Production of biodiesel from waste fish fat through ultrasound-assisted transesterification using petro-diesel as cosolvent and optimization of process parameters using response surface methodology.

Biodiesel is a highly promising and viable alternative to fossil-based diesel that also addresses the urgent need for effective waste management. It can be synthesized by the chemical modification of triglycerides sourced from vegetable origin, animal fat, or algal oil. The transesterification reaction is the preferred method of producing biodiesel. However, the non-miscibility of alcohol and oil layer causes excessive utilization of alcohol, catalyst, and a substantial reacting time and temperature. In the current investigation, transesterification of waste fish oil was performed with petro-diesel as cosolvent, under the influence of ultrasound energy. The combination of both techniques is a unique and efficient way to minimize the mass transfer limitations considerably and hence reduces the parameters of the reaction. It is also a sincere effort to comply with the principles of green chemistry. The optimum reaction conditions were obtained using response surface methodology (RSM) that were as follows: molar ratio of methanol to oil 9.09:1, catalyst concentration of 0.97 wt%, cosolvent concentration of 29.1 wt%, temperature 60.1℃, and a reacting time 30 min. Under these listed conditions, 98.1% biodiesel was achievable, which was in close agreement with the expected result. In addition, the cosolvent removal step from the crude biodiesel was also eliminated as it could be employed as a blended fuel in CI engines.

The hologenome of Daphnia magna reveals possible DNA methylation and microbiome-mediated evolution of the host genome.

Properties that make organisms ideal laboratory models in developmental and medical research are often the ones that also make them less representative of wild relatives. The waterflea Daphnia magna is an exception, by both sharing many properties with established laboratory models and being a keystone species, a sentinel species for assessing water quality, an indicator of environmental change and an established ecotoxicology model. Yet, Daphnia's full potential has not been fully exploited because of the challenges associated with assembling and annotating its gene-rich genome. Here, we present the first hologenome of Daphnia magna, consisting of a chromosomal-level assembly of the D. magna genome and the draft assembly of its metagenome. By sequencing and mapping transcriptomes from exposures to environmental conditions and from developmental morphological landmarks, we expand the previously annotates gene set for this species. We also provide evidence for the potential role of gene-body DNA-methylation as a mutagen mediating genome evolution. For the first time, our study shows that the gut microbes provide resistance to commonly used antibiotics and virulence factors, potentially mediating Daphnia's environmental-driven rapid evolution. Key findings in this study improve our understanding of the contribution of DNA methylation and gut microbiota to genome evolution in response to rapidly changing environments.

Trait evolution during a rapid global weed invasion despite little genetic differentiation.

Invasive species often possess a great capacity to adapt to novel environments in the form of spatial trait variation, as a result of varying selection regimes, genetic drift, or plasticity. We explored the geographic differentiation in several phenotypic traits related to plant growth, reproduction, and defense in the highly invasive Centaurea solstitialis by measuring neutral genetic differentiation (F ST), and comparing it with phenotypic differentiation (P ST), in a common garden experiment in individuals originating from regions representing the species distribution across five continents. Native plants were more fecund than non-native plants, but the latter displayed considerably larger seed mass. We found indication of divergent selection for these two reproductive traits but little overall genetic differentiation between native and non-native ranges. The native versus invasive P ST-F ST comparisons demonstrated that, in several invasive regions, seed mass had increased proportionally more than the genetic differentiation. Traits displayed different associations with climate variables in different regions. Both capitula numbers and seed mass were associated with winter temperature and precipitation and summer aridity in some regions. Overall, our study suggests that rapid evolution has accompanied invasive success of C. solstitialis and provides new insights into traits and their genetic bases that can contribute to fitness advantages in non-native populations.

Historical exposure to chemicals reduces tolerance to novel chemical stress in Daphnia (waterflea).

Until the last few decades, anthropogenic chemicals used in most production processes have not been comprehensively assessed for their risk and impact on wildlife and humans. They are transported globally and usually end up in the environment as unintentional pollutants, causing long-term adverse effects. Modern toxicology practices typically use acute toxicity tests of unrealistic concentrations of chemicals to determine their safe use, missing pathological effects arising from long-term exposures to environmentally relevant concentrations. Here, we study the transgenerational effect of environmentally relevant concentrations of five chemicals on the priority list of international regulatory frameworks on the keystone species Daphnia magna. We expose Daphnia genotypes resurrected from the sedimentary archive of a lake with a known history of chemical pollution to the five chemicals to understand how historical exposure to chemicals influences adaptive responses to novel chemical stress. We measure within- and transgenerational plasticity in fitness-linked life history traits following exposure of "experienced" and "naive" genotypes to novel chemical stress. As the revived Daphnia originate from the same genetic pool sampled at different times in the past, we are able to quantify the long-term evolutionary impact of chemical pollution by studying genome-wide diversity and identifying functional pathways affected by historical chemical stress. Our results suggest that historical exposure to chemical stress causes reduced genome-wide diversity, leading to lower cross-generational tolerance to novel chemical stress. Lower tolerance is underpinned by reduced gene diversity at detoxification, catabolism and endocrine genes in experienced genotypes. We show that these genes sit within pathways that are conserved and potential chemical targets in other species, including humans.

Y chromosomal noncoding RNAs regulate autosomal gene expression via piRNAs in mouse testis.

BACKGROUND: Deciphering the functions of Y chromosome in mammals has been slow owing to the presence of repeats. Some of these repeats transcribe coding RNAs, the roles of which have been studied. Functions of the noncoding transcripts from Y chromosomal repeats however, remain unclear. While a majority of the genes expressed during spermatogenesis are autosomal, mice with different deletions of the long arm of the Y chromosome (Yq) were previously also shown to be characterized by subfertility, sterility and sperm abnormalities, suggesting the presence of effectors of spermatogenesis at this location. Here we report a set of novel noncoding RNAs from mouse Yq and explore their connection to some of the autosomal genes expressed in testis. RESULTS: We describe a set of novel mouse male-specific Y long arm (MSYq)-derived long noncoding (lnc) transcripts, named Pirmy and Pirmy-like RNAs. Pirmy shows a large number of splice variants in testis. We also identified Pirmy-like RNAs present in multiple copies at different loci on mouse Y chromosome. Further, we identified eight differentially expressed autosome-encoded sperm proteins in a mutant mouse strain, XYRIIIqdel (2/3 Yq-deleted). Pirmy and Pirmy-like RNAs have homology to 5'/3'UTRs of these deregulated autosomal genes. Several lines of experiments show that these short homologous stretches correspond to piRNAs. Thus, Pirmy and Pirmy-like RNAs act as templates for several piRNAs. In vitro functional assays reveal putative roles for these piRNAs in regulating autosomal genes. CONCLUSIONS: Our study elucidates a set of autosomal genes that are potentially regulated by MSYq-derived piRNAs in mouse testis. Sperm phenotypes from the Yq-deleted mice seem to be similar to that reported in inter-specific male-sterile hybrids. Taken together, this study provides novel insights into possible role of MSYq-derived ncRNAs in male sterility and speciation.

Extensive standing genetic variation from a small number of founders enables rapid adaptation in Daphnia.

We lack a thorough understanding of the origin and maintenance of standing genetic variation that enables rapid evolutionary responses of natural populations. Whole genome sequencing of a resurrected Daphnia population shows that standing genetic variation in over 500 genes follows an evolutionary trajectory that parallels the pronounced and rapid adaptive evolution of multiple traits in response to predator-driven natural selection and its subsequent relaxation. Genetic variation carried by only five founding individuals from the regional genotype pool is shown to suffice at enabling the observed evolution. Our results provide insight on how natural populations can acquire the genomic variation, through colonization by a few regional genotypes, that fuels rapid evolution in response to strong selection pressures. While these evolutionary responses in our study population involved hundreds of genes, we observed no evidence of genetic erosion.

The methylome of the model arbuscular mycorrhizal fungus, Rhizophagus irregularis, shares characteristics with early diverging fungi and Dikarya.

Early-diverging fungi (EDF) are distinct from Dikarya and other eukaryotes, exhibiting high N6-methyldeoxyadenine (6mA) contents, rather than 5-methylcytosine (5mC). As plants transitioned to land the EDF sub-phylum, arbuscular mycorrhizal fungi (AMF; Glomeromycotina) evolved a symbiotic lifestyle with 80% of plant species worldwide. Here we show that these fungi exhibit 5mC and 6mA methylation characteristics that jointly set them apart from other fungi. The model AMF, R. irregularis, evolved very high levels of 5mC and greatly reduced levels of 6mA. However, unlike the Dikarya, 6mA in AMF occurs at symmetrical ApT motifs in genes and is associated with their transcription. 6mA is heterogeneously distributed among nuclei in these coenocytic fungi suggesting functional differences among nuclei. While far fewer genes are regulated by 6mA in the AMF genome than in EDF, most strikingly, 6mA methylation has been specifically retained in genes implicated in components of phosphate regulation; the quintessential hallmark defining this globally important symbiosis.

Adaptive Divergence under Gene Flow along an Environmental Gradient in Two Coexisting Stickleback Species.

There is a general and solid theoretical framework to explain how the interplay between natural selection and gene flow affects local adaptation. Yet, to what extent coexisting closely related species evolve collectively or show distinctive evolutionary responses remains a fundamental question. To address this, we studied the population genetic structure and morphological differentiation of sympatric three-spined and nine-spined stickleback. We conducted genotyping-by-sequencing and morphological trait characterisation using 24 individuals of each species from four lowland brackish water (LBW), four lowland freshwater (LFW) and three upland freshwater (UFW) sites in Belgium and the Netherlands. This combination of sites allowed us to contrast populations from isolated but environmentally similar locations (LFW vs. UFW), isolated but environmentally heterogeneous locations (LBW vs. UFW), and well-connected but environmentally heterogenous locations (LBW vs. LFW). Overall, both species showed comparable levels of genetic diversity and neutral genetic differentiation. However, for all three spatial scales, signatures of morphological and genomic adaptive divergence were substantially stronger among populations of the three-spined stickleback than among populations of the nine-spined stickleback. Furthermore, most outlier SNPs in the two species were associated with local freshwater sites. The few outlier SNPs that were associated with the split between brackish water and freshwater populations were located on one linkage group in three-spined stickleback and two linkage groups in nine-spined stickleback. We conclude that while both species show congruent evolutionary and genomic patterns of divergent selection, both species differ in the magnitude of their response to selection regardless of the geographical and environmental context.

Evolutionary epidemiology of schistosomiasis: linking parasite genetics with disease phenotype in humans.

Here we assess the role of parasite genetic variation in host disease phenotype in human schistosomiasis by implementing concepts and techniques from environmental association analysis in evolutionary epidemiology. Schistosomiasis is a tropical disease that affects more than 200 million people worldwide and is caused by parasitic flatworms belonging to the genus Schistosoma. While the role of host genetics has been extensively studied and demonstrated, nothing is yet known on the contribution of parasite genetic variation to host disease phenotype in human schistosomiasis. In this study microsatellite genotypes of 1561 Schistosoma mansoni larvae collected from 44 human hosts in Senegal were linked to host characteristics such as age, gender, infection intensity, liver and bladder morbidity by means of multivariate regression methods (on each parasite locus separately). This revealed a highly significant association between allelic variation at the parasite locus L46951 and host infection intensity and bladder morbidity. Locus L46951 is located in the 3' untranslated region of the cGMP-dependent protein kinase gene that is expressed in reproductive organs of adult schistosome worms and appears to be linked to egg production. This putative link between parasite genetic variation and schistosomiasis disease phenotype sets the stage for further functional research.

Adaptive and non-adaptive divergence in a common landscape.

Species in a common landscape often face similar selective environments. The capacity of organisms to adapt to these environments may be largely species specific. Quantifying shared and unique adaptive responses across species within landscapes may thus improve our understanding of landscape-moderated biodiversity patterns. Here we test to what extent populations of two coexisting and phylogenetically related fishes-three-spined and nine-spined stickleback-differ in the strength and nature of neutral and adaptive divergence along a salinity gradient. Phenotypic differentiation, neutral genetic differentiation and genomic signatures of adaptation are stronger in the three-spined stickleback. Yet, both species show substantial phenotypic parallelism. In contrast, genomic signatures of adaptation involve different genomic regions, and are thus non-parallel. The relative contribution of spatial and environmental drivers of population divergence in each species reflects different strategies for persistence in the same landscape. These results provide insight in the mechanisms underlying variation in evolutionary versatility and ecological success among species within landscapes.The three-spined stickleback is a model species for the study of adaptive divergence. Here, Raeymaekers et al. compare how the three-spined stickleback and its relative the nine-spined stickleback vary at the phenotypic and genomic levels in response to the same spatial and environmental drivers.

Can Evolution Supply What Ecology Demands?

A simplistic view of the adaptive process pictures a hillside along which a population can climb: when ecological 'demands' change, evolution 'supplies' the variation needed for the population to climb to a new peak. Evolutionary ecologists point out that this simplistic view can be incomplete because the fitness landscape changes dynamically as the population evolves. Geneticists meanwhile have identified complexities relating to the nature of genetic variation and its architecture, and the importance of epigenetic variation is under debate. In this review, we highlight how complexity in both ecological 'demands' and the evolutionary 'supply' influences organisms' ability to climb fitness landscapes that themselves change dynamically as evolution proceeds, and encourage new synthetic effort across research disciplines towards ecologically realistic studies of adaptation.

Temporal genetic stability in natural populations of the waterflea Daphnia magna in response to strong selection pressure.

Studies monitoring changes in genetic diversity and composition through time allow a unique understanding of evolutionary dynamics and persistence of natural populations. However, such studies are often limited to species with short generation times that can be propagated in the laboratory or few exceptional cases in the wild. Species that produce dormant stages provide powerful models for the reconstruction of evolutionary dynamics in the natural environment. A remaining open question is to what extent dormant egg banks are an unbiased representation of populations and hence of the species' evolutionary potential, especially in the presence of strong environmental selection. We address this key question using the water flea Daphnia magna, which produces dormant stages that accumulate in biological archives over time. We assess temporal genetic stability in three biological archives, previously used in resurrection ecology studies showing adaptive evolutionary responses to rapid environmental change. We show that neutral genetic diversity does not decline with the age of the population and it is maintained in the presence of strong selection. In addition, by comparing temporal genetic stability in hatched and unhatched populations from the same biological archive, we show that dormant egg banks can be consulted to obtain a reliable measure of genetic diversity over time, at least in the multidecadal time frame studied here. The stability of neutral genetic diversity through time is likely mediated by the buffering effect of the resting egg bank.

Characterization of genome-wide SNPs for the water flea Daphnia pulicaria generated by genotyping-by-sequencing (GBS).

The keystone aquatic herbivore Daphnia has been studied for more than 150 years in the context of evolution, ecology and ecotoxicology. Although it is rapidly becoming an emergent model for environmental and population genomics, there have been limited genome-wide level studies in natural populations. We report a unique resource of novel Single Nucleotide Polymorphic (SNP) markers for Daphnia pulicaria using the reduction in genomic complexity with the restriction enzymes approach, genotyping-by-sequencing. Using the genome of D. pulex as a reference, SNPs were scored for 53 clones from five natural populations that varied in lake trophic status. Our analyses resulted in 32,313 highly confident and bi-allelic SNP markers. 1,364 outlier SNPs were mapped on the annotated D. pulex genome, which identified 2,335 genes, including 565 within functional genes. Out of 885 EuKaryotic Orthologous Groups that we found from outlier SNPs, 294 were involved in three metabolic and four regulatory pathways. Bayesian-clustering analyses showed two distinct population clusters representing the possible combined effects of geography and lake trophic status. Our results provide an invaluable tool for future population genomics surveys in Daphnia targeting informative regions related to physiological processes that can be linked to the ecology of this emerging eco-responsive taxon.

Daphnia magna transcriptome by RNA-Seq across 12 environmental stressors.

The full exploration of gene-environment interactions requires model organisms with well-characterized ecological interactions in their natural environment, manipulability in the laboratory and genomic tools. The waterflea Daphnia magna is an established ecological and toxicological model species, central to the food webs of freshwater lentic habitats and sentinel for water quality. Its tractability and cyclic parthenogenetic life-cycle are ideal to investigate links between genes and the environment. Capitalizing on this unique model system, the STRESSFLEA consortium generated a comprehensive RNA-Seq data set by exposing two inbred genotypes of D. magna and a recombinant cross of these genotypes to a range of environmental perturbations. Gene models were constructed from the transcriptome data and mapped onto the draft genome of D. magna using EvidentialGene. The transcriptome data generated here, together with the available draft genome sequence of D. magna and a high-density genetic map will be a key asset for future investigations in environmental genomics.

An SNP-based second-generation genetic map of Daphnia magna and its application to QTL analysis of phenotypic traits.

BACKGROUND: Although Daphnia is increasingly recognized as a model for ecological genomics and biomedical research, there is, as of yet, no high-resolution genetic map for the genus. Such a map would provide an important tool for mapping phenotypes and assembling the genome. Here we estimate the genome size of Daphnia magna and describe the construction of an SNP array based linkage map. We then test the suitability of the map for life history and behavioural trait mapping. The two parent genotypes used to produce the map derived from D. magna populations with and without fish predation, respectively and are therefore expected to show divergent behaviour and life-histories. RESULTS: Using flow cytometry we estimated the genome size of D. magna to be about 238 mb. We developed an SNP array tailored to type SNPs in a D. magna F2 panel and used it to construct a D. magna linkage map, which included 1,324 informative markers. The map produced ten linkage groups ranging from 108.9 to 203.6 cM, with an average distance between markers of 1.13 cM and a total map length of 1,483.6 cM (Kosambi corrected). The physical length per cM is estimated to be 160 kb. Mapping infertility genes, life history traits and behavioural traits on this map revealed several significant QTL peaks and showed a complex pattern of underlying genetics, with different traits showing strongly different genetic architectures. CONCLUSIONS: The new linkage map of D. magna constructed here allowed us to characterize genetic differences among parent genotypes from populations with ecological differences. The QTL effect plots are partially consistent with our expectation of local adaptation under contrasting predation regimes. Furthermore, the new genetic map will be an important tool for the Daphnia research community and will contribute to the physical map of the D. magna genome project and the further mapping of phenotypic traits. The clones used to produce the linkage map are maintained in a stock collection and can be used for mapping QTLs of traits that show variance among the F2 clones.

A comparison of τ-MnAl particulates produced via different routes.

MnAl alloys are very promising rare-earth-free permanent magnets. Nanocrystalline microstructures can have beneficial effects on the properties of magnetic MnAl alloys. In the present work we examined multiple routes to process MnAl alloys and studied the effects of milling on Mn-46 at.% Al powders. Mn54Al46 was produced via gas atomization, melt spinning, and rapid solidification rate processing. It was then mechanically milled using a water-cooled Union Process attritor for times up to 20 h. X-ray diffraction patterns showed the presence of mostly the high-temperature ε-phase with significant amounts of the equilibrium γ2 and ß phases in both the cast and milled particulates. The powders were annealed for various temperatures and times in order to obtain the ferromagnetic τ-phase. Magnetic measurements of the optimally annealed powders showed a coercivity of 3.62 kOe and saturation magnetization of 59.8 emu g(-1) for mechanically milled gas-atomized powder annealed at 500 °C for 30 min.

Computational identification of miRNAs, their targets and functions in three-spined stickleback (Gasterosteus aculeatus).

An intriguing question in biology is how the evolution of gene regulation is shaped by natural selection in natural populations. Among the many known regulatory mechanisms, regulation of gene expression by microRNAs (miRNAs) is of critical importance. However, our understanding of their evolution in natural populations is limited. Studying the role of miRNAs in three-spined stickleback, an important natural model for speciation research, may provide new insights into adaptive polymorphisms. However, lack of annotation of miRNA genes in its genome is a bottleneck. To fill this research gap, we used the genome of three-spined stickleback to predict miRNAs and their targets. We predicted 1486 mature miRNAs using the homology-based miRNA prediction approach. We then performed functional annotation and enrichment analysis of these targets, which identified over-represented motifs. Further, a database resource (GAmiRdb) has been developed for dynamically searching miRNAs and their targets exclusively in three-spined stickleback. Finally, the database was used in two case studies focusing on freshwater adaptation in natural populations. In the first study, we found 44 genomic regions overlapping with predicted miRNA targets. In the second study, we identified two SNPs altering the MRE seed site of sperm-specific glyceraldehyde-3-phosphate gene. These findings highlight the importance of the GAmiRdb knowledge base in understanding adaptive evolution.

RiDs db: Repeats in diseases database.

UNLABELLED: The non-coding fraction of the human genome, which is approximately 98%, is mainly constituted by repeats. Transpositions, expansions and deletions of these repeat elements contribute to a number of diseases. None of the available databases consolidates information on both tandem and interspersed repeats with the flexibility of FASTA based homology search with reference to disease genes. Repeats in diseases database (RiDs db) is a web accessible relational database, which aids analysis of repeats associated with Mendelian disorders. It is a repository of disease genes, which can be searched by FASTA program or by limitedor free- text keywords. Unlike other databases, RiDs db contains the sequences of these genes with access to corresponding information on both interspersed and tandem repeats contained within them, on a unified platform. Comparative analysis of novel or patient sequences with the reference sequences in RiDs db using FASTA search will indicate change in structure of repeats, if any, with a particular disorder. This database also provides links to orthologs in model organisms such as zebrafish, mouse and Drosophila. AVAILABILITY: The database is available for free at http://115.111.90.196/ridsdb/index.php.