Christopher S. Willett

Proline biosynthesis genes and their regulation under salinity stress in the euryhaline copepod Tigriopus californicus.

Diverse organisms regulate concentrations of intracellular organic osmolytes in response to changes in environmental salinity or desiccation. In marine crustaceans, accumulation of high concentrations of proline is a dominant component of response to hyperosmotic stress. In the euryhaline copepod Tigriopus californicus, synthesis of proline from its metabolic precursor glutamate is tightly regulated by changes in environmental salinity. Here, for the first time in a marine invertebrate, the genes responsible for this pathway have been cloned and characterized. The two proteins display the sequence features of homologous enzymes identified from other eukaryotes. One of the cloned genes, delta1-pyrroline-5-carboxylase reductase (P5CR), is demonstrated to have the reductase enzyme activity when expressed in proline-auxotroph bacteria, while the second, delta1-pyrroline-5-carboxylase synthase (P5CS), does not rescue proline-auxotroph bacteria. In contrast to results from higher plants, neither levels of P5CS nor P5CR mRNAs increase in response to salinity stress in T. californicus. Hence, regulation of proline synthesis during osmotic stress in T. californicus is likely mediated by some form of post-transcriptional regulation of either P5CS or P5CR. Understanding the regulation this pathway may elucidate the mechanisms limiting the salinity ranges of marine taxa.

VIABILITY OF CYTOCHROME C GENOTYPES DEPENDS ON CYTOPLASMIC BACKGROUNDS IN TIGRIOPUS CALIFORNICUS

Abstract.— Because of their extensive functional interaction, mitochondrial DNA (mtDNA) and nuclear genes may evolve to form coadapted complexes within reproductively isolated populations. As a consequence of coadaptation, the fitness of particular nuclear alleles may depend on mtDNA genotype. Among populations of the copepod Tigriopus californicus, there are high levels of amino acid substitutions in both the mtDNA genes encoding subunits of cytochrome c oxidase (COX) and the nuclear gene encoding cytochrome c (CYC), the substrate for COX. Because of the functional interaction between enzyme and substrate proteins, we hypothesized that the fitness of CYC genotypes would depend on mtDNA genotype. To test this hypothesis, segregation ratios for CYC and a second nuclear marker (histone H1) unrelated to mitochondrial function were scored in F2 progeny of several reciprocal interpopulation crosses. Genotypic ratios at the CYC locus (but not the H1 locus) differed between reciprocal crosses and differed from expected Mendelian ratios, suggesting that CYC genotypic fitnesses were strongly influenced by cytoplasmic (including mtDNA) background. However, in most cases the nature of the deviations from Mendelian ratios and differences between reciprocal crosses are not consistent with simple coevolution between CYC and mtDNA background. In a cross in which both newly hatched larvae and adults were sampled, only the adult sample showed deviations from Mendelian ratios, indicating that genotypic viabilities differed. In two of six crosses, large genotypic ratio differences for CYC were observed between the sexes. These results suggest that significant variation in nuclear‐mtDNA coadaptation may exist between T. californicus populations and that the relative viability of specific cytonuclear allelic combinations is somehow affected by sex.

Can we predict ectotherm responses to climate change using thermal performance curves and body temperatures

Thermal performance curves (TPCs), which quantify how an ectotherms body temperature (Tb ) affects its performance or fitness, are often used in an attempt to predict organismal responses to climate change. Here, we examine the key - but often biologically unreasonable - assumptions underlying this approach; for example, that physiology and thermal regimes are invariant over ontogeny, space and time, and also that TPCs are independent of previously experienced Tb. We show how a critical consideration of these assumptions can lead to biologically useful hypotheses and experimental designs. For example, rather than assuming that TPCs are fixed during ontogeny, one can measure TPCs for each major life stage and incorporate these into stage-specific ecological models to reveal the life stage most likely to be vulnerable to climate change. Our overall goal is to explicitly examine the assumptions underlying the integration of TPCs with Tb , to develop a framework within which empiricists can place their work within these limitations, and to facilitate the application of thermal physiology to understanding the biological implications of climate change.

ENVIRONMENTAL INFLUENCES ON EPISTATIC INTERACTIONS: VIABILITIES OF CYTOCHROME C GENOTYPES IN INTERPOPULATION CROSSES

Abstract .The genetic incompatibilities that underlie F2 hybrid breakdown and reproductive isolation between al‐lopatric populations may be susceptible to environmental interactions. Here we show that epistatic interactions between cytochrome c (CYC) alleles and mitochondrial DNA (mtDNA) variation are dramatically influenced by environmental temperature in interpopulation hybrids of the copepod Tigriopus californicus. CYC is a nuclear‐encoded gene that functionally interacts with electron transport system (ETS) complexes composed in part of mtDNA‐encoded proteins. Previous studies have provided evidence for functional coadaptation between CYC and ETS complex IV (cytochrome c oxidase) and for cytoplasmic effects on the fitness of CYC genotype in copepod hybrids. In this study, selection on CYC genotype is shown to continue into advanced generation hybrids (F2‐F8) increasing the likelihood that CYC itself is involved in the interaction (and not a linked factor). Relative viabilities varied markedly between copepods raised in two different temperature/light regimes. These results suggest that both intrinsic coadaptation and extrinsic selection will influence the outcome of natural hybridizations between populations. Furthermore, the results indicate that the fitness of particular hybrid genotypes depends on additional non‐mtDNA encoded genes that interact with CYC.

POTENTIAL FITNESS TRADE-OFFS FOR THERMAL TOLERANCE IN THE INTERTIDAL COPEPOD TIGRIOPUS CALIFORNICUS

Thermal adaptation to spatially varying environmental conditions occurs in a wide range of species, but what is less clear is the nature of fitness trade‐offs associated with this temperature adaptation. Here, populations of the intertidal copepod Tigriopus californicus are examined at both local and latitudinal scales to determine whether these populations have evolved differences in their survival under high temperature stress. A clear pattern of increasing high temperature stress tolerance is seen with decreasing latitude, consistent with temperature adaptation. Additionally, there is also evidence for significant variation in thermal tolerance on a smaller scale. The competitive fitness of pairs of northern and southern copepod populations were also examined under a series of lower, more moderate temperatures. These fitness assays show that the southern populations that have the best survival under extreme high temperatures have lowered competitive fitness at the lower temperatures tested, whereas the fitness of the southern populations exceeded that of the northern populations at the highest temperatures tested. Combined, these results suggest that there may be evolutionary trade‐offs between performance at high and stressful temperatures and fitness at moderate temperatures in this species.

Pheromone binding proteins in the European and Asian corn borers: no protein change associated with pheromone differences

Pheromone binding proteins (PBPs) are thought to play a role in the recognition of sex pheromone in male moth antennae. By binding selectively to different components of pheromone blends, these PBPs could play a role in differentiating between structurally related compounds. In this study we have characterized the pheromone binding proteins of two pheromone strains of the European corn borer (Ostrinia nubilalis) and also the closely related Asian corn borer (O. furnacalis). We have been able to detect only one PBP gene, which encodes a mature protein that is identical in amino acid sequence in individuals from different pheromone strains and different species. This result suggests that the PBP is not detecting differences between the two isomeric compounds of the European corn borer pheromone or the difference in double bond position between the pheromone molecules of the European and Asian corn borers.

Investigations of fine-scale phylogeography in Tigriopus californicus reveal historical patterns of population divergence

BackgroundThe intertidal copepod Tigriopus californicus is a model for studying the process of genetic divergence in allopatry and for probing the nature of genetic changes that lead to reproductive isolation. Although previous studies have revealed a pattern of remarkably high levels of genetic divergence between the populations of this species at several spatial scales, it is not clear what types of historical processes are responsible. Particularly lacking are data that can yield insights into population history from the finest scales of geographic resolution.ResultsSequence variation in both cytochrome b (CYTB, mtDNA) and the rieske iron-sulfur protein (RISP, nuclear) are examined at a fine scale within four different regions for populations of T. californicus. High levels of genetic divergence are seen for both genes at the broader scale, and genetic subdivision is apparent at nearly all scales in these populations for these two genes. Patterns of polymorphism and divergence in both CYTB and RISP suggest that selection may be leading to non-neutral evolution of these genes in several cases but a pervasive pattern of neither selection nor coadaptation is seen for these markers.ConclusionThe use of sequence data at a fine-scale of resolution in this species has provided novel insights into the processes that have resulted in the accumulation of genetic divergence among populations. This divergence is likely to result from an interplay between a limited dispersal ability for this copepod and the temporal instability of copepod habitat. Both shorter-term processes such as the extinction/recolonization dynamics of copepod pools and longer-term processes such as geological uplift of coastline and sea level changes appear to have impacted the patterns of differentiation. Some patterns of sequence variation are consistent with selection acting upon the loci used in this study; however, it appears that most phylogeographic patterns are the result of history and not selection on these genes in this species.

Deleterious Epistatic Interactions Between Electron Transport System Protein-Coding Loci in the Copepod Tigriopus californicus

The nature of epistatic interactions between genes encoding interacting proteins in hybrid organisms can have important implications for the evolution of postzygotic reproductive isolation and speciation. At this point very little is known about the fitness differences caused by specific closely interacting but evolutionarily divergent proteins in hybrids between populations or species. The intertidal copepod Tigriopus californicus provides an excellent model in which to study such interactions because the species range includes numerous genetically divergent populations that are still capable of being crossed in the laboratory. Here, the effect on fitness due to the interactions of three complex III proteins of the electron transport system in F2 hybrid copepods resulting from crosses of a pair of divergent populations is examined. Significant deviations from Mendelian inheritance are observed for each of the three genes in F2 hybrid adults but not in nauplii (larvae). The two-way interactions between these genes also have a significant impact upon the viability of these hybrid copepods. Dominance appears to play an important role in mediating the interactions between these loci as deviations are caused by heterozygote/homozygote deleterious interactions. These results suggest that the fitness consequences of the interactions of these three complex III-associated genes could influence reproductive isolation in this system.

Characterization of the glutamate dehydrogenase gene and its regulation in a euryhaline copepod

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of free amino acids (FAA) in crustaceans and other metazoans. Glutamate synthesized by GDH via reductive amination is the amino group donor for alanine synthesis and the precursor required for proline synthesis. Since both proline and alanine are important intracellular osmolytes in many marine invertebrates, GDH has been widely implicated as playing a central role in response to hyperosmotic stress in these organisms. We have isolated the gene encoding a GDH homolog from the euryhaline copepod Tigriopus californicus and examined the regulation of GDH under salinity stress. The gene encodes a protein of 557 residues with 76% amino acid identity with Drosophila melanogaster GDH. The gene encodes an N-terminal mitochondrial signal sequence peptide. Only a single intron of 71 bp was found in the GDH gene in T. californicus when genomic sequences and cDNA sequences were compared. The levels of GDH mRNA do not increase during hyperosmotic stress in this copepod. The effects of salt and hyperosmotic stress on GDH enzyme activity were also investigated. GDH activities decrease with increasing NaCl concentrations in in vitro enzyme assays, while live animals exposed to hyperosmotic stress showed no change in GDH enzyme activities. Combined, these results indicate that GDH transcription and enzyme activity do not appear to function in the regulation of alanine and proline accumulation during hyperosmotic stress in T. californicus. The manner in which this important physiological process is regulated remains unknown.

Do pheromone binding proteins converge in amino acid sequence when pheromones converge

Abstract. Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same pheromone component.