Mitochondrial Enzyme Activities and Body Condition of Naturally Infected Sunfish (Lepomis gibbosus).

AbstractParasites can affect host behavior, cognition, locomotion, body condition, and many other physiological traits. Changes to host aerobic metabolism may be responsible for these parasite-induced performance alterations. Whole-organism metabolic rate is underpinned by cellular energy metabolism driven most prominently by mitochondria. However, few studies have explored how mitochondrial enzymatic activity relates to body condition and parasite infection, despite it being a putative site for metabolic disruptions related to health status. We studied correlations among natural parasite infection, host body condition, and activity of key mitochondrial enzymes in target organs from wild-caught pumpkinseed sunfish (Lepomis gibbosus) to better understand the cellular responses of fish hosts to endoparasite infection. Enzymatic activities in the gills, spleen, and brain of infected fish were not significantly related to parasite infection or host body condition. However, the activity of cytochrome c oxidase, an enzyme involved in oxidative phosphorylation, in fish hearts was higher in individuals with a lower body condition. Activities of citrate synthase, electron transport system (complexes I and III), and carnitine palmitoyltransferase were also significantly different among organ types. These results provide preliminary information regarding the likely mitochondrial pathways affecting host body condition, the maintenance energetic requirements of different organs, and the organs' specific dependency on particular mitochondrial pathways. These results help pave the way for future studies on the effects of parasite infection on mitochondrial metabolism.

A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics.

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

Evolutionary Trajectories are Contingent on Mitonuclear Interactions.

Critical mitochondrial functions, including cellular respiration, rely on frequently interacting components expressed from both the mitochondrial and nuclear genomes. The fitness of eukaryotic organisms depends on a tight collaboration between both genomes. In the face of an elevated rate of evolution in mtDNA, current models predict that the maintenance of mitonuclear compatibility relies on compensatory evolution of the nuclear genome. Mitonuclear interactions would therefore exert an influence on evolutionary trajectories. One prediction from this model is that the same nuclear genome evolving with different mitochondrial haplotypes would follow distinct molecular paths toward higher fitness. To test this prediction, we submitted 1,344 populations derived from 7 mitonuclear genotypes of Saccharomyces cerevisiae to >300 generations of experimental evolution in conditions that either select for a mitochondrial function or do not strictly require respiration for survival. Performing high-throughput phenotyping and whole-genome sequencing on independently evolved individuals, we identified numerous examples of gene-level evolutionary convergence among populations with the same mitonuclear background. Phenotypic and genotypic data on strains derived from this evolution experiment identify the nuclear genome and the environment as the main determinants of evolutionary divergence, but also show a modulating role for the mitochondrial genome exerted both directly and via interactions with the two other components. We finally recapitulated a subset of prominent loss-of-function alleles in the ancestral backgrounds and confirmed a generalized pattern of mitonuclear-specific and highly epistatic fitness effects. Together, these results demonstrate how mitonuclear interactions can dictate evolutionary divergence of populations with identical starting nuclear genotypes.

Bioenergetic consequences of sex-specific mitochondrial DNA evolution.

Doubly uniparental inheritance (DUI) represents a notable exception to the general rule of strict maternal inheritance (SMI) of mitochondria in metazoans. This system entails the coexistence of two mitochondrial lineages (F- and M-type) transmitted separately through oocytes and sperm, thence providing an unprecedented opportunity for the mitochondrial genome to evolve adaptively for male functions. In this study, we explored the impact of a sex-specific mitochondrial evolution upon gamete bioenergetics of DUI and SMI bivalve species, comparing the activity of key enzymes of glycolysis, fermentation, fatty acid metabolism, tricarboxylic acid cycle, oxidative phosphorylation and antioxidant metabolism. Our findings suggest reorganized bioenergetic pathways in DUI gametes compared to SMI gametes. This generally results in a decreased enzymatic capacity in DUI sperm with respect to DUI oocytes, a limitation especially prominent at the terminus of the electron transport system. This bioenergetic remodelling fits a reproductive strategy that does not require high energy input and could potentially link with the preservation of the paternally transmitted mitochondrial genome in DUI species. Whether this phenotype may derive from positive or relaxed selection acting on DUI sperm is still uncertain.

A Naturally Heteroplasmic Clam Provides Clues about the Effects of Genetic Bottleneck on Paternal mtDNA.

Mitochondrial DNA (mtDNA) is present in multiple copies within an organism. Since these copies are not identical, a single individual carries a heterogeneous population of mtDNAs, a condition known as heteroplasmy. Several factors play a role in the dynamics of the within-organism mtDNA population: among them, genetic bottlenecks, selection, and strictly maternal inheritance are known to shape the levels of heteroplasmy across mtDNAs. In Metazoa, the only evolutionarily stable exception to the strictly maternal inheritance of mitochondria is the doubly uniparental inheritance (DUI), reported in 100+ bivalve species. In DUI species, there are two highly divergent mtDNA lineages, one inherited through oocyte mitochondria (F-type) and the other through sperm mitochondria (M-type). Having both parents contributing to the mtDNA pool of the progeny makes DUI a unique system to study the dynamics of mtDNA populations. Since, in bivalves, the spermatozoon has few mitochondria (4-5), M-type mtDNA faces a tight bottleneck during embryo segregation, one of the narrowest mitochondrial bottlenecks investigated so far. Here, we analyzed the F- and M-type mtDNA variability within individuals of the DUI species Ruditapes philippinarum and investigated for the first time the effects of such a narrow bottleneck affecting mtDNA populations. As a potential consequence of this narrow bottleneck, the M-type mtDNA shows a large variability in different tissues, a condition so pronounced that it leads to genotypes from different tissues of the same individual not to cluster together. We believe that such results may help understanding the effect of low population size on mtDNA bottleneck.

Effects of mitonuclear combination and thermal acclimation on the energetic phenotype.

Activity of the oxidative phosphorylation complexes rely on intimately associated subunits encoded by the mitochondrial and nuclear genomes. Given the key role of this system in adenosine triphosphate production, genes from both genomes must coevolve. A combination of northern redbelly dace (Chrosomus eos) or finescale dace (C. neogaeus) mitochondrial genome with a C. eos nuclear genome allows for a close examination of a naturally occurring disruption of mitonuclear coevolution. We, therefore, investigated the combined effect of mitonuclear genotypes, acclimation, and temperature on the activity of enzymes linked with the energy metabolism in a sympatric population of wild type and cybrid. As expected, the activity of the nuclear-encoded citrate synthase was only influenced by temperature while the cytochrome c oxidase (composed of nuclear and mitochondrial subunits from wild type and cybrid individuals) responded differently to temperature. This study provides clear evidence of the extent by which mitonuclear coadaptation could influence aerobic metabolism.

Mitochondrial thermo-sensitivity in invasive and native freshwater mussels.

Climate change is impacting many, if not all, forms of life. Increases in extreme temperature fluctuations and average temperatures can cause stress, particularly in aquatic sessile ectotherms such as freshwater mussels. However, some species seem to thrive more than others in face of temperature-related stressors. Thermal tolerance may, for example, explain the success of invasive species. It is also known that mitochondria can play a key role in setting an ectothermic species' thermal tolerance. In this study, we aimed to characterize the mitochondrial thermo-tolerance in invasive and endemic freshwater mussels. With the use of high-resolution respirometry, we analyzed the mitochondrial respiration of two freshwater bivalve species exposed to a broad range of temperatures. We noticed that the invasive dreissenid Dreissena bugensis possessed a less thermo-tolerant mitochondrial metabolism than the endemic unionid Elliptio complanata This lack of tolerance was linked with a more noticeable aerobic metabolic depression at elevated temperatures. This decrease in mitochondrial metabolic activity was also linked with an increase in leak oxygen consumption as well as a stable maintenance of the activity of cytochrome c oxidase in both species. These findings may be associated both with the species' life history characteristics, as D. bugensis is more adapted to unstable habitats, in which selection pressures for resistance adaptations are reduced. Our findings add to the growing body of literature characterizing the mitochondrial metabolism of many aquatic ectotherms in our changing world.

Linking paternally inherited mtDNA variants and sperm performance.

Providing robust links between mitochondrial genotype and phenotype is of major importance given that mitochondrial DNA (mtDNA) variants can affect reproductive success. Because of the strict maternal inheritance (SMI) of mitochondria in animals, haplotypes that negatively affect male fertility can become fixed in populations. This phenomenon is known as 'mother's curse'. Doubly uniparental inheritance (DUI) of mitochondria is a stable exception in bivalves, which entails two mtDNA lineages that evolve independently and are transmitted separately through oocytes and sperm. This makes the DUI mitochondrial lineages subject to different sex-specific selective sieves during mtDNA evolution, thus DUI is a unique model to evaluate how direct selection on sperm mitochondria could contribute to male reproductive fitness. In this study, we tested the impact of mtDNA variants on sperm performance and bioenergetics in DUI and SMI species. Analyses also involved measures of sperm performance following inhibition of main energy pathways and sperm response to oocyte presence. Compared to SMI, DUI sperm exhibited (i) low speed and linearity, (ii) a strict OXPHOS-dependent strategy of energy production, and (iii) a partial metabolic shift towards fermentation following egg detection. Discussion embraces the adaptive value of mtDNA variation and suggests a link between male-energetic adaptation, fertilization success and paternal mitochondria preservation. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Metabolic remodelling associated with mtDNA: insights into the adaptive value of doubly uniparental inheritance of mitochondria.

Mitochondria produce energy through oxidative phosphorylation (OXPHOS), which depends on the expression of both nuclear and mitochondrial DNA (mtDNA). In metazoans, a striking exception from strictly maternal inheritance of mitochondria is doubly uniparental inheritance (DUI). This unique system involves the maintenance of two highly divergent mtDNAs (F- and M-type, 8-40% of nucleotide divergence) associated with gametes, and occasionally coexisting in somatic tissues. To address whether metabolic differences underlie this condition, we characterized the OXPHOS activity of oocytes, spermatozoa, and gills of different species through respirometry. DUI species express different gender-linked mitochondrial phenotypes in gametes and partly in somatic tissues. The M-phenotype is specific to sperm and entails (i) low coupled/uncoupled respiration rates, (ii) a limitation by the phosphorylation system, and (iii) a null excess capacity of the final oxidases, supporting a strong control over the upstream complexes. To our knowledge, this is the first example of a phenotype resulting from direct selection on sperm mitochondria. This metabolic remodelling suggests an adaptive value of mtDNA variations and we propose that bearing sex-linked mitochondria could assure the energetic requirements of different gametes, potentially linking male-energetic adaptation, mitotype preservation and inheritance, as well as resistance to both heteroplasmy and ageing.

The effect of cryopreservation on mitochondrial function in freshwater mussel tissue samples (Bivalvia: Unionida).

In non-thermoregulating and sessile organisms, such as the imperiled freshwater mussels (Bivalvia: Unionida), thermal sensitivity of mitochondria is a key factor for survival to global warming. Given the protected status of many unionids, non-destructive biopsies and subsequent cryopreservation are advisable procedures for further investigation of their mitochondrial function. To address whether long-term cryopreservation affects mitochondria in freshwater mussels, the mitochondrial respiration in permeabilized somatic cells of Elliptio complanata has been fully characterized through high-resolution respirometry. Our results indicate that cryopreservation does affect the absolute rate of respiration, which significantly decrease compared to fresh tissues, independently of substrates combination, respiratory state and normalizing factor. However, the negative impact is not reflected at the level of flux control ratios, suggesting that, even in front of a sharp decline in the aerobic capacity, cryopreserved tissues preserve the mitochondrial organization and could be thus employed for the qualitative analysis of mitochondrial function.

The extremely divergent maternally- and paternally-transmitted mitochondrial genomes are co-expressed in somatic tissues of two freshwater mussel species with doubly uniparental inheritance of mtDNA.

Freshwater mussel species with doubly uniparental inheritance (DUI) of mtDNA are unique because they are naturally heteroplasmic for two extremely divergent mtDNAs with ~50% amino acid differences for protein-coding genes. The paternally-transmitted mtDNA (or M mtDNA) clearly functions in sperm in these species, but it is still unknown whether it is transcribed when present in male or female soma. In the present study, we used PCR and RT-PCR to detect the presence and expression of the M mtDNA in male and female somatic and gonadal tissues of the freshwater mussel species Venustaconcha ellipsiformis and Utterbackia peninsularis (Unionidae). This is the first study demonstrating that the M mtDNA is transcribed not only in male gonads, but also in male and female soma in freshwater mussels with DUI. Because of the potentially deleterious nature of heteroplasmy, we suggest the existence of different mechanisms in DUI species to deal with this possibly harmful situation, such as silencing mechanisms for the M mtDNA at the transcriptional, post-transcriptional and/or post-translational levels. These hypotheses will necessitate additional studies in distantly-related DUI species that could possess different mechanisms of action to deal with heteroplasmy.

The Complete Female- and Male-Transmitted Mitochondrial Genome of Meretrix lamarckii.

Bivalve mitochondrial genomes show many uncommon features, like additional genes, high rates of gene rearrangement, high A-T content. Moreover, Doubly Uniparental Inheritance (DUI) is a distinctive inheritance mechanism allowing some bivalves to maintain and transmit two separate sex-linked mitochondrial genomes. Many bivalve mitochondrial features, such as gene extensions or additional ORFs, have been proposed to be related to DUI but, up to now, this topic is far from being understood. Several species are known to show this unusual organelle inheritance but, being widespread only among Unionidae and Mytilidae, DUI distribution is unclear. We sequenced and characterized the complete female- (F) and male-transmitted (M) mitochondrial genomes of Meretrix lamarckii, which, in fact, is the second species of the family Veneridae where DUI has been demonstrated so far. The two mitochondrial genomes are comparable in length and show roughly the same gene content and order, except for three additional tRNAs found in the M one. The two sex-linked genomes show an average nucleotide divergence of 16%. A 100-aminoacid insertion in M. lamarckii M-cox2 gene was found; moreover, additional ORFs have been found in both F and M Long Unassigned Regions of M. lamarckii. Even if no direct involvement in DUI process has been demonstrated so far, the finding of cox2 insertions and supernumerary ORFs in M. lamarckii both strengthens this hypothesis and widens the taxonomical distribution of such unusual features. Finally, the analysis of inter-sex genetic variability shows that DUI species form two separate clusters, namely Unionidae and Mytilidae+Veneridae; this dichotomy is probably due to different DUI regimes acting on separate taxa.