are 50% or more in enteric bacteria and Drosophila. In contrast, recent estimates of alpha for hominids have been at most 13%. Here, we estimate alpha for protein sequences of murid rodents based on nucleotide polymorphism data from multiple genes in a population of the house mouse subspecies Mus musculus castaneus, which inhabits the ancestral range of the Mus species complex and nucleotide divergence between M. m. castaneus and M. famulus or the rat. We estimate that 57% of amino acid substitutions in murids have been driven by positive selection. Hominids, therefore, are exceptional in having low apparent levels of adaptive protein evolution. The high frequency of adaptive amino acid substitutions in wild mice is consistent with their large effective population size, leading to effective natural selection at the molecular level. Effective natural selection also manifests itself as a paucity of effectively neutral nonsynonymous mutations in M. m. castaneus compared to humans

Sequencing of Pooled DNA Samples (Pool-Seq) Uncovers Complex Dynamics of Transposable Element Insertions in Drosophila melanogaster

Transposable elements (TEs) are mobile genetic elements that parasitize genomes by semi-autonomously increasing their own copy number within the host genome. While TEs are important for genome evolution, appropriate methods for performing unbiased genome-wide surveys of TE variation in natural populations have been lacking. Here, we describe a novel and cost-effective approach for estimating population frequencies of TE insertions using paired-end Illumina reads from a pooled population sample. Importantly, the method treats insertions present in and absent from the reference genome identically, allowing unbiased TE population frequency estimates. We apply this method to data from a natural Drosophila melanogaster population from Portugal. Consistent with previous reports, we show that low recombining genomic regions harbor more TE insertions and maintain insertions at higher frequencies than do high recombining regions. We conservatively estimate that there are almost twice as many “novel” TE insertion sites as sites known from the reference sequence in our population sample (6,824 novel versus 3,639 reference sites, with on average a 31-fold coverage per insertion site). Different families of transposable elements show large differences in their insertion densities and population frequencies. Our analyses suggest that the history of TE activity significantly contributes to this pattern, with recently active families segregating at lower frequencies than those active in the more distant past. Finally, using our high-resolution TE abundance measurements, we identified 13 candidate positively selected TE insertions based on their high population frequencies and on low Tajima's D values in their neighborhoods

Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70 null flies

<p>Abstract</p> <p>Background</p> <p>The heat shock protein Hsp70 promotes inducible thermotolerance in nearly every organism examined to date. Hsp70 interacts with a network of other stress-response proteins, and dissecting the relative roles of these interactions in causing thermotolerance remains difficult. Here we examine the effect of <it>Hsp70 </it>gene copy number modification on thermotolerance and the expression of multiple stress-response genes in <it>Drosophila melanogaster</it>, to determine which genes may represent mechanisms of stress tolerance independent of Hsp70.</p> <p>Results</p> <p><it>Hsp70 </it>copy number in four strains is positively associated with <it>Hsp70 </it>expression and inducible thermotolerance of severe heat shock. When assayed at carefully chosen temperatures, <it>Hsp70 </it>null flies are almost entirely deficient in thermotolerance. In contrast to expectations, increasing <it>Hsp70 </it>expression levels induced by thermal pretreatment are associated with increasing levels of seven other inducible <it>Hsps </it>across strains. In addition, complete <it>Hsp70 </it>loss causes upregulation of the inducible <it>Hsps </it>and six constitutive stress-response genes following severe heat shocks.</p> <p>Conclusion</p> <p>Modification of <it>Hsp70 </it>copy number quantitatively and qualitatively affects the expression of multiple other stress-response genes. A positive association between absolute expression levels of <it>Hsp70 </it>and other <it>Hsps </it>after thermal pretreatment suggests novel regulatory mechanisms. Severe heat shocks induce both novel gene expression patterns and almost total mortality in the <it>Hsp70 </it>null strain: alteration of gene expression in this strain does not compensate for <it>Hsp70 </it>loss but suggests candidates for overexpression studies.</p

Molecular evidence for increased regulatory conservation during metamorphosis, and against deleterious cascading effects of hybrid breakdown in Drosophila

<p>Abstract</p> <p>Background</p> <p>Speculation regarding the importance of changes in gene regulation in determining major phylogenetic patterns continues to accrue, despite a lack of broad-scale comparative studies examining how patterns of gene expression vary during development. Comparative transcriptional profiling of adult interspecific hybrids and their parental species has uncovered widespread divergence of the mechanisms controlling gene regulation, revealing incompatibilities that are masked in comparisons between the pure species. However, this has prompted the suggestion that misexpression in adult hybrids results from the downstream cascading effects of a subset of genes improperly regulated in early development.</p> <p>Results</p> <p>We sought to determine how gene expression diverges over development, as well as test the cascade hypothesis, by profiling expression in males of <it>Drosophila melanogaster</it>, <it>D. sechellia</it>, and <it>D. simulans</it>, as well as the <it>D. simulans </it>(♀) × <it>D. sechellia </it>(♂) male F1 hybrids, at four different developmental time points (3rd instar larval, early pupal, late pupal, and newly-emerged adult). Contrary to the cascade model of misexpression, we find that there is considerable stage-specific autonomy of regulatory breakdown in hybrids, with the larval and adult stages showing significantly more hybrid misexpression as compared to the pupal stage. However, comparisons between pure species indicate that genes expressed during earlier stages of development tend to be more conserved in terms of their level of expression than those expressed during later stages, suggesting that while Von Baer's famous law applies at both the level of nucleotide sequence and expression, it may not apply necessarily to the underlying overall regulatory network, which appears to diverge over the course of ontogeny and which can only be ascertained by combining divergent genomes in species hybrids.</p> <p>Conclusion</p> <p>Our results suggest that complex integration of regulatory circuits during morphogenesis may lead to it being more refractory to divergence of underlying gene regulatory mechanisms - more than that suggested by the conservation of gene expression levels between species during earlier stages. This provides support for a 'developmental hourglass' model of divergence of gene expression in <it>Drosophila </it>resulting in a highly conserved pupal stage.</p

Evolutionary Dynamics of the Ty3/Gypsy LTR Retrotransposons in the Genome of Anopheles gambiae

Ty3/gypsy elements represent one of the most abundant and diverse LTR-retrotransposon (LTRr) groups in the Anopheles gambiae genome, but their evolutionary dynamics have not been explored in detail. Here, we conduct an in silico analysis of the distribution and abundance of the full complement of 1045 copies in the updated AgamP3 assembly. Chromosomal distribution of Ty3/gypsy elements is inversely related to arm length, with densities being greatest on the X, and greater on the short versus long arms of both autosomes. Taking into account the different heterochromatic and euchromatic compartments of the genome, our data suggest that the relative abundance of Ty3/gypsy LTRrs along each chromosome arm is determined mainly by the different proportions of heterochromatin, particularly pericentric heterochromatin, relative to total arm length. Additionally, the breakpoint regions of chromosomal inversion 2La appears to be a haven for LTRrs. These elements are underrepresented more than 7-fold in euchromatin, where 33% of the Ty3/gypsy copies are associated with genes. The euchromatin on chromosome 3R shows a faster turnover rate of Ty3/gypsy elements, characterized by a deficit of proviral sequences and the lowest average sequence divergence of any autosomal region analyzed in this study. This probably reflects a principal role of purifying selection against insertion for the preservation of longer conserved syntenyc blocks with adaptive importance located in 3R. Although some Ty3/gypsy LTRrs show evidence of recent activity, an important fraction are inactive remnants of relatively ancient insertions apparently subject to genetic drift. Consistent with these computational predictions, an analysis of the occupancy rate of putatively older insertions in natural populations suggested that the degenerate copies have been fixed across the species range in this mosquito, and also are shared with the sibling species Anopheles arabiensis

Contrasting Patterns of Transposable Element Insertions in Drosophila Heat-Shock Promoters

The proximal promoter regions of heat-shock genes harbor a remarkable number of P transposable element (TE) insertions relative to both positive and negative control proximal promoter regions in natural populations of Drosophila melanogaster. We have screened the sequenced genomes of 12 species of Drosophila to test whether this pattern is unique to these populations. In the 12 species' genomes, transposable element insertions are no more abundant in promoter regions of single-copy heat-shock genes than in promoters with similar or dissimilar architecture. Also, insertions appear randomly distributed across the promoter region, whereas insertions clustered near the transcription start site in promoters of single-copy heat-shock genes in D. melanogaster natural populations. Hsp70 promoters exhibit more TE insertions per promoter than all other genesets in the 12 species, similarly to in natural populations of D. melanogaster. Insertions in the Hsp70 promoter region, however, cluster away from the transcription start site in the 12 species, but near it in natural populations of D. melanogaster. These results suggest that D. melanogaster heat-shock promoters are unique in terms of their interaction with transposable elements, and confirm that Hsp70 promoters are distinctive in TE insertions across Drosophila

Ancestral Inference and the Study of Codon Bias Evolution: Implications for Molecular Evolutionary Analyses of the Drosophila melanogaster Subgroup

Reliable inference of ancestral sequences can be critical to identifying both patterns and causes of molecular evolution. Robustness of ancestral inference is often assumed among closely related species, but tests of this assumption have been limited. Here, we examine the performance of inference methods for data simulated under scenarios of codon bias evolution within the Drosophila melanogaster subgroup. Genome sequence data for multiple, closely related species within this subgroup make it an important system for studying molecular evolutionary genetics. The effects of asymmetric and lineage-specific substitution rates (i.e., varying levels of codon usage bias and departures from equilibrium) on the reliability of ancestral codon usage was investigated. Maximum parsimony inference, which has been widely employed in analyses of Drosophila codon bias evolution, was compared to an approach that attempts to account for uncertainty in ancestral inference by weighting ancestral reconstructions by their posterior probabilities. The latter approach employs maximum likelihood estimation of rate and base composition parameters. For equilibrium and most non-equilibrium scenarios that were investigated, the probabilistic method appears to generate reliable ancestral codon bias inferences for molecular evolutionary studies within the D. melanogaster subgroup. These reconstructions are more reliable than parsimony inference, especially when codon usage is strongly skewed. However, inference biases are considerable for both methods under particular departures from stationarity (i.e., when adaptive evolution is prevalent). Reliability of inference can be sensitive to branch lengths, asymmetry in substitution rates, and the locations and nature of lineage-specific processes within a gene tree. Inference reliability, even among closely related species, can be strongly affected by (potentially unknown) patterns of molecular evolution in lineages ancestral to those of interest