The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

Transcriptomes of Clusterin- and S100B-transfected neuronal cells elucidate protective mechanisms against hypoxia and oxidative stress in the hooded seal (Cystophora cristata) brain.

BACKGROUND: The hooded seal (Cystophora cristata) exhibits impressive diving skills and can tolerate extended durations of asphyxia, hypoxia and oxidative stress, without suffering from irreversible neuronal damage. Thus, when exposed to hypoxia in vitro, neurons of fresh cortical and hippocampal tissue from hooded seals maintained their membrane potential 4-5 times longer than neurons of mice. We aimed to identify the molecular mechanisms underlying the intrinsic neuronal hypoxia tolerance. Previous comparative transcriptomics of the visual cortex have revealed that S100B and clusterin (apolipoprotein J), two stress proteins that are involved in neurological disorders characterized by hypoxic conditions, have a remarkably high expression in hooded seals compared to ferrets. When overexpressed in murine neuronal cells (HN33), S100B and clusterin had neuroprotective effects when cells were exposed to hypoxia. However, their specific roles in hypoxia have remained largely unknown. METHODS: In order to shed light on potential molecular pathways or interaction partners, we exposed HN33 cells transfected with either S100B, soluble clusterin (sCLU) or nuclear clusterin (nCLU) to normoxia, hypoxia and oxidative stress for 24 h. We then determined cell viability and compared the transcriptomes of transfected cells to control cells. Potential pathways and upstream regulators were identified via Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA). RESULTS: HN33 cells transfected with sCLU and S100B demonstrated improved glycolytic capacity and reduced aerobic respiration at normoxic conditions. Additionally, sCLU appeared to enhance pathways for cellular homeostasis to counteract stress-induced aggregation of proteins. S100B-transfected cells sustained lowered energy-intensive synaptic signaling. In response to hypoxia, hypoxia-inducible factor (HIF) pathways were considerably elevated in nCLU- and sCLU-transfected cells. In a previous study, S100B and sCLU decreased the amount of reactive oxygen species and lipid peroxidation in HN33 cells in response to oxidative stress, but in the present study, these functional effects were not mirrored in gene expression changes. CONCLUSIONS: sCLU and S100B overexpression increased neuronal survival by decreasing aerobic metabolism and synaptic signaling in advance to hypoxia and oxidative stress conditions, possibly to reduce energy expenditure and the build-up of deleterious reactive oxygen species (ROS). Thus, a high expression of CLU isoforms and S100B is likely beneficial during hypoxic conditions.

Male-female relatedness at specific SNP-linkage groups influences cryptic female choice in Chinook salmon (Oncorhynchus tshawytscha).

In a range of taxa, the relatedness between mates influences both pre- and post-mating processes of sexual selection. However, relatively little is known about the genetic loci facilitating such a bias, with the exception of the major histocompatibility complex. Here, we performed tightly controlled replicated in vitro fertilization trials to explore the impact of relatedness on two possible mechanisms of cryptic female choice (CFC) in Chinook salmon (Oncorhynchus tshawytscha). We tested (i) whether relatedness of mates, assessed using 682 single nucleotide polymorphisms (SNPs) on 29 SNP-linkage groups (LGs), biases a male's sperm velocity in ovarian fluid (a parameter previously shown to predict male fertilization success), and (ii) whether relatedness of mates governs fertilization success via other mechanisms, probably via sperm-egg interactions. We found that relatedness on three LGs explained the variation in sperm velocity, and relatedness on two LGs explained fertilization success, which might indicate the presence of genes important in sperm-ovarian fluid and sperm-egg interactions in these genomic regions. Mapping of the SNPs on these LGs to the rainbow trout genome revealed two genes that affect fertility in humans and represent candidate genes for further studies. Our results thereby provide a novel contribution to the understanding of the mechanism of CFC.

Elevated antioxidant defence in the brain of deep-diving pinnipeds.

While foraging, marine mammals undertake repetitive diving bouts. When the animal surfaces, reperfusion makes oxygen readily available for the electron transport chain, which leads to increased production of reactive oxygen species and risk of oxidative damage. In blood and several tissues, such as heart, lung, muscle and kidney, marine mammals generally exhibit an elevated antioxidant defence. However, the brain, whose functional integrity is critical to survival, has received little attention. We previously observed an enhanced expression of several antioxidant genes in cortical neurons of hooded seals (Cystophora cristata). Here, we studied antioxidant gene expression and enzymatic activity in the visual cortex, cerebellum and hippocampus of harp seals (Pagophilus groenlandicus) and hooded seals. Moreover, we tested several genes for positive selection. We found that antioxidants in the first line of defence, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione (GSH) were constitutively enhanced in the seal brain compared to mice (Mus musculus), whereas the glutaredoxin and thioredoxin systems were not. Possibly, the activity of the latter systems is stress-induced rather than constitutively elevated. Further, some, but not all members, of the glutathione-s-transferase (GST) family appear more highly expressed. We found no signatures of positive selection, indicating that sequence and function of the studied antioxidants are conserved in pinnipeds.

Transcriptomes Suggest That Pinniped and Cetacean Brains Have a High Capacity for Aerobic Metabolism While Reducing Energy-Intensive Processes Such as Synaptic Transmission.

The mammalian brain is characterized by high energy expenditure and small energy reserves, making it dependent on continuous vascular oxygen and nutritional supply. The brain is therefore extremely vulnerable to hypoxia. While neurons of most terrestrial mammals suffer from irreversible damage after only short periods of hypoxia, neurons of the deep-diving hooded seal (Cystophora cristata) show a remarkable hypoxia-tolerance. To identify the molecular mechanisms underlying the intrinsic hypoxia-tolerance, we excised neurons from the visual cortices of hooded seals and mice (Mus musculus) by laser capture microdissection. A comparison of the neuronal transcriptomes suggests that, compared to mice, hooded seal neurons are endowed with an enhanced aerobic metabolic capacity, a reduced synaptic transmission and an elevated antioxidant defense. Publicly available whole-tissue brain transcriptomes of the bowhead whale (Balaena mysticetus), long-finned pilot whale (Globicephala melas), minke whale (Balaenoptera acutorostrata) and killer whale (Orcinus orca), supplemented with 2 newly sequenced long-finned pilot whales, suggest that, compared to cattle (Bos taurus), the cetacean brain also displays elevated aerobic capacity and reduced synaptic transmission. We conclude that the brain energy balance of diving mammals is preserved during diving, due to reduced synaptic transmission that limits energy expenditure, while the elevated aerobic capacity allows efficient use of oxygen to restore energy balance during surfacing between dives.

Cell Culture Experiments Reveal that High S100B and Clusterin Levels may Convey Hypoxia-tolerance to the Hooded Seal (Cystophora cristata) Brain.

While the brain of most mammals suffers from irreversible damage after only short periods of low oxygen levels (hypoxia), marine mammals are excellent breath-hold divers that have adapted to hypoxia. In addition to physiological adaptations, such as large oxygen storing capacity and strict oxygen economy during diving, the neurons of the deep-diving hooded seal (Cystophora cristata) have an intrinsic tolerance to hypoxia. We aim to understand the molecular basis of this neuronal hypoxia tolerance. Previously, transcriptomics of the cortex of the hooded seal have revealed remarkably high expression levels of S100B and clusterin (apolipoprotein J) when compared to the ferret, a non-diving carnivore. Both genes have much-debated roles in hypoxia and oxidative stress. Here, we evaluated the effects of S100B and of two isoforms of clusterin (soluble and nucleus clusterin) on the survival, metabolic activity and the amount of reactive oxygen species (ROS) in HN33 neuronal mouse cells exposed to hypoxia and oxidative stress. S100B and soluble clusterin had neuroprotective effects, with reduced ROS-levels and retention of normoxic energy status of cells during both stress conditions. The protective effects of nucleus clusterin were restricted to hypoxia. S100B and clusterin showed purifying selection in marine and terrestrial mammals, indicating a functional conservation across species. Immunofluorescence revealed identical cellular distributions of S100B and clusterin in mice, ferrets and hooded seals, further supporting the functional conservation. Taken together, our data suggest that the neuroprotective effects of all three proteins are exclusively facilitated by their increased expression in the brain of the hooded seal.

Three QTL in the honey bee Apis mellifera L. suppress reproduction of the parasitic mite Varroa destructor.

Varroa destructor is a highly virulent ectoparasitic mite of the honey bee Apis mellifera and a major cause of colony losses for global apiculture. Typically, chemical treatment is essential to control the parasite population in the honey bee colony. Nevertheless a few honey bee populations survive mite infestation without any treatment. We used one such Varroa mite tolerant honey bee lineage from the island of Gotland, Sweden, to identify quantitative trait loci (QTL) controlling reduced mite reproduction. We crossed a queen from this tolerant population with drones from susceptible colonies to rear hybrid queens. Two hybrid queens were used to produce a mapping population of haploid drones. We discriminated drone pupae with and without mite reproduction, and screened the genome for potential QTL using a total of 216 heterozygous microsatellite markers in a bulk segregant analysis. Subsequently, we fine mapped three candidate target regions on chromosomes 4, 7, and 9. Although the individual effect of these three QTL was found to be relatively small, the set of all three had significant impact on suppression of V. destructor reproduction by epistasis. Although it is in principle possible to use these loci for marker-assisted selection, the strong epistatic effects between the three loci complicate selective breeding programs with the Gotland Varroa tolerant honey bee stock.