Mitonuclear effects on sex ratio persist across generations in interpopulation hybrids.

Eukaryotic energy production requires tight coordination between nuclear and mitochondrial gene products. Because males and females often have different energetic strategies, optimal mitonuclear coordination may be sex-specific. Previous work found evidence for sex-specific mitonuclear effects in the copepod Tigriopus californicus by comparing two parental lines and their reciprocal F1 crosses. However, an alternative hypothesis is that the patterns were driven by the parental source of nuclear alleles. Here we test this alternative hypothesis by extending the same cross to F2 hybrids, who receive both maternal and paternal nuclear alleles from F1 hybrids. Results confirm mitonuclear effects on sex ratio, with distorted ratios persisting from the F1 to F2 generations, despite reduced fitness in F2 hybrids. No sex by cross interactions were found for other phenotypic traits measured. Mitochondrial DNA content was higher in females. Both routine metabolic rate and oxidative DNA damage were lower in F2 hybrids than in parentals. The persistence of sex-specific mitonuclear effects, even in the face of F2 hybrid breakdown, attests to the magnitude of these effects, which contribute to the maintenance of within-population mitochondrial DNA polymorphisms.

Optimal outbreeding is shaped during larval life-history in the splash pool copepod Tigriopus californicus.

Inbreeding and outbreeding depression are dynamic forms of selection critical to mating system evolution and the efficacy of conservation biology. Most evidence on how the relative severity and timing of these forces are shaped is confined to self-fertilization, distant outcrossing, and intermediate 'optimal outcrossing' in hermaphrodites. We tested the notion that closed population demographics may reduce and delay the costs of inbreeding relative to distant outbreeding in an intertidal copepod with separate sexes and a biphasic larval / post-metamorphic life-history (Tigriopus californicus). At three lifecycle stages (fecundity, metamorphosis, and post-metamorphosis), we quantified the effects of inbreeding and outbreeding in crosses with varying degrees of recent common ancestry. Although inbreeding and outbreeding depression have distinct genetic mechanisms, both manifested the same stage-specific consequences for fitness. Inbreeding and outbreeding depression were not apparent for fecundity, post-metamorphic survival, sex ratio, or the ability to acquire mates, but inbreeding between full siblings and outbreeding between interpopulation hybrids reduced the fraction of offspring that completed metamorphosis by 32% and 47%, respectively. On average, the effects of inbreeding on metamorphic rate were weaker and nearly twice as variable among families than those of outbreeding, suggesting genetic load was less pervasive than the incompatibilities accrued between divergent populations. Overall, our results indicate the transition from larval to juvenile life stages is markedly susceptible to both inbreeding and outbreeding depression in T. californicus. We suggest stage-specific selection acting concurrently with the timing of metamorphosis may be an instrumental factor shaping reproductive optima in species with complex life-histories.

Genomic Architecture of Hybrid Male Sterility in a Species Without Sex Chromosomes (Tigriopus californicus, Copepoda: Harpacticoida).

Sterility among hybrids is one of the most prevalent forms of reproductive isolation delineating species boundaries and is expressed disproportionately in heterogametic XY males. While hybrid male sterility (HMS) due to the "large X effect" is a well-recognized mechanism of reproductive isolation, it is less clear how HMS manifests in species that lack heteromorphic sex chromosomes. We evaluated differences in allele frequencies at approximately 460,000 SNPs between fertile and sterile F2 interpopulation male hybrids to characterize the genomic architecture of HMS in a species without sex chromosomes (Tigriopus californicus). We tested associations between HMS and mitochondrial-nuclear and/or nuclear-nuclear signatures of incompatibility. Genomic regions associated with HMS were concentrated on a single chromosome with the same primary 2-Mbp regions identified in one pair of reciprocal crosses. Gene Ontology analysis revealed that annotations associated with spermatogenesis were the most overrepresented within the implicated region, with nine protein-coding genes connected with this process found in the quantitative trait locus of chromosome 2. Our results indicate that a narrow genomic region was associated with the sterility of male hybrids in T. californicus and suggest that incompatibilities among select nuclear loci may replace the large X effect when sex chromosomes are absent.

Tissue Clearing To Localize Microplastics via Three-Dimensional Imaging of Whole Organisms.

Understanding the biological impacts of plastic pollution requires an effective methodology to detect unlabeled microplastics in environmental samples. Detecting unlabeled microplastics in an organism generally requires a digestion protocol, which results in the loss of spatial information on the distribution of microplastic within the organism and could lead to the disappearance of the smaller plastics. Fluorescence microscopy allows visualization of ingested microplastics but many labeling strategies are nonspecific and label biomass, thus limiting our ability to distinguish internalized plastics. While prelabeled plastics can be used to avoid nonspecific labeling, this approach precludes the detection of environmental microplastics in organisms. Also, using prelabeled microplastics can affect the viability of the organism and impact plastic uptake. Thus, a method was developed that employs nonspecific labeling with a tissue-clearing technique. Briefly, unlabeled microplastics are stained with a fluorescent dye after ingestion by the organism. The tissue-clearing technique then removes tissue-bound dye while rendering the structurally intact organism transparent. The internalized plastics remain stained and can be visualized in the cleared tissue with fluorescence microscopy. The technique is demonstrated using polystyrene beads in living aquatic organismsTigriopus californicusandDaphnia magnaand by spiking a model vertebrate (Cephalochordata) with different microplastics.

Loss of mitochondrial performance at high temperatures is correlated with upper thermal tolerance among populations of an intertidal copepod.

Environmental temperatures have pervasive effects on the performance and tolerance of ectothermic organisms, and thermal tolerance limits likely play key roles underlying biogeographic ranges and responses to environmental change. Mitochondria are central to metabolic processes in eukaryotic cells, and these metabolic functions are thermally sensitive; however, potential relationships between mitochondrial function, thermal tolerance limits and local thermal adaptation in general remain unresolved. Loss of ATP synthesis capacity at high temperatures has recently been suggested as a mechanistic link between mitochondrial function and upper thermal tolerance limits. Here we use a common-garden experiment with seven locally adapted populations of intertidal copepods (Tigriopus californicus), spanning approximately 21.5° latitude, to assess genetically based variation in the thermal performance curves of maximal ATP synthesis rates in isolated mitochondria. These thermal performance curves displayed substantial variation among populations with higher ATP synthesis rates at lower temperatures (20-25 °C) in northern populations than in southern populations. In contrast, mitochondria from southern populations maintained ATP synthesis rates at higher temperatures than the temperatures that caused loss of ATP synthesis capacity in mitochondria from northern populations. Additionally, there was a tight correlation between the thermal limits of ATP synthesis and previously determined variation in upper thermal tolerance limits among populations. This suggests that mitochondria may play an important role in latitudinal thermal adaptation in T. californicus, and supports the hypothesis that loss of mitochondrial performance at high temperatures is linked to whole-organism thermal tolerance limits in this ectotherm.

Exogenous oxidative stressors elicit differing age and sex effects in Tigriopus californicus.

As organisms age, cellular function declines in a time-dependent manner. Oxidative stress induced by reactive oxygen species damages cellular machinery and contributes to senescence which narrows the homeostatic window needed to maintain function and survive stress. Sex differences in longevity are apparent in many species and may be related to sex-specific homeostatic responses. Here we use the emerging aging model system Tigriopus californicus, the splashpool copepod, to estimate sex- and age-specific tolerances to two chemical oxidants, hydrogen peroxide and paraquat. Sex-specific tolerance was estimated for both oxidants simultaneously for 15 age-classes. As animals aged, hydrogen peroxide tolerance decreased but paraquat tolerance increased. Also, we observed no sex difference for hydrogen peroxide tolerance, while females were more tolerant of paraquat. Our results demonstrate that oxidative stressors can have dramatically different sex and age effects in Tigriopus californicus. These findings underscore the challenges ahead in understanding relationships among oxidative stressors, sex, and aging.

A combination of red structural and pigmentary coloration in the eyespot of a copepod.

While the specific mechanisms of colour production in biological systems are diverse, the mechanics of colour production are straightforward and universal. Colour is produced through the selective absorption of light by pigments, the scattering of light by nanostructures or a combination of both. When Tigriopus californicus copepods were fed a carotenoid-limited diet of yeast, their orange-red body coloration became faint, but their eyespots remained unexpectedly bright red. Raman spectroscopy indicated a clear signature of the red carotenoid pigment astaxanthin in eyespots; however, refractive index matching experiments showed that eyespot colour disappeared when placed in ethyl cinnamate, suggesting a structural origin for the red coloration. We used transmission electron microscopy to identify consecutive nanolayers of spherical air pockets that, when modelled as a single thin film layer, possess the correct periodicity to coherently scatter red light. We then performed microspectrophotometry to quantify eyespot coloration and confirmed a distinct colour difference between the eyespot and the body. The observed spectral reflectance from the eyespot matched the reflectance predicted from our models when considering the additional absorption by astaxanthin. Together, this evidence suggests the persistence of red eyespots in copepods is the result of a combination of structural and pigmentary coloration.

Mitonuclear interactions alter sex-specific longevity in a species without sex chromosomes.

Impaired mitochondrial function can lead to senescence and the ageing phenotype. Theory predicts degenerative ageing phenotypes and mitochondrial pathologies may occur more frequently in males due to the matrilineal inheritance pattern of mitochondrial DNA observed in most eukaryotes. Here, we estimated the sex-specific longevity for parental and reciprocal F1 hybrid crosses for inbred lines derived from two allopatric Tigriopus californicus populations with over 20% mitochondrial DNA divergence. T. californicus lacks sex chromosomes allowing for more direct testing of mitochondrial function in sex-specific ageing. To better understand the ageing mechanism, we estimated two age-related phenotypes (mtDNA content and 8-hydroxy-20-deoxyguanosine (8-OH-dG) DNA damage) at two time points in the lifespan. Sex differences in lifespan depended on the mitochondrial and nuclear backgrounds, including differences between reciprocal F1 crosses which have different mitochondrial haplotypes on a 50 : 50 nuclear background, with nuclear contributions coming from alternative parents. Young females showed the highest mtDNA content which decreased with age, while DNA damage in males increased with age and exceed that of females 56 days after hatching. The adult sex ratio was male-biased and was attributed to complex mitonuclear interactions. Results thus demonstrate that sex differences in ageing depend on mitonuclear interactions in the absence of sex chromosomes.

Ultraviolet irradiation alters the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions in copepod myocytes.

The efficient production of energy via oxidative phosphorylation is essential to the growth, survival, and reproduction of eukaryotes. The behavior (position of, and communication between, mitochondria) and morphology of mitochondria play key roles in efficient energy production and are influenced by oxidative stressors such as ultraviolet (UV) radiation. We tested the hypothesis that mitochondria change their behavior and morphology to meet energetic demands of responding to changes in oxidative stress. Specifically, we predicted that UV irradiation would increase the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions to influence whole-animal metabolic rate. Using transmission electron microscopy, we found that both three and six hours of UV-A/B irradiation (0.5 W/m2) increased the proportion of inter-mitochondrial junctions (with increasing mitochondrial aspect ratio) and the density of inner mitochondrial membrane in myocytes of Tigriopus californicus copepods. Mitochondrial density increased following both irradiation treatments, but mitochondrial size decreased under the six hour treatment. Metabolic rate was maintained under three hours of irradiation but decreased following six hours of exposure. These observations demonstrate that the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions can play formative roles in maintaining whole-animal metabolic rate, and ultimately organismal performance, under exposure to an oxidative stressor.

Ultraviolet irradiation increases size of the first clutch but decreases longevity in a marine copepod.

An important component of life history theory is understanding how natural variation arises in populations. Both endogenous and exogenous factors contribute to organism survival and reproduction, and therefore, it is important to understand how such factors are both beneficial and detrimental to population dynamics. One ecologically relevant factor that influences the life history of aquatic organisms is ultraviolet (UV) radiation. While the majority of research has focused on the potentially detrimental effects that UV radiation has on aquatic organisms, few studies have evaluated hormetic responses stimulated by radiation under select conditions. The goal of this study was to evaluate the impact of UV-A/B irradiation on life history characteristics in Tigriopus californicus copepods. After exposing copepods to UV-A/B irradiation (control, 1-, and 3-hr UV treatments at 0.5 W/m2), we measured the impact of exposure on fecundity, reproductive effort, and longevity. We found that UV irradiation increased the size of the first clutch among all reproducing females in both the 1- and 3-hr experimental groups and decreased longevity among all females that mated in the 1-hr treatment. UV irradiation had no effect on the number of clutches females produced. These findings indicate a potential benefit of UV irradiation on reproductive performance early in life, although the same exposure came at a cost to longevity.

Novel microRNAs are associated with population divergence in transcriptional response to thermal stress in an intertidal copepod.

The role of gene expression in adaptation to differing thermal environments has been assayed extensively. Yet, in most natural systems, analyses of gene expression reveal only one level of the complexity of regulatory machineries. MicroRNAs (miRNAs) are small noncoding RNAs which are key components of many gene regulatory networks, and they play important roles in a variety of cellular pathways by modulating post-transcriptional quantities of mRNA available for protein synthesis. The characterization of miRNA loci and their regulatory dynamics in nonmodel systems are still largely understudied. In this study, we examine the role of miRNAs in response to high thermal stress in the intertidal copepod Tigriopus californicus. Allopatric populations of this species show varying levels of local adaptation with respect to thermal regimes, and previous studies showed divergence in gene expression between populations from very different thermal environments. We examined the transcriptional response to temperature stress in two populations separated by only 8 km by utilizing RNA-seq to quantify both mRNA and miRNA levels. Using the currently available genome sequence, we first describe the repertoire of miRNAs in T. californicus and assess the degree to which transcriptional response to temperature stress is governed by miRNA activity. The two populations showed large differences in the number of genes involved, the magnitude of change in commonly used genes and in the number of miRNAs involved in transcriptional modulation during stress. Our results suggest that an increased level of regulatory network complexity may underlie improved survivorship under thermal stress in one of the populations.

Latitudinal Clines in Temperature and Salinity Tolerance in Tidepool Copepods.

Local adaptation has been understudied in marine systems, but might be expected to be pronounced in the tidepool copepod Tigriopus californicus, which has a broad geographic range and extremely restricted dispersal. Tolerance to temperature and salinity was assessed in 14 populations over a 20° latitudinal range. Adaptive differentiation to temperature and salinity was found at scales as low as 5.6 km. Latitudinal clines were significant, with northern populations being more tolerant of low salinity and less tolerant of high temperature and high salinity. Both temperature and salinity tolerance were more closely associated with long-term thermal maxima than with long-term precipitation data. Hyperthermal and hyposmotic tolerance were inversely correlated, a pattern that could potentially slow adaptation to future conditions. Together, these studies of intraspecific geographic patterns in resistance to multiple stressors are important in predicting how environmental change may effect range shifts and local extinctions.

Locally adapted populations of a copepod can evolve different gene expression patterns under the same environmental pressures.

As populations diverge in allopatry, but under similar thermal conditions, do similar thermal performance phenotypes evolve by maintaining similar gene expression patterns, or does genetic divergence lead to divergent patterns of gene expression between these populations? We used genetically divergent populations of the copepod Tigriopus californicus, whose performance at different thermal conditions is well characterized, to investigate transcriptome-wide expression responses under two different thermal regimes: (1) a nonvariable temperature regime and (2) a regime with variable temperature. Our results show the expression profiles of the response to these regimes differed substantially among populations, even for populations that are geographically close. This pattern was accentuated when populations were raised in the variable temperature environment. Less heat-tolerant populations mounted strong but divergent responses to the different thermal regimes, with a large heat-shock response observed in one population, and an apparent reduction in the expression of genes involved in basic cellular processes in the other. Our results suggest that as populations diverge in allopatry, they may evolve starkly different responses to changes in temperature, at the gene expression level, while maintaining similar thermal performance phenotypes.

Maintenance of polygenic sex determination in a fluctuating environment: an individual-based model.

R. A. Fisher predicted that individuals should invest equally in offspring of both sexes, and that the proportion of males and females produced (the primary sex ratio) should evolve towards 1:1 when unconstrained. For many species, sex determination is dependent on sex chromosomes, creating a strong tendency for balanced sex ratios, but in other cases, multiple autosomal genes interact to determine sex. In such cases, the maintenance of multiple sex-determining alleles at multiple loci and the consequent among-family variability in sex ratios presents a puzzle, as theory predicts that such systems should be unstable. Theory also predicts that environmental influences on sex can complicate outcomes of genetic sex determination, and that population structure may play a role. Tigriopus californicus, a copepod that lives in splash-pool metapopulations and exhibits polygenic and environment-dependent sex determination, presents a test case for relevant theory. We use this species as a model for parameterizing an individual-based simulation to investigate conditions that could maintain polygenic sex determination. We find that metapopulation structure can delay the degradation of polygenic sex determination and that periods of alternating frequency-dependent selection, imposed by seasonal fluctuations in environmental conditions, can maintain polygenic sex determination indefinitely.

Recovery from hybrid breakdown in a marine invertebrate is faster, stronger and more repeatable under environmental stress.

Understanding how environmental stress alters the consequences of hybridization is important, because the rate of hybridization and the likelihood of hybrid speciation both appear elevated in harsh, disturbed or marginal habitats. We assessed fitness, morphometrics and molecular genetic composition over 14 generations of hybridization between two highly divergent populations of the marine copepod Tigriopus californicus. Replicated, experimental hybrid populations in both control and high-salinity conditions showed a decline in fitness, followed by a recovery. Recovery was faster in the salinity stress treatment, returning to parental levels up to two generations earlier than in the control. This recovery was stable in the high-salinity treatment, whereas in the control treatment, fitness dropped back below parental levels at the final time point. Recovery in the high-salinity treatment was also stronger in terms of competitive fitness and heat-shock tolerance. Finally, consequences of hybridization were more repeatable under salinity stress, where among-replicate variance for survivorship and molecular genetic composition was lower than in the control treatment. In a system with low effective population sizes (estimates ranged from 17 to 63), where genetic drift might be expected to be the predominate force, strong selection under harsh environmental conditions apparently promoted faster, stronger and more repeatable recovery from depressed hybrid fitness.

Sex without sex chromosomes: genetic architecture of multiple loci independently segregating to determine sex ratios in the copepod Tigriopus californicus.

Sex-determining systems are remarkably diverse and may evolve rapidly. Polygenic sex-determination systems are predicted to be transient and evolutionarily unstable, yet examples have been reported across a range of taxa. Here, we provide the first direct evidence of polygenic sex determination in Tigriopus californicus, a harpacticoid copepod with no heteromorphic sex chromosomes. Using genetically distinct inbred lines selected for male- and female-biased clutches, we generated a genetic map with 39 SNPs across 12 chromosomes. Quantitative trait locus mapping of sex ratio phenotype (the proportion of male offspring produced by an F2 female) in four F2 families revealed six independently segregating quantitative trait loci on five separate chromosomes, explaining 19% of the variation in sex ratios. The sex ratio phenotype varied among loci across chromosomes in both direction and magnitude, with the strongest phenotypic effects on chromosome 10 moderated to some degree by loci on four other chromosomes. For a given locus, sex ratio phenotype varied in magnitude for individuals derived from different dam lines. These data, together with the environmental factors known to contribute to sex determination, characterize the underlying complexity and potential lability of sex determination, and confirm the polygenic architecture of sex determination in T. californicus.

Variation in tolerance to common marine pollutants among different populations in two species of the marine copepod Tigriopus.

Geographical variation in chemical tolerance within a species can significantly influence results of whole animal bioassays, yet a literature survey showed that the majority of articles using bioassays did not provide detail on the original field collection site of their test specimens confounding the ability for accurate replication and comparison of results. Biological variation as a result of population-specific tolerance, if not addressed, can be misinterpreted as experimental error. Our studies of two marine copepod species, Tigriopus japonicus and Tigriopus californicus, found significant intra- and inter-specific variation in tolerance to copper and tributyltin. Because both species tolerate copper concentrations orders of magnitude higher than those found in coastal waters, difference in copper tolerance may be a by-product of adaptation to other stressors such as high temperature. Controlling for inter-population tolerance variation will greatly strengthen the application of bioassays in chemical toxicity tests.

Multigenerational response to artificial selection for biased clutch sex ratios in Tigriopus californicus populations.

Polygenic sex determination (PSD) is relatively rare and theoretically evolutionary unstable, yet has been reported across a range of taxa. Evidence for multilocus PSD is provided by (i) large between-family variance in sex ratio, (ii) paternal and maternal effects on family sex ratio and (iii) response to selection for family sex ratio. This study tests the polygenic hypothesis of sex determination in the harpacticoid copepod Tigriopus californicus using the criterion of response to selection. We report the first multigenerational quantitative evidence that clutch sex ratio responds to artificial selection in both directions (selection for male- and female-biased families) and in multiple populations of T. californicus. In the five of six lines that showed a response to selection, realized heritability estimated by multigenerational analysis ranged from 0.24 to 0.58. Divergence of clutch sex ratio between selection lines is rapid, with response to selection detectable within the first four generations of selection.

Interpopulational variation in the cold tolerance of a broadly distributed marine copepod.

Latitudinal trends in cold tolerance have been observed in many terrestrial ectotherms, but few studies have investigated interpopulational variation in the cold physiology of marine invertebrates. Here, the intertidal copepod Tigriopus californicus was used as a model system to study how local adaptation influences the cold tolerance of a broadly distributed marine crustacean. Among five populations spanning 18° in latitude, the following three metrics were used to compare cold tolerance: the temperature of chill-coma onset, the chill-coma recovery time and post-freezing recovery. In comparison to copepods from warmer southern latitudes, animals from northern populations exhibited lower chill-coma onset temperatures, shorter chill-coma recovery times and faster post-freezing recovery rates. Importantly, all three metrics showed a consistent latitudinal trend, suggesting that any single metric could be used equivalently in future studies investigating latitudinal variation in cold tolerance. Our results agree with previous studies showing that populations within a single species can display strong local adaptation to spatially varying climatic conditions. Thus, accounting for local adaptation in bioclimate models will be useful for understanding how broadly distributed species like T. californicus will respond to anthropogenic climate change.

CYTOCHROME C OXIDASE ACTIVITY IN INTERPOPULATION HYBRIDS OF A MARINE COPEPOD: A TEST FOR NUCLEAR-NUCLEAR OR NUCLEAR-CYTOPLASMIC COADAPTATION.

The respiratory enzyme cytochrome c oxidase (COX) is composed of subunits encoded by both nuclear and mitochondrial genes; thus, COX activity reflects, to some extent, the coordinated function of the two genomes. Because extensive mtDNA differentiation exists between populations of the copepod Tigriopus californicus, we hypothesized that laboratory hybridizations that disrupt natural combinations of nuclear and mitochondrial genes might negatively impact COX activity. Although experimental results varied greatly among different crosses, replicate sets of crosses between two particular populations showed consistent evidence for nuclear-cytoplasmic coadaptation.