Detection of Minute virus of mice strains in different cell lines: Implications for adventitious agent testing.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests. An MMV-specific polymerase chain reaction was evaluated as a more encompassing method and was shown as suitable replacement for cell culture-based detection of the different MMV strains, with the additional benefit that detection is more rapid and can be extended to other rodent parvoviruses that might contaminate biotechnological processes. Although no MMV contamination event of human-derived cell lines has happened in the past, biotechnological processes that are based on these also need to consider MMV-specific testing, as, for example, HEK293, a human-derived cell line commonly used in biopharmaceutical manufacturing, was shown as susceptible to productive MMV infection in the current work.

Characteristics of the spiny dogfish (Squalus acanthias) nuclear genome.

Sequenced shark nuclear genomes are underrepresented, with reference genomes available for only four out of nine orders so far. Here, we present the nuclear genome, with annotations, of the spiny dogfish (Squalus acanthias), a shark of interest to biomedical and conservation efforts, and the first representative of the second largest order of sharks (Squaliformes) with nuclear genome annotations available. Using Pacific Biosciences Continuous Long Read data in combination with Illumina paired-end and Hi-C sequencing, we assembled the genome de novo, followed by RNA-Seq-supported annotation. The final chromosome-level assembly is 3.7 Gb in size, has a BUSCO completeness score of 91.6%, and an error rate of less than 0.02%. Annotation predicted 33,283 gene models in the spiny dogfish's genome, of which 31,979 are functionally annotated.

A closed, autologous bioprocess optimized for TCR-T cell therapies.

Autologous cell therapy has proven to be an effective treatment for hematological malignancies. Cell therapies for solid tumors are on the horizon, however the high cost and complexity of manufacturing these therapies remain a challenge. Routinely used open steps to transfer cells and reagents through unit operations further burden the workflow reducing efficiency and increasing the chance for human error. Here we describe a fully closed, autologous bioprocess generating engineered TCR-T cells. This bioprocess yielded 5-12 × 10e9 TCR-expressing T cells, transduced at low multiplicity of infections, within 7-10 days, and cells exhibited an enriched memory T-cell phenotype and enhanced metabolic fitness. It was demonstrated that activating, transducing, and expanding leukapheresed cells in a bioreactor without any T-cell or peripheral blood mononuclear cell enrichment steps had a high level of T-cell purity (~97%). Several critical process parameters of the bioreactor, including culturing at a high cell density (7e6 cells/mL), adjusting rocking agitations during phases of scale-up, lowering glycolysis through the addition of 2-deoxy- d-glucose, and modulating interleukin-2 levels, were investigated on their roles in regulating transduction efficiency, cell growth, and T-cell fitness such as T-cell memory phenotype and resistance to activation-induced cell death. The bioprocess described herein supports scale-out feasibility by enabling the processing of multiple patients' batches in parallel within a Grade C cleanroom.

Differences by origin in methylome suggest eco-phenotypes in the kelp Saccharina latissima.

Most kelp species are of high ecological and economic importance worldwide, but are highly susceptible to rising ocean temperatures due to their sessile lifestyle. Due to interference with reproduction, development and growth, natural kelp forests have vanished in multiple regions after extreme summer heat waves. Furthermore, increasing temperatures are likely to decrease biomass production and, thus, reduce production security of farmed kelp. Epigenetic variation, and cytosine methylation as a heritable epigenetic trait, is a rapid means of acclimation and adaptation to environmental conditions, including temperature. While the first methylome of brown macroalgae has been recently described in the kelp Saccharina japonica, its functional relevance and contribution to environmental acclimation is currently unknown. The main objective of our study was to identify the importance of the methylome in the congener kelp species Saccharina latissima for temperature acclimation. Our study is the first to compare DNA methylation in kelp between wild populations of different latitudinal origin, and the first to investigate the effect of cultivation and rearing temperature on genome-wide cytosine methylation. Origin appears to determine many traits in kelp, but it is unknown to what extent the effects of thermal acclimation may be overruled by lab-related acclimation. Our results suggest that seaweed hatchery conditions have strong effects on the methylome and, thus, putatively on the epigenetically controlled characteristics of young kelp sporophytes. However, culture origin could best explain epigenetic differences in our samples suggesting that epigenetic mechanisms contribute to local adaptation of eco-phenotypes. Our study is a first step to understand whether DNA methylation marks (via their effect on gene regulation) may be used as biological regulators to enhance production security and kelp restoration success under rising temperatures, and highlights the importance to match hatchery conditions to origin.

Evolutionary dynamics of sex-biased gene expression in a young XY system: insights from the brown alga genus Fucus.

Sex-biased gene expression is considered to be an underlying cause of sexually dimorphic traits. Although the nature and degree of sex-biased expression have been well documented in several animal and plant systems, far less is known about the evolution of sex-biased genes in more distant eukaryotic groups. Here, we investigate sex-biased gene expression in two brown algal dioecious species, Fucus serratus and Fucus vesiculosus, where male heterogamety (XX/XY) has recently emerged. We find that in contrast to evolutionary distant plant and animal lineages, male-biased genes do not experience high turnover rates, but instead reveal remarkable conservation of bias and expression levels between the two species, suggesting their importance in sexual differentiation. Genes with consistent male bias were enriched in functions related to gamete production, along with sperm competition and include three flagellar proteins under positive selection. We present one of the first reports, outside of the animal kingdom, showing that male-biased genes display accelerated rates of coding sequence evolution compared with female-biased or unbiased genes. Our results imply that evolutionary forces affect male and female sex-biased genes differently on structural and regulatory levels, resulting in unique properties of differentially expressed transcripts during reproductive development in Fucus algae.

Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

Ancient DNA Reveals Maternal Philopatry of the Northeast Eurasian Brown Bear (Ursus arctos) Population during the Holocene.

Significant palaeoecological and paleoclimatic changes that took place during Late Pleistocene-Early Holocene transition are considered important factors that led to megafauna extinctions. Unlike many other species, the brown bear (Ursus arctos) has survived this geological time. Despite the fact that several mitochondrial DNA clades of brown bears became extinct at the end of the Pleistocene, this species is still widely distributed in Northeast Eurasia. Here, using the ancient DNA analysis of a brown bear individual that inhabited Northeast Asia in the Middle Holocene (3460 ± 40 years BP) and comparative phylogenetic analysis, we show a significant mitochondrial DNA similarity of the studied specimen with modern brown bears inhabiting Yakutia and Chukotka. In this study, we clearly demonstrate the maternal philopatry of the Northeastern Eurasian U. arctos population during the several thousand years of the Holocene.

Exploring the potential of mRNA for taxonomic delineation of marine benthic eukaryotes.

Direct sequencing of mRNA isolated from environmental samples has been commonly used to analyze the functional activity of ambient communities and, occasionally used for taxonomic identification. Here we assess the viability of using mRNA for investigating the species composition of marine benthic eukaryotes. Total RNA was extracted from sediments sampled close to fish farms and mRNA was sequenced after poly-A enrichment on an Illumina MiSeq sequencer. We investigated the origin of both raw reads and assembled contigs by aligning them to the NCBI non-redundant (nr/nt) nucleotide database. Although sequences were predominantly of eukaryotic origin, the analyses were complicated both by experimental and database artefacts. These issues were addressed by applying filtering procedures that removed the majority of ambiguous sequences from downstream analyses. These processes resulted in a set of 436 high-confidence contigs, the vast majority of which mapped to benthic organisms. Our alignments were dominated by annelids, consistent with burrowing groups found in a parallel morphological analysis. This study shows that it is possible to obtain adequate taxonomic information from the RNA of an eukaryotic community from a limited sample at a moderate cost, demonstrates how both laboratory and in silico artefacts can be overcome through appropriate bioinformatic procedures, and finally highlights some of the drawbacks and caveats of using NCBI as a reference database for such a dataset.

Insights into the species evolution of Calanus copepods in the northern seas revealed by de novo transcriptome sequencing.

Copepods of the zooplankton genus Calanus play a key role in marine ecosystems in the northern seas. Although being among the most studied organisms on Earth, due to their ecological importance, genomic resources for Calanus spp. remain scarce, mostly due to their large genome size (from 6 to 12 Gbps). As an alternative to whole-genome sequencing in Calanus spp., we sequenced and de novo assembled transcriptomes of five Calanus species: Calanus glacialis, C. hyperboreus, C. marshallae, C. pacificus, and C. helgolandicus. Functional assignment of protein families based on clusters of orthologous genes (COG) and gene ontology (GO) annotations showed analogous patterns of protein functions across species. Phylogenetic analyses using maximum likelihood (ML) of 191 protein-coding genes mined from RNA-seq data fully resolved evolutionary relationships among seven Calanus species investigated (five species sequenced for this study and two species with published datasets), with gene and site concordance factors showing that 109 out of 191 protein-coding genes support a separation between three groups: the C. finmarchicus group (including C. finmarchicus, C. glacialis, and C. marshallae), the C. helgolandicus group (including C. helgolandicus, C. sinicus, and C. pacificus) and the monophyletic C. hyperboreus group. The tree topology obtained in ML analyses was similar to a previously proposed phylogeny based on morphological criteria and cleared certain ambiguities from past studies on evolutionary relationships among Calanus species.

Adaptation of Temperate Seagrass to Arctic Light Relies on Seasonal Acclimatization of Carbon Capture and Metabolism.

Due to rising global surface temperatures, Arctic habitats are becoming thermally suitable for temperate species. Whether a temperate species can immigrate into an ice-free Arctic depends on its ability to tolerate extreme seasonal fluctuations in daylength. Thus, understanding adaptations to polar light conditions can improve the realism of models predicting poleward range expansions in response to climate change. Plant adaptations to polar light have rarely been studied and remain unknown in seagrasses. If these ecosystem engineers can migrate polewards, seagrasses will enrich biodiversity, and carbon capture potential in shallow coastal regions of the Arctic. Eelgrass (Zostera marina) is the most widely distributed seagrass in the northern hemisphere. As the only seagrass species growing as far north as 70°N, it is the most likely candidate to first immigrate into an ice-free Arctic. Here, we describe seasonal (and diurnal) changes in photosynthetic characteristics, and in genome-wide gene expression patterns under strong annual fluctuations of daylength. We compared PAM measurements and RNA-seq data between two populations at the longest and shortest day of the year: (1) a Mediterranean population exposed to moderate annual fluctuations of 10-14 h daylength and (2) an Arctic population exposed to high annual fluctuations of 0-24 h daylength. Most of the gene expression specificities of the Arctic population were found in functions of the organelles (chloroplast and mitochondrion). In winter, Arctic eelgrass conserves energy by repressing respiration and reducing photosynthetic energy fluxes. Although light-reactions, and genes involved in carbon capture and carbon storage were upregulated in summer, enzymes involved in CO2 fixation and chlorophyll-synthesis were upregulated in winter, suggesting that winter metabolism relies not only on stored energy resources but also on active use of dim light conditions. Eelgrass is unable to use excessive amounts of light during summer and demonstrates a significant reduction in photosynthetic performance under long daylengths, possibly to prevent photoinhibition constrains. Our study identified key mechanisms that allow eelgrass to survive under Arctic light conditions and paves the way for experimental research to predict whether and up to which latitude eelgrass can potentially migrate polewards in response to climate change.

Revisiting mouse minute virus inactivation by high temperature short time treatment.

In recent years, high temperature short time (HTST) treatment technology has been increasingly adopted for medium treatment to mitigate the potential risk of viral contamination in mammalian cell culture GMP manufacturing facilities. Mouse minute virus (MMV), also called minute virus of mice (MVM), implicated in multiple viral contamination events is commonly used as a relevant model virus to assess the effectiveness of HTST treatment of cell culture media. However, results from different studies vary broadly in inactivation kinetics as well as log reduction factors (LRFs) achieved under given treatment conditions. To determine whether the reported discrepancies stemmed from differences in MMV strains, laboratory-scale HTST devices, medium matrices, and/or experimental designs, we have taken a collaborative approach to systematically assess the effectiveness of HTST treatment for MMV inactivation. This effort was conceptualized based on a media treatment gap analysis conducted by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) under the MIT Center for Biomedical Innovation (CBI). Specifically, two different MMV strains were used to evaluate the effectiveness of HTST at various treatment conditions with regard to exposure temperature and hold time duration by two independent laboratories within two different companies. To minimize experimental variations, the two sites used the same batches of MMV stocks, the same commercially purchased medium, and the same model of thermocyclers as the laboratory-scale HTST device. The two independent laboratories yielded similar MMV inactivation kinetics and comparable LRF. No significant differences were observed between the two MMV strains evaluated, suggesting that the variations from prior studies were likely due to differences in equipment, medium matrices, or other factors. The data presented here indicate that MMV inactivation by HTST treatment obeys first-order kinetics and can be mathematically modeled using an Arrhenius equation. The model-based extrapolation provides a quantitative estimate of MMV inactivation by the current industry standard HTST condition (102°C for a hold time of 10 s) used for medium treatment. Finally, based on the data from the current study and the industry experience, it is recommended that any alternative virus barrier technologies adopted for medium treatment should provide a clearance of at least 3.0 LRF based on a worst-case model virus to effectively mitigate potential risks of viral contamination.

The Seagrass Methylome Is Associated With Variation in Photosynthetic Performance Among Clonal Shoots.

Evolutionary theory predicts that clonal organisms are more susceptible to extinction than sexually reproducing organisms, due to low genetic variation and slow rates of evolution. In agreement, conservation management considers genetic variation as the ultimate measure of a population's ability to survive over time. However, clonal plants are among the oldest living organisms on our planet. Here, we test the hypothesis that clonal seagrass meadows display epigenetic variation that complements genetic variation as a source of phenotypic variation. In a clonal meadow of the seagrass Zostera marina, we characterized DNA methylation among 42 shoots. We also sequenced the whole genome of 10 shoots to correlate methylation patterns with photosynthetic performance under exposure to and recovery from 27°C, while controlling for somatic mutations. Here, we show for the first time that clonal seagrass shoots display DNA methylation variation that is independent from underlying genetic variation, and associated with variation in photosynthetic performance under experimental conditions. It remains unknown to what degree this association could be influenced by epigenetic responses to transplantation-related stress, given that the methylomes showed a strong shift under acclimation to laboratory conditions. The lack of untreated control samples in the heat stress experiment did not allow us to distinguish methylome shifts induced by acclimation from such induced by heat stress. Notwithstanding, the co-variation in DNA methylation and photosynthetic performance may be linked via gene expression because methylation patterns varied in functionally relevant genes involved in photosynthesis, and in the repair and prevention of heat-induced protein damage. While genotypic diversity has been shown to enhance stress resilience in seagrass meadows, we suggest that epigenetic variation plays a similar role in meadows dominated by a single genotype. Consequently, conservation management of clonal plants should consider epigenetic variation as indicator of resilience and stability.

Proteomic and Transcriptomic Patterns during Lipid Remodeling in Nannochloropsis gaditana.

Nutrient limited conditions are common in natural phytoplankton communities and are often used to increase the yield of lipids from industrial microalgae cultivations. Here we studied the effects of bioavailable nitrogen (N) and phosphorus (P) deprivation on the proteome and transcriptome of the oleaginous marine microalga Nannochloropsis gaditana. Turbidostat cultures were used to selectively apply either N or P deprivation, controlling for variables including the light intensity. Global (cell-wide) changes in the proteome were measured using Tandem Mass Tag (TMT) and LC-MS/MS, whilst gene transcript expression of the same samples was quantified by Illumina RNA-sequencing. We detected 3423 proteins, where 1543 and 113 proteins showed significant changes in abundance in N and P treatments, respectively. The analysis includes the global correlation between proteomic and transcriptomic data, the regulation of subcellular proteomes in different compartments, gene/protein functional groups, and metabolic pathways. The results show that triacylglycerol (TAG) accumulation under nitrogen deprivation was associated with substantial downregulation of protein synthesis and photosynthetic activity. Oil accumulation was also accompanied by a diverse set of responses including the upregulation of diacylglycerol acyltransferase (DGAT), lipase, and lipid body associated proteins. Deprivation of phosphorus had comparatively fewer, weaker effects, some of which were linked to the remodeling of respiratory metabolism.

Novel genomic resources for shelled pteropods: a draft genome and target capture probes for Limacina bulimoides, tested for cross-species relevance.

BACKGROUND: Pteropods are planktonic gastropods that are considered as bio-indicators to monitor impacts of ocean acidification on marine ecosystems. In order to gain insight into their adaptive potential to future environmental changes, it is critical to use adequate molecular tools to delimit species and population boundaries and to assess their genetic connectivity. We developed a set of target capture probes to investigate genetic variation across their large-sized genome using a population genomics approach. Target capture is less limited by DNA amount and quality than other genome-reduced representation protocols, and has the potential for application on closely related species based on probes designed from one species. RESULTS: We generated the first draft genome of a pteropod, Limacina bulimoides, resulting in a fragmented assembly of 2.9 Gbp. Using this assembly and a transcriptome as a reference, we designed a set of 2899 genome-wide target capture probes for L. bulimoides. The set of probes includes 2812 single copy nuclear targets, the 28S rDNA sequence, ten mitochondrial genes, 35 candidate biomineralisation genes, and 41 non-coding regions. The capture reaction performed with these probes was highly efficient with 97% of the targets recovered on the focal species. A total of 137,938 single nucleotide polymorphism markers were obtained from the captured sequences across a test panel of nine individuals. The probes set was also tested on four related species: L. trochiformis, L. lesueurii, L. helicina, and Heliconoides inflatus, showing an exponential decrease in capture efficiency with increased genetic distance from the focal species. Sixty-two targets were sufficiently conserved to be recovered consistently across all five species. CONCLUSION: The target capture protocol used in this study was effective in capturing genome-wide variation in the focal species L. bulimoides, suitable for population genomic analyses, while providing insights into conserved genomic regions in related species. The present study provides new genomic resources for pteropods and supports the use of target capture-based protocols to efficiently characterise genomic variation in small non-model organisms with large genomes.

The complete mitochondrial genome of the blue skate Dipturus batis.

The complete mitochondrial genome of the blue skate Dipturus batis is described from shotgun sequencing on an Illumina next-generation sequencing platform. We report a 16,911 bp long sequence similar in size to other members of the genus, containing 13 protein-coding regions, 22 tRNA genes, 2 rRNA genes, and 2 non-coding areas. Phylogenetic analysis was performed using the complete mitochondrial genomes of 17 related species, placing D. batis within the Rajini tribe of the Rajidae family, consistent with current taxonomy. The new resource adds to a growing database of rajid mitogenomes which will help resolve phylogenetic relationships within the family.

Towards population genomics in non-model species with large genomes: a case study of the marine zooplankton Calanus finmarchicus.

Advances in next-generation sequencing technologies and the development of genome-reduced representation protocols have opened the way to genome-wide population studies in non-model species. However, species with large genomes remain challenging, hampering the development of genomic resources for a number of taxa including marine arthropods. Here, we developed a genome-reduced representation method for the ecologically important marine copepod Calanus finmarchicus (haploid genome size of 6.34 Gbp). We optimized a capture enrichment-based protocol based on 2656 single-copy genes, yielding a total of 154 087 high-quality SNPs in C. finmarchicus including 62 372 in common among the three locations tested. The set of capture probes was also successfully applied to the congeneric C. glacialis. Preliminary analyses of these markers revealed similar levels of genetic diversity between the two Calanus species, while populations of C. glacialis showed stronger genetic structure compared to C. finmarchicus. Using this powerful set of markers, we did not detect any evidence of hybridization between C. finmarchicus and C. glacialis. Finally, we propose a shortened version of our protocol, offering a promising solution for population genomics studies in non-model species with large genomes.

Circulating small non-coding RNAs provide new insights into vitamin K nutrition and reproductive physiology in teleost fish.

BACKGROUND: Vitamin K (VK) is a fat-soluble vitamin known for its essential role in blood coagulation, but also on other biological processes (e.g. reproduction, brain and bone development) have been recently suggested. Nevertheless, the molecular mechanisms behind its particular function on reproduction are not yet fully understood. METHODS: The potential role of VK on reproduction through nutritional supplementation in Senegalese sole (Solea senegalensis) was assessed by gonadal maturation and 11-ketosterone, testosterone and estriol plasma levels when fed with control or VK supplemented (1250 mg kg-1 of VK1) diets along a six month trial. At the end, sperm production and quality (viability and DNA fragmentation) were evaluated. Circulating small non-coding RNAs (sncRNAs) in blood plasma from males were also studied through RNA-Seq. RESULTS: Fish fed with dietary VK supplementation had increased testosterone levels and lower sperm DNA fragmentation. SncRNAs from blood plasma were found differentially expressed when nutritional and sperm quality conditions were compared. PiR-675//676//4794//5462 and piR-74614 were found up-regulated in males fed with dietary VK supplementation. Let-7g, let-7e(18nt), let-7a-1, let-7a-3//7a-2//7a-1, let-7e(23nt) and piR-675//676//4794//5462 were found to be up-regulated and miR-146a and miR-146a-1//146a-2//146a-3 down-regulated when fish with low and high sperm DNA fragmentation were compared. Bioinformatic analyses of predicted mRNAs targeted by sncRNAs revealed the potential underlying pathways. CONCLUSIONS: VK supplementation improves fish gonad maturation and sperm quality, suggesting an unexpected and complex regulation of the nutritional status and reproductive performance through circulating sncRNAs. GENERAL SIGNIFICANCE: The use of circulating sncRNAs as reliable and less-invasive physiological biomarkers in fish nutrition and reproduction has been unveiled.

The Intestinal Mycobiota in Wild Zebrafish Comprises Mainly Dothideomycetes While Saccharomycetes Predominate in Their Laboratory-Reared Counterparts.

As an integral part of the resident microbial community of fish intestinal tract, the mycobiota is expected to play important roles in health and disease resistance of the host. The composition of the diverse fungal communities, which colonize the intestine, is greatly influenced by the host, their diet and geographic origin. Studies of fungal communities are rare and the majority of previous studies have relied on culture-based methods. In particular, fungal communities in fish are also poorly characterized. The aim of this study was to provide an in-depth overview of the intestinal mycobiota in a model fish species (zebrafish, Danio rerio) and to determine differences in fungal composition between wild and captive specimens. We have profiled the intestinal mycobiota of wild-caught (Sharavati River, India), laboratory-reared (Bodø, Norway) and wild-caught-laboratory-kept (Uttara, India) zebrafish by sequencing the fungal internal transcribed spacer 2 region on the Illumina MiSeq platform. Wild fish were exposed to variable environmental factors, whereas both laboratory groups were kept in controlled conditions. There were also differences in husbandry practices at Bodø and Uttara, particularly diet. Zebrafish from Bodø were reared in the laboratory for over 10 generations, while wild-caught-laboratory-kept fish from Uttara were housed in the laboratory for only 2 months before sample collection. The intestine of zebrafish contained members of more than 15 fungal classes belonging to the phyla Ascomycota, Basidiomycota, and Zygomycota. Fungal species richness and diversity distinguished the wild-caught and laboratory-reared zebrafish communities. Wild-caught zebrafish-associated mycobiota comprised mainly Dothideomycetes in contrast to their Saccharomycetes-dominated laboratory-reared counterparts. The predominant Saccharomycetes in laboratory-reared fish belonged to the saprotrophic guild. Another characteristic feature of laboratory-reared fish was the significantly higher abundance of Cryptococcus (Tremellomycetes) compared to wild fish. This pioneer study has shed light into the differences in the intestinal fungal communities of wild-caught and laboratory-reared zebrafish and the baseline data generated will enrich our knowledge on fish mycobiota.

Low incubation temperature during early development negatively affects survival and related innate immune processes in zebrafish larvae exposed to lipopolysaccharide.

In many fish species, the immune system is significantly constrained by water temperature. In spite of its critical importance in protecting the host against pathogens, little is known about the influence of embryonic incubation temperature on the innate immunity of fish larvae. Zebrafish (Danio rerio) embryos were incubated at 24, 28 or 32 °C until first feeding. Larvae originating from each of these three temperature regimes were further distributed into three challenge temperatures and exposed to lipopolysaccharide (LPS) in a full factorial design (3 incubation × 3 challenge temperatures). At 24 h post LPS challenge, mortality of larvae incubated at 24 °C was 1.2 to 2.6-fold higher than those kept at 28 or 32 °C, regardless of the challenge temperature. LPS challenge at 24 °C stimulated similar immune-related processes but at different levels in larvae incubated at 24 or 32 °C, concomitantly with the down-regulation of some chemokine and lysozyme transcripts in the former group. Larvae incubated at 24 °C and LPS-challenged at 32 °C exhibited a limited immune response with up-regulation of hypoxia and oxidative stress processes. Annexin A2a, S100 calcium binding protein A10b and lymphocyte antigen-6, epidermis were identified as promising candidates for LPS recognition and signal transduction.

Dynamics of miRNA transcriptome during gonadal development of zebrafish.

Studies in non-teleost vertebrates have found microRNAs (miRNAs) to be essential for proper gonadal development. However, comparatively little is known about their role during gonadal development in teleost fishes. So far in zebrafish, a model teleost, transcript profiling throughout gonadal development has not been established because of a tiny size of an organ in juvenile stages and its poor distinguishability from surrounding tissues. We performed small RNA sequencing on isolated gonads of See-Thru-Gonad line, from the undifferentiated state at 3 weeks post fertilization (wpf) to fully mature adults at 24 wpf. We identified 520 gonadal mature miRNAs; 111 of them had significant changes in abundance over time, while 50 miRNAs were either testis- or ovary-enriched significantly in at least one developmental stage. We characterized patterns of miRNA abundance over time including isomiR variants. We identified putative germline versus gonadal somatic miRNAs through differential small RNA sequencing of isolated gametes versus the whole gonads. This report is the most comprehensive analysis of the miRNA repertoire in zebrafish gonads during the sexual development to date and provides an important database from which functional studies can be performed.