The role of mitochondria in sex- and age-specific gene expression in a species without sex chromosomes.

Mitochondria perform an array of functions, many of which involve interactions with gene products encoded by the nucleus. These mitochondrial functions, particularly those involving energy production, can be expected to differ between sexes and across ages. Here, we measured mitochondrial effects on sex- and age-specific gene expression in parental and reciprocal F1 hybrids between allopatric populations of Tigriopus californicus with over 20% mitochondrial DNA divergence. Because the species lacks sex chromosomes, sex-biased mitochondrial effects are not confounded by the effects of sex chromosomes. Results revealed pervasive sex differences in mitochondrial effects, including effects on energetics and aging involving nuclear interactions throughout the genome. Using single-individual RNA sequencing, sex differences were found to explain more than 80% of the variance in gene expression. Males had higher expression of mitochondrial genes and mitochondrially targeted proteins (MTPs) involved in oxidative phosphorylation (OXPHOS), while females had elevated expression of non-OXPHOS MTPs, indicating strongly sex-dimorphic energy metabolism at the whole organism level. Comparison of reciprocal F1 hybrids allowed insights into the nature of mito-nuclear interactions, showing both mitochondrial effects on nuclear expression, and nuclear effects on mitochondrial expression. While based on a small set of crosses, sex-specific increases in mitochondrial expression with age were associated with longer life. Network analyses identified nuclear components of strong mito-nuclear interactions and found them to be sexually dimorphic. These results highlight the profound impact of mitochondria and mito-nuclear interactions on sex- and age-specific gene expression.

Mother's Curse effects on lifespan and aging.

The Mother's Curse hypothesis posits that mothers curse their sons with harmful mitochondria, because maternal mitochondrial inheritance makes selection blind to mitochondrial mutations that harm only males. As a result, mitochondrial function may be evolutionarily optimized for females. This is an attractive explanation for ubiquitous sex differences in lifespan and aging, given the prevalence of maternal mitochondrial inheritance and the established relationship between mitochondria and aging. This review outlines patterns expected under the hypothesis, and traits most likely to be affected, chiefly those that are sexually dimorphic and energy intensive. A survey of the literature shows that evidence for Mother's Curse is limited to a few taxonomic groups, with the strongest support coming from experimental crosses in Drosophila. Much of the evidence comes from studies of fertility, which is expected to be particularly vulnerable to male-harming mitochondrial mutations, but studies of lifespan and aging also show evidence of Mother's Curse effects. Despite some very compelling studies supporting the hypothesis, the evidence is quite patchy overall, with contradictory results even found for the same traits in the same taxa. Reasons for this scarcity of evidence are discussed, including nuclear compensation, factors opposing male-specific mutation load, effects of interspecific hybridization, context dependency and demographic effects. Mother's Curse effects may indeed contribute to sex differences, but the complexity of other contributing factors make Mother's Curse a poor general predictor of sex-specific lifespan and aging.

The role of mitochondria in sex- and age-specific gene expression in a species without sex chromosomes.

Mitochondria perform an array of functions, many of which involve interactions with gene products encoded by the nucleus. These mitochondrial functions, particularly those involving energy production, can be expected to differ between sexes and across ages. Here we measured mitochondrial effects on sex- and age-specific gene expression in parental and reciprocal F1 hybrids between allopatric populations of Tigriopus californicus with over 20% mitochondrial DNA divergence. Because the species lacks sex chromosomes, sex-biased mitochondrial effects are not confounded by the effects of sex chromosomes. Using single-individual RNA sequencing, sex differences were found to explain more than 80% of the variance in gene expression. Males had higher expression of mitochondrial genes and mitochondrially targeted proteins (MTPs) involved in oxidative phosphorylation (OXPHOS), while females had elevated expression of non-OXPHOS MTPs, indicating strongly sex-dimorphic energy metabolism at the whole organism level. Comparison of reciprocal F1 hybrids allowed insights into the nature of mito-nuclear interactions, showing both mitochondrial effects on nuclear expression, as well as nuclear effects on mitochondrial expression. Across both sexes, increases in mitochondrial expression with age were associated with longer life. Network analyses identified nuclear components of strong mito-nuclear interactions, and found them to be sexually dimorphic. These results highlight the profound impact of mitochondria and mito-nuclear interactions on sex- and age-specific gene expression.

Genomic recovery lags behind demographic recovery in bottlenecked populations of the Channel Island fox, Urocyon littoralis.

With continued global change, recovery of species listed under the Endangered Species Act is increasingly challenging. One rare success was the recovery and delisting of the Channel Island fox (Urocyon littoralis) after 90%-99% population declines in the 1990s. While their demographic recovery was marked, less is known about their genetic recovery. To address genetic changes, we conducted the first multi-individual and population-level direct genetic comparison of samples collected before and after the recent bottlenecks. Using whole-exome sequencing, we found that already genetically depauperate populations were further degraded by the 1990s declines and remain low, particularly on San Miguel and Santa Rosa Islands, which underwent the most severe bottlenecks. The two other islands that experienced recent bottlenecks (Santa Cruz, and Santa Catalina islands) showed mixed results based on multiple metrics of genetic diversity. Previous island fox genomics studies showed low genetic diversity before the declines and no change after the demographic recovery, thus this is the first study to show a decrease in genetic diversity over time in U. littoralis. Additionally, we found that divergence between populations consistently increased over time, complicating prospects for using inter-island translocation as a conservation tool. The Santa Catalina subspecies is now federally listed as threatened, yet other de-listed subspecies are still recovering genetic variation which may limit their ability to adapt to changing environmental conditions. This study further demonstrates that species conservation is more complex than population size and that some island fox populations are not yet 'out of the woods'.

Mitochondrial effects on fertility and longevity in Tigriopus californicus contradict predictions of the mother's curse hypothesis.

Strict maternal inheritance of mitochondria favours the evolutionary accumulation of sex-biased fitness effects, as mitochondrial evolution occurs exclusively in female lineages. The 'mother's curse' hypothesis proposes that male-harming mutations should accumulate in mitochondrial genomes when they have neutral or beneficial effects on female fitness. Rigorous empirical tests have largely focused on Drosophila, where support for the predictions of mother's curse has been mixed. We investigated the impact of mother's curse mutations in Tigriopus californicus, a minute crustacean. Using non-recombinant backcrosses, we introgressed four divergent mitochondrial haplotypes into two nuclear backgrounds and recorded measures of fertility and longevity. We found that the phenotypic effects of mitochondrial mutations were context dependent, being influenced by the nuclear background in which they were expressed, as well as the sex of the individual and rearing temperature. Mitochondrial haplotype effects were greater for fertility than longevity, and temperature effects were greater for longevity. However, in opposition to mother's curse expectations, females had higher mitochondrial genetic variance than males for fertility and longevity, little evidence of sexual antagonism favouring females was found, and the impacts of mitonuclear mismatch harmed females but not males. Together, this indicates that selection on mitochondrial variation has not resulted in the accumulation of male mutation load in Tigriopus californicus.

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.

Food deprivation exposes sex-specific trade-offs between stress tolerance and life span in the copepod Tigriopus californicus.

Long life is standardly assumed to be associated with high stress tolerance. Previous work shows that the copepod Tigriopus californicus breaks this rule, with longer life span under benign conditions found in males, the sex with lower stress tolerance. Here, we extended this previous work, raising animals from the same families in food-replete conditions until adulthood and then transferring them to food-limited conditions until all animals perished. As in previous work, survivorship under food-replete conditions favored males. However, under food deprivation life span strongly favored females in all crosses. Compared to benign conditions, average life span under nutritional stress was reduced by 47% in males but only 32% in females. Further, the sex-specific mitonuclear effects previously found under benign conditions were erased under food limited conditions. Results thus demonstrate that sex-specific life span, including mitonuclear interactions, are highly dependent on nutritional environment.

Data and Diversity in the Development of Acute Water Quality Criteria in the United States.

The US Environmental Protection Agency (USEPA) is responsible for the development of water quality criteria, regulatory standards that protect aquatic organisms from harmful chemical exposure. Although these criteria are intended to be broadly protective of aquatic life, the data used to derive criteria do not necessarily reflect the actual diversity of natural communities nor are they available for most chemicals. In addition, although the USEPA's current procedures emphasize using toxicity data with a certain minimum amount of biological diversity, the quantitative impact of such diversity on criteria is unclear. In the present study we assessed the changes to acute toxicity data over time, determined the prevalence of significant taxonomic differences in sensitivity, and investigated the effect of biological diversity on criteria. We found major gaps in existing toxicity data that we hypothesize have contributed to the absence of acute criteria for the majority of chemical pollutants. Taxonomic patterns of sensitivity in these data are abundant, although the resolution of the patterns is relatively poor. In addition, we found that the amount of biological diversity in a toxicity data set and the data set's taxonomic composition does not quantitatively affect criteria in most cases. Because the USEPA has published acute criteria for fewer than 20% of priority pollutants and the persistence of major gaps in toxicity data over the last 37 years, we recommend that the USEPA consider revisions to their water quality criteria guidelines that will expedite the criteria development process and advance the responsible management of pollutants in the aquatic environment. Environ Toxicol Chem 2022;41:1333-1343. © 2022 SETAC.

Effect of Geography and Captivity on Scat Bacterial Communities in the Imperiled Channel Island Fox.

With developing understanding that host-associated microbiota play significant roles in individual health and fitness, taking an interdisciplinary approach combining microbiome research with conservation science is increasingly favored. Here we establish the scat microbiome of the imperiled Channel Island fox (Urocyon littoralis) and examine the effects of geography and captivity on the variation in bacterial communities. Using high throughput 16S rRNA gene amplicon sequencing, we discovered distinct bacterial communities in each island fox subspecies. Weight, timing of the sample collection, and sex contributed to the geographic patterns. We uncovered significant taxonomic differences and an overall decrease in bacterial diversity in captive versus wild foxes. Understanding the drivers of microbial variation in this system provides a valuable lens through which to evaluate the health and conservation of these genetically depauperate foxes. The island-specific bacterial community baselines established in this study can make monitoring island fox health easier and understanding the implications of inter-island translocation clearer. The decrease in bacterial diversity within captive foxes could lead to losses in the functional services normally provided by commensal microbes and suggests that zoos and captive breeding programs would benefit from maintaining microbial diversity.

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.

Sex Ratios in a Warming World: Thermal Effects on Sex-Biased Survival, Sex Determination, and Sex Reversal.

Rising global temperatures threaten to disrupt population sex ratios, which can in turn cause mate shortages, reduce population growth and adaptive potential, and increase extinction risk, particularly when ratios are male biased. Sex ratio distortion can then have cascading effects across other species and even ecosystems. Our understanding of the problem is limited by how often studies measure temperature effects in both sexes. To address this, the current review surveyed 194 published studies of heat tolerance, finding that the majority did not even mention the sex of the individuals used, with <10% reporting results for males and females separately. Although the data are incomplete, this review assessed phylogenetic patterns of thermally induced sex ratio bias for 3 different mechanisms: sex-biased heat tolerance, temperature-dependent sex determination (TSD), and temperature-induced sex reversal. For sex-biased heat tolerance, documented examples span a large taxonomic range including arthropods, chordates, protists, and plants. Here, superior heat tolerance is more common in females than males, but the direction of tolerance appears to be phylogenetically fluid, perhaps due to the large number of contributing factors. For TSD, well-documented examples are limited to reptiles, where high temperature usually favors females, and fishes, where high temperature consistently favors males. For temperature-induced sex reversal, unambiguous cases are again limited to vertebrates, and high temperature usually favors males in fishes and amphibians, with mixed effects in reptiles. There is urgent need for further work on the full taxonomic extent of temperature-induced sex ratio distortion, including joint effects of the multiple contributing mechanisms.

Sex differences in early transcriptomic responses to oxidative stress in the copepod Tigriopus californicus.

BACKGROUND: Patterns of gene expression can be dramatically different between males and females of the same species, in part due to genes on sex chromosomes. Here we test for sex differences in early transcriptomic response to oxidative stress in a species which lacks heteromorphic sex chromosomes, the copepod Tigriopus californicus. RESULTS: Male and female individuals were separately exposed to control conditions and pro-oxidant conditions (hydrogen peroxide and paraquat) for periods of 3 hours and 6 hours. Variance partitioning showed the greatest expression variance among individuals, highlighting the important information that can be obscured by the common practice of pooling individuals. Gene expression variance between sexes was greater than that among treatments, showing the profound effect of sex even when males and females share the same genome. Males exhibited a larger response to both pro-oxidants, differentially expressing more than four times as many genes, including up-regulation of more antioxidant genes, heat shock proteins and protease genes. While females differentially expressed fewer genes, the magnitudes of fold change were generally greater, indicating a more targeted response. Although females shared a smaller fraction of differentially expressed genes between stressors and time points, expression patterns of antioxidant and protease genes were more similar between stressors and more GO terms were shared between time points. CONCLUSIONS: Early transcriptomic responses to the pro-oxidants H2O2 and paraquat in copepods revealed substantial variation among individuals and between sexes. The finding of such profound sex differences in oxidative stress response, even in the absence of sex chromosomes, highlights the importance of studying both sexes and the potential for developing sex-specific strategies to promote optimal health and aging in humans.

Effects of oxidative stress on sex-specific gene expression in the copepod Tigriopus californicus revealed by single individual RNA-seq.

Oxidative stress reflects the imbalance of pro-oxidants and antioxidants. Prolonged oxidative stress can induce cellular damage, diseases and aging, and the effects may be sex-specific. Tigriopus californicus has recently been proposed as an alternative model system for sex-specific studies due to the absence of sex chromosomes. In this study, we used comparative transcriptomic analyses to assess sex-specific transcriptional responses to oxidative stress. Male and female individuals were maintained separately in one of three treatments: 1) control conditions with an algae diet, 2) pro-oxidant (H2O2) conditions with an algae diet or 3) decreased antioxidant conditions (reduced carotenoids due to a yeast diet). Single individual RNA-seq was then conducted for twenty-four libraries using Ligation Mediated RNA sequencing (LM-Seq). Variance in gene expression was partitioned into 62.3% between sexes, 26.85% among individuals and 10.85% among treatments. Within each of the three treatments, expression was biased toward females. However, compared to the control treatment, males in both pro-oxidant and decreased antioxidant treatments differentially expressed more genes while females differentially expressed fewer genes but with a greater magnitude of fold change. As the first study of copepods to apply single individual RNA-seq, the findings will contribute to a better understanding of transcriptomic variation among individuals as well as sex-specific response mechanisms to oxidative stress in the absence of sex chromosomes.

Sex-specific stress tolerance, proteolysis, and lifespan in the invertebrate Tigriopus californicus.

Because stress tolerance and longevity are mechanistically and phenotypically linked, the sex with higher acute stress tolerance might be expected to also live longer. On the other hand, the association between stress tolerance and lifespan may be complicated by tradeoffs between acute tolerance and long-term survival. Here we use the copepod Tigriopus californicus to test for sex differences in stress resistance, proteolytic activity and longevity. Unlike many model organisms, this species does not have sex chromosomes. However, substantial sex differences were still observed. Females were found to have superior tolerance to a range of acute stressors (high temperature, high salinity, low salinity, copper and bisphenol A (BPA)) across a variety of treatments including different populations, pure vs. hybrid crosses, and different shading environments. Upregulation of proteolytic capacity - one molecular mechanism for responding to acute stress - was also found to be sexually dimorphic. In the combined stress treatment of chronic copper exposure followed by acute heat exposure, proteolytic capacity was suppressed for males. Females, however, maintained a robust proteolytic stress response. While females consistently showed greater tolerance to short-term stress, lifespan was largely equivalent between the two sexes under both benign conditions and mild thermal stress. Our findings indicate that short-term stress tolerance does not predict long-term survival under relatively mild conditions.

Genomic signatures of mitonuclear coevolution across populations of Tigriopus californicus.

The copepod Tigriopus californicus shows extensive population divergence and is becoming a model for understanding allopatric differentiation and the early stages of speciation. Here, we report a high-quality reference genome for one population (~190 megabases across 12 scaffolds, and ~15,500 protein-coding genes). Comparison with other arthropods reveals 2,526 genes presumed to be specific to T. californicus, with an apparent proliferation of genes involved in ion transport and receptor activity. Beyond the reference population, we report re-sequenced genomes of seven additional populations, spanning the continuum of reproductive isolation. Populations show extreme mitochondrial DNA divergence, with higher levels of amino acid differentiation than observed in other taxa. Across the nuclear genome, we find elevated protein evolutionary rates and positive selection in genes predicted to interact with mitochondrial DNA and the proteins and RNA it encodes in multiple pathways. Together, these results support the hypothesis that rapid mitochondrial evolution drives compensatory nuclear evolution within isolated populations, thereby providing a potentially important mechanism for causing intrinsic reproductive isolation.

Long-term laboratory culture causes contrasting shifts in tolerance to two marine pollutants in copepods of the genus Tigriopus.

Organismal chemical tolerance is often used to assess ecological risk and monitor water quality, yet tolerance can differ between field- and lab-raised organisms. In this study, we examined how tolerance to copper (Cu) and tributyltin oxide (TBTO) in two species of marine copepods, Tigriopus japonicus and T. californicus, changed across generations under benign laboratory culture (in the absence of pre-exposure to chemicals). Both copepod species exhibited similar chemical-specific changes in tolerance, with laboratory maintenance resulting in increased Cu tolerance and decreased TBTO tolerance. To assess potential factors underlying these patterns, chemical tolerance was measured in conjunction with candidate environmental variables (temperature, UV radiation, diet type, and starvation). The largest chemical-specific effect was found for starvation, which decreased TBTO tolerance but had no effect on Cu tolerance. Understanding how chemical-specific tolerance can change in the laboratory will be critical in strengthening bioassays and their applications for environmental protection and chemical management.

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.

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.

Acclimation and adaptation to common marine pollutants in the copepod Tigriopus californicus.

Establishing water quality criteria using bioassays is complicated by variation in chemical tolerance between populations. Two major contributors to this variation are acclimation and adaptation, which are both linked to exposure history, but differ in how long their effects are maintained. Our study examines how tolerance changes over multiple generations of exposure to two common marine pollutants, copper (Cu) and tributyltin oxide (TBTO), in a sexually reproducing marine copepod, Tigriopus californicus. Lines of T. californicus were chronically exposed to sub-lethal levels of Cu and TBTO for 12 generations followed by a recovery period of 3 generations in seawater control conditions. At each generation, the average number of offspring produced and survived to 28 d was determined and used as the metric of tolerance. Lines exposed to Cu and TBTO showed an overall increase in tolerance over time. Increased Cu tolerance arose by generation 3 in the chronically exposed lines and was lost after 3 generations in seawater control conditions. Increased TBTO tolerance was detected at generation 7 and was maintained even after 3 generations in seawater control conditions. It was concluded from this study that tolerance to Cu is consistent with acclimation, a quick gain and loss of tolerance. In contrast, TBTO tolerance is consistent with adaptation, in which onset of tolerance was delayed relative to an acclimation response and maintained in the absence of exposure. These findings illustrate that consideration of exposure history is necessary when using bioassays to measure chemical tolerance.