Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25 °C, E. huxleyi produces mainly 14:0, 18:4n − 3, 18:5n − 3 and 22:6n − 3. When the cells were transferred from 25 °C to 15 °C, the amount of unsaturated fatty acids, i.e. 18:1n − 9, 18:3n − 3 and 18:5n − 3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ?15 fatty acid desaturase, EhDES15, involved in the production of n − 3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ?6 desaturase-like domain, but it does not contain a cytochrome b₅ domain nor typical His-boxes found in ?15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n − 3 fatty acid species, which are produced at low levels in wild-type cells grown at 30 °C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ?15 desaturases. These results suggested the gene encodes a novel ?15 desaturase responsible for the synthesis of 18:3n − 3 from 18:2n − 6 in E. huxleyi.     

Influence of static and fluctuating salinity on cadmium uptake and metallothionein expression by the dogwhelk Nucella lapillus (L.)

The aim of this study was to investigate the effect of salinity on cadmium (Cd) accumulation and metallothionein (MT) expression in the dogwhelk Nucella lapillus (L.). Adult dogwhelks (shell length: 23.4±1.3 mm) were acclimated to salinity of 33 psu (control), 22 or 11 psu under controlled laboratory conditions (9.5 °C; pH 7.9) for 10 days in a stepwise manner by reducing the salinity by 5.5 psu day⁻¹. Ten treatment groups were used and comprised five salinity regimes (three fixed salinity [33, 22 or 11 psu] and two fluctuating salinity [varied daily between 33 and 22 psu or 33 and 11 psu in a cyclic manner]) at each of two Cd concentrations (control: <0.001 μg Cd l⁻¹ or treatment: 500 μg Cd l⁻¹). After acclimation, groups of 20 dogwhelks were exposed to each of the 10 Cd/salinity combinations. All the control and Cd-exposed dogwhelks exposed to 11 psu were dead within 5 days of exposure due to hypo-osmotic stress. Twenty days after exposure to all other treatments, concentrations of Cd and MTs in the tissues of surviving dogwhelks were quantified using atomic absorption spectrophotometry and the silver saturation method, respectively. Both Cd accumulation and MT induction in control or Cd-exposed N. lapillus were significantly influenced by changes in salinity, especially at a prolonged and fixed low salinity (22 psu), although such influences of salinity on the concentration of MTs were dependent on the tissue type. The study highlights that salinity should be considered when monitoring trace metals and/or MTs in intertidal molluscs, particularly in estuarine or transplanted biomonitors.     

The environmental effects of salinity and temperature on the oxygen consumption and total body osmolality of the marine flatworm Procerodes littoralis

The present study examined the effect of salinity and temperature on the rate of oxygen consumption and total body osmolality of the triclad turbellarian Procerodes littoralis, a common marine flatworm normally found in areas where freshwater streams run out over intertidal areas. Extremes in environmental factors encountered by P. littoralis were recorded at the study site. These were salinity (0-44 psu), temperature (2.7-24.9 °C) and oxygen concentration (2.8-16.1 mg l⁻¹). Respirometry experiments showed minimal oxygen consumption rates at the salinity extremes encountered by the study species (0 and 40 psu). Further experiments showed relatively constant oxygen consumption rates over the temperature range 5-20 °C and elevated consumption rates at temperatures above 25 °C. Total body osmolality of P. littoralis increased with increasing salinity. The study illustrates how a marine flatworm uses integrated physiological and behavioural mechanisms to successfully inhabit an environment that is predominantly freshwater for up to 75% of the tidal cycle.     

Metabolic response of juvenile gray snapper (Lutjanus griseus) to temperature and salinity: Physiological cost of different environments

Juvenile gray snapper (Lutjanus griseus) occupy a wide range of estuarine and nearshore habitats that differ in physico-chemical properties. To quantify the energetic cost of inhabiting these different habitats, routine metabolism of individual gray snapper was measured in the laboratory at 20 combinations of temperature (18, 23, 28, and 33 °C) and salinity (5, 15, 25, 35, and 45 psu). An open, flow-through respirometer was used, enabling trials to be run for long periods (∼16 h), while maintaining water quality (dissolved O₂>70% saturation), and providing fish sufficient time to habituate to the chambers undisturbed. Video recordings of fish in the respirometer chambers were analyzed to quantify the spontaneous activity rate of individuals. Analysis of covariance, using fish weight and mean activity rate as covariates, indicated significant temperature and salinity effects on oxygen consumption. Oxygen consumption was significantly higher at high salinities, and the salinity effect was temperature dependent. A polynomial equation describing oxygen consumption as a function of temperature and salinity indicated the increase due to salinity from 5 to 45 psu at high temperatures (30-33 °C) was equivalent to a 3 °C increase in temperature. At intermediate temperatures (24-26 °C), the increase due to salinity from 5 to 45 psu was less dramatic, equivalent to a 2 °C increase in temperature. At the lowest temperatures (18 °C), salinity did not have a significant effect on oxygen consumption. The increased metabolic costs in high salinities (∼7% at the high temperature) represent a significant energy cost for juveniles, that would need to be balanced by lower predation risk or greater food availability to result in similar juvenile production compared to lower salinity environments.     

Interacting effects of temperature and salinity on Bonamia sp. parasitism in the Asian oyster Crassostrea ariakensis

The proposition to introduce the Asian oyster Crassostrea ariakensis to the mid-Atlantic region of the USA is being considered with caution, particularly after the discovery of a novel microcell haplosporidian parasite, Bonamia sp., in North Carolina. Although this parasite was found to be pathogenic in C. ariakensis under warm euhaline conditions, its persistence in C. ariakensis exposed to various temperature and salinity combinations remained unresolved. In this laboratory experiment, we tested the influence of temperature in combination with a wide range of salinities (10, 20 and 30 psu) on Bonamia sp. Temperature was either changed from warm (>20 °C) to cold (6 °C for 6 weeks) and back to warm or maintained constant and warm. Warm temperature was associated with higher host mortality than cold temperature, suggesting that temperature influenced Bonamia sp. pathogenicity. The effect of salinity was revealed under warm temperature with highest mortality levels observed in infected C. ariakensis exposed to 30 psu. When temperature was increased following low-temperature exposure, Bonamia sp. was not detected; however sub-optimal experimental conditions may have contributed to this result, making it difficult to draw conclusions regarding the reemergence of the parasite after low-temperature exposure. Although the overwintering of Bonamia sp. in C. ariakensis will need to be further investigated, the results presented here suggest that Bonamia sp. may be able to persist in C. ariakensis under a combination of low temperature and meso- to euhaline salinities.     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

Development and mortality of the first naupliar stages of Eurytemora affinis (Copepoda, Calanoida) under different conditions of salinity and temperature

As a prevalent species complex in temperate estuaries and salt marshes of the Northern Hemisphere, populations of Eurytemora affinis that inhabit these environments must be adapted to salinity fluctuations. Some populations have invaded freshwater environments. In this work, we focus on the combined effects of temperature and salinity fluctuations on mortality rates and development time of the first naupliar stages under starvation. Two temperatures (10 and 15 °C) and eight salinities, ranging from 0 to 35 psu are investigated. We show (i) that among all experimental conditions the optimal temperature and salinity for naupliar survival and development are 15 psu and 15 °C, and (ii) that only the most extreme salinities (i.e. 0 and 35 psu) have a negative effect on naupliar survival. Nauplii develop faster and reach a higher developmental stage at 15 than at 10 °C, independent of salinity. The relevance of this metabolic adaptive pattern is discussed in the general framework of in situ behavior, tidal forcing and biogeographic variability, as well as the potential sources of the observed individual variability.     

Environmental influences on the formation and germination of hibernacula in the brackish-water bryozoan Victorella pavida Saville Kent, 1870 (Ctenostomata: Victorellidae)

Brackish-water and fresh-water bryozoans produce asexually derived dormant propagules that allow survival of unfavourable conditions and provide a potential means of dispersal. The propagules of brackish-water ctenostome bryozoans are called hibernacula. We monitored the life-cycle of the brackish-water ctenostome Victorella pavida Saville Kent, 1870 in its natural habitat and investigated, in laboratory cultures, the influence of temperature and salinity on the production and germination of hibernacula and on subsequent colony growth. V. pavida is a protected species in the UK, where its only locality is at Swanpool lagoon, Falmouth. Colonies were collected from Swanpool monthly from January 2004 to January 2005. Hibernaculum germination appeared to be triggered by increased water temperature (c. 13 °C) in the lagoon in March and April. In culture, germination was triggered by transfer from 5 °C to 19 °C in a range of salinities; subsequent colony growth was affected by salinity, with strongest growth at 13, 18 and 36 psu, and reduced growth at 5 and 9 psu. At 3.5 psu, hibernacula germinated, but there was no further development. At 36 psu there was an initial lag in growth, but after 30 d the colonies were comparable with those kept at 18 psu. Hibernaculum formation by colonies occurred from June to October, with production increasing towards October. Hibernacula appear not to have long-term viability but merely to permit survival from one year to the next. The results suggest that any changes in the hydrographic regime at Swanpool could have significant consequences for the survival of V. pavida.     

Combined effects of temperature and salinity on larval development and attachment of the subtidal barnacle Balanus trigonus Darwin

The combined effects of temperature and salinity on larval development and attachment of Balanus trigonus Darwin (Cirripedia, Balanidae) was examined under controlled laboratory conditions. Whilst larval survivorship was not affected (>70%), the duration of larval development was significantly affected by temperature and salinity. The effect of temperature was comparatively stronger than that of salinity. The majority of nauplius II larvae metamorphosed into cypris stage after 4-5 and 10-11 days at 28 °C (22-34‰) and 18 °C (22-34‰), respectively. Temperature, salinity and the duration of assay had a significant effect on cypris attachment with significant interaction among these main effects. Maximum (>80% in 6 days) and minimum percent attachment (0% in 6 days) on polystyrene surfaces were observed at 24 °C (34‰) and 18 °C (22‰), respectively. At high temperature (28 °C) and low salinity (22-26‰), larvae rapidly (4 days) developed into cyprids, but less than 33% attached. These results suggest that low larval attachment rates may lead to the low recruitment of B. trigonus in Hong Kong waters during summer when the water temperature is high (about 28 °C) and salinity is low (<26‰).     

Effects of salinity, temperature and pH on the survival of the nemertean Procephalothrix simulus Iwata, 1952

The salinity, temperature and pH tolerance of Procephalothrix simulus Iwata, 1952, were experimentally studied. In hypo-media, the nemerteans could survive 96 h in 3.3‰ solution at 10 °C (median lethal salinity [LS₅₀] was not determined at this temperature), and 96 h LS₅₀ were 7.3‰ and 13.5‰ at 20 °C and 30 °C, respectively. In hyper-media, 96 h LS₅₀ values were 53.9‰, 47.1‰ and 41.4‰ at 10 °C, 20 °C and 30 °C, respectively. The trend of body weight changes in diluted media indicated that this nemertean is a volume regulator. During a 96-h exposure in media at 0 °C, worms were thanatoid but could recover if the temperature was gradually elevated to 20 °C. In thermal tolerance experiments, the nemertean survived 96 h in seawater of 30 °C, and worms suffered high mortalities when the temperature exceeded 32 °C. Present results suggest that the interaction of temperature and salinity on the lethal effects on P. simulus is significant (P < 0.05). Elevated temperature (range 10-30 °C) decreased the worm's solute tolerance, and elevated salinity (range 18-38‰) decreased the worm's thermal tolerance. The survival pH level for this nemertean ranged from 5.00 to 9.20.     

Influence of abiotic factors on bacterial proliferation and anoxic survival of the sea mussel Mytilus edulis L.

The effect of several abiotic factors (salinity, temperature and pH) on bacterial proliferation and survival time of the sea mussel Mytilus edulis L. were studied under anoxic incubations. In addition, the presence in the incubation media of ammonium and the volatile fatty acids propionate and acetate, both excreted fermentation products of the bivalve, was tested.Anoxic incubations with seawater diluted with demineralised water showed at the lowest salinity (50% seawater, SW) a significant increase in the capacity of M. edulis to survive anoxia as compared to both 75% SW and control [100% SW, corresponding to 32 practical salinity units (psu)]. Formation of biotic sulphide and ammonium occurred in all incubations. However, bacterial proliferation was postponed by 2-3 days at lowest salinity and accordingly, concentrations of both compounds were lower. Anoxic survival profiles of mussels collected from different habitats in the Dutch Scheldt area, characterised by differences in salinity (range from 17 to 31 psu), corresponded with the above salinity effect. Walsoorden mussels (17 psu) showed the longest (P<0.001) survival time under anoxia (LT₅₀=17.2 days) as compared with Paulina (27 psu) and Wemeldinge (31 psu) mussels (LT₅₀=12.8 and 9.8 days, respectively). Condition index (ratio of soft body weight to shell volume) was not correlated with anoxic survival time in untreated mussels, although this was clearly the case when the antibiotic chloramphenicol was added to the anoxic seawater.Acidification of the anoxic incubation medium had a positive effect on survival time. LT₅₀ values significantly (P<0.001) increased from 10.2 days at pH 8.1 to 11.6 and 11.5 days at pH 7.3 and 6.5, respectively. Biotic sulphide and ammonium accumulation as well as bacterial numbers were significantly lower at pH 7.3 and 6.5 as compared with pH 8.1. Anoxic incubations at 10 °C (LT₅₀=12.0 days) strongly increased survival time as compared to 18 °C (LT₅₀=5.9 days). The benefit of antibiotic addition was also stronger at lower temperature (10 °C).Addition of both propionate and acetate (0.5 mM) displayed no effect on mortality of mussels under anoxia, but ammonium (0.5 mM) caused a negative effect (P<0.001). Biotic sulphide and ammonium concentrations measured in both volatile fatty acid incubations were lower than the control situation, as well as total bacterial numbers.This study shows that environmental factors play a significant role in determining the course of bacterial infection and death of bivalves exposed to anoxia.     

Effects of temperature, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea

The effects of temperature, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea were examined in the laboratory. The irradiance at the light compensation point (I₀) was 14.40 μmol m^− 2 s^− 1 and the irradiance at growth saturation (I_s) was 114 μmol m^− 2 s^− 1. We exposed A. sanguinea to 48 combinations of temperature (5-30 °C) and salinity (5-40) under saturating irradiance; it exhibited its maximum growth rate of 1.13 divisions/day at a combination of 25 °C and salinity of 20. A. sanguinea was able to grow at temperatures from 10 to 30 °C and salinities from 10 to 40. This study revealed that A. sanguinea was a eurythermal and euryhaline organism; in Japan it should have formed blooms in early summer, when salinity was relatively low. In addition, it was noteworthy that A. sanguinea had markedly cold-durability, retaining the motile form of vegetative cells for more than 50 days at 5 °C and at salinities of 25-30.     

Embryonic encapsulation and maternal incubation: Requirements for survival of the early stages of the estuarine gastropod Crepipatella dilatata

During their reproductive period, females of Crepipatella dilatata deposit their embryos in capsules that they then brood in the pallial cavity until juveniles emerge several weeks later, after passing through a transient veliger “larval” stage. Artificially excapsulated veligers of this species experimentally exposed to a wide range of salinities (5, 10, 15, 20, 25, and 30 psu) for six hours showed reduced activity at salinities of 15 and 20 psu, whereas encapsulated veligers exposed to those same salinities showed no reduction of activity. Artificially excapsulated veligers showed high mortality at salinities of 5 and 10 psu; encapsulated embryonic stages also showed high mortalities at 5 psu and serious sublethal effects at 10 psu in tests excluding maternal protection, showing that encapsulation alone does not provide complete protection from low salinity stress. Natural tidal cycles in the Quempillén River estuary also reduced embryonic survival at salinities of ≤ 10 psu when the capsules were exposed without maternal protection. In contrast, encapsulated embryos protected by their mothers survived well regardless of the salinity to which they were exposed, under both natural and laboratory-simulated estuarine tidal cycles. C. dilatata are able to develop in the estuary only because of maternal protection, since salinity levels in this environment sometimes decline to as low as 7 psu. Successful embryonic development in this estuary reflects the capacity of C. dilatata adults to detect dangerously low salinity levels and then seal themselves off from the environment for up to 50 hrs (O. Chaparro pers. obs.) when the salinity drops below 22.5 psu, allowing salinity to remain above this level within the pallial cavity despite continued salinity declines in the surrounding seawater.     

Combined effect of temperature and salinity on the Thermotolerance and osmotic pressure of juvenile white shrimp litopenaeus vannamei (Boone)

Thermotolerance (CTMax) was determined in L. vannamei in three salinities and five acclimation temperatures 20, 23, 26, 29 and 32 °C. In white shrimp, the CTMax was not significantly affected by salinity (P>0.05). A direct relationship was obtained between CTMax and acclimation temperature. The end point of the CTMax in L. vannamei exposed to different combinations of temperature and salinity was defined as the loss of the righting response (LRR). The acclimation response ratio (ARR) for the juveniles of white shrimp ranged from 0.42 to 0.49; values in agreement with other crustaceans from tropical and sub tropical climates. The osmotic pressure of the hemolymph was measured in control organisms and in organisms exposed to CTMax; significant differences were found in organisms maintained in 10 and 40 psu, but there were no significant differences in hemolymph osmotic pressure in those that were acclimated to 26 psu.     

The Effect of Temperature on the Lipid Composition of the Green Alga Botryococcus

The lipid composition of the green alga Botryococcus was studied at three different cultivation temperatures: suboptimal (18°C), optimal (25°C), and supraoptimal (32°C). Cultivation at the supraoptimal temperature was found to considerably inhibit the synthesis of nearly all intracellular lipids, except for triacylglycerides, and to influence their fatty acid composition. In particular, the content of trienoic fatty acids was significantly lower at the supraoptimal than at the optimal cultivation temperature. At the same time, the fatty acid composition of the extracellular lipids of the alga virtually did not depend on cultivation temperature.     

Effects of temperature, salinity, and air exposure on development of the estuarine pulmonate gastropod Amphibola crenata

The primitive pulmonate snail Amphibola crenata embeds embryos within a smooth mud collar on exposed estuarine mudflats in New Zealand. Development through hatching of free-swimming veliger larvae was monitored at 15 salinity and temperature combinations covering the range of 2-30 ppt salinity and 15-25 °C. The effect of exposure to air on developmental rate was also assessed. There were approximately 18,000 embryos in each egg collar. The total number of veligers released from standard-sized egg collar fragments varied with both temperature and salinity: embryonic survival was generally higher at 15 and 20 °C than at 25 °C; moreover, survival was generally highest at intermediate salinities, and greatly reduced at 2 ppt salinity regardless of temperature. Even at 2 ppt salinity, however, about one-third of embryos were able to develop successfully to hatching. Embryonic tolerance to low salinity was apparently a property of the embryos themselves, or of the surrounding egg capsules; there was no indication that the egg collars protected embryos from exposure to environmental stress. Mean hatching times ranged between 7 and 22 days, with reduced developmental rates both at lower temperature and lower salinity. At each salinity tested, developmental rate to hatching was similar at 20 and 25 °C. At 15 °C, time to hatching was approximately double that recorded at the two higher exposure temperatures. Exposing the egg collars to air for 6-9 h each day at 20 °C (20 ppt salinity) accelerated hatching by about 24 h, suggesting that developmental rate in this species is limited by the rates at which oxygen or wastes can diffuse into and from intact collars, respectively. Similarly, veligers from egg capsules that were artificially separated from egg collars at 20 °C developed faster than those within intact egg collars. The remarkable ability of embryos of A. crenata to hatch over such a wide range of temperatures and salinities, and to tolerate a considerable degree of exposure to air, explains the successful colonization of this species far up into New Zealand estuaries.     

Does scope for growth change as a result of salinity stress in the amphipod Gammarus oceanicus?

Physiological performance (feeding, metabolism, growth and excretion) across a broad range of salinity (5-30 psu) were determined for the benthic amphipod Gammarus oceanicus, a species of marine origin inhabiting brackish waters of the southern Baltic Sea. Feeding rates decreased with increasing salinity, whereas the nutritive absorption efficiency increased. Faeces production and ammonia excretion rates decreased strongly from the lowest to the highest salinity by 60% and 58%, respectively. Increasing salinity was accompanied by a reduction in the metabolic rate from 438 J g^− 1 dry wt d^− 1 (5.1 mW g^− 1) at 5 psu to 245 J g^− 1 (2.8 mW g^− 1) at 30 psu. Individuals were able to maintain a positive energy balance at all experimental salinities. The greatest values for scope for growth were recorded at the environmental salinity (7 psu) with a mean of 769 J g^− 1 dry wt d^− 1 (8.7 mW g^− 1).     

Tolerance of Sargassum thunbergii germlings to thermal, osmotic and desiccation stress

The construction of artificial seaweed beds in the intertidal zone is a challenge due to extreme levels of physical stress. In order to provide a basis for the construction using the dispersal of microscopic juveniles, a three-way factorial experimental design was used to evaluate the tolerance of Sargassum thunbergii germlings shortly released from fertile thalli to temperature, salinity and desiccation in this study. Results revealed that temperature, salinity and desiccation significantly affected the growth and survival of germlings. Germlings showed rapid growth with relative growth rate (RGR, % day⁻¹) over 16% when cultured at 25 °C and full immersion in normal seawater. Although growths of germlings subjected to moderate conditions were significantly inhibited, RGRs over 13% were obtained. The RGRs of germlings below 10% were observed only at 35 °C and 9 h desiccation treatments. In comparison to growth, survival was less affected by physical stress. Germlings showed low mortalities below 10% under appropriate conditions (25 °C and 30 °C combined with full immersion), and below 60% under moderate conditions, by the end of experiment. However, the mortality rates increased to over 90% under extreme conditions (9 h desiccation and 35 °C combined with full immersion in salinity of 12). These results showed that S. thunbergii germlings had high tolerance to physical stresses. In addition to the main effects, both two-way and three-way interactions between temperature, salinity and desiccation were significant. Based on the magnitude of effect, desiccation was the predominant factor affecting both growth and survival. According to the results, construction of artificial tanks in natural habitat to minimize desiccation may be an effective strategy for S. thunbergii restoration using germlings.     

Combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam Ruditapes philippinarum

The combined effects of temperature and salinity on both immune responses and survival in air of the clam, Ruditapes philippinarum, were evaluated for the first time. The animals were kept for 7 days at three differing temperature (5 °C, 15 °C, 30 °C) and salinity values (18 psu, 28 psu, 38 psu), and effects of the resulting 9 experimental conditions on total haemocyte count (THC), Neutral Red uptake (NRU), haemolymph protein concentration, and lysozyme activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. The survival-in-air test was also performed. Two-way ANOVA analysis revealed that temperature influenced significantly THC and NRU, whereas salinity and temperature/salinity interaction affected NRU only. Temperature and salinity did not influence significantly HL and CFH lysozyme activity, as well as haemolymph total protein content. Survival-in-air test is widely used to evaluate general stress conditions in clams. In the present study, temperature and salinity were shown to influence the resistance to air exposure of R. philippinarum. The highest LT₅₀ (air exposure time resulting in 50% mortality) value was recorded in clams kept at 18 psu and 15 °C, whereas the lowest value was observed in clams kept at 28 psu and 30 °C. Overall, results obtained demonstrated that temperature and salinity can affect some functional responses of haemocytes from R. philippinarum, and suggested a better physiological condition for animals kept at 15 °C temperature and 18 psu salinity.     

The effects of environmental factors on the embryonic survival of the Patagonian squid Loligo gahi

Seawater temperature and salinity are environmental variables that impose physiological limits for the embryonic development of marine invertebrates. For cephalopod species, these limits have rarely been established. This work presents experimental results on the embryonic survival of the Patagonian squid Loligo gahi, which is the last decades' most important loliginid species in terms of volume of commercial catches worldwide, as a function of seawater temperature and salinity. Reference magnitudes of surface seawater temperature and salinity within the area of distribution of the species were explored by analysis of satellite databases and published information. Embryos were incubated under eight constant regimes of temperature within 4-22 °C and four constant salinity regimes within 20-34.33‰ (12 °C). Also, to determine the effects of sudden temperature changes on embryonic survival, embryos were incubated at four variable regimes of temperature, with thermal shifts (6-day long 2-°C magnitude alterations of the incubation temperature) applied both at early and late stages of embryonic development. Embryonic survival was zero in incubations at constant temperature regimes ≤5° and at 22 °C, low at 6 °C, and high within 8-20 °C. A function was fitted by nonlinear regression to relate embryonic survival and mean incubation temperature. Thermal shifts applied in incubations at 20-22-20 °C variable regime of temperature provoked low embryonic survival compared to that observed in incubation at 20 °C constant regime. Embryonic survival was zero in incubations conducted at 20.0‰ and 34.3‰ salinity, and high at 26.4‰ and 32.8‰ salinities.