Heat stress response and evolution of thermal tolerance in the copepod Tigriopus californicus

With the warming trend due to climate change, conservation of species requires knowledge in ecological and evolutionary aspects of thermal tolerance and adaptation. In this dissertation, I use tidepool copepod, Tigriopus californicus, as a model for studying both aspects of thermal tolerance. For ecological aspect, identifying the most sensitive life stage can help us predict future responses especially in marine organisms with complex life history. I examined different survivorship to acute heat stress among life stages and across populations of T. californicus. Results revealed early life stages of T. californicus survived acute heat stress at higher temperatures than adults in contrast to popular belief. However, heat stress during larval stage of T. californicus resulted in developmental delay. Survivorship in larval and juvenile stages across populations also conform with a pattern previously observed in adults with more heat tolerant populations toward southern range of the species. In order to uncover the evolutionary basis underlying thermal tolerant in T. californicus, I examined allele specific expression in F1 hybrid between populations from San Diego (SD) and Santa Cruz (SC). RNA sequencing revealed regulatory divergence in several gene ontology categories that potentially contribute to thermal tolerance including, electron carrier genes, genes involved in muscle and cuticle assembly, genes involved in proteolysis and Heat Shock Protein (HSP) genes. Heat Shock Protein Beta 1 (HSPB1) is one of the highest expressed HSPs in response to heat stress. HSPB1 Allelic imbalance suggested divergence in cis regulatory element underlying heat stress induced expression. HSPB1 promotor sequencing revealed polymorphisms in the Heat Shock Elements (HSEs), a binding site for Heat Shock Transcription Factor (HSF), where heat tolerant southern populations contain 2 canonical HSEs while northern populations have substitutions in the conserved motif of HSEs. Allele specific expression in more F1 crosses confirmed biased expression favoring alleles from populations with 2 canonical HSEs. Functional assays comparing recombinant SD and SC HSPB1 demonstrated that SD HSPB1 has a better capacity for preventing protein aggregation and preserving enzymatic function under high temperature. Overall, results from this dissertation provide insights on both ecological and evolutionary perspectives of thermal tolerance

Heat stress response and evolution of thermal tolerance in the copepod Tigriopus californicus

With the warming trend due to climate change, conservation of species requires knowledge in ecological and evolutionary aspects of thermal tolerance and adaptation. In this dissertation, I use tidepool copepod, Tigriopus californicus, as a model for studying both aspects of thermal tolerance. For ecological aspect, identifying the most sensitive life stage can help us predict future responses especially in marine organisms with complex life history. I examined different survivorship to acute heat stress among life stages and across populations of T. californicus. Results revealed early life stages of T. californicus survived acute heat stress at higher temperatures than adults in contrast to popular belief. However, heat stress during larval stage of T. californicus resulted in developmental delay. Survivorship in larval and juvenile stages across populations also conform with a pattern previously observed in adults with more heat tolerant populations toward southern range of the species. In order to uncover the evolutionary basis underlying thermal tolerant in T. californicus, I examined allele specific expression in F1 hybrid between populations from San Diego (SD) and Santa Cruz (SC). RNA sequencing revealed regulatory divergence in several gene ontology categories that potentially contribute to thermal tolerance including, electron carrier genes, genes involved in muscle and cuticle assembly, genes involved in proteolysis and Heat Shock Protein (HSP) genes. Heat Shock Protein Beta 1 (HSPB1) is one of the highest expressed HSPs in response to heat stress. HSPB1 Allelic imbalance suggested divergence in cis regulatory element underlying heat stress induced expression. HSPB1 promotor sequencing revealed polymorphisms in the Heat Shock Elements (HSEs), a binding site for Heat Shock Transcription Factor (HSF), where heat tolerant southern populations contain 2 canonical HSEs while northern populations have substitutions in the conserved motif of HSEs. Allele specific expression in more F1 crosses confirmed biased expression favoring alleles from populations with 2 canonical HSEs. Functional assays comparing recombinant SD and SC HSPB1 demonstrated that SD HSPB1 has a better capacity for preventing protein aggregation and preserving enzymatic function under high temperature. Overall, results from this dissertation provide insights on both ecological and evolutionary perspectives of thermal tolerance

Multiple Modes of Adaptation: Regulatory and Structural Evolution in a Small Heat Shock Protein Gene.

Thermal tolerance is a key determinant of species distribution. Despite much study, the genetic basis of adaptive evolution of thermal tolerance, including the relative contributions of transcriptional regulation versus protein evolution, remains unclear. Populations of the intertidal copepod Tigriopus californicus are adapted to local thermal regimes across their broad geographic range. Upon thermal stress, adults from a heat tolerant southern population, San Diego (SD), upregulate several heat shock proteins (HSPs) to higher levels than those from a less tolerant northern population, Santa Cruz (SC). Suppression of a specific HSP, HSPB1, significantly reduces T. californicus survival following acute heat stress. Sequencing of HSPB1 revealed population specific nucleotide substitutions in both promoter and coding regions of the gene. HSPB1 promoters from heat tolerant populations contain two canonical heat shock elements (HSEs), the binding sites for heat shock transcription factor (HSF), whereas less tolerant populations have mutations in these conserved motifs. Allele specific expression of HSPB1 in F1 hybrids between tolerant and less tolerant populations showed significantly biased expression favoring alleles from tolerant populations and supporting the adaptive divergence in these cis-regulatory variants. The functional impact of population-specific nonsynonymous substitutions in HSPB1 coding sequences was tested by assessing the thermal stabilization properties of SD versus SC HSPB1 protein variants. Recombinant HSPB1 from the southern SD population showed greater capacity for protecting protein structure under elevated temperature. Our results indicate that both regulatory and protein coding sequence evolution within a single gene appear to contribute to thermal tolerance phenotypes and local adaptation among conspecific populations