OXPHOS capacity is diminished and the phosphorylation system inhibited during diapause in an extremophile, embryos of Artemia franciscana.

Diapause exhibited by embryos of Artemia franciscana is accompanied by severe arrest of respiration. A large fraction of this depression is attributable to downregulation of trehalose catabolism that ultimately restricts fuel to mitochondria. This study now extends knowledge on the mechanism by revealing metabolic depression is heightened by inhibitions within mitochondria. Compared with that in embryo lysates during post-diapause, oxidative phosphorylation (OXPHOS) capacity P is depressed during diapause when either NADH-linked substrates (pyruvate and malate) for electron transfer (electron transfer capacity, E) through respiratory Complex I or the Complex II substrate succinate are used. When pyruvate, malate and succinate were combined, respiratory inhibition by the phosphorylation system in diapause lysates was discovered as judged by P/E flux control ratios (two-way ANOVA; F1,24=38.78; P<0.0001). Inhibition was eliminated as the diapause extract was diluted (significant interaction term; F2,24=9.866; P=0.0007), consistent with the presence of a diffusible inhibitor. One candidate is long-chain acyl-CoA esters known to inhibit the adenine nucleotide translocator. Addition of oleoyl-CoA to post-diapause lysates markedly decreased the P/E ratio to 0.40±0.07 (mean±s.d.; P=0.002) compared with 0.79±0.11 without oleoyl-CoA. Oleoyl-CoA inhibits the phosphorylation system and may be responsible for the depressed P/E in lysates from diapause embryos. With isolated mitochondria, depression of P/E by oleoyl-CoA was fully reversed by addition of l-carnitine (control versus recovery with l-carnitine, P=0.338), which facilitates oleoyl-CoA transport into the matrix and elimination by ß-oxidation. In conclusion, severe metabolic arrest during diapause promoted by restricting glycolytic carbon to mitochondria is reinforced by depression of OXPHOS capacity and the phosphorylation system.

Vertically migrating swimmers generate aggregation-scale eddies in a stratified column.

Biologically generated turbulence has been proposed as an important contributor to nutrient transport and ocean mixing1-3. However, to produce non-negligible transport and mixing, such turbulence must produce eddies at scales comparable to the length scales of stratification in the ocean. It has previously been argued that biologically generated turbulence is limited to the scale of the individual animals involved 4 , which would make turbulence created by highly abundant centimetre-scale zooplankton such as krill irrelevant to ocean mixing. Their small size notwithstanding, zooplankton form dense aggregations tens of metres in vertical extent as they undergo diurnal vertical migration over hundreds of metres3,5,6. This behaviour potentially introduces additional length scales-such as the scale of the aggregation-that are of relevance to animal interactions with the surrounding water column. Here we show that the collective vertical migration of centimetre-scale swimmers-as represented by the brine shrimp Artemia salina-generates aggregation-scale eddies that mix a stable density stratification, resulting in an effective turbulent diffusivity up to three orders of magnitude larger than the molecular diffusivity of salt. These observed large-scale mixing eddies are the result of flow in the wakes of the individual organisms coalescing to form a large-scale downward jet during upward swimming, even in the presence of a strong density stratification relative to typical values observed in the ocean. The results illustrate the potential for marine zooplankton to considerably alter the physical and biogeochemical structure of the water column, with potentially widespread effects owing to their high abundance in climatically important regions of the ocean 7 .

Prepared microplastics interaction with Artemia salina under low pH conditions representing ocean acidification; a simulated environmental exposure.

Ocean acidification and microplastic pollution are two of the major ecological concerns. The distribution of large quantities of plastic debris and microplastics all across the oceans emphasises the need to determine the influence of microplastics in ocean acidification and to evaluate its concomitant toxicological effects on aquatic life forms. Studies on the combined impact of both the stressors are very limited, but much needed in the current scenario. Where most of the present-day research use purchased microplastics of defined size and morphology (microspheres, fibres, rods, etc.), the present study employs prepared "true to life microplastics" that resemble the environmental microplastic pollutants in morphology and size heterogeneity. The present study focusses on evaluating the fate and impact of oceanic microplastics on the physiology and development of Artemia salina (Brine shrimp), one among the most ecologically significant zooplankton species. Natural sea water was acidified by controlled perturbation of carbon dioxide using a valve system. The hatching rate of A. salina cysts receded significantly (p < 0.05) upon singular exposures to microplastics and low pH (7.80), whereas combined effect was insignificant. The reactive oxygen species (ROS) elevated as a result of individual exposures to microplastics and low pH. However, only in 0.5 mg mL-1 PE treatments at pH 7.80, an additive impact was reported for ROS activity (p < 0.05). The SOD activities increased significantly but it can be attributed as the individual responses towards exposure to both the stressors. A significant additive impact was not observed for SOD activity (p > 0.05). But during the development, significant morphological anomalies were observed. Changes in the appendages of nauplii and juveniles as a result of combined exposure to microplastics and low pH treatments are significant findings. Our observations suggest that coupled exposure to microplastics and low pH could induce significant oxidative stress in the marine zooplanktons and also adversely affect their normal development. Findings from the current study emphasise the need for further research to understand the coupled toxicological impacts of ocean acidification and predominant pollutants such as microplastics to other marine animals as well.

Lethal, behavioral, growth and developmental toxicities of alkyl-PAHs and non-alkyl PAHs to early-life stage of brine shrimp, Artemia parthenogenetica.

Alkyl-PAHs are the predominant form of PAHs in crude oils which are supposed to demonstrate different toxicities compared to non-alkyl PAHs. Little information is available about the toxicity of alkyl-PAHs on marine Artemia. This study addressed and compared the lethal, behavioral, growth and developmental toxicities of three alkyl-PAHs, namely 3-methyl phenanthrene (3-mPhe), retene (Ret) and 2-methyl anthracene (2-mAnt), to their non-alkyl forms, phenanthrene (Phe) and anthracene (Ant) using Artemia parthenogenetica (nauplii, <24 h) as test organism following a 48 h and a 7 d of exposure, respectively. Benzo-a-pyrene (Bap) was selected as a reference toxicant for the comparison with the above alkyl-PAHs and non-alkyl PAHs. Results showed that for all tested endpoints, A. parthenogenetica nauplii had the highest sensitivity to Bap while Ant had no significant effect on nauplii survival or development within given concentrations. Considering the aqueous freely dissolved PAH concentrations, the 48 h-LC50 (survival), 48 h-EC50 (immobility) and 7 d-LC10 (survival) of Bap were calculated as 0.321, 0.285 and 0.027 µg/L, respectively, which were twofold to fivefold lower than those of Phe, 3-mPhe, Ret, Ant and 2-mAnt. A higher acute toxicity of alkyl-PAHs (3-mPhe and 2-mAnt) than their non-alkyl forms (Phe and Ant) was observed. Not limited to Phe, the common non-polar narcotic mode of action was also observed for Bap, 3-mPhe, Ret and 2-mAnt, which was evident by the inhibited mobility of nauplii. The decreased body lengths were found for all PAH treatments compared to the solvent control, whereas instar retardations were only found in nauplii exposed to Bap, Phe and Ret. Our findings emphasized the sensitivity differences of A. parthenogenetica nauplii to selected alkyl PAHs and non-alkyl PAHs and confirmed the application of lethal, behavioral and growth indicators in the toxicity evaluation of selected PAHs other than Ant. However, the distinct toxicities of these PAHs suggested other toxic modes of action may play more important roles apart from narcotic mode of action and need to be elucidated in future studies. In addition, a strong correlation between the body length and the instar of A. parthenogenetica nauplii was observed for each PAH exposure, suggesting that body length can be representative for both growth and developmental indicators during biological monitoring of PAH pollution in marine environment.

Short exposure of Artemia salina to group-12 metals: Comparing hatchability, mortality, lipid peroxidation, and swimming speed.

The hatchability, mortality rate, lipid peroxide levels, and swimming speed of Artemia salina have been compared based on short exposures of ZnCl2, CdCl2, and HgCl2 in artificial seawater. The hatching tests were carried out for 12, 24, 36, and 48 h at 28 °C. Mortality rate and lipid peroxide (LPO) levels were determined after 24 h of exposure at 28 °C, in the dark, and on living larvae using the FOX method. The swimming speed was determined after 24 h using a microcomputer coupled to a digital camera, with simultaneous treatment of the recorded images every 25 s, at 25 °C, under red-light irradiation. Results showed that Zn caused a gradual inhibition of the hatching for concentrations <900 µmol L-1; however, Cd and Hg displayed almost complete inhibition for concentrations ≤100 µmol L-1. Also, the heavy metals caused a dose-dependent increase of mortality (LD50) in the following order: Zn = 3290 µmol L-1 < Cd = 2206 µmol L-1 < Hg = 15.6 µmol L-1. Furthermore, significant LPO levels were found for Cd (1500-2000 µmol L-1, p < 0.001) and Hg (5-20 µmol L-1, p < 0.001). Finally, the swimming speed values increased significantly, for Zn ≈ 2.5 mm s-1 (1500 µmol L-1, p < 0.001), Cd ≈ 3.5 mm s-1 (2000 µmol L-1, p < 0.05), and Hg ≈ 4.0 mm s-1 (15 µmol L-1, p < 0.05), after 24 h exposure. There is a clear dose-dependent toxicity, indicating that Zn, Cd and Hg can induce significant changes in hatchability, mortality, and ethological and biochemical parameters.

Effects of commercial formulations of glyphosate on marine crustaceans and implications for risk assessment under temperature changes.

Glyphosate-based formulations are the most commonly used herbicides worldwide with the risk of potential contamination of aquatic bodies. The present study assessed the response of four marine crustaceans to three different brands of herbicides Roundup®Platinum, Efesto® and Taifun® MK CL.T, under two selected temperatures of 20 °C and 30 °C. The harpacticoid copepod Tigriopus fulvus, the anostracan Artemia franciscana, the amphipod Corophium insidiosum and the isopod Sphaeroma serratum were chosen as testing organisms. Effects of herbicides and temperatures were assessed by estimating lethal concentrations. The results showed that the high temperature rises the toxicity of glyphosate with an increase of mortality of all the tested species. This is an important aspect for future risk assessments of pesticides under global climate change scenarios. Efesto® resulted the most toxic brand, showing C. insidiosum the most sensitive with 96 h-LC50 values of 3.25 mg/L acid equivalent (a.e.) at 30 °C and 7.94 mg/L a.e. at 20 °C followed by T. fulvus while A. franciscana and S. serratum were the less sensitive. This study provides important information for assessing the toxic effects of three different brands of glyphosate-based herbicides on non-target marine organisms suggesting that they should be carefully managed to minimize any negative impact on marine organisms.

Ingestion and bioaccumulation of polystyrene nanoplastics and their effects on the microalgal feeding of Artemia franciscana.

Nanoplastics (NPs) have become one of the most serious environmental problems nowadays. The environmental issues linked to NPs are attributed to the effects after ingestion in marine organisms. Due to the incipient and controversial information about the effects of PS NPs on the feeding of organisms, the aim of this work is to assess (i) digestion dynamics of Artemia franciscana when exposed to PS NPs as the lowest concentration of PS NPs reported in toxicity test [0 (control), 0.006 and 0.6 mg·L-1] and possible interferences in the ingestion of microalgae and (ii) the accumulation and depuration of PS NPs by A. franciscana. Artemia were subjected to ingestion experiments [24 h and 3.5 h], in which the organisms were exposed to PS NPs or to PS NPs + microalgae. Post-exposure feeding (24 h exposure and 2 h feeding) and depuration (24 h exposure and 24 h of depuration) were also carried out. More than 90% of the PS NPs were ingested by Artemia and bioaccumulated in the mandible, stomach, gut, tail gut and appendages after 24 h. The ingestion of microalgae was not affected by the presence of the PS NPs. Data of post-exposure feeding indicated that Artemia previously exposed to plastic and/or microalgae presented similar microalgal ingestion (around 70%); the highest microalgal consumption (around 90%) was recorded in the treatment in which Artemia were previously starved (no plastic and no microalgae). The presence of PS NPs in the gut after the depuration experiments indicates that 24 h was not enough to eliminate the PS NPs.

Assessment of the use of cooked Araucaria angustifolia seed coats extract as food for brine shrimp.

The use of vegetable waste and its screening for potential cytotoxicity is of utmost importance to ensure its safe use in the feed industry for fish and other animals. We evaluated aqueous and ethanolic extracts of cooked Araucaria angustifolia seed coats. The Stiasny index for the aqueous and ethanolic extracts was 2.87% ± 0.03% and 60.53% ± 4.79%, respectively. Condensed tannins were 11-fold higher in the ethanolic extract than the aqueous extract. The flavonoid and polyphenol contents were 1.7- and 1.8-fold higher in the ethanolic extract than in the aqueous extract, respectively. The 36 h EC50 for brine shrimp hatchability was 300.32 µg/mL for the aqueous extract, and 76.60 µg/mL for the ethanolic extract. The 24 h LC50 was 1405.96 µg/mL for the aqueous extract, and it was 356.32 µg/mL for the ethanolic extract. The aqueous extract was nontoxic to A. salina nauplii, and therefore, it can be used as a possible food additive in fish feed. The results also demonstrated that the different solvents used in the extraction affected the yield and the total phenolic, total flavonoid, and condensed tannin content. Further in vivo and cell line cytotoxicity testing is recommended to substantiate these findings.

An Evaluation Research About Effects of Characterized Cadmium Selenide (CdSe) and Lead Selenide (PbSe) Quantum Dots on Brine Shrimp (Artemia salina).

Quantum dots (QDs), such as cadmium selenide (CdSe) and lead selenide (PbSe) exhibit excellent optical, magnetic and chemical properties due to their extremely size (ca. 1-10 nm) and are attractive semiconductor nanomaterials for optical studies and energy storage. In this study, aqueous synthesis of CdSe and PbSe QDs in a size range of 2-10 nm was described. Synthesized QDs were characterized using SEM and TEM, DLS, zeta potential, FTIR, EDX and XRD. Highest accumulation (72.5 ± 5.8 mg L-1) of PbSe QDs occurred at 10 ppm suspensions. In general accumulation increased up to 48 h exposure then fluctuate tended to decline. For CdSe QDs, accumulation tended to decrease for 72 h exposure except that for 5 ppm groups. For the elimination period, in general, the elimination levels of PbSe and CdSe QDs from exposed individuals decreased (p < 0.05) even it has some fluctuate.

When Parasites Are Good for Health: Cestode Parasitism Increases Resistance to Arsenic in Brine Shrimps.

Parasites and pollutants can both affect any living organism, and their interactions can be very important. To date, repeated studies have found that parasites and heavy metals or metalloids both have important negative effects on the health of animals, often in a synergistic manner. Here, we show for the first time that parasites can increase host resistance to metalloid arsenic, focusing on a clonal population of brine shrimp from the contaminated Odiel and Tinto estuary in SW Spain. We studied the effect of cestodes on the response of Artemia to arsenic (acute toxicity tests, 24h LC50) and found that infection consistently reduced mortality across a range of arsenic concentrations. An increase from 25°C to 29°C, simulating the change in mean temperature expected under climate change, increased arsenic toxicity, but the benefits of infection persisted. Infected individuals showed higher levels of catalase and glutathione reductase activity, antioxidant enzymes with a very important role in the protection against oxidative stress. Levels of TBARS were unaffected by parasites, suggesting that infection is not associated with oxidative damage. Moreover, infected Artemia had a higher number of carotenoid-rich lipid droplets which may also protect the host through the "survival of the fattest" principle and the antioxidant potential of carotenoids. This study illustrates the need to consider the multi-stress context (contaminants and temperature increase) in which host-parasite interactions occur.

Kairomones from an estuarine fish increase visual sensitivity in brine shrimp (Artemia franciscana) from Great Salt Lake, Utah, USA.

Chemical cues from fish, or kairomones, often impact the behavior of zooplankton. These behavioral changes are thought to improve predator avoidance. For example, marine and estuarine crustacean zooplankton become more sensitive to light after kairomone exposure, which likely deepens their vertical distribution into darker waters during the day and thereby reduces their visibility to fish predators. Here, we show that kairomones from an estuarine fish induce similar behavioral responses in adult brine shrimp (Artemia franciscana) from an endorheic, hypersaline lake, Great Salt Lake, Utah, USA. Given downwelling light stimuli, kairomone-exposed A. franciscana induce a descent response upon dimmer light flashes than they do in the absence of kairomones. Using extracellular electroretinogram (ERG) recordings, we also find that kairomones induce physiological changes in the retina that may lead to increased visual sensitivity, suggesting that kairomone-induced changes to photobehavior are mediated at the photoreceptor level. However, kairomones did not induce structural changes within the eye. Although A. franciscana inhabit endorheic environments that are too saline for most fish, kairomones from an estuarine fish amplify photobehavior in these branchiopod crustaceans. The mechanism for this behavioral change has both similarities to and differences from that described in marine malacostracan crustaceans.

ArHsp40 and ArHsp40-2 contribute to stress tolerance and longevity in Artemia franciscana, but only ArHsp40 influences diapause entry.

Embryos of the crustacean Artemia franciscana develop either ovoviviparously or oviparously, yielding swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Nauplii moult several times and become adults whereas cysts enter diapause, a state of dormancy characterized by exceptionally low metabolism and high stress tolerance. Synthesis of molecular chaperones such as the J-domain proteins ArHsp40 and ArHsp40-2 occurs during embryo development and post-diapause growth of A. franciscana and they influence development and stress tolerance. To further investigate J-domain protein function, ArHsp40 and ArHsp40-2 were each knocked down by RNA interference. Reductions in ArHsp40 and ArHsp40-2 had no effect on adult survival, time to release of cysts and nauplii from females and first-brood size. However, knockdown of both A. franciscana J-domain proteins reduced the longevity and heat tolerance of nauplii, with the loss of ArHsp40 having a greater effect. The knockdown of ArHsp40, but not of ArHsp40-2, caused approximately 50% of cysts to abort diapause entry and hatch without exposure to an exogenous signal such as low temperature and/or desiccation. Cysts lacking ArHsp40 that entered diapause exhibited decreased stress tolerance as did cysts with reduced ArHsp40-2, the latter to a lesser degree. The longevity of nauplii hatching prematurely from cysts was less than for nauplii arising by other means. The results expand our understanding of Hsp40 function in A. franciscana stress tolerance and development, especially during diapause, and they provide the first example of a molecular chaperone that influences diapause entry.

Can physiological engineering/programming increase multi-generational thermal tolerance to extreme temperature events?

Organisms increasingly encounter higher frequencies of extreme weather events as a consequence of global climate change. Currently, few strategies are available to mitigate climate change effects on animals arising from acute extreme high-temperature events. We tested the capacity of physiological engineering to influence the intra- and multi-generational upper thermal tolerance capacity of a model organism, Artemia, subjected to extreme high temperatures. Enhancement of specific physiological regulators during development could affect thermal tolerance or life-history attributes affecting subsequent fitness. Using experimental Artemia populations, we exposed F0 individuals to one of four treatments: heat hardening (28°C to 36°C, 1°C per 10 min), heat hardening plus serotonin (0.056 µg ml-1), heat hardening plus methionine (0.79 mg ml-1) and a control treatment. Regulator concentrations were based on previous literature. Serotonin may promote thermal tolerance, acting upon metabolism and life history. Methionine acts as a methylation agent across generations. For all groups, measurements were collected for three performance traits of individual thermal tolerance (upper sublethal thermal limit, lethal limit and dysregulation range) over two generations. The results showed that no treatment increased the upper thermal limit during acute thermal stress, although serotonin-treated and methionine-treated individuals outperformed controls across multiple thermal performance traits. Additionally, some effects were evident across generations. Together, these results suggest that phenotypic engineering provides complex outcomes, and if implemented with heat hardening can further influence performance in multiple thermal tolerance traits, within and across generations. Potentially, such techniques could be up-scaled to provide resilience and stability in populations susceptible to extreme temperature events.

Distribution of Defensive Metabolites in Nudibranch Molluscs.

Many plants and animals store toxic or unpalatable compounds in tissues that are easily encountered by predators during attack. Defensive compounds can be produced de novo, or obtained from dietary sources and stored directly without selection or modification, or can be selectively sequestered or biotransformed. Storage strategies should be optimized to produce effective defence mechanisms but also prevent autotoxicity of the host. Nudibranch molluscs utilize a diverse range of chemical defences, and we investigated the accumulation and distribution of defensive secondary metabolites in body tissues of 19 species of Chromodorididae nudibranchs. We report different patterns of distribution across tissues, where: 1) the mantle had more or different (but structurally related) compounds than the viscera; 2) all compounds in the mantle were also in the viscera; and 3) the mantle had fewer compounds than the viscera. We found no further examples of species that selectively store a single compound, previously reported in Chromodoris species. Consistent with other studies, we found high concentrations of metabolites in mantle rim tissues compared to the viscera. Using bioassays, compounds in the mantle were more toxic than compounds found in the viscera for Glossodoris vespa Rudman, 1990 and Ceratosoma brevicaudatum Abraham, 1876. In G. vespa, compounds in the mantle were also more unpalatable to palaemonid shrimp than compounds found in the viscera. This indicates that these species may modify compounds to increase bioactivity for defensive purposes and/or selectively store more toxic compounds. We highlight clear differences in the storage of sequestered chemical defences, which may have important implications for species to employ effective defences against a range of predators.

Toxicity assessment of zero valent iron nanoparticles on Artemia salina.

The present study deals with the toxicity assessment of two differently synthesized zero valent iron nanoparticles (nZVI, chemical and biological) as well as Fe2+ ions on Artemia salina at three different initial concentrations of 1, 10, and 100 mg/L of these particles. The assessment was done till 96 h at time intervals of 24 h. EC50 value was calculated to evaluate the 50% mortality of Artemia salina at all exposure time durations. Between chemically and biologically synthesized nZVI nanoparticles, insignificant differences in the level of mortality were demonstrated. At even 24 h, Fe2+ ion imparted complete lethality at the highest exposure concentration (100 mg/L). To understand intracellular oxidative stress because of zero valent iron nanoparticles, ROS estimation, SOD activity, GSH activity, and catalase activity was performed which demonstrated that ionic form of iron is quite lethal at high concentrations as compared with the same concentration of nZVI exposure. Lower concentrations of nZVI were more toxic as compared with the ionic form and was in order of CS-nZVI > BS-nZVI > Fe2+ . Cell membrane damage and bio-uptake of nanoparticles were also evaluated for all three concentrations of BS-nZVI, CS-nZVI, and Fe2+ using adult Artemia salina in marine water; both of which supported the observations made in toxicity assessment. This study can be further explored to exploit Artemia salina as a model organism and a biomarker in an nZVI prone aquatic system to detect toxic levels of these nanoparticles. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1617-1627, 2017.

Effect of biosolid hydrochar on toxicity to earthworms and brine shrimp.

The hydrothermal carbonization of sewage sludge has been studied as an alternative technique for the conversion of sewage sludge into value-added products, such as soil amendments. We tested the toxicity of biosolid hydrochar (Sewchar) to earthworms. Additionally, the toxicity of Sewchar process water filtrate with and without pH adjustment was assessed, using brine shrimps as a model organism. For a Sewchar application of 40 Mg ha-1, the earthworms significantly preferred the side of the vessel with the reference soil (control) over side of the vessel with the Sewchar treatments. There was no acute toxicity of Sewchar to earthworms within the studied concentration range (up to 80 Mg ha-1). Regarding the Sewchar process water filtrate, the median lethal concentration (LC50) to the shrimps was 8.1% for the treatments in which the pH was not adjusted and 54.8% for the treatments in which the pH was adjusted to 8.5. The lethality to the shrimps significantly increased as the amount of Sewchar process water filtrate increased. In the future, specific toxic substances in Sewchar and its process water filtrate, as well as their interactions with soil properties and their impacts on organisms, should be elucidated. Additionally, it should be identified whether the amount of the toxic compounds satisfies the corresponding legal requirements for the safe application of Sewchar and its process water filtrate.

Monte Carlo estimation of stage structured development from cohort data.

Cohort data are frequently collected to study stage-structured development and mortalities of many organisms, particularly arthropods. Such data can provide information on mean stage durations, among-individual variation in stage durations, and on mortality rates. Current statistical methods for cohort data lack flexibility in the specification of stage duration distributions and mortality rates. In this paper, we present a new method for fitting models of stage-duration distributions and mortality to cohort data. The method is based on a Monte Carlo within MCMC algorithm and provides Bayesian estimates of parameters of stage-structured cohort models. The algorithm is computationally demanding but allows for flexible specifications of stage-duration distributions and mortality rates. We illustrate the algorithm with an application to data from a previously published experiment on the development of brine shrimp from Mono Lake, California, through nine successive stages. In the experiment, three different food supply and temperature combination treatments were studied. We compare the mean duration of the stages among the treatments while simultaneously estimating mortality rates and among-individual variance of stage durations. The method promises to enable more detailed studies of development of both natural and experimental cohorts. An R package implementing the method and which allows flexible specification of stage duration distributions is provided.

Impacts of harvesting on brine shrimp (Artemia franciscana) in Great Salt Lake, Utah, USA.

Selective harvesting can cause evolutionary responses in populations via shifts in phenotypic characteristics, especially those affecting life history. Brine shrimp (Artemia franciscana) cysts in Great Salt Lake (GSL), Utah, USA are commercially harvested with techniques that select against floating cysts. This selective pressure could cause evolutionary changes over time. Our objectives are to (1) determine if there is a genetic basis to cyst buoyancy, (2) determine if cyst buoyancy and nauplii mortality have changed over time, and (3) to examine GSL environmental conditions over time to distinguish whether selective harvesting pressure or a trend in environmental conditions caused changes in cyst buoyancy and nauplii mortality. Mating crosses between floating and sinking parental phenotypes with two food concentrations (low and high) indicated there is a genetic basis to cyst buoyancy. Using cysts harvested from 1991-2011, we found cyst buoyancy decreased and nauplii mortality increased over time. Data on water temperature, salinity, and chlorophyll a concentration in GSL from 1994 to 2011 indicated that although water temperature has increased over time and chlorophyll a concentration has decreased over time, the selective harvesting pressure against floating cysts is a better predictor of changes in cyst buoyancy and nauplii mortality over time than trends in environmental conditions. Harvesting of GSL A. franciscana cysts is causing evolutionary changes, which has implications for the sustainable management and harvesting of these cysts. Monitoring phenotypic characteristics and life-history traits of the population should be implemented and appropriate responses taken to reduce the impacts of the selective harvesting.

The ontogeny of tolerance curves: habitat quality vs. acclimation in a stressful environment.

Stressful environments affect life-history components of fitness through (i) instantaneous detrimental effects, (ii) historical (carry-over) effects and (iii) history-by-environment interactions, including acclimation effects. The relative contributions of these different responses to environmental stress are likely to change along life, but such ontogenic perspective is often overlooked in studies of tolerance curves, precluding a better understanding of the causes of costs of acclimation, and more generally of fitness in temporally fine-grained environments. We performed an experiment in the brine shrimp Artemia to disentangle these different contributions to environmental tolerance, and investigate how they unfold along life. We placed individuals from three clones of A. parthenogenetica over a range of salinities during a week, before transferring them to a (possibly) different salinity for the rest of their lives. We monitored individual survival at repeated intervals throughout life, instead of measuring survival or performance at a given point in time, as commonly done in acclimation experiments. We then designed a modified survival analysis model to estimate phase-specific hazard rates, accounting for the fact that individuals may share the same treatment for only part of their lives. Our approach allowed us to distinguish effects of salinity on (i) instantaneous mortality in each phase (habitat quality effects), (ii) mortality later in life (history effects) and (iii) their interaction. We showed clear effects of early salinity on late survival and interactions between effects of past and current environments on survival. Importantly, analysis of the ontogenetic dynamics of the tolerance curve reveals that acclimation affects different parts of the curve at different ages. Adopting a dynamical view of the ontogeny of tolerance curve should prove useful for understanding niche limits in temporally changing environments, where the full sequence of environments experienced by an individual determines its overall environmental tolerance, and how it changes throughout life.

Differential protein expression using proteomics from a crustacean brine shrimp (Artemia sinica) under CO2-driven seawater acidification.

Gradually increasing atmospheric CO2 partial pressure (pCO2) has caused an imbalance in carbonate chemistry and resulted in decreased seawater pH in marine ecosystems, termed seawater acidification. Anthropogenic seawater acidification is postulated to affect the physiology of many marine calcifying organisms. To understand the possible effects of seawater acidification on the proteomic responses of a marine crustacean brine shrimp (Artemia sinica) three groups of cysts were hatched and further raised in seawater at different pH levels (8.2 as control and 7.8 and 7.6 as acidification stress levels according to the predicted levels at the end of this century and next century, respectively) for 1, 7 and 14 days followed by examination of the protein expression changes via two-dimensional gel electrophoresis. Searches of protein databases revealed that 67 differential protein spots were altered due to lower pH level (7.6 and 7.8) stress in comparison to control groups (pH 8.2) by mass spectrometry. Generally, these differentially expressed proteins included the following: 1) metabolic process-related proteins involved in glycolysis and glucogenesis, nucleotide/amino acid/fatty acid metabolism, protein biosynthesis, DNA replication and apoptosis; 2) stress response-related proteins, such as peroxiredoxin, thioredoxin peroxidase, 70-kDa heat shock protein, Na/K ATPase, and ubiquinol-cytochrome c reductase; 3) immune defence-related proteins, such as prophenoloxidase and ferritin; 4) cytoskeletal-related proteins, such as myosin light chain, TCP1 subunit 2, tropomyosin and tubulin alpha chain; and 5) signal transduction-related proteins, such as phospholipase C-like protein, 14-3-3 zeta, translationally controlled tumour protein and RNA binding motif protein. Taken together, these data support the idea that CO2-driven seawater acidification may affect protein expression in the crustacean A. sinica and possibly also in other species that feed on brine shrimp in the ecosystem, particularly marine food webs.