#psubiology

Most animal cells rely on aerobic metabolism for survival and are damaged or die within minutes without oxygen. Embryos of the annual killifish Austrofundulus limnaeus, however, survive months without oxygen. Determining how their cells survive without oxygen has the potential to revolutionize our understanding of the cellular mechanisms supporting vertebrate anoxia tolerance and the evolution of such tolerance. Therefore, we aimed to establish and characterize an anoxia-tolerant cell line from A. limnaeus for investigating mechanisms of vertebrate anoxia tolerance. The PSU-AL-WS40NE cell line of neuroepithelial identity was established from embryonic tissue of A. limnaeus using a tissue explant. The cells can survive for at least 49 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for two mammalian cell lines. PSU-AL-WS40NE cells accumulate lactate during anoxia, indicating use of common metabolic pathways for anaerobic metabolism. Additionally, they express many of the same small noncoding RNAs that are stress-responsive in whole embryos of A. limnaeus and mammalian cells, as well as anoxia-responsive small noncoding RNAs derived from the mitochondrial genome (mitosRNAs). The establishment of the cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology.

Zebrafish (Danio rerio) are a widely utilized model system for human disorders, but common laboratory strains have distinct behavioral and physiological differences. Accompanying these known strain differences, commonly used “wildtype” zebrafish strains have both shared and unique suites of single nucleotide polymorphisms and copy number variants (CNVs). Despite this, genomic variation is often ignored in study design, and the actual strain used is often not adequately reported. The goal of this study was to assess CNVs across three common laboratory strains of zebrafish—AB, Tubingen (TU), and WIK—and provide these data as a tool for the zebrafish community. Herein we identified 1351 CNV regions within the most recent genome assembly (GRCz11) covering 1.9% of the zebrafish genome (31.7 Mb). CNVs were found across all chromosomes, and 2200 genes (5121 transcripts) lie within ±5 kb of identified CNVs, pointing to likely cis regulatory actions of CNVs on nearby gene neighbors. We have created a Public Session accessible on the UCSC Genome Browser to view CNVs from this study titled “danRer11 zebrafish CNV across strains” as a tool for the zebrafish community.

Copy number variants (CNVs) represent a substantial source of genomic variation in vertebrates and have been associated with numerous human diseases. Despite this, the extent of CNVs in the zebrafish, an important model for human disease, remains unknown. Using 80 zebrafish genomes, representing three commonly used laboratory strains and one native population, we constructed a genome-wide, high-resolution CNV map for the zebrafish comprising 6,080 CNV elements and encompassing 14.6% of the zebrafish reference genome. This amount of copy number variation is four times that previously observed in other vertebrates, including humans. Moreover, 69% of the CNV elements exhibited strain specificity, with the highest number observed for Tubingen. This variation likely arose, in part, from Tubingen's large founding size and composite population origin. Additional population genetic studies also provided important insight into the origins and substructure of these commonly used laboratory strains. This extensive variation among and within zebrafish strains may have functional effects that impact phenotype and, if not properly addressed, such extensive levels of germ-line variation and population substructure in this commonly used model organism can potentially confound studies intended for translation to human diseases.

The unique life form of plants promotes the accumulation of large numbers of somatic mutations that can be passed on to offspring in the next generation. However, rates of mutation accumulation remain similar in plants and animals. The solution to this paradox may be that plants have the potential to filter somatic mutations prior to seed dispersal via three forms of intra-generation selection. To provide an experimental test of this hypothesis, we used plants of Mimulus guttatus to compare the performance of offspring from self-pollinations made within the same flower (autogamy), to offspring from self-pollinations between stems on the same plant (geitonogamy). The fitness effects of somatic mutations are expected to differ between progeny from these crosses, as autogamy will result in homozygosity of a proportion of somatic mutations, but geitonogamous progeny will remain heterozygous. We developed a novel analytical approach to test whether the homozygous effects of somatic mutations were evident from differences in the means and variances of fitness between autogamous and geitonogamous progeny. Consistent with our predictions, the variance in progeny fitness among stems was significantly greater for autogamous compared to geitonogamous progeny from the same stem. Surprisingly, several autogamous progeny groups displayed increased fitness compared to geitonogamous controls, indicating that the effects of beneficial somatic mutations were prevalent in progeny. Phenotypic effects of somatic mutations were also evident from the positive association between mean fitness and the within-group variance in fitness for autogamous progeny. These results support the hypothesis that somatic mutations accumulate during vegetative growth, but they are filtered by the different forms of intra-generation selection, resulting in the culling of expressed deleterious mutations and the retention of beneficial mutations.

Viruses that infect thermophilic Archaea are unique in both their structure and genetic makeup. The lemon-shaped fuselloviruses—which infect members of the order Sulfolobales, growing optimally at 80 °C and pH 3—are some of the most ubiquitous and best studied viruses of the thermoacidophilic Archaea. Nonetheless, much remains to be learned about these viruses. In order to investigate fusellovirus evolution, we have isolated and characterized a novel fusellovirus, Sulfolobus spindle-shaped virus 10 (formerly SSV-L1). Comparative genomic analyses highlight significant similarity with both SSV8 and SSV9, as well as conservation of promoter elements within the Fuselloviridae. SSV10 encodes five ORFs with no homology within or outside of the Fuselloviridae, as well as a putatively functional Cas4-like ORF, which may play a role in evading CRISPR-mediated host defenses. Moreover, we demonstrate the ability of SSV10 to withstand mutation in a fashion consistent with mutagenesis in SSV1.

Thermophilic Nanoarchaeota form symbiotic associations with Archaea and three nanoarchaeotal systems have been described. To explore the range of nanoarchaeotal-host associations further, we enriched for nanoarchaeotes from a hot spring at Hell’s Gate, Tikitere, New Zealand. A stable anaerobic enrichment culture was obtained at 80 °C, pH 6.0. Transmission electron microscopy (TEM) revealed small (∼200 nm) flagellated cocci that were often attached to larger cocci. The nanoarchaeote (strain Ncl-1) and its host (strain NZ3T) were isolated in co-culture and their genomes assembled. Based on 16S rRNA gene similarity (88.4%) and average amino acid identity (AAI; 52%), Ncl-1 is closely related to Candidatus Nanopusillus acidilobi and their genomes both encode for archaeal flagella and partial glycolysis and gluconeogenesis pathways, but lack ATP synthase genes. Like Nanoarchaeum equitans, Ncl-1 has a CRISPR-Cas system. Similar to all nanoarchaeotes, Ncl-1 relies on its crenarchaeotal host for most of its biosynthetic needs. The host NZ3T was isolated in pure culture and grows on complex proteinaceous substrates but not on sugars, alcohols, or fatty acids. NZ3T requires thiosulfate and grows best at 82 °C and pH 6.0. 16S rRNA gene and concatenated ribosomal protein phylogenies placed strain NZ3T in the Desulfurococcaceae, most closely related to Ignisphaera aggregans (∼92% 16S rRNA gene sequence similarity, 45% AAI). Based on phylogenetic, physiological and genomic data, Ncl-1 and NZ3T represent novel genera in the Nanoarchaeota and the Desulfurococcaceae, respectively, with the proposed names Candidatus Nanoclepta minutus and Zestosphaera tikiterensis gen. nov., sp. nov., type strain NZ3T (=DSM 107634T =OCM 1213T).

Abstract: The potential for rapid evolution in invasive species makes them useful for studying adaptive responses of populations to novel environments. However, phenotypic divergence during invasion is not necessarily due to selection, but may be a product of neutral processes resulting from population bottlenecks during colonization and range expansion. We investigated phenotypic adaptation during the establishment and range expansion of the invasive bunchgrass, slender false brome (Brachypodium sylvaticum; Poaceae). Utilizing a novel approach, we made robust comparisons of functional traits using genetic similarity based on unique alleles to determine the genetic probability of contribution from native source regions, and integrated these probabilities into our QST‐FST comparisons for twelve physiological and anatomical traits associated with drought stress in the introduced range. Our results indicate phenotypic divergence greater than neutral expectations in five traits between native and invasive populations, indicating selective divergence occurred during invasive species establishment. Results indicate that the majority of divergence in B. sylvaticum occurred after introduction to the novel environment, but prior to invasive range expansion. This study provides evidence for adaptive genetic differentiation during the establishment of an invasive species, while also describing a robust method for detection of selective processes after species introduction to a novel environment.

Significance and Impact of the Study: The possibility of genetic variation controlling intestinal microbiome diversity could have significant impacts on strain selection for rainbow trout aquaculture, especially given the consistent rearing conditions experienced in our sample populations likely result in differences in intramyocellular lipid storage.