Search

Sperm velocity is a key trait that predicts the outcome of sperm competition. By promoting or impeding sperm velocity, females can control fertilization via postcopulatory cryptic female choice. In Chinook salmon, ovarian fluid (OF), which surrounds the ova, mediates sperm velocity according to male and female identity, biasing the outcome of sperm competition towards males with faster sperm. Past investigations have revealed proteome variation in OF, but the specific components of OF that differentially mediate sperm velocity have yet to be characterized. Here we use quantitative proteomics to investigate whether OF protein composition explains variation in sperm velocity and fertilization success. We found that OF proteomes from six females robustly clustered into two groups and that these groups are distinguished by the abundance of a restricted set of proteins significantly associated with sperm velocity. Exposure of sperm to OF from females in group I had faster sperm compared to sperm exposed to the OF of group II females. Overall, OF proteins that distinguished between these groups were enriched for vitellogenin and calcium ion interactions. Our findings suggest that these proteins may form the functional basis for cryptic female choice via the biochemical and physiological mediation of sperm velocity.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabruscelidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17β-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

The Nidovirales comprise a genetically diverse group of positive-sense single-stranded RNA virus families that infect a range of invertebrate and vertebrate hosts. Recent metagenomic studies have identified nido-like virus sequences, particularly those related to the Coronaviridae, in a range of aquatic hosts including fish, amphibians and reptiles. We sought to identify additional members of the Coronaviridae in both bony and jawless fish through a combination of total RNA sequencing (meta-transcriptomics) and data mining of published RNA sequencing data, and from this reveal more of the long-term patterns and processes of coronavirus evolution. Accordingly, we identified a number of divergent viruses that fell within the Letovirinae subfamily of the Coronaviridae, including those in a jawless fish – the pouched lamprey. By mining fish transcriptome data we identified additional virus transcripts matching these viruses in bony fish from both marine and freshwater environments. These new viruses retained sequence conservation in the RNA-dependant RNA polymerase across the Coronaviridae, but formed a distinct and diverse phylogenetic group. Although there are broad-scale topological similarities between the phylogenies of the major groups of coronaviruses and their vertebrate hosts, the evolutionary relationships of viruses within the Letovirinae does not mirror that of their hosts. For example, the coronavirus found in the pouched lamprey fell within the phylogenetic diversity of bony fish letoviruses, indicative of past host switching events. Hence, despite possessing a phylogenetic history that likely spans the entire history of the vertebrates, coronavirus evolution has been characterised by relatively frequent cross-species transmission, particularly in hosts that reside in aquatic habitats.

Background In a time of rapid environmental change, understanding how the challenges experienced by one generation can influence the fitness of future generations is critically needed. Using tolerance assays and transcriptomic and methylome approaches, we use zebrafish as a model to investigate cross-generational acclimation to hypoxia. Results We show that short-term paternal exposure to hypoxia endows offspring with greater tolerance to acute hypoxia. We detected two hemoglobin genes that are significantly upregulated by more than 6-fold in the offspring of hypoxia exposed males. Moreover, the offspring which maintained equilibrium the longest showed greatest upregulation in hemoglobin expression. We did not detect differential methylation at any of the differentially expressed genes, suggesting that other epigenetic mechanisms are responsible for alterations in gene expression. Conclusions Overall, our findings suggest that an epigenetic memory of past hypoxia exposure is maintained and that this environmentally induced information is transferred to subsequent generations, pre-acclimating progeny to cope with hypoxic conditions.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings because sex change has been challenging to achieve in captivity. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using either aromatase inhibition or manipulation of social groups. Complete transition from female to male occurred over 60 days and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy using radioimmunoassay, nanoString nCounter mRNA and histological analyses, respectively. Early-stage decreases in plasma 17β-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Expression of male-related genes responsible for androgen production cyp11c1 and hsd11b2 increased from mid sex change. Gonadal expression of the glucocorticoid and mineralocorticoid receptors nr3c1 and nr3c2, putative mediators of the stress hormone cortisol, increased in late stages of sex change. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.Summary statementThe spotty wrasse, Notolabrus celidotus, is a new temperate model for the study of vertebrate sex change, this work characterises endocrine and genetic markers based on laboratory induced sex change.

Establishing the sex of individuals in wild systems can be challenging and often requires genetic testing. Genotyping-by-sequencing (GBS) and other reduced-representation DNA sequencing (RRS) protocols (e.g., RADseq, ddRAD) have enabled the analysis of genetic data on an unprecedented scale. Here, we present a novel approach for the discovery and statistical validation of sex-specific loci in GBS data sets. We used GBS to genotype 166 New Zealand fur seals (NZFS, Arctocephalus forsteri) of known sex. We retained monomorphic loci as potential sex-specific markers in the locus discovery phase. We then used (i) a sex-specific locus threshold (SSLT) to identify significantly male-specific loci within our data set; and (ii) a significant sex-assignment threshold (SSAT) to confidently assign sex in silico the presence or absence of significantly male-specific loci to individuals in our data set treated as unknowns (98.9% accuracy for females; 95.8% for males, estimated via cross-validation). Furthermore, we assigned sex to 86 individuals of true unknown sex using our SSAT and assessed the effect of SSLT adjustments on these assignments. From 90 verified sex-specific loci, we developed a panel of three sex-specific PCR primers that we used to ascertain sex independently of our GBS data, which we show amplify reliably in at least two other pinniped species. Using monomorphic loci normally discarded from large SNP data sets is an effective way to identify robust sex-linked markers for nonmodel species. Our novel pipeline can be used to identify and statistically validate monomorphic and polymorphic sex-specific markers across a range of species and RRS data sets.

Phenotypic plasticity represents an elegant adaptive response of individuals to a change in their environment. Bluehead wrasses (Thalassoma bifasciatum) exhibit astonishing sexual plasticity, including female-to-male sex change and discrete male morphs that differ strikingly in behavior, morphology, and gonadal investment. Using RNA-seq transcriptome profiling, we examined the genes and physiological pathways underlying flexible behavioral and gonadal differences among female, dominant (bourgeois) male, and female-mimic (sneaker) male blueheads. For the first time in any organism, we find that female mimicry by sneaker males has a transcriptional signature in both the brain and the gonad. Sneaker males shared striking similarity in neural gene expression with females, supporting the idea that males with alternative reproductive phenotypes have "female-like brains." Sneaker males also overexpressed neuroplasticity genes, suggesting that their opportunistic reproductive strategy requires a heightened capacity for neuroplasticity. Bourgeois males overexpressed genes associated with socio-sexual behaviors (e.g., isotocin), but also neuroprotective genes and biomarkers of oxidative stress and aging, indicating a hitherto unexplored cost to these males of attaining the reproductively privileged position at the top of the social hierarchy. Our novel comparison of testicular transcriptomes in a fish with male sexual polymorphism associates greater gonadal investment by sneaker males with overexpression of genes involved in cell proliferation and sperm quality control. We propose that morphological female-mimicry by sneaker male teleosts entails pervasive downregulation of androgenesis genes, consistent with low androgen production in males lacking well-developed secondary sexual characters.

Background Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation. Results At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf). Conclusion Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3915-z) contains supplementary material, which is available to authorized users.

L-DOPA is the primary pharmacological treatment for relief of the motor symptoms of Parkinson’s disease (PD). With prolonged treatment (⩾5 years) the majority of patients will develop abnormal involuntary movements as a result of L-DOPA treatment, known as L-DOPA-induced dyskinesia. Understanding the underlying mechanisms of dyskinesia is a crucial step toward developing treatments for this debilitating side effect. We used the 6-hydroxydopamine (6-OHDA) rat model of PD treated with a three-week dosing regimen of L-DOPA plus the dopa decarboxylase inhibitor benserazide (4 mg/kg and 7.5 mg/kg s.c., respectively) to induce dyskinesia in 50% of individuals. We then used RNA-seq to investigate the differences in mRNA expression in the striatum of dyskinetic animals, non-dyskinetic animals, and untreated parkinsonian controls at the peak of dyskinesia expression, 60 min after L-DOPA administration. Overall, 255 genes were differentially expressed; with significant differences in mRNA expression observed between all three groups. In dyskinetic animals 129 genes were more highly expressed and 14 less highly expressed when compared with non-dyskinetic and untreated parkinsonian controls. In L-DOPA treated animals 42 genes were more highly expressed and 95 less highly expressed when compared with untreated parkinsonian controls. Gene set cluster analysis revealed an increase in expression of genes associated with the cytoskeleton and phosphoproteins in dyskinetic animals compared with non-dyskinetic animals, which is consistent with recent studies documenting an increase in synapses in dyskinetic animals. These genes may be potential targets for drugs to ameliorate L-DOPA-induced dyskinesia or as an adjunct treatment to prevent their occurrence.

The New Zealand sea lion (NZSL) is of high conservation concern due to its limited distribution and its declining population size. Historically, it occupied most of coastal New Zealand, but is now restricted to a few coastal sites in southern mainland New Zealand and the sub-Antarctic Islands. NZSLs have experienced a recent reduction in population size due to sealing in the 1900s, which is expected to have resulted in increased inbreeding and a loss of genetic variation, potentially reducing the evolutionary capacity of the species and negatively impacting on its long-term prospects for survival. We used 17 microsatellite loci, previously shown to have cross-species applications in pinnipeds, to determine locus- and population-specific statistics for 1205 NZSLs from 7 consecutive breeding seasons. We show that the NZSL population has a moderate level of genetic diversity in comparison to other pinnipeds. We provide genetic evidence for a population reduction, likely caused by historical sealing, and a measure of allele sharing/parental relatedness (internal relatedness) that is suggestive of increased inbreeding in pups that died during recent epizootic episodes. We hypothesize that population bottlenecks and nonrandom mating have impacted on the population genetic architecture of NZSLs, affecting its population recovery.

Models of ageing predict that sperm function and fertility should decline with age as sperm are exposed to free radical damage and mutation accumulation. However, theory also suggests that mating with older males should be beneficial for females because survival to old age is a demonstration of a male's high genetic and/or phenotypic quality. Consequently, declines in sperm fitness may be offset by indirect fitness benefits exhibited in offspring. While numerous studies have investigated age-based declines in male fertility, none has taken the integrated approach of studying age-based effects on both male fertility and offspring fitness. Here, using a cohort-based longitudinal study of zebrafish ( Danio rerio ), we report a decline in male mating success and fertility with male age but also compensating indirect benefits. Using in vitro fertilization, we show that offspring from older males exhibit superior early survival compared to those from their youngest counterparts. These findings suggest that the high offspring fitness observed for the subset of males that survive to an old age (approx. 51% in this study) may represent compensating benefits for declining fertility with age, thus challenging widely held views about the fitness costs of mating with older males.

Aim We used endemic frogs to test the hypothesis that New Zealand was fullysubmerged during the Oligocene marine transgression (c.25–35 Ma, the Oligo-cene drowning) and that all the land biota subsequently arrived by dispersal –including the poorly dispersing frogs.Location New Zealand.Methods The complete mitochondrial genome of the New Zealand nativefrog Leiopelma hochstetteri was sequenced, assembled and analysed, and com-pared with that of L. archeyi.Results Using many phylogenetic analyses we examined the evolutionary rela-tionships of the two species of Leiopelma, both for the deepest divergencewithin New Zealand and relative to other frogs. Our results give estimates ofwell over 65 Ma for the first divergence of leiopelmatid frogs within NewZealand, and over 150 Ma for the divergence of Leiopelma from its closest rela-tive, the North American tailed frog Ascaphus truei.Main conclusions The identification of such a deep divergence among theextant Leiopelma species provides some of the strongest support yet for amixed-age model for the origin of New Zealand’s terrestrial biota, where someelements are of vicariant origin and others have arisen by long-distance dis-persal. This contradicts a full drowning model and supports the view that theremust have been some continuous land through the Oligocene.KeywordsDivergence times, frogs, Leiopelmatidae, long-range dispersal, New Zealand,Oligocene drowning, vicariance.INTRODUCTIONLeiopelma is a genus of frogs native to New Zealand thatconsists of four extant and three recently extinct species, aswell as Miocene fossils. It is an ancient lineage of frogs thatpossesses morphological features not found in most otherfrogs – such as the absence of vocal sacs, lack of toe pads,absence of external ear drums, and the presence of nine ver-tebrae instead of eight (Hay et al., 1995). Two of the species,Leiopelma hochstetteri Fitzinger, 1861 and Leiopelma archeyiTurbott, 1942, are found on the North Island in remnantmontane forests, while Leiopelma pakeka Bell, Daugherty &Hay, 1998 is found on Maud’s Island and Leiopelma hamil-toni McCullough, 1919 on Stephen’s Island, both smallislands in Cook Strait. The latter two species differphenotypically and genetically only marginally from oneanother (Holyoake et al., 2001) and together with L. archeyishow molecular divergences expected between related specieswithin a genus. Physically, L. hamiltoni and L. archeyi haveidentical bone shape, differing only in size. However, L.hochstetteri is reasonably distinct from the other three speciesby being more aquatic, having partially webbed toes (Gibbs,2006, p. 154), lacking parental care, and having some differ-ences in bone shape. All the New Zealand frogs show similarmorphologies and traditionally all have been regarded as arelated group of species within the limits of a single genus.It has been suggested (see Nielson et al., 2006) that theclosest relative of Leiopelma is Ascaphus truei (hereafter Asca-phus), also an ancient lineage of frogs, now found along thewest coast of North America from British Columbia to

Males of many animal species fight to establish social dominance and control access to females so that they have more opportunities to reproduce than their competitors. Males with lower social status will struggle to directly compete for mates, thus they attempt to mate with females by stealth. This often leads to more than one male mating with the same female so that the sperm from each male end up competing to fertilise that female’s eggs, a phenomenon known as sperm competition. Males suspend their sperm in a fluid to make a mixture known as semen. It has been shown that, compared to high status males, low status males will produce higher quality semen that contains greater numbers of faster swimming sperm, giving them an advantage in sperm competition. Growing evidence from several species indicates that males can quickly adjust how fast their sperm swim in response to social cues that signal changing risks of sperm competition. However, how these rapid adjustments occur remains largely unknown, and whether they alter a male’s reproductive success against a competitor has seldom been examined. Chinook salmon usually live in the North Pacific Ocean but they swim up rivers in North America and Asia to reproduce. They have also been introduced to several other countries including New Zealand where they are farmed commercially. The fish are highly prized by sport fishermen and are also of cultural significance to certain groups of indigenous people in North America. Barlett et al. studied the semen of chinook salmon, undertaking a series of experiments in which males switched between high and low social status. The experiments show that, as predicted, the sperm of males that changed from high to low social status started to swim faster. These changes in speed were caused by the fluid in the semen and altered the number of eggs that the male’s sperm fertilised when competing against sperm from another male. In their natural range some populations of chinook salmon are declining due to overfishing combined with habitat loss and alteration. The findings of Barlett et al. contribute to a better understanding of how this fish species reproduces, which may lead to the introduction of measures that help natural populations to recover or help to improve commercial farming. Improved knowledge of how the fluid in semen affects sperm activity may also have important consequences for our wider understanding of male fertility in humans and other animals.

Sexual behavior in teleost fish is highly plastic. It can be attributed to the relatively few sex differences found in adult brain transcriptomes. Environmental and hormonal factors can influence sex-specific behavior. Androgen treatment stimulates behavioral masculinization. Sex dimorphic gene expression in developing teleost brains and the molecular basis for androgen-induced behavioral masculinization are poorly understood. In this study, juvenile zebrafish (Danio rerio) were treated with 100 ng/L of 17 alpha-methyltestosterone (MT) during sexual development from 20 days post fertilization to 40 days and 60 days post fertilization. We compared brain gene expression patterns in MT-treated zebrafish with control males and females using RNA-Seq to shed light on the dynamic changes in brain gene expression during sexual development and how androgens affect brain gene expression leading to behavior masculinization. We found modest differences in gene expression between juvenile male and female zebrafish brains. Brain aromatase (cyp19a1b), prostaglandin 3a synthase (ptges3a), and prostaglandin reductase 1 (ptgr1) were among the genes with sexually dimorphic expression patterns. MT treatment significantly altered gene expression relative to both male and female brains. Fewer differences were found among MT-treated brains and male brains compared to female brains, particularly at 60 dpf. MT treatment upregulated the expression of hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), deiodinase, iodothyronine, type II (dio2), and gonadotrophin releasing hormones (GnRH) 2 and 3 (gnrh2 and gnrh3) suggesting local synthesis of 11-ketotestosterone, triiodothyronine, and GnRHs in zebrafish brains which are influenced by androgens. Androgen, estrogen, prostaglandin, thyroid hormone, and GnRH signaling pathways likely interact to modulate teleost sexual behavior.

Cortisol is the main glucocorticoid (GC) in fish and the hormone most directly associated with stress. Recent research suggests that this hormone may act as a key factor linking social environmental stimuli and the onset of sex change by initiating a shift in steroidogenesis from estrogens to androgens. For many teleost fish, sex change occurs as a usual part of the life cycle. Changing sex is known to enhance the lifetime reproductive success of these fish and the modifications involved (behavioral, gonadal and morphological) are well studied. However, the exact mechanism behind the transduction of the environmental signals into the molecular cascade that underlies this singular process remains largely unknown. We here synthesize current knowledge regarding the role of cortisol in teleost sex change with a focus on two well-described transformations: temperature-induced masculinization and socially regulated sex change. Three non-mutually exclusive pathways are considered when describing the potential role of cortisol in mediating teleost sex change: cross-talk between GC and androgen pathways, inhibition of aromatase expression and upregulation ofamh(the gene encoding anti-Müllerian hormone). We anticipate that understanding the role of cortisol in the initial stages of sex change will further improve our understanding of sex determination and differentiation across vertebrates, and may lead to new tools to control fish sex ratios in aquaculture.

Pests are a global threat to biodiversity, ecosystem function, and human health. Pest control approaches are thus numerous, but their implementation costly, damaging to non-target species, and ineffective at low population densities. The Trojan Female Technique (TFT) is a prospective self-perpetuating control technique that is species-specific and predicted to be effective at low densities. The goal of the TFT is to harness naturally occurring mutations in the mitochondrial genome that impair male fertility while having no effect on females. Here, we provide proof-of-concept for the TFT, by showing that introduction of a male fertility-impairing mtDNA haplotype into replicated populations of Drosophila melanogaster causes numerical population suppression, with the magnitude of effect positively correlated with its frequency at trial inception. Further development of the TFT could lead to establishing a control strategy that overcomes limitations of conventional approaches, with broad applicability to invertebrate and vertebrate species, to control environmental and economic pests. DOI: http://dx.doi.org/10.7554/eLife.23551.001, eLife digest Insect and other animal pests pose some of the greatest challenges to biodiversity, global economies and human health. The environmental and agricultural losses caused by pests have been estimated at 120 billion US dollars a year in the US alone. Many pests also spread diseases, such as dengue fever and malaria. A variety of different strategies are used to control pests, but their effects are generally short-lived and they are often ineffective when pest numbers are low. Furthermore, many of these strategies are harmful to other wildlife, such as bees. Most of the DNA within an animal cell is contained within a structure called the nucleus. However, some DNA is also found within other compartments called mitochondria. The Trojan Female technique has been proposed as a new strategy to control insect pests that harnesses naturally-occurring changes (known as mutations) in this mitochondrial DNA (or mtDNA for short). Introducing mutations that lower the fertility of males, but have no effects on females, into a pest population should, in theory, lead to a long-lasting decline in the size of the population, even if it is relatively small to begin with. Wolff et al. tested this theory in fruit flies, which are often used as models of insects and other animals in research projects. Adding female fruit flies carrying a mutation in mtDNA that lowers male fertility (known as “Trojan Females”) into populations of fruit flies reduced the size of the population over several generations. The mutation was maintained in the population for at least ten generations, and no “rescue” mutations evolved in the nuclear DNA to compensate for the mtDNA mutation. This indicates that the Trojan Female technique could be effective at controlling pests, without the need for Trojan Females to be repeatedly released into the populations. The next steps following on from this work are to test this approach in economically important pest species, and to find out whether the approach is effective in various environments outside the laboratory. If these findings do indeed translate into these pests, then the Trojan Female technique may have the potential to be used to control a wide variety of different pest species from mosquitos through to rats. DOI: http://dx.doi.org/10.7554/eLife.23551.002

Cryptic female choice (CFC), a form of sexual selection during or post mating, describes processes of differential sperm utilization by females to bias fertilization outcomes towards certain males. In Chinook salmon (Oncorhynchus tshawytscha) the ovarian fluid surrounding the ova of a given female differently enhances the sperm velocity of males. Sperm velocity is a key ejaculate trait that determines fertilization success in externally fertilizing fishes, thus the differential effect on sperm velocity might bias male fertilization outcomes and represent a mechanism of CFC. Once sperm reach the oocyte, CFC could potentially be further facilitated by sperm–egg interactions, which are well understood in externally fertilizing marine invertebrates. Here, we explored the potential genetic basis of both possible mechanisms of CFC by examining whether the genotypic combinations of mates (amino-acid divergence, number of shared alleles) at the major histocompatibility complex (MHC) class I and II explain the variation in sperm velocity and/or male fertilization success that is not explained by sperm velocity, which might indicate MHC-based sperm–egg interactions. We recorded sperm velocity in ovarian fluid, employed paired-male fertilization trials and evaluated the fertilization success of each male using microsatellite-based paternity assignment. We showed that relative sperm velocity was positively correlated with fertilization success, confirming that the differential effect on sperm velocity may be a mechanism of CFC in Chinook salmon. The variation in sperm velocity was independent of MHC class I and II. However, the MHC class II divergence of mates explained fertilization success, indicating that this locus might influence sperm–egg interactions.

Background Cannabis has been very much in the news of late, with controversy and debate surrounding legalisation and medicinal uses purposes. However, there is also strong evidence for negative consequences of prolonged cannabis use. Significant contributions in this area have been made by the Christchurch Health and Development Study (CHDS), a longitudinal study of a birth cohort of 1265 children born in 1977 in Christchurch (New Zealand), who have been studied on 23 occasions from birth to the age of 35 (n=987). Methods It is now clear that many environmental factors, including drugs, impact on the pattern of epigenetic marks throughout the genome. We sought to explore the impacts of heavy cannabis use on the methylome in participants of the CHDS. We selected DNA (purified from peripheral blood samples) of 96 CHDS subjects including 49 heavy long-term cannabis smokers (25 of whom also smoked tobacco), and 47 non-smoking, non-cannabis using controls. Epigenome wide analysis of methylation was carried out using Illumina 850 K EPIC arrays. Results Our key observations were that for the cannabis plus tobacco smokers compared with non-smoking controls, the most differentially hypermethylated sites were in the AHRR and F2RL3 genes (adjusted P 4.4×10^-8, 4.9×10^-6 respectively), replicating multiple previous studies on methylomic effects of tobacco. For those sites (~15000) significantly showing greater than 5% differential methylation after multiple comparison adjustment, 99% were hypermethylated. Comparing cannabis only users with non-smoking controls, for those sites (~4500) significantly showing greater than 5% differential methylation after multiple comparison adjustment, 99% were hypomethylated. The differentially methylated sites which were hypermethylated in tobacco smokers but hypomethylated in cannabis only users were most strongly represented in PI3K-Akt and oxytocin KEGG pathways (FDR corrected p values Discussion These data suggest that cannabis has quite different methylome effects to tobacco smoking, and that there may be interaction effects which modify the methylome impacts when both cannabis and tobacco are smoked. We are seeking to validate a proportion of these methylation changes using bisulfite-amplicon sequencing in a wider selection of samples from the CHDS and other cohorts. These analyses may help to identify epigenome impacts of cannabis use, and point to gene regulatory processes that underlie both potentially positive as well as adverse consequences of its use.

The mechanisms by which paternal inheritance of mitochondrial DNA (mtDNA) (paternal leakage) and, subsequently, recombination of mtDNA are prevented vary in a species-specific manner with one mechanism in common: paternally derived mtDNA is assumed to be vastly outnumbered by maternal mtDNA in the zygote. To date, this dilution effect has only been described for two mammalian species, human and mouse. Here, we estimate the mtDNA content of chinook salmon oocytes to evaluate the dilution effect operating in another vertebrate; the first such study outside a mammalian system. Employing real-time PCR, we determined the mtDNA content of chinook salmon oocytes to be 3.2 x 10(9)+/-1.0 x 10(9), and recently, we determined the mtDNA content of chinook salmon sperm to be 5.73+/-2.28 per gamete. Accordingly, the ratio of paternal-to-maternal mtDNA if paternal leakage occurs is estimated to be 1:5.5 x 10(8). This contribution of paternal mtDNA to the overall mtDNA pool in salmon zygotes is three to five orders of magnitude smaller than those revealed for the mammalian system, strongly suggesting that paternal inheritance of mtDNA per offspring will be much less likely in this system than in mammals.

Animal mitochondrial DNA (mtDNA) is predominantly inherited maternally. Various mechanisms to avoid the transmission of paternal mtDNA to offspring have been proposed, including the dilution of paternal mtDNA by maternal mtDNA in the zygote. The effectiveness of dilution as a barrier will be determined by the number of mtDNA molecules contributed by each parental gamete, and is expected to be highly variable among different taxa due to interspecific differences in mating systems and gamete investment. Estimates of this ratio are currently limited to few mammalian species, and data from other taxa are therefore needed to better understand the mechanisms of mitochondrial inheritance. The present study estimates mtDNA content in salmon sperm, the first nonmammalian vertebrate to be examined. Although highly divergent, it appears that the mtDNA content may be conserved within vertebrate taxa, indicating that the reduction of mtDNA is a key factor of spermatogenesis to ensure mitochondrial functionality on the one hand, and to avoid paternal leakage at a significant or detectable level on the other hand. We employ quantitative real-time PCR (Q-PCR) and demonstrate the accuracy and high reproducibility of our experiments. Furthermore, we compare and evaluate two standard approaches used for the quantification of genes, Q-PCR and blotting methods, in regard to their utility in the accurate quantification of mitochondrial genes.

Socially-controlled functional sex change in fishes is a dramatic example of adaptive reproductive plasticity. Functional gonadal sex change can occur within a week while behavioral sex change can begin within minutes. Significant progress has been made in understanding the neuroendocrine bases of this phenomenon at both the gonadal and the neurobiological levels, but a detailed mechanistic understanding remains elusive. We are working with sex-changing wrasses to identify evolutionarily-conserved neuroendocrine pathways underlying this reproductive adaptation. One key model is the bluehead wrasse (Thalassoma bifasciatum), in which sex change is well studied at the behavioral, ecological, and neuroendocrine levels. Bluehead wrasses show rapid increases in aggressive and courtship behaviors with sex change that do not depend on the presence of gonads. The display of male-typical behavior is correlated with the expression of arginine vasotocin, and experiments support a role for this neuropeptide. Estrogen synthesis is also critical in the process. Female bluehead wrasses have higher abundance of aromatase mRNA in the brain and gonads, and estrogen implants block behavioral sex change. While established methods have advanced our understanding of sex change, a full understanding will require new approaches and perspectives. First, contributions of other neuroendocrine systems should be better characterized, particularly glucocorticoid and thyroid signaling. Second, advances in genomics for non-traditional model species should allow conserved mechanisms to be identified with a key next-step being manipulative tests of these mechanisms. Finally, advances in genomics now also allow study of the role of epigenetic modifications and other regulatory mechanisms in the dramatic alterations across the sex-change process.

Current Tris-based solutions for DNA electrophoresis produce a positive feedback loop between current and temperature at high voltage, resulting in long running times for the separation of even small DNA fragments. We optimized the separation of small DNA fragments (90–300 bp) in polyacrylamide-based electrophoresis at high voltages (200volts/ cm) by substituting Tris with low concentration alkali salts (e.g. 1 m M LiCl and CsCl). These media reduced the heat produced during electrophoresis, enhanced the DNA fragment resolution, and allowed gels to be run at higher voltages, reducing gel running times by 25%. In addition, the elimination of Tris and EDTA from the buffer reduced material costs approximately 10-fold.

The mitochondrial genome is considered generally to be an innocent bystander in adaptive evolution; however, there is increasing evidence that mitochondrial DNA (mtDNA) is an important contributor to viability and fecundity. Some of this evidence is now well documented, with mtDNA mutations having been shown to play a causal role in degenerative diseases, ageing, and cancer. However, most research on mtDNA has ignored the possibility that other instances exist where mtDNA mutations could have profound fitness consequences. Recent work in humans and other species now indicates that mtDNA mutations play an important role in sperm function, male fertility, and male fitness. Ironically, deleterious mtDNA mutations that affect only males, such as those that impair sperm function, will not be subject to natural selection because mitochondria are generally maternally inherited and could reach high frequencies in populations if the mutations are not disadvantageous in females. Here, we review how such mtDNA mutations might affect the viability of natural populations. We consider factors that increase or decrease the strength of the effect of mtDNA mutations on population viability and discuss what mechanisms exist to mitigate deleterious mtDNA effects.

Reduced fertility typically occurs among women in their late 30s, but increasing evidence indicates that advanced paternal age is associated with changes in reproduction as well. Numerous studies have investigated age-based declines in semen traits, but the impact of paternal age on semen parameter values remains inconclusive. Using data from 90 studies (93,839 subjects), we conducted a systematic review and meta-analysis to quantify the effect of male age on seven ejaculate traits (semen volume, sperm concentration, total sperm count, morphology, total motility, progressive motility and DNA fragmentation). Age-associated declines in semen volume, percentage motility, progressive motility, normal morphology and unfragmented cells were statistically significant and results generally seemed to be robust against confounding factors. Unexpectedly, sperm concentration did not decline with increasing male age, even though we found that sperm concentration declined over time. Our findings indicate that male age needs more recognition as a potential contributor to the negative pregnancy outcomes and reduced offspring health associated with delayed first reproduction. We suggest that greater focus on collection of DNA fragmentation and progressive motility in a clinical setting may lead to better patient outcomes during fertility treatments of aging couples.

The ovarian, or coelomic, fluid that is released with the egg mass of many fishes is increasingly found to play an important role in several biological processes crucial for reproductive success. These include maintenance of oocyte fertility and developmental competence, prolonging of sperm motility, and enhancing sperm swimming speed. Here we examined if and how the proteome of chinook salmon (Oncorhynchus tshawytscha) ovarian fluid varied among females and then sought to examine the composition of this fluid. Ovarian fluid in chinook salmon was analyzed using 1D SDS PAGE and LC-MS/MS tryptic digest screened against Mascot and Sequest databases. We found marked differences in the number and concentrations of proteins in salmon ovarian fluid across different females. A total of 174 proteins were identified in ovarian fluid, 47 of which were represented by six or more peptides, belonging to one of six Gene Ontology pathways. The response to chemical stimulus and response to hypoxia pathways were best represented, accounting for 26 of the 174 proteins. The current data set provides a resource that furthers our understanding of those factors that influence successful egg production and fertilisation in salmonids and other species.

Next-generation sequencing (NGS) on pooled samples has already been broadly applied in human medical diagnostics and plant and animal breeding. However, thus far it has been only sparingly employed in ecology and conservation, where it may serve as a useful diagnostic tool for rapid assessment of species genetic diversity and structure at the population level. Here we undertake a comprehensive evaluation of the accuracy, practicality and limitations of parallel tagged amplicon NGS on pooled population samples for estimating species population diversity and structure. We obtained 16S and Cyt b data from 20 populations of Leiopelma hochstetteri, a frog species of conservation concern in New Zealand, using two approaches – parallel tagged NGS on pooled population samples and individual Sanger sequenced samples. Data from each approach were then used to estimate two standard population genetic parameters, nucleotide diversity (π) and population differentiation (FST), that enable population genetic inference in a species conservation context. We found a positive correlation between our two approaches for population genetic estimates, showing that the pooled population NGS approach is a reliable, rapid and appropriate method for population genetic inference in an ecological and conservation context. Our experimental design also allowed us to identify both the strengths and weaknesses of the pooled population NGS approach and outline some guidelines and suggestions that might be considered when planning future projects.

Background Commercial viewing and swimming with dusky dolphins (Lagenorhynchus obscurus) near Kaikoura, New Zealand began in the late 1980s and researchers have previously described changes in vocalisation, aerial behaviour, and group spacing in the presence of vessels. This study was conducted to assess the current effects that tourism has on the activity budget of dusky dolphins to provide wildlife managers with information for current decision-making and facilitate development of quantitative criteria for management of this industry in the future. Methodology/Principal Findings First-order time discrete Markov chain models were used to assess changes in the behavioural state of dusky dolphin pods targeted by tour vessels. Log-linear analysis was conducted on behavioural state transitions to determine whether the likelihood of dolphins moving from one behavioural state to another changed based on natural and anthropogenic factors. The best-fitting model determined by Akaike Information Criteria values included season, time of day, and vessel presence within 300 m. Interactions with vessels reduced the proportion of time dolphins spent resting in spring and summer and increased time spent milling in all seasons except autumn. Dolphins spent more time socialising in spring and summer, when conception occurs and calves are born, and the proportion of time spent resting was highest in summer. Resting decreased and traveling increased in the afternoon. Conclusions/Significance Responses to tour vessel traffic are similar to those described for dusky dolphins elsewhere. Disturbance linked to vessels may interrupt social interactions, carry energetic costs, or otherwise affect individual fitness. Research is needed to determine if increased milling is a result of acoustic masking of communication due to vessel noise, and to establish levels at which changes to behavioural budgets of dusky dolphins are likely to cause long-term harm. Threshold values from these studies would allow managers to set appropriate operational conditions based on quantifiable criteria.

Around 1 in 200 children born each year have mutations in the mitochondrial DNA (mtDNA)—the genome of the cellular powerhouse. In most cases this causes only mild disease, often without any symptoms. However, for about 1 in 6,500 individuals, mitochondrial disease causes serious and often fatal conditions, which include blindness, muscular weakness, and heart failure. Currently available therapies for individuals carrying mitochondrial disease are not curative, but at best supportive in nature. Mitochondrial replacement (MR), via three-way in vitro fertilization (IVF) [1–3], may be a tractable intervention to circumvent what are clearly devastating diseases. The approach differs from traditional IVF in that embryos effectively have three parents who each make a genetic contribution, with the embryo possessing nuclear DNA from the mother and father and mtDNA from a donor female that has healthy mtDNA [1–3]. The key merit of the approach is that it limits the carryover of disease carrying mtDNA, while enabling both the mother and the father to have a genetic connection to their offspring, which would otherwise be lost if standard egg donation was used. These therapies promise a way to reduce the risk of having offspring with potentially devastating mitochondrial disease and affected families eagerly await such treatments. However, MR approaches are technically challenging, have low rates of success [1–3], and are not risk free even if healthy offspring can be produced [4, 5]. Despite growing optimism based on successful mitochondrial replacement experiments [1–3], we remain ignorant of the broad effects that the small mitochondrial genome (mtDNA) might have on phenotype, both directly through its role in cellular energetics and indirectly as a manipulator or moderator of nuclear genetic events [6]. A growing literature shows that mtDNA can have wide effects on phenotypes, and that these effects are particularly prominent in males. Experiments in mice and fruit flies have shown that simply varying the mtDNA, while keeping the nuclear genetics constant, can result in major alterations to longevity, fertility, and behavior [7]. In addition, the long-held view that mtDNA was a fairly minor player on phenotype is currently being drastically overhauled: new work shows that mtDNA haplotype has direct and indirect effects on nuclear gene regulation [6]. Faced with this evidence, it seems prudent to move towards clinical application of mitochondria replacement with caution. One of the key considerations will be to determine whether outcomes from such procedures are affected by the similarity between the donor and recipient mtDNAs. A strong prediction is that as mtDNA donor and recipient mtDNAs diverge, offspring survival and health may be reduced as a consequence of the breakdown in co-evolved mtDNA– nuclear interactions [8]. The literature is already rich with examples of how mitochondrial genome variation maintained within the human population alters cellular physiology and disease susceptibility of carriers depending on the nuclear background alongside which this variation is expressed. Reinforcing these concerns is a recent study showing that mismatched mitochondria in nuclear-transfer-derived cells—a technique akin to pronuclear transfer (PNT) suggested for MR—possess alloantigenecity, triggering an immune response in a murine model [5]. Likely, establishing the principle of replacing mutant mtDNA with the most similar non-disease carrying mtDNA is a good one to aspire to, and given advances in sequencing technologies such matching would add a trivial cost to what is a significant manipulation, while likely improving outcomes for both the offspring and their parents. The other issue that requires further thought and experimentation is that a large number of mtDNA disorders appear late in life, and we simply do not yet know what the longer term effects of such replacements might be. While flies, mice, and non-human primates may give us some insights into the utility of such therapies, they are unlikely to help us know what transpires in the later life of humans. One potentially complicating issue here is that mtDNA replacement is not 100%, thus there will be potential for carryover of disease-causing mutant mtDNAs, and these may increase in frequency within an individual over time. Pre-clinical trials applying MR therapies to mammalian model systems led to embryos containing varying amounts of carryover mtDNAs [1, 2]. Perhaps the most comprehensive estimate comes from a recent comparative study applying PNT, spindle-chromosome transfer (ST), and polar body transfer (PBT1, PBT2) in parallel to a mouse model [2]. Probing for heteroplasmy (the presence of more than one mtDNA type in a cell) in F1 infants (tail tips), this study found an average carryover of over 5% for ST individuals, over 20% for PNT individuals, and 0 (i.e. below detection limit of 1%) to 2% for DOI 10.1002/bies.201500008

In most species mitochondrial DNA (mtDNA) is inherited maternally in an apparently clonal fashion, although how this is achieved remains uncertain. Population genetic studies show not only that individuals can harbor more than one type of mtDNA (heteroplasmy) but that heteroplasmy is common and widespread across a diversity of taxa. Females harboring a mixture of mtDNAs may transmit varying proportions of each mtDNA type (haplotype) to their offspring. However, mtDNA variants are also observed to segregate rapidly between generations despite the high mtDNA copy number in the oocyte, which suggests a genetic bottleneck acts during mtDNA transmission. Understanding the size and timing of this bottleneck is important for interpreting population genetic relationships and for predicting the inheritance of mtDNA based disease, but despite its importance the underlying mechanisms remain unclear. Empirical studies, restricted to mice, have shown that the mtDNA bottleneck could act either at embryogenesis, oogenesis or both. To investigate whether the size and timing of the mitochondrial bottleneck is conserved between distant vertebrates, we measured the genetic variance in mtDNA heteroplasmy at three developmental stages (female, ova and fry) in chinook salmon and applied a new mathematical model to estimate the number of segregating units (N(e)) of the mitochondrial bottleneck between each stage. Using these data we estimate values for mtDNA Ne of 88.3 for oogenesis, and 80.3 for embryogenesis. Our results confirm the presence of a mitochondrial bottleneck in fish, and show that segregation of mtDNA variation is effectively complete by the end of oogenesis. Considering the extensive differences in reproductive physiology between fish and mammals, our results suggest the mechanism underlying the mtDNA bottleneck is conserved in these distant vertebrates both in terms of it magnitude and timing. This finding may lead to improvements in our understanding of mitochondrial disorders and population interpretations using mtDNA data.

International audience; The solitary ascidian Styela clava Herdman, 1882 is considered to be native to Japan, Korea, northern China and the Russian Federation in the NW Pacific, but it has spread globally over the last 80 years and is now established as an introduced species on the east and west coasts of North America, Europe, Australia and New Zealand. In eastern Canada it reaches sufficient density to be a serious pest to aquaculture concerns. We sequenced a fragment of the cytochrome oxidase subunit I mitochondrial gene (COI) from a total of 554 individuals to examine the genetic relationships of 20 S. clava populations sampled throughout the introduced and native ranges, in order to investigate invasive population characteristics. The data presented here show a moderate level of genetic diversity throughout the northern hemisphere. The southern hemisphere (particularly New Zealand) displays a greater amount of haplotype and nucleotide diversity in comparison. This species, like many other invasive species, shows a range of genetic diversities among introduced populations independent of the age of incursion. The successful establishment of this species appears to be associated with multiple incursions in many locations, while other locations appear to have experienced rapid expansion from a potentially small population with reduced genetic diversity. These contrasting patterns create difficulties when attempting to manage and mitigate a species that continues to spread among ports and marinas around the world.

The relationships between the compositions of ovarian, seminal fluids and sperm function are not well known in teleostean fish species. The objective of the present study was to determine the concentration of the major inorganic ions (Na + , K + , Ca 2+ , Mg, Cl − ), osmolality, and pH of ovarian and seminal fluid of sexually mature chinook salmon ( Oncorhynchus tshawytscha ), and to determine if the composition of these fluids influences sperm motility traits (swimming speed, duration of forward mobility, swimming path trajectory, and percent motility). Cation concentrations and osmolality were significantly different in the two fluids. The ionic composition of ovarian fluid differed among individual females, and also among samples collected at different times through the spawning season. Carbonate and bicarbonate were the principal buffer ions in ovarian fluid, and its viscosity was considerably greater than that of water and was shear-dependent. The duration of forward motility (longevity) of spermatozoa, swimming speed, percent motility, and path trajectory were measured using milt from 10 males activated in the ovarian fluid from 7 females whose ion concentrations were known. No significant correlations were observed between the composition of the seminal fluid and sperm traits. However, in ovarian fluid, sperm longevity was negatively correlated with variation in [Ca 2+ ] and [Mg 2+ ], while percent motility increased with increasing [Mg 2+ ]. These observations provide a possible chemical basis for cryptic female mate choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males, and thus their fertilization success.

Supplementary feeding is often a key tool in the intensive management of captive and threatened species. Although it can increase such parameters as breeding frequency and individual survival, supplementary feeding may produce undesirable side effects that increase overall extinction risk. Recent attempts to increase breeding frequency and success in the kakapo Strigops habroptilus using supplementary feeding inadvertently resulted in highly male-biased chick sex ratios. Here, we describe how the inclusion of sex allocation theory has remedied this conservation dilemma. Our study is the first to manipulate chick sex ratios in an endangered species by altering maternal condition and highlights the importance of incorporating evolutionary theory into modern conservation practice.

The initiation of mammalian puberty is underpinned by an increase in Kisspeptin (Kiss1) signaling via its receptor (Kiss1r/GPR54) on gonadotropin-releasing hormone (GnRH) neurons. Animals and humans with loss-of-function mutations in Kiss1 or Kiss1r fail to go through puberty. The timing of puberty is dependent on environmental factors, and malleability in puberty timing suggests a mechanism that can translate environmental signals into patterns of Kiss1/Kiss1r gene expression. Epigenetics is a powerful mechanism that can control gene expression in an environment-dependent manner. We investigated whether epigenetic DNA methylation is associated with gene expression changes at puberty. We used bisulfite-PCR-pyrosequencing to define the methylation in the promoters of Kiss1 and Kiss1r before and after puberty in female rats. Both Kiss1 and Kiss1r showed highly significant puberty-specific differential promoter methylation patterns. By identifying key differentially methylated residues associated with puberty, these findings will be important for further studies investigating the control of gene expression across the pubertal transition.

We examined the publication records of a cohort of 168 life scientists in the field of ecology and evolutionary biology to assess gender differences in research performance. Clear discrepancies in publication rate between men and women appear very early in their careers and this has consequences for the subsequent citation of their work. We show that a recently proposed index designed to rank scientists fairly is in fact strongly biased against female researchers, and advocate a modified index to assess men and women on a more equitable basis.