Incubation media modify silver nanoparticle toxicity for whitefish (Coregonus lavaretus) and roach (Rutilus rutilus) embryos.

Toxicological studies were performed to examine silver nanoparticle (AgNP, size: 14.4 ± 2.5 nm) transformation within three different test media and consequent effects on embryos of whitefish (Coregonus lavaretus) and roach (Rutilus rutilus). The test media, namely ASTM very hard water, ISO standard dilution medium, and natural lake water differed predominantly in ionic strength. Total silver was determined using inductively coupled plasma mass spectrometry (ICP-MS), while AgNPs were characterized by transmission electron microscopy and single particle ICP-MS. Silver species distributions were estimated via thermodynamic speciation calculations. Data demonstrated that increased AgNP dissolution accompanied by decreasing ionic strength of the test medium did not occur as noted in other studies. Further, other physicochemical parameters including AgNP size and metallic species distribution did not markedly affect AgNP-induced toxicity. Irrespective of the test medium, C. lavaretus were more sensitive to AgNP exposure (median lethal concentration after 8 weeks: 0.51-0.73 mg/L) compared to R. rutilus, where adverse effects were only observed at 5 mg/L in natural lake water. In addition, AgNP-induced toxicity was lower in the two standard test media compared to natural lake water. Currently, there are no apparent studies assessing simultaneously the sensitivity of C. lavaretus and R. rutilus to AgNP exposure. Therefore, the aim of this study was to (1) investigate AgNP-induced toxicity in C. lavaretus and R. rutilus cohabiting in the same aquatic environment and (2) the role played by test media in the observed effects of AgNPs on these aquatic species.

Sulfate toxicity to early life stages of European whitefish (Coregonus lavaretus) in soft freshwater.

Sulfate occurs naturally in the aquatic environment but its elevated levels can be toxic to aquatic life in freshwater environments. We investigated the toxicity of sulfate in humic, soft freshwater to whitefish (Coregonus lavaretus) from fertilization of eggs to hatching i.e. during the critical phases of whitefish early development. Anadromous Kokemäenjoki whitefish eggs and sperm during fertilization, embryos and larvae were exposed in the long-term 175-day incubation to seven different sodium sulfate (Na2SO4) concentrations from 44 to 2 000 mg SO4 L-1. Endpoint variables were the fertilization success, offspring survival and larval growth. Egg fertilization and early embryonic development were the most sensitive developmental stages of whitefish to sulfate, although the fertilization success and survival of embryos decreased only in the highest concentration of 2 000 mg SO4 L-1. The survival during late embryonic period, hatching and the 5-day larval period was high and no difference between the control and sulfate treatments were observed. LC50-values of sulfate for early embryonic period and for the entire embryonic and larval period was 1 413 and 1 161 mg L-1, respectively. The NOEC (No-observed Effect Concentration) of sulfate for the both periods was 1 207 mg L-1. The tolerance of whitefish early stages to sulfate toxicity seems to be on the same level as the tolerance of other salmonids' early stages.

The heat shock response shows plasticity in embryonic lake whitefish (Coregonus clupeaformis) exposed to repeated thermal stress.

We examined the impact of repeated thermal stress on the heat shock response (HSR) of thermally sensitive lake whitefish (Coregonus clupeaformis) embryos. Our treatments were designed to mimic temperature fluctuations in the vicinity of industrial thermal effluents. Embryos were either maintained at control temperatures (3 oC) or exposed to a repeated thermal stress (TS) of 3 or 6 oC above control temperature every 3 or 6 days throughout embryonic development. At 82 days post-fertilisation, repeated TS treatments were stopped and embryos received either a high level TS of 12, 15, or 18 oC above ambient temperature for 1 or 4 h, or no additional TS. These treatments were carried out after a 6 h recovery from the last repeated TS. Embryos in the no repeated TS group responded, as expected, with increases in hsp70 mRNA in response to 12, 15 and 18 oC high-level TS. However, exposure to repeated TS of 3 or 6 °C every 6 days also resulted in a significant upregulation of hsp70 mRNA relative to the controls. Importantly, these repeated TS events and the associated elevations in hsp70 attenuated the upregulation of hsp70 in response to a 1 h, high-level TS of 12 oC above ambient, but not to either longer (4 h) or higher (15 or 18 oC) TS events. Conversely, hsp90α mRNA levels were not consistently elevated in the no repeated TS groups exposed to high-level TS. In some instances, hsp90α levels appeared to decrease in embryos exposed to repeated TS followed by a high-level TS. The observed attenuation of the HSR in lake whitefish embryos demonstrates that embryos of this species have plasticity in their HSR and repeated TS may protect against high-level TS, but the response differs based on repeated TS treatment, high-level TS temperature and duration, and the gene of interest.

Hypoxia affects the ontogeny of the hypothalamus-pituitary-interrenal axis functioning in the lake whitefish (Coregonus clupeaformis).

Early life stages are sensitive to environmental insults and changes during critical developmental periods; this can often result in altered adult behaviour and physiology. Examining the development of the hypothalamus-pituitary-interrenal (HPI) axis and its responsiveness, or lack thereof, during development are important for understanding the short- and long-term impacts of stressors on embryonic and larval fish. We examined the ontogeny of the HPI axis in embryonic (21, 38, 63, 83 and 103 days post-fertilisation (dpf)) and larval (1, 2, 3 and 4 weeks post-hatch (wph)) lake whitefish (Coregonus clupeaformis) by quantifying changes in mRNA levels of several genes associated with HPI axis functioning and whole animal cortisol levels throughout development and in response to a severe or mild hypoxic stress. Cortisol, and crh, crhbp1, pomc and star transcripts were detected from the earliest embryonic age studied. Cortisol levels in control embryos decreased between 21 and 63 dpf, suggesting the utilisation of maternal cortisol deposits. However, by 83 dpf (70% developed) endogenous de novo synthesis had generated a 4.5-fold increase in whole embryo cortisol. Importantly, we provide novel data showing that the HPI axis can be activated even earlier. Whole body cortisol increased in eyed lake whitefish embryos (38 dpf; ~32% developed) in response to hypoxia stress. Coincident with this hypoxia-induced increase in cortisol in 38 dpf embryos were corresponding increases in crh, crhbp1, pomc and star transcript levels. Beyond 38 dpf, the HPI axis in lake whitefish embryos was hyporesponsive to hypoxia stress at all embryonic ages examined (63, 83 and 103 dpf; 54, 72 and 85% developed, respectively). Post-hatch, larvae responded to hypoxia with an increase in cortisol levels and HPI axis genes at 1 wph, but this response was lost and larvae appeared hyporesponsive at subsequent ages (2, 3 and 4 wph). Collectively our work demonstrates that during fish embryogenesis and the larval stage there are windows where the HPI axis is responsive and windows where it is truly hyporesponsive; both could be beneficial in ensuring undisrupted development particularly in the face of increasing environmental changes.

Modifying effects of a cobble substrate on thermal environments and implications for embryonic development in lake whitefish (Coregonus clupeaformis).

A laboratory flume was constructed to examine substrate effects on aquatic development. The flume was designed as a once-through system with a submerged cobble-filled corebox. Lake whitefish (Coregonus clupeaformis) embryos and temperature probes were deployed at multiple sites within the cobble and in the open water channel. Embryos were incubated in the flume for two different experimental periods: one to examine substrate impacts during natural lake cooling (37 days: 5 December 2016 to 10 January 2017) and the second to investigate substrate effects while administering a twice weekly 1 h heat shock (51 days: 11 January to 2 March 2017). During incubation, no significant difference was found in the average temperature between locations; however, temperatures were more stable within the cobble. Following both incubation periods, embryos retrieved from the cobble were significantly smaller in both dry mass and body length by up to 20%. These results demonstrate differences between embryos submerged in a cobble substrate and in the open water column, highlighting the need to consider the physical influences from the incubation environment when assessing development effects as part of any scientific study or environmental assessment.

Polyunsaturated Fatty Acid Content in Muscle Tissue Is Associated with the Duration of Embryo Development in Salmonoid Fishes (Salmonoidei).

A hypothesis was advanced and grounded that the total content of eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3) acids in fish muscle tissue is associated with the species-specific (taxon-specific) duration of embryo development. A meta-analysis of the original and published data was performed using fishes of the families Coregonidae and Salmonidae as an example. Fishes with longer embryo development times, which are observed at lower temperatures, were found to have significantly higher EPA + DHA contents in muscles as compared with the species that belong to the same families but have shorter embryo development times. This association was explained by the fact that an embryo forms more cells per unit tissue volume at lower temperatures, which requires a greater specific amount of cell membranes and, therefore, greater amounts of EPA and DHA to produce them.

Sex-specific changes in gene expression in response to estrogen pollution around the onset of sex differentiation in grayling (Salmonidae).

The synthetic 17α-ethinylestradiol (EE2) is a common estrogenic pollutant that has been suspected to affect the demography of river-dwelling salmonids. One possibility is that exposure to EE2 tips the balance during initial steps of sex differentiation, so that male genotypes show female-specific gene expression and gonad formation. Here we study EE2 effects on gene expression around the onset of sex differentiation in a population of European grayling (Thymallus thymallus) that suffers from sex ratio distortions. We exposed singly-raised embryos to one dose of 1 ng/L EE2, studied gene expression 10 days before hatching, at the day of hatching, and around the end of the yolk-sac stage, and related it to genetic sex (sdY genotype). We found that exposure to EE2 affects expression of a large number of genes, especially around hatching. These effects were strongly sex-dependent. We then raised fish for several months after hatching and found no evidence of sex reversal in the EE2-exposed fish. We conclude that ecologically relevant (i.e. low) levels of EE2 pollution do not cause sex reversal by simply tipping the balance at early stages of sex differentiation, but that they interfere with sex-specific gene expression.

Expression and activity of lipid and oxidative metabolism enzymes following elevated temperature exposure and thyroid hormone manipulation in juvenile lake whitefish (Coregonus clupeaformis).

Temperature has unequivocal effects on several aspects of fish physiology, but the full extent of its interaction with key endocrine signaling systems to influence metabolic function remains unknown. The aim of the current study was to assess the individual and combined effects of elevated temperature and hyperthyroidism on hepatic metabolism in juvenile lake whitefish by quantifying mRNA abundance and activity of key metabolic enzymes. Fish were exposed to 13 (control), 17 or 21 °C for 0, 4, 8 or 24 days in the presence or absence of low-T4 (1 µg × g body weight-1) or high-T4 (10 µg × g body weight-1) treatment. Our results demonstrate moderate sensitivity to elevated temperature in this species, characterized by short-term changes in mRNA abundance of several metabolic enzymes and long-term declines in citrate synthase (CS) and cytochrome c oxidase (COX) activities. T4-induced hyperthyroidism also had several short-term effects on mRNA abundance of metabolic transcripts, including depressions in acetyl-coA carboxylase ß (accß) and carnitine palmitoyltransferase 1ß (cpt1ß), and stabilization of cs mRNA levels; however, these effects were primarily limited to elevated temperature groups, indicating temperature-dependent effects of exogenous T4 treatment in this species. In contrast, maximal CS and COX activities were not altered by hyperthyroidism at any temperature. Collectively, our data suggest that temperature has the potential to manipulate thyroid hormone physiology in juvenile lake whitefish and, under warm-conditions, hyperthyroidism may suppress certain elements of the ß-oxidation pathway without substantial impacts on overall cellular oxidative capacity.

Hypoxia alters the expression of hif-1a mRNA and downstream HIF-1 response genes in embryonic and larval lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis) embryos and larvae were exposed to hypoxia at different developmental ages to determine when the cellular response to hypoxia could be initiated. mRNA levels of hypoxia-inducible factor 1α (hif-1α), hsp70, and several HIF-1 target genes were quantified in embryos at 21, 38, 63, 83- and 103-days post fertilisation (dpf) and in larvae at 1, 2, 3- and 4-weeks post hatch (wph) following a 6-hour hypoxia exposure. hsp70 mRNA levels were increased in response to hypoxia at all embryonic ages. By comparison, the first observed change in hif-1α mRNA in response to hypoxia was at 38 dpf, where it was down-regulated from high basal levels, with this response persisting through to 83 dpf. Interestingly, this decrease in hif-1α mRNA coincided with increases in the mRNA levels of the HIF-1 target genes: vegfa (vascular endothelial growth factor A), igfbp1 (insulin-like growth factor binding protein 1), ldha (lactate dehydrogenase a), gapdh (glyceraldehyde-3-phosphate dehydrogenase) and epo (erythropoietin) at select ages. Collectively, this suggests a possible HIF-1-mediated response to hypoxia despite a decrease in hif-1α mRNA. Coinciding with a decrease in basal levels, increases in hif-1α were measured in response to hypoxia at 103 dpf and in larval fish at 1, 2 and 3 wph but there were no consistent increases in HIF-1 target genes at these ages. Overall, our findings indicate that lake whitefish can mount a response to hypoxia early in embryogenesis which may mitigate some of the damaging effects of exposure to low oxygen levels at these critical life history stages.

Assessment of Fish Embryo Survival and Growth by In Situ Incubation in Acidic Boreal Streams Undergoing Biomining Effluents.

The applicability of an in situ incubation method in monitoring the effects of metal mining on early life stages of fish was evaluated by investigating the impacts of a biomining technology utilizing mine on the mortality, growth, and yolk consumption of brown trout (Salmo trutta) and whitefish (Coregonus lavaretus) embryos. Newly fertilized eggs were incubated from autumn 2014 to spring 2015 in six streams under the influence of the mine located in North-Eastern Finland and in six reference streams. Although the impacted streams clearly had elevated concentrations of several metals and sulfate, the embryonic mortality of the two species did not differ between the impacted and the reference streams. Instead, particle accumulation to some cylinders had a significant impact on the embryonic mortality of both species. In clean cylinders, mortality was higher in streams with lower minimum pH. However, low pH levels were evident in both the reference and the mine-impacted groups. The embryonic growth of neither species was impacted by the mining activities, and the growth and yolk consumption of the embryos was mainly regulated by water temperature. Surprisingly, whitefish embryos incubated in streams with lower minimum pH had larger body size. In general, the applied in situ method is applicable in boreal streams for environmental assessment and monitoring, although in our study, we did not observe a specific mining impact differing from the effects of other environmental factors related to catchment characteristics.

Daily, repeating fluctuations in embryonic incubation temperature alter metabolism and growth of Lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis) utilize overwintering embryonic development (up to 180 days), and such stenothermic, cold-water embryos may be particularly susceptible to thermal shifts. We incubated whitefish embryos in temperature treatments that were constant temperature (2.0 ±â€¯0.1 °C, 5.0 ±â€¯0.1 °C, and 8.0 ±â€¯0.1 °C; mean ±â€¯SD) or variable temperature (VT, mean = 5.0 ±â€¯0.3 °C). In the VT, a daily 2 °C temperature change followed a continuous pattern throughout development: 2-4-6-8-6-4-2 °C. Hatchling survival proportion from fertilization to hatch was significantly impacted by incubation temperature (P < 0.001): 2 °C (0.88 ±â€¯0.01) and 5 °C (0.91 ±â€¯0.01) showed higher survival than both the VT (0.83 ±â€¯0.02) and 8 °C groups (0.15 ±â€¯0.06), which were statistically distinct from each other. Time to hatch (dpf) was significantly different across all treatments (P < 0.001): 8 °C (68 ±â€¯2 dpf), VT (111 ±â€¯4 dpf), 5 °C (116 ±â€¯4 dpf), 2 °C (170 ±â€¯3 dpf). Likewise, hatchling yolk-free dry mass (mg) and total body length (mm) were significantly different across all treatments (P < 0.001): 8 °C (0.66 ±â€¯0.08 mg; 11.1 ±â€¯0.08 mm), VT (0.97 ±â€¯0.06 mg; 11.7 ±â€¯0.05 mm), 5 °C (1.07 ±â€¯0.03 mg; 12.0 ±â€¯0.02 mm), 2 °C (1.36 ±â€¯0.04 mg; 12.8 ±â€¯0.05 mm). Oxygen consumption rate (V̇o2) was significantly affected by the interaction between treatment and measurement temperature (P < 0.001). Hatchling VT whitefish showed mean V̇o2 that was higher compared to the 2 °C group measured at 2 °C, and lower compared to the 2 °C and 5 °C group measured at 8 °C. This study demonstrates that the VT incubation treatment produced fewer (increased mortality), smaller embryos that hatched earlier than 2 °C and 5 °C embryos. The plasticity of V̇o2 for this stenothermic-incubating fish species under variable incubation conditions reveals a metabolic cost to cycling thermal incubation conditions.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

The effects of fluctuating temperature regimes on the embryonic development of lake whitefish (Coregonus clupeaformis).

Fluctuating incubation temperatures may have significant effects on fish embryogenesis; yet most laboratory-based studies use constant temperatures. For species that experience large, natural seasonal temperature changes during embryogenesis, such as lake whitefish (Coregonus clupeaformis), seasonal temperature regimes are likely optimal for development. Anthropogenic activities can increase average and/or variability of natural incubation temperatures over large (e.g. through climate change) or smaller (e.g. thermal effluent discharge) geographic scales. To investigate this, we incubated lake whitefish embryos under constant (2, 5, or 8°C) and fluctuating temperature regimes. Fluctuating temperature regimes had a base temperature of 2°C with: 1) seasonal temperature changes that modeled natural declines/inclines; 2) tri-weekly +3°C, 1h temperature spikes; or 3) both seasonal temperature changes and temperature spikes. We compared mortality to hatch, morphometrics, and heart rate at three developmental stages. Mortality rate was similar for embryos incubated at constant 2°C, constant 5°C, or with seasonal temperatures, but was significantly greater at constant 8°C. Embryos incubated constantly at >2°C had reduced body growth and yolk consumption compared to embryos incubated with seasonal temperature changes. When measured at the common base temperature of 2°C, embryos incubated at constant 2°C had lower heart rates than embryos incubated with both seasonal temperature changes and temperature spikes. Our study suggests that incubating lake whitefish embryos with constant temperatures may significantly alter development, growth, and heart rate compared to incubating with seasonal temperature changes, emphasizing the need to include seasonal temperature changes in laboratory-based studies.

Lipid content and fatty acid profile during lake whitefish embryonic development at different incubation temperatures.

Lipids serve as energy sources, structural components, and signaling molecules during fish embryonic development, and utilization of lipids may vary with temperature. Embryonic energy utilization under different temperatures is an important area of research in light of the changing global climate. Therefore, we examined percent lipid content and fatty acid profiles of lake whitefish (Coregonus clupeaformis) throughout embryonic development at three incubation temperatures. We sampled fertilized eggs and embryos at gastrulation, eyed and fin flutter stages following chronic incubation at temperatures of 1.8, 4.9 and 8.0°C. Hatchlings were also sampled following incubation at temperatures of 3.3, 4.9 and 8.0°C. Fertilized eggs had an initial high percentage of dry mass composed of lipid (percent lipid content; ~29%) consisting of ~20% saturated fatty acids (SFA), ~32% monounsaturated fatty acids (MUFA), ~44% polyunsaturated fatty acids (PUFA), and 4% unidentified. The most abundant fatty acids were 16:0, 16:1, 18:1(n-9c), 20:4(n-6), 20:5(n-3) and 22:6(n-3). This lipid profile matches that of other cold-water fish species. Percent lipid content increased during embryonic development, suggesting protein or other yolk components were preferentially used for energy. Total percentage of MUFA decreased during development, which indicated MUFA were the primary lipid catabolized for energy during embryonic development. Total percentage of PUFA increased during development, driven largely by an increase in 22:6(n-3). Temperature did not influence percent lipid content or percent MUFA at any development stage, and had inconsistent effects on percent SFA and percent PUFA during development. Thus, lake whitefish embryos appear to be highly adapted to low temperatures, and do not alter lipids in response to temperature within their natural incubation conditions.

Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage lake whitefish (Coregonus clupeaformis) embryos.

Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12°C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2h followed by a 2h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6°C TS for 1h every 6 days or +9°C TS for 1h every 6 days. Following a 2h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12°C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1h TS and 2h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development.

Salmonid fish: model organisms to study cardiovascular morphogenesis in conjoined twins?

BACKGROUND: There is a gap in knowledge regarding the cardiovascular system in fish conjoined twins, and regarding the cardiovascular morphogenesis of conjoined twins in general. We examined the cardiovascular system in a pair of fully developed ventrally conjoined salmonid twins (45.5 g body weight), and the arrangement of the blood vessels during early development in ventrally conjoined yolk sac larvae salmonid twins (<0.5 g body weight). RESULTS: In the fully developed twins, one twin was normal, while the other was small and severely malformed. The mouth of the small twin was blocked, inhibiting respiration and feeding. Both twins had hearts, but these were connected through a common circulatory system. They were joined by the following blood vessels: (i) arteria iliaca running from arteria caudalis of the large twin to the kidney of the small twin; (ii) arteria subclavia running from aorta dorsalis of the large twin to aorta dorsalis of the small twin; (iii) vena hepatica running from the liver of the small twin into the sinus venosus of the large twin. Among the yolk sac larvae twins investigated, distinct vascular connections were found in some individuals through a joined v. vitellina hepatica. CONCLUSIONS: Ventrally conjoined fish twins can develop cardiovascular connections during early development, enabling a normal superior twin to supply a malfunctioning twin with oxygen and nutrients. Since the yolk sac in salmonids is transparent, twinning in salmonids may be a useful model in which to study cardiovascular morphogenesis in conjoined twins.

Successful cryopreservation of spermatogonia in critically endangered Manchurian trout (Brachymystax lenok).

Because of the lack of cryopreservation techniques for fish eggs and embryos, cryopreservation of fish spermatogonia and subsequent generation of eggs and sperm would be an exit strategy for the long-term preservation of genetic resources. This study aimed to optimize cryoprotectants, cooling rates, and thawing temperatures for slow freezing of spermatogonia from endangered Manchurian trout (Brachymystax lenok). Whole testes were frozen with a cryomedium containing 1.3 M methanol, 0.2 M trehalose, and 10% egg yolk at a cooling rate of -1 °C/min and then stored in liquid nitrogen for 2 days. After thawing at 30 °C in a water bath, testicular cells from thawed testes were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted spermatogonia migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency did not significantly differ between frozen and fresh testes, demonstrating that Manchurian trout spermatogonia can be successfully cryopreserved in liquid nitrogen.

RNA-seq reveals transcriptomic shock involving transposable elements reactivation in hybrids of young lake whitefish species.

Identifying the molecular basis of reproductive isolation among diverging lineages represents an essential step toward understanding speciation in natural populations. Postzygotic barriers can lead to hybrid breakdown, a syndrome that has been documented in several systems, potentially involving the reactivation of transposable elements. In northeastern North America, two lake whitefish lineages have repeatedly colonized postglacial lakes ~12,000 years ago, and a dwarf limnetic species has evolved multiple times from the normal benthic species. Reproductive isolation is incomplete between them; viable hybrids can be generated in the laboratory but significant mortality occurs and is associated with a malformed phenotype in backcross embryos, thus revealing a hybrid breakdown syndrome. By means of RNA-seq analyses, the objective of this study was to determine which genes were misregulated in hybrids and rigorously test the hypothesis of transposable element reactivation. We compared the transcriptomic landscape in pure embryos, F1-hybrids, and healthy and malformed backcrosses at the late embryonic stage. Extensive expression differences consistent with previously documented adaptive divergence between pure normal and dwarf embryos were identified for the first time. Pronounced transcriptome-wide deregulation in malformed backcrosses was observed, with over 15% of transcripts differentially expressed in all comparisons, compared with 1.5% between pure parental forms. Convincing evidence of transposable elements and noncoding transcripts reactivation in malformed backcrosses is presented. We propose that hybrid breakdown likely results from extensive genomic incompatibilities, plausibly encompassing transposable elements. Combined with previous studies, these results reveal synergy among many reproductive barriers, thus maintaining divergence between these two young whitefish species.

Reproductive isolation in a nascent species pair is associated with aneuploidy in hybrid offspring.

Speciation may occur when the genomes of two populations accumulate genetic incompatibilities and/or chromosomal rearrangements that prevent inter-breeding in nature. Chromosome stability is critical for survival and faithful transmission of the genome, and hybridization can compromise this. However, the role of chromosomal stability on hybrid incompatibilities has rarely been tested in recently diverged populations. Here, we test for chromosomal instability in hybrids between nascent species, the 'dwarf' and 'normal' lake whitefish (Coregonus clupeaformis). We examined chromosomes in pure embryos, and healthy and malformed backcross embryos. While pure individuals displayed chromosome numbers corresponding to the expected diploid number (2n = 80), healthy backcrosses showed evidence of mitotic instability through an increased variance of chromosome numbers within an individual. In malformed backcrosses, extensive aneuploidy corresponding to multiples of the haploid number (1n = 40, 2n = 80, 3n = 120) was found, suggesting meiotic breakdown in their F1 parent. However, no detectable chromosome rearrangements between parental forms were identified. Genomic instability through aneuploidy thus appears to contribute to reproductive isolation between dwarf and normal lake whitefish, despite their very recent divergence (approx. 15-20 000 generations). Our data suggest that genetic incompatibilities may accumulate early during speciation and limit hybridization between nascent species.

Embryonic development of lake whitefish Coregonus clupeaformis: a staging series, analysis of growth and effects of fixation.

A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development.