Rapid evolution of body fluid regulation following independent invasions into freshwater habitats

Colonizations from marine to freshwater environments constitute among the most dramatic evolutionary transitions in the history of life. Colonizing dilute environments poses great challenges for acquiring essential ions against steep concentration gradients. This study explored the evolution of body fluid regulation following freshwater invasions by the copepod Eurytemora affinis. The goals of this study were to determine (1) whether invasions from saline to freshwater habitats were accompanied by evolutionary shifts in body fluid regulation (hemolymph osmolality) and (2) whether parallel shifts occurred during independent invasions. We measured hemolymph osmolality for ancestral saline and freshwater invading populations reared across a range of common-garden salinities (0.2-25 PSU). Our results revealed the evolution of increased hemolymph osmolality (by 16-31%) at lower salinities in freshwater populations of E. affinis relative to their saline ancestors. Moreover, we observed the same evolutionary shifts across two independent freshwater invasions. Such increases in hemolymph osmolality are consistent with evidence of increased ion uptake in freshwater populations at low salinity, found in a previous study, and are likely to entail increased energetic costs upon invading freshwater habitats. Our findings are consistent with the evolution of increased physiological regulation accompanying transitions into stressful environments.     

Zonal migration and trail‐following of an intertidal gastropod analyzed by led tracking in the field

A new technique is described for the continuous recording of the movements of intertidal molluscs in the field. Photographic monitoring of snails equipped with LEDs powered by small batteries revealed that the prosobranch gastropod Nerita textilis adopts a hybrid (zonal‐homing) pattern including a definite trail‐following behaviour. The method allowed the study of individual feeding migrations in terms of speed and orientation of the snails at different moments of the tidal cycle. The application of the LED technique to other molluscs and nocturnal animals is discussed. Nocturnal arena tests performed to complete the field observations confirmed the presence of interindividual and self‐trailing in N. textilis but not in the related sympatric species N. plicata. The behaviour of N. textilis is compared to that of other intertidal molluscs.     

盐碱胁迫对青海湖裸鲤耗氧率、血浆渗透浓度和离子浓度的影响

采用呼吸生理和血液生理的方法,探讨了性成熟青海湖裸鲤(Gymnocypris przewalskii)对盐度、碳酸盐碱度的适应性以及青海湖裸鲤在盐碱胁迫下的渗透调节和离子调节规律。将裸鲤从淡水突变到盐水(盐度14.01±0.01)、碱水(碳酸盐碱度33.03±0.01mmol·L-1)和青海湖水(盐度12.12±0.02、碳酸盐碱度29.07±0.09mmol·L-1)中,测定了在不同水质胁迫下裸鲤耗氧率、血浆渗透压和离子浓度的变化。结果表明:与淡水对照组比较,呼吸耗氧率除了盐度组在3h有显著性升高(P<0.05),其他实验组96h内均未出现显著性变化(P>0.05);裸鲤的窒息点为0.14～0.17mg·L-1,各实验组窒息点均没有显著性差异(P>0.05);碱度组血浆的渗透浓度和Na+、Cl-、Ca2+离子浓度在96h内未出现变化(P>0.05),盐度组和青海湖水组在所测时间段有显著的升高(P<0.05),并且随着胁迫时间的延长,在72h达到峰值。各实验组血浆K+浓度变化没有血浆Na+、Cl-明显,K+浓度有增高的趋势,且都在72h达到了峰值。青海湖裸鲤具有较强的盐碱耐受性,能通过渗透调节和离子调控适应高盐碱环境,而消耗能量较少。     

Cortisol stimulates hypo-osmoregulatory ability in Atlantic salmon, Salmo salar L.

Hypo-osmoregulatory ability in juvenile Atlantic salmon, Sulrno salur L., was improved by cortisol treatment. Implantation of a vegetable shortening pellet containing cortisol (50 mg kg⁻¹) resulted in elevated plasma cortisol titres. Maximum cortisol levels (160–170ngml⁻¹) were observed at days 6 and 12 after the implantation and dropped significantly by day 55. Cortisol-implanted fish in fresh water developed a twofold increase in gill Na⁺/K⁺-ATPase activity at days 6 and 12, and a threefold increase by day 55. Intestinal mucosa Na⁺/K⁺-ATPase activity was not affected by cortisol. Cortisol-implanted fish exposed to 28 ppt sea water for 48 h tended to show an improved ability to regulate their plasma osmolarity and reduce their ionic load. The osmo-regulatory ability attained at days 12 and 55 was further evaluated by exposing fish to 37 ppt sea water for 96 h. While all the control fish died relatively early in these tests, cortisol-implanted fish showed a clear reduction in their mortality rate. These results indicate that cortisol can induce biochemical and organismal changes during winter months that typify preadaptive events normally occurring in the spring.     

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 μmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.     

Effect of elevated temperature on osmotic and ionic regulation in a salt-tolerant caddisfly from Death Valley,California

The effects of temperature (8–10 or 20°C) on regulation of haemolymph osmotic and ionic concentrations were investigated over a range of salinities (0–25‰) in fifth-instar larvae of the Death Valley caddisfly Limnephilus assimilis. At low temperatures, levels of chloride and sodium in the haemolymph are regulated over a wide range of salinities corresponding to the salinities at which larvae occur in nature and at which they can complete development into adults. In contrast, haemolymph osmolality is constant at low salinities (<14‰) but approaches conformity with the medium at higher salinities. High temperature reduces the larva's ability to maintain low chloride concentrations in its haemolymph and also leads to a reduction in haemolymph osmotic pressure; thus, at high temperatures ions account for more of the haemolymph osmotic concentration than at low temperatures. These data suggest that the absence of larvae from thermal pools and from all Death Valley waters in summer can be explained by the effects of high water temperatures on hydromineral regulation.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

Lethal oxygen levels at different temperatures and the preferred temperature during hypoxia of the Atlantic cod, Gadus morhua L.

The behavioural thermoregulation of Atlantic cod Gadus morhua L. was investigated in a shuttlebox at normoxia and at three levels of hypoxia: 30, 20 and 15% oxygen saturation.
The preferred temperatures at normoxia, 30, 20 and 15% oxygen saturation were 13·9, 13·1. 10·0 and 8·8° C, respectively.
A decrease in metabolism and an increased blood oxygen affinity are among the physiological advantages of selecting a lower temperature during hypoxia. Furthermore the chances of surviving low oxygen saturations are better at low temperatures.
In natural environments, this behaviour may result in habitat shifts of fish living in heterothermal environments with changing oxygen saturations, especially in coastal areas with eutrophication, as for example the Baltic Sea.     

Exposure of goldsinny, rock cook and corkwing wrasse to low temperature and low salinity: survival, blood physiology and seasonal variation

Wrasse used as cleaner fish with farmed Atlantic salmon Salmo salar can be subjected to large and rapid temperature and salinity fluctuations in late autumn and early winter, when summer-warmed surface water is affected by early snowmelt episodes. Because of their containment in sea cages, wrasse which are essentially acclimated to summer temperatures may be rapidly exposed to winter conditions. Short-term tolerance of low temperature and low salinity by three species of wrasse, goldsinny Ctenolabrus rupestris rock cook Centrolabrus exoletus corkwing Crenilabrus melops caught during the summer, and winter-caught corkwing, was investigated. A 3–day period at 30 or 32‰ salinity and temperature 8, 6 or 4° C (for summer-caught fish; 4° C only for winter-caught) was followed by a decline in salinity to 24, 16 or 8‰ over c. 36 h, followed by a further 24 h at these salinities held constant, at each of the three temperatures. Controls in 30 or 32‰ were maintained at 8, 6 or 4° C. Mortality of summer-caught corkwing and rock cook was high at 4° C, whereas the influence of salinity on mortality was small. Mortality of goldsinny was low or zero in all treatments. Surviving corkwing and rock cook after 3 days at 4° C and 32‰ salinity had elevated plasma osmolality: in summer-caught corkwing, plasma [Cl°] and [Na⁺] were high, whereas in rock cook only [Na⁺] was high. Haematocrit was low in summer-caught corkwing, high in rock cook. In survivors of all three species at the end of the experiment, values of all these parameters were comparable with those of fish at the beginning of the experiment, except that survivors at low salinity (8, 16‰) had low plasma osmolality, at all temperatures, and in surviving rock cook in these treatments haematocrit was high and plasma [Cl^?] was low. Winter-caught corkwing had higher osmolality, [Na⁺] and [Cl^?] than summer-caught corkwing; there was no difference in haematocrit. Survival of wintercaught corkwing exposed to four salinities at 4° C was much higher than that of summercaught corkwing under the same conditions. Little change in blood physiology was recorded for winter-caught corkwing, with only fish subjected to 8‰ and 4° C showing signs of osmoregulatory stress. The interspecific and seasonal differences in survival and blood physiology at low temperature and low salinity are discussed in relation to wrasse survival over winter, both in the field and in salmon farms.     

Parallel adaptive evolution of Atlantic cod on both sides of the Atlantic Ocean in response to temperature

Despite the enormous economic and ecological importance of marine organisms, the spatial scales of adaptation and biocomplexity remain largely unknown. Yet, the preservation of local stocks that possess adaptive diversity is critical to the long-term maintenance of productive stable fisheries and ecosystems. Here, we document genomic evidence of range-wide adaptive differentiation in a broadcast spawning marine fish, Atlantic cod (Gadus morhua), using a genome survey of single nucleotide polymorphisms. Of 1641 gene-associated polymorphisms examined, 70 (4.2%) tested positive for signatures of selection using a Bayesian approach. We identify a subset of these loci (n = 40) for which allele frequencies show parallel temperature-associated clines (p < 0.001, r² = 0.89) in the eastern and western north Atlantic. Temperature associations were robust to the statistical removal of geographic distance or latitude effects, and contrasted ‘neutral’ loci, which displayed no temperature association. Allele frequencies at temperature-associated loci were significantly correlated, spanned three linkage groups and several were successfully annotated supporting the involvement of multiple independent genes. Our results are consistent with the evolution and/or selective sweep of multiple genes in response to ocean temperature, and support the possibility of a new conservation paradigm for non-model marine organisms based on genomic approaches to resolving functional and adaptive diversity.     

Hypoxia tolerance in Atlantic cod

Oxygen saturation levels that killed 50 and 5% of cod Gadus morhua over 96 h averaged 21·2 and 27·7%, respectively. No fish survived at 10% saturation and only a few survived at 16% saturation, whereas no mortality occurred at 34 and 40% oxygen saturation. Since metabolic rate and oxygen consumption increase with increasing temperature, we hypothesized that cod would be less tolerant to hypoxic conditions at 6 than at 2° C. However, temperature (2 and 6° C) had no measurable impact on cod survival. Small (mean & S.D.; 45·2 ± 4·2 cm) and large (57·5 ± 3·8 cm) cod had the same tolerance to hypoxia. At the end of the experiments, hypoxia had a significant effect on blood haematocrit, mean cellular haemoglobin content, liver lactate, plasma glucose and plasma lactate, but accounted for only a small fraction (< 10%) of the variation, except for plasma lactate which exhibited a strong response with concentrations increasing progressively with decreasing levels of oxygen saturation. Temperature had a significant effect on most variates in normoxia and hypoxia. Variates also affected by oxygen level showed significant interactions between oxygen and size or temperature effects. However, these interactions accounted for only a small proportion of the variation. Physiological parameters indicated that extending the duration of our tests beyond 96 h would not have changed our estimates of the lethal thresholds. Hypoxic conditions are a permanent feature of the deep waters of the Gulf of St Lawrence. This study shows that a significant portion of the benthic habitats in the Gulf are uninhabitable for cod which would be expected to avoid waters below 28% oxygen saturation.     

Drinking and water absorption by the larvae of herring (Clupea harengus) and turbot (Scophthalmus maximus)

The efficiencies of water absorption from the guts of the larvae of herring ( Clupea harengus L.) and turbot ( Scophthalmus maximus L.) were estimated by two methods, The first method was based on the differences in the rates of accumulation, by drinking, and clearance from the gut of radiolabelled inert markers. The second method used the equilibrium level of radioactivity in the larvae to measure the concentration of the markers in the gut above background as water is absorbed from the gut. Water absorption efficiencies for herring larvae drinking sea water were estimated to be 77% using both methods. When external salinity was reduced to 50% sea water, drinking rates and water absorption efficiency in herring larvae fell substantially.
Estimates of water absorption efficiency of seawater-adapted turbot larvae were similar (71–84%) to that of herring using both methods. Although temperature had a marked effect on both the rate of drinking and water absorption, there was no significant thermal effect on the efficiency of absorption from the guts of turbot larvae. The limitations of the techniques and the implications of the estimates in terms of water balance in fish larvae are discussed.     

Characterization of 18 new microsatellite loci in Atlantic cod (Gadus morhua L.)

Eighteen new microsatellite loci consisting of 10 di‐, 5 tri‐, 2 tetra‐ and 1 heptanucleotide repeats are introduced for the Atlantic cod (Gadus morhua L.). All loci were co‐amplified in two polymerase chain reactions (plus two previously published microsatellites) and all products were typed clearly. The number of alleles per locus ranged from six (PGmo130) to 45 (PGmo76) and the observed heterozygosity ranged from 0.356 (PGmo130) to 0.957 (PGmo95). All loci except one followed Hardy–Weinberg expectations. Genetic linkage disequilibrium analysis between all pairs of loci did not yield any significant values.     

Modelling regional differences in 'sealworm', Pseudoterranova decipiens (Nematoda, Ascaridoidea), infections in some North Atlantic cod, Gadus morhua, stocks

The ascaridoid nematode Pseudoterranova decipiens has a complex life-cycle. Adult worms reproduce in the stomach of grey seals, Halichoerus grypus , and other pinnipeds, and the larvae are found encysted in the musculature of Atlantic cod, a common host. An increase in infection levels in Canadian, Icelandic and Norwegian cod stocks over the last decade has been attributed to recent increases in seal population numbers. A simple model is proposed for the accumulation of larval parasites in the fish through its food. The model describes how regional differences in fish growth can also explain some differences in infection levels.