Intracellular Expression of an Ice Nucleation Protein Reduces Cryoinjury in Insect Cells

Exposure of insect cells to subzero temperatures typically leads to cell membrane disruption and lethal intracellular ice formation. This study seeks to examine the cryoprotective value of trangenically expressing a bacterial ice nucleation protein (INP) in Spodoptera frugiperda (Sf-21) cells. The bacterium Pseudomonas syringae naturally produces a membrane-bound INP (inaZ), capable of structuring water and initiating ice formation at temperatures as high as -2 °C. I hypothesized that intracellular expression of an altered form of inaZ (PsINP) in Sf-21 cells will mediate highly regulated ice nucleation when cells are cooled to -80 °C in a slow, controlled manner, and that cells expressing PsINP (Sf-21-PsINP) will maintain cell membrane integrity in greater proportions than wildtype cells (Sf-21-WT). Following one freeze-thaw cycle, 60% of Sf-21-WT cell membranes remained intact, while 72% of Sf-21-PsINP cells maintained membrane integrity. This difference is statistically significant, and suggests that PsINP expression helps to prevent cryoinjury during freezing, and positively impacts cell viability following thawing

Incipient resistance to an effective pesticide results from genetic adaptation and the canalization of gene expression

The resistance of pest species to chemical controls has vast ecological, economic, and societal costs. In most cases, resistance is only detected after spreading throughout an entire population. Detecting resistance in its incipient stages, by comparison, provides time to implement preventative strategies. Incipient resistance can be detected by coupling standard toxicology assays with large-scale gene expression experiments. We apply this approach to a system where an invasive parasite, sea lamprey (Petromyzon marinus), has been treated with the highly effective pesticide 3-trifluoromethyl-4-nitrophenol (TFM) for 60 years. Toxicological experiments revealed that lamprey from treated populations did not have higher survival to TFM exposure than lamprey from untreated populations, demonstrating that full-fledged resistance has not yet evolved. In contrast, we find hundreds of genes differentially expressed in response to TFM in the population with the longest history of exposure, many of which relate to TFM's primary mode of action, the uncoupling of oxidative phosphorylation, and subsequent depletion of ATP. Three genes critical to oxidative phosphorylation, ATP5PB, PLCB1, and NDUFA9, were nearly fixed for alternative alleles in comparisons of SNPs between treated and untreated populations (FST > 5 SD from the mean). ATP5PB encodes subunit b of ATP synthase and an additional subunit, ATP5F1B, was canalized for high expression in treated populations, but remained plastic in response to TFM treatment in individuals from the untreated population. These combined genomic and transcriptomic results demonstrate that an adaptive, genetic response to TFM is likely driving incipient resistance in a damaging pest species

Intracellular Expression of an Ice Nucleation Protein Reduces Cryoinjury in Insect Cells

Exposure of insect cells to subzero temperatures typically leads to cell membrane disruption and lethal intracellular ice formation. This study seeks to examine the cryoprotective value of trangenically expressing a bacterial ice nucleation protein (INP) in Spodoptera frugiperda (Sf-21) cells. The bacterium Pseudomonas syringae naturally produces a membrane-bound INP (inaZ), capable of structuring water and initiating ice formation at temperatures as high as -2 °C. I hypothesized that intracellular expression of a genetically altered form of inaZ (PsINP) in Sf-21 cells will mediate highly regulated ice nucleation when cells are cooled to -80 °C in a slow, controlled manner. I also predicted that cells expressing PsINP (Sf-21-PsINP) will maintain cell membrane integrity in greater proportions than wildtype cells (Sf-21-WT). Following one freeze-thaw cycle, 60% of Sf-21-WT cell membranes remained intact, while 72% of Sf-21-PsINP cells maintained membrane integrity. This difference is statistically significant, and indicates that PsINP expression helps to prevent cryoinjury during freezing, and positively impacts cell viability following thawing

Declining Populations in Changing Environments: Adaptive Responses, Genetic Diversity, and Conservation

Many salmonid populations are supported through captive breeding programs in which hatchery production supplies fish for reintroduction or supplementation efforts. In Lake Champlain, Atlantic salmon (Salmo salar) are the subject of a reintroduction effort that is complicated by the occurrence of thiamine (vitamin B1) deficiency in adult salmon returning to spawn. This deficiency results in high offspring mortality rates that must be mitigated by hatchery interventions (reviewed in Chapter 1). I used an experimental transcriptomics approach coupled with survival analyses to assess genetic variation in thiamine deficiency outcomes (i.e., survival at the family level) and identified candidate genes that may comprise a putatively adaptive response to selection imposed by thiamine deficiency (Chapter 2). Using sequence data from this study, I next compared patterns of genetic variation in the Lake Champlain population against two other populations to identify signatures of selection associated with hatchery rearing environment and differences in life history strategies (Chapter 3). Finally, I surveyed salmonid populations for density-dependent effects of adult spawning density on per capita fitness and found that in many cases, hatchery releases can contribute to decreased individual fitness. Using genotype data for returning adults in multiple populations, I also tested for reductions in effective population size (Ne) associated with hatchery supplementation and describe how increasing hatchery contribution to a population decreases Ne(Chapter 4). Together, my results demonstrate the powerful influences of hatchery supplementation on salmonid populations and suggest that specific modifications to hatchery practices can limit negative impacts of captive breeding on population genetic and demographic characteristics

Genomic signatures of adaptation to novel environments: hatchery and life-history associated loci in landlocked and anadromous Atlantic salmon (Salmo salar)

The adaptation of marine and anadromous fishes to novel, freshwater environments requires major physiological shifts in functions related to osmoregulation, immunity, and metabolism. For Atlantic salmon (Salmo salar) populations, such changes have occurred independently in many landlocked populations that were formed as a result of extensive hydrological shifts in North America around 10,000 years ago. We compared patterns of genetic variation between two landlocked and one anadromous population of Atlantic salmon to identify loci that may have played an important evolutionary role in facilitating the transition from an anadromous to an entirely freshwater life history. Outlier loci included single nucleotide polymorphisms (SNPs) in genes related to functions including immunity, growth, and osmoregulation. We also used these same populations to characterize loci associated with distinct hatchery rearing environments. This additional comparison identified outlier SNPs annotated to genes related to wound healing, consistent with findings from other genetic studies of domestication selection in fishes. Together, our results highlight putative responses to both natural selection imposed by major environmental changes and artificial selection levied by differing hatchery environments.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Regional differentiation and extensive hybridization between mitochondrial clades of the Southern Ocean giant sea spiderColossendeis megalonyx

Assessing the enormous diversity of Southern Ocean benthic species and their evolutionary histories 2 is a central task in the era of global climate change. Based on mitochondrial markers, it was recently suggested that the circumpolar giant sea spider Colossendeis megalonyx comprises a complex of at least six cryptic species with mostly small and non-overlapping distribution ranges. Here, we expand the sampling to include over 500 mitochondrial COI sequences of specimens from around the Antarctic. Using multiple species delimitation approaches, the number of distinct mitochondrial OTUs increased from six to 15–20 with our larger dataset. In contrast to earlier studies, many of these clades show almost circumpolar distributions. Additionally, analysis of the nuclear internal transcribed spacer region for a subset of these specimens showed incongruence between nuclear and mitochondrial results. These mito-nuclear discordances suggest that several of the divergent mitochondrial lineages can hybridize and should not be interpreted as cryptic species. Our results suggest survival of C. megalonyx during Pleistocene glaciations in multiple refugia, some of them probably located on the Antarctic shelf, and emphasize the importance of multi-gene datasets to detect the presence of cryptic species, rather than their inference based on mitochondrial data alon