Disparate responses to salinity across species and organizational levels in anchialine shrimps.

Environmentally induced plasticity in gene expression is one of the underlying mechanisms of adaptation to habitats with variable environments. For example, euryhaline crustaceans show predictable changes in the expression of ion-transporter genes during salinity transfers, although studies have typically been limited to specific genes, taxa and ecosystems of interest. Here, we investigated responses to salinity change at multiple organizational levels in five species of shrimp representing at least three independent invasions of the anchialine ecosystem, defined as habitats with marine and freshwater influences with spatial and temporal fluctuations in salinity. Although all five species were generally strong osmoregulators, salinity-induced changes in gill physiology and gene expression were highly species specific. While some species exhibited patterns similar to those of previously studied euryhaline crustaceans, instances of distinct and atypical patterns were recovered from closely related species. Species-specific patterns were found when examining: (1) numbers and identities of differentially expressed genes, (2) salinity-induced expression of genes predicted a priori to play a role in osmoregulation, and (3) salinity-induced expression of orthologs shared among all species. Notably, ion transport genes were unchanged in the atyid Halocaridina rubra while genes normally associated with vision and light perception were among those most highly upregulated. Potential reasons for species-specific patterns are discussed, including variation among anchialine habitats in salinity regimes and divergent evolution in anchialine taxa. Underexplored mechanisms of osmoregulation in crustaceans revealed here by the application of transcriptomic approaches to ecologically and taxonomically understudied systems are also explored.

Salinity-induced changes in gene expression from anterior and posterior gills of Callinectes sapidus (Crustacea: Portunidae) with implications for crustacean ecological genomics.

Decapods represent one of the most ecologically diverse taxonomic groups within crustaceans, making them ideal to study physiological processes like osmoregulation. However, prior studies have failed to consider the entire transcriptomic response of the gill - the primary organ responsible for ion transport - to changing salinity. Moreover, the molecular genetic differences between non-osmoregulatory and osmoregulatory gill types, as well as the hormonal basis of osmoregulation, remain underexplored. Here, we identified and characterized differentially expressed genes (DEGs) via RNA-Seq in anterior (non-osmoregulatory) and posterior (osmoregulatory) gills during high to low salinity transfer in the blue crab Callinectes sapidus, a well-studied model for crustacean osmoregulation. Overall, we confirmed previous expression patterns for individual ion transport genes and identified novel ones with salinity-mediated expression. Notable, novel DEGs among salinities and gill types for C. sapidus included anterior gills having higher expression of structural genes such as actin and cuticle proteins while posterior gills exhibit elevated expression of ion transport and energy-related genes, with the latter likely linked to ion transport. Potential targets among recovered DEGs for hormonal regulation of ion transport between salinities and gill types included neuropeptide Y and a KCTD16-like protein. Using publically available sequence data, constituents for a "core" gill transcriptome among decapods are presented, comprising genes involved in ion transport and energy conversion and consistent with salinity transfer experiments. Lastly, rarefication analyses lead us to recommend a modest number of sequence reads (~10-15M), but with increased biological replication, be utilized in future DEG analyses of crustaceans.

Taking their breath away: metabolic responses to low-oxygen levels in anchialine shrimps (Crustacea: Atyidae and Alpheidae).

Crustaceans generally act as oxy-regulators, maintaining constant oxygen uptake as oxygen partial pressures decrease, but when a critical low level is reached, ventilation and aerobic metabolism shut down. Cave-adapted animals, including crustaceans, often show a reduced metabolic rate possibly owing in part to the hypoxic nature of such environments. However, metabolic rates have not been thoroughly explored in crustaceans from anchialine habitats (coastal ponds and caves), which can experience variable oxygenic regimes. Here, an atypical oxy-conforming pattern of oxygen uptake is reported in the Hawaiian anchialine atyid Halocaridina rubra, along with other unusual metabolic characteristics. Ventilatory rates are near-maximal in normoxia and did not increase appreciably as PO2 declined, resulting in a decline in VO2 during progressive hypoxia. Halocaridina rubra maintained in anoxic waters survived for seven days (the duration of the experiment) with no measureable oxygen uptake, suggesting a reliance on anaerobic metabolism. Supporting this, lactate dehydrogenase activity was high, even in normoxia, and oxygen debts were quickly repaid by an unusually extreme increase in oxygen uptake upon exposure to normoxia. In contrast, four related anchialine shrimp species from the Ryukyu Islands, Japan, exhibited physiological properties consistent with previously studied crustaceans. The unusual respiratory patterns found in H. rubra are discussed in the context of a trade-off in gill morphology for osmoregulatory ion transport vs. diffusion of respiratory gasses. Future focus on anchialine species may offer novel insight into the diversity of metabolic responses to hypoxia and other physiological challenges experienced by crustaceans.

Osmoregulation in the Hawaiian anchialine shrimp Halocaridina rubra (Crustacea: Atyidae): expression of ion transporters, mitochondria-rich cell proliferation and hemolymph osmolality during salinity transfers.

Studies of euryhaline crustaceans have identified conserved osmoregulatory adaptions allowing hyper-osmoregulation in dilute waters. However, previous studies have mainly examined decapod brachyurans with marine ancestries inhabiting estuaries or tidal creeks on a seasonal basis. Here, we describe osmoregulation in the atyid Halocaridina rubra, an endemic Hawaiian shrimp of freshwater ancestry from the islands' anchialine ecosystem (coastal ponds with subsurface freshwater and seawater connections) that encounters near-continuous spatial and temporal salinity changes. Given this, survival and osmoregulatory responses were examined over a wide salinity range. In the laboratory, H. rubra tolerated salinities of ~0-56‰, acting as both a hyper- and hypo-osmoregulator and maintaining a maximum osmotic gradient of ~868 mOsm kg(-1) H2O in freshwater. Furthermore, hemolymph osmolality was more stable during salinity transfers relative to other crustaceans. Silver nitrate and vital mitochondria-rich cell staining suggest all gills are osmoregulatory, with a large proportion of each individual gill functioning in ion transport (including when H. rubra acts as an osmoconformer in seawater). Additionally, expression of ion transporters and supporting enzymes that typically undergo upregulation during salinity transfer in osmoregulatory gills (i.e. Na(+)/K(+)-ATPase, carbonic anhydrase, Na(+)/K(+)/2Cl(-) cotransporter, V-type H(+)-ATPase and arginine kinase) were generally unaltered in H. rubra during similar transfers. These results suggest H. rubra (and possibly other anchialine species) maintains high, constitutive levels of gene expression and ion transport capability in the gills as a means of potentially coping with the fluctuating salinities that are encountered in anchialine habitats. Thus, anchialine taxa represent an interesting avenue for future physiological research.

Carbonic anhydrase induction in euryhaline crustaceans is rate-limited at the post-transcriptional level.

The transfer of euryhaline crustaceans from full-strength seawater to low salinity results in both a rapid up-regulation of carbonic anhydrase (CA; EC 4.2.1.1) mRNA and a slow induction of CA activity. There is a delay of several days between the two processes, which is attributed to the time required to synthesize new enzyme. These delays may also be due to limitations in the cellular uptake of Zn, which is a required post-translational active site modification to CA. To investigate these processes, the euryhaline crabs, Callinectes sapidus and Carcinus maenas, were acclimated to salinities below their isosmotic points (22.5 and 25 ppt, respectively) for 7 days to activate the physiological and molecular mechanisms of osmoregulation. CA mRNA increased 90-fold in C. sapidus and 2-fold in C. maenas within 6h; whereas it took 48 h for the initial increases in CA activity (120% and 31%), and 4 to 7 days for new acclimated levels (300% and 100%, respectively). Crabs were then transferred to lower salinities (10 and 15 ppt) to induce further CA activity and to determine if previous increases in CA mRNA reduced the time required for subsequent CA induction. Additionally, the expression of the Zn transporter ZIP1 was examined in C. sapidus at 35 and 22.5 ppt. In both species, prior CA mRNA elevation failed to accelerate the rate of CA induction. Levels of CA mRNA did not change in either crab following transfer from intermediate to low salinity. Taken together, these results show that the timecourse of CA induction at low salinity is not limited by the expression of CA mRNA, but by the synthesis of new enzyme from an existing pool of mRNA. No increases in ZIP1 expression occurred at low salinity, therefore these delays may be due to the limits of cellular Zn uptake.

Functional characterization of neuroendocrine regulation of branchial carbonic anhydrase induction in the euryhaline crab Callinectes sapidus.

Carbonic anhydrase (CA) plays an essential role as a provider of counterions for Na(+)/H(+) and Cl(-)/HCO3 (-) exchange in branchial ionic uptake processes in euryhaline crustaceans. CA activity and gene expression are low in crabs acclimated to full-strength seawater, with transfer to low salinity resulting in large-scale inductions of mRNA and subsequent enzyme activity in the posterior ion-regulating gills (e.g., G7). In the green crab Carcinus maenas, CA has been shown to be under inhibitory neuroendocrine control by a putative hormone in the x-organ-sinus gland complex (XOSG), located in the eyestalk. This study characterizes the neuroendocrine regulation of CA induction in the blue crab Callinectes sapidus, a commonly used experimental organism for crustacean osmoregulation. In crabs acclimated to full-strength seawater, eyestalk ligation (ESL) triggered a 1.8- and 100-fold increase in CA activity and mRNA, respectively. Re-injection with eyestalk homogenates abolished increases in CA activity and fractionally reduced CA gene expression. ESL also enhanced CA induction by 33% after 96 h in crabs transferred to 15 ppt salinity. Injection of eyestalk homogenates into intact crabs transferred from 35 to 15 ppt diminished by 43% the CA induction stimulated by low salinity. These results point to the presence of a repressor hormone in the eyestalk. Separate injections of medullary tissue (MT) and sinus gland (SG), two components of the eyestalk, reduced salinity-stimulated CA activity by 22% and 49%, suggesting that the putative repressor is localized to the SG. Crabs injected with SG extract harvested from crabs acclimated to 5 ppt showed no decrease in CA activity, demonstrating that the hormone is down-regulated at low salinity. Our results show the presence in the XOSG of an inhibitory compound that regulates salinity-stimulated CA induction.

Altered expression of Na(+)/K(+)-ATPase and other osmoregulatory genes in the gills of euryhaline animals in response to salinity transfer: a meta-analysis of 59 quantitative PCR studies over 10 years.

Recent advances in molecular techniques have allowed gene expression in euryhaline animals to be quantified during salinity transfers. As these investigations transition from studying single genes to utilizing genomics-based methodologies, it is an appropriate time to summarize single gene studies. Therefore, a meta-analysis was performed on 59 published studies that used quantitative polymerase chain reaction (qPCR) to examine expression of osmoregulatory genes (the Na(+)/K(+)-ATPase, NKA; the Na(+)/K(+)/2Cl(-) cotransporter, NKCC; carbonic anhydrase, CA; the cystic fibrosis transmembrane regulator, CFTR; and the H(+)-ATPase, HAT) in response to salinity transfer. Based on 887 calculated effect sizes, NKA, NKCC, CA, and HAT are up-regulated after salinity transfer, while surprisingly, CFTR is unchanged. Meta-analysis also identified influential factors contributing to these changes. For example, expression was highest: 1) during transfers from higher to lower salinities comprising a physiological transition from osmoconformity to osmoregulation, 2) 1-3 days following transfer, 3) during dissimilar transfers, and 4) in crustaceans rather than teleosts. Methodological characteristics (e.g., types of controls) were not important. Experiments lacking in the current literature were also identified. Meta-analyses are powerful tools for quantitatively synthesizing a large body of literature, and this report serves as a template for their application in other areas of comparative physiology.

Anemonefish oxygenate their anemone hosts at night.

Many stony coral-dwelling fishes exhibit adaptations to deal with hypoxia among the branches of their hosts; however, no information exists on the respiratory ecophysiology of obligate fish associates of non-coral organisms such as sea anemones and sponges. This study investigated metabolic and behavioral interactions between two-band anemonefish (Amphiprion bicinctus) and bulb-tentacle sea anemones (Entacmaea quadricolor) at night. We measured the net dark oxygen uptake ( , µmol O2 h(-1)) of fish-anemone pairs when partners were separate from each other, together as a unit, and together as a unit but separated by a mesh screen that prevented physical contact. We also measured the effects of water current on sea anemone and quantified the nocturnal behaviors of fish in the absence and presence of host anemones in order to discern the impacts of anemone presence on fish behavior. Net of united pairs was significantly higher than that of both separated pairs and united pairs that were separated by a mesh screen. Anemone increased with flow rate from 0.5 to 2.0 cm s(-1), after which remained constant up to a water flow rate of 8.0 cm s(-1). Furthermore, the percentage time and bout frequency of flow-modulating behaviors by fish increased significantly when anemones were present. We conclude that physical contact between anemonefish and sea anemones elevates the of at least one of the partners at night, and anemonefish behavior at night appears to oxygenate sea anemone hosts and to augment the metabolism of both partners.

Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals.

The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail.

Microarray-detected changes in gene expression in gills of green crabs (Carcinus maenas) upon dilution of environmental salinity.

The interaction between environmental salinity and gene expression was studied in gills of the euryhaline green shore crab Carcinus maenas. A 4462-feature oligonucleotide microarray was used to analyze changes in transcript abundance in posterior ion-transporting gills at 8 time periods following transfer of animals from 32 to 10 or 15 ppt salinity. Transcripts encoding Na(+)/K(+)-ATPase α-subunit and cytoplasmic carbonic anhydrase were upregulated with significant changes between 6 and 24h post-transfer. Other transport proteins showing similar transcriptional upregulation were an organic cation transporter, a sodium/glucose cotransporter, an endomembrane protein associated with regulating plasma membrane protein composition, and a voltage-gated calcium channel. Transport proteins showing little transcriptional response included Na(+)/H(+) exchanger, Na(+)/K(+)/2Cl(-) cotransporter, and V-type H(+)-ATPase B subunit, all of which have been implicated in osmoregulatory ion transport across crustacean gill. Interestingly, there was little affect of salinity dilution on transcriptional expression of stress proteins, suggesting that salinity acclimation is part of normal physiology for C. maenas. Expression of transcripts encoding a variety of mitochondrial proteins was significantly upregulated between 4 days and 7 days post-transfer, consistent with the proliferation of mitochondria-rich cells previously observed at this time.

Down-regulation of activity and expression of three transport-related proteins in the gills of the euryhaline green crab, Carcinus maenas, in response to high salinity acclimation.

The euryhaline green crab, Carcinus maenas, undergoes an annual cycle of salinity exposure, having to adapt to low salinity during its annual spring migration into estuaries, and then having to re-adapt to high salinity when it moves off-shore at the end of summer. Most studies have focused on low salinity acclimation, the activation of osmoregulatory mechanisms, and the induction of transport protein and transport-related enzyme activity and gene expression. In this study we followed the changes in hemolymph osmolality, carbonic anhydrase activity, and mRNA expression of three proteins through a complete cycle of low (15 ppt) and high (32 ppt) salinity acclimation. One week of low salinity acclimation resulted in hemolymph osmoregulation and a four-fold induction of branchial carbonic anhydrase activity. Relative mRNA expression increased for two CA isoforms (CAc 100-fold, and CAg 7-fold) and the α-subunit of the Na/K-ATPase (8-fold). Upon re-exposure to high salinity, hemolymph osmolality increased to 32 ppt acclimated levels by 6 h, and mRNA levels returned to high salinity, baseline levels within 1 week. However, CA activity remained unchanged in response to high salinity exposure for the first week and then gradually declined to baseline levels over 4 weeks. The relative timing of these changes suggests that while whole-organism physiological adaptations and regulation at the gene level can be very rapid, changes at the level of protein expression and turnover are much slower. It is possible that the high metabolic cost of protein synthesis and/or processing could be the underlying reason for long biological life spans of physiologically important proteins.

Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and functional genomics.

Carcinus maenas, commonly known as the European green crab, is one of the best-known and most successful marine invasive species. While a variety of natural and anthropogenic mechanisms are responsible for the geographic spread of this crab, its ability to adapt physiologically to a broad range of salinities, temperatures and other environmental factors has enabled its successful establishment in new habitats. To extend our understanding of hormonal control in C. maenas, including factors that allow for its extreme adaptability, we have undertaken a mass spectral/functional genomics investigation of the neuropeptides used by this organism. Via a strategy combining MALDI-based high resolution mass profiling, biochemical derivatization, and nanoscale separation coupled to tandem mass spectrometric sequencing, 122 peptide paracrines/hormones were identified from the C. maenas central nervous system and neuroendocrine organs. These peptides include 31 previously described Carcinus neuropeptides (e.g. NSELINSILGLPKVMNDAamide [beta-pigment dispersing hormone] and PFCNAFTGCamide [crustacean cardioactive peptide]), 49 peptides only described in species other than the green crab (e.g. pQTFQYSRGWTNamide [Arg(7)-corazonin]), and 42 new peptides de novo sequenced here for the first time (e.g. the pyrokinins TSFAFSPRLamide and DTGFAFSPRLamide). Of particular note are large collections of FMRFamide-like peptides (25, including nine new isoforms sequenced de novo) and A-type allatostatin peptides (25, including 10 new sequences reported here for the first time) in this study. Also of interest is the identification of two SIFamide isoforms, GYRKPPFNGSIFamide and VYRKPPFNGSIFamide, the latter peptide known previously only from members of the astacidean genus Homarus. Using transcriptome analyses, 15 additional peptides were characterized, including an isoform of bursicon beta and a neuroparsin-like peptide. Collectively, the data presented in this study not only greatly expand the number of identified C. maenas neuropeptides, but also provide a framework for future investigations of the physiological roles played by these molecules in this highly adaptable species.

Acute physiological responses of the freshwater snail Elimia flava (Mollusca: Pleuroceridae) to environmental pH and calcium.

The individual and interactive effects of environmental pH (7 [control], 6, 5, and 4) and calcium (0, 5, and 50 mg/L) were studied on hemolymph ions (pH, Ca(2+), total CO(2), Na(+), K(+)) and osmolality in the freshwater snail, Elimia flava, over a 72-h exposure. All hemolymph factors strongly differed with environmental pH. Snails exposed to pH 4 were inactive and experienced more dramatic ionic disturbances than snails at pH 5, 6, and 7, including reduced hemolymph pH, depressed Na(+) concentrations, and increased Ca(2+) and total CO(2) concentrations. There was an initial but transient increase in hemolymph K(+) over the 72 h exposure period. Environmental calcium ameliorated effects of acidification on hemolymph pH and Na(+), reducing the degree of depression in both variables irrespective of environmental pH or exposure time. In a separate experiment, effects of acidification on snail respiration were examined in which VO(2) was measured over 24 h in snails exposed to pH 7 and 4. Exposure to pH 4 caused a 64% reduction in oxygen uptake at 2 h and a maximum reduction (81%) at 11 h. Our results suggest that snails exposed to pH 4 cease interacting with the surrounding medium and use internal stores of CaCO(3) to buffer hemolymph acidification, whereas snails at pH 5 and higher appear to use environmental calcium as a buffer source. These results suggest an important role of environmental calcium in ameliorating the impacts of short-term, sublethal acidification.

Differential expression and induction of two carbonic anhydrase isoforms in the gills of the euryhaline green crab, Carcinus maenas, in response to low salinity.

Two isoforms of the enzyme carbonic anhydrase (CA) from the gills of the euryhaline green crab were sequenced and identified; these were found to match the cytoplasmic (CAc) and membrane-associated (CAg) isoforms known from other species. The mRNA of the membrane-associated isoform is present in significantly higher levels of abundance in gills of crabs acclimated to 32 ppt, at which the crab is an osmotic and ionic conformer. Upon transfer to low salinity (15 ppt), in which the crab is an osmoregulator, however, the cytoplasmic isoform undergoes a rapid 100-fold increase in abundance in the posterior gills, becoming the dominant isoform. CAg increases 3-fold initially and then remains elevated through 14 days of low salinity acclimation. The induction of CAc mRNA is believed to be the molecular basis for the 20 fold increase in CA protein-specific activity during low salinity acclimation. The initial increase in CAc mRNA takes place at 6 h, and maximal levels of expression are achieved by 24 h; this precedes the induction of CA activity and is within the time in which hemolymph osmotic and ionic concentrations stabilize at new acclimated levels. The increase in expression of the CAg isoform is believed to be more closely related to changes in the population of branchial chloride cells. Changes in the relative abundance of mRNA for the alpha-subunit of the Na(+)/K(+)-ATPase were smaller in magnitude than those for CAc, but the timing was similar. There were no changes in expression of a control gene, arginine kinase (AK) in posterior gills, and there were no significant changes in expression in anterior gills for any of the genes measured here. These results support the use of a control tissue (anterior gills) in addition to a control gene for expression studies.

Recent advances in crustacean genomics.

Crustaceans are a diverse and ancient group of arthropods that have long been studied as interesting model systems in biology, especially for understanding animal evolution and physiology and for environmentally relevant studies. Like many model systems, advances in DNA-sequencing methodologies have led to a large amount of genomics-related projects. The purpose of this article is to highlight the genome projects and functional genomics (transcriptomics) projects that are currently underway in crustacean biology. Specifically, we have surveyed the amount of publicly available DNA sequence data (both genomic and EST data) across all crustacean taxa for which a significant number of DNA sequences have been generated. Several ongoing projects are presented including the ecology of invasive species, thermal physiology, ion and water balance, ecology and evolutionary biology, and developmental biology.

Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crab Callinectes sapidus.

Two isoforms of the enzyme carbonic anhydrase (CA) in the blue crab gill, CasCAg and CasCAc, were identified, sequenced, and found to match the membrane-associated and cytoplasmic isoforms, respectively. The membrane-associated isoform is present in much higher levels of mRNA expression in both anterior and posterior gills in crabs acclimated to high salinity (35 p.p.t.), but expression of the cytoplasmic isoform in the posterior gill undergoes a significantly greater degree of up-regulation after exposure to low salinity (15 p.p.t.). CasCAc has the largest scope of induction (100-fold) reported for any transport-related protein in the gill, and this may be necessary to overcome diffusion limitations between gill cytoplasm and the apical boundary layer. Furthermore, the timing of the changes in expression of CasCAc corresponds to the timing of the induction of protein-specific CA activity and CA protein concentration. No changes in CA mRNA expression or activity occur in the anterior gills. The pattern of up-regulation of expression of mRNA of the alpha-subunit of the Na+/K+-ATPase is similar to that for CasCAc, and both precede the establishment of the new acclimated physiological state of the crab in low salinity. A putative ;housekeeping' gene, arginine kinase, also showed about a threefold increase in expression in response to low salinity, but only in the posterior gills. These results suggest that for studies of expression in crustacean gill tissue, a control tissue, such as the anterior gill, be used until an adequate control gene is identified.

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities.

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.

Functional evidence for the presence of a carbonic anhydrase repressor in the eyestalk of the euryhaline green crab Carcinus maenas.

Carbonic anhydrase (CA) activity and relative expression of CA mRNA were measured in the gills of the euryhaline green crab Carcinus maenas in response to eyestalk ablation (ESA), injection of eyestalk extract and exposure to low salinity. For crabs acclimated to 32 p.p.t. salinity, ESA alone resulted in an increase in both CA activity and relative mRNA expression in the posterior, ion-transporting gills, but not in the anterior, respiratory gills. The ESA-stimulated increase in CA activity was abolished by injections of extracts of eyestalks taken from crabs acclimated to 32 p.p.t. salinity. Transfer of intact crabs from 32 to 10 p.p.t. salinity for 7 days resulted in an eightfold increase in CA activity and a sixfold increase in mRNA expression in posterior gills. ESA potentiated the normal low salinity-mediated CA induction by 23%. Daily injections of eyestalk extract reduced low salinity-stimulated CA induction by nearly 50% in intact crabs and by almost 75% in eyestalk ablated crabs. A 4-day transfer to 10 p.p.t. salinity also caused significant increases in both CA activity and mRNA expression in posterior gills, and ESA resulted in a 32% increase in the normal degree of CA induction. Daily injections of eyestalk extracts reduced CA induction in a dose-dependent manner over the 4-day time course. When CA induction was reduced by 66%, hemolymph osmotic regulation was also disrupted. These results are functional evidence for the presence of a CA repressor in the major endocrine complex of the crab, the eyestalk. This compound appears to function in keeping CA expression at low, baseline levels in crabs at high salinity. Exposure to low salinity removes the effects of the putative repressor, allowing CA expression, and thus CA activity, to increase.

Quantitative changes in branchial carbonic anhydrase activity and expression in the euryhaline green crab, Carcinus maenas, in response to low salinity exposure.

Hemolymph osmolality, and changes in gill carbonic anhydrase (CA) activity, relative mRNA expression, and CA protein concentration were measured in the green crab Carcinus maenas acclimated to 32 ppt salinity and transferred to 10 ppt. Hemolymph osomolality stabilized at new, acclimated values, by 24 hr after transfer. There was a large increase in CA mRNA concentrations, as measured by quantitative PCR, in the posterior gills by 24 hr post-transfer that remained elevated through 4 days. By 7 days, however, CA mRNA levels began to decline. CA activity, on the other hand, did not begin to increase until 48 hr after transfer to 10 ppt, but it continued to increase through 7 days. CA protein concentration increased by 5-fold in posterior gills in crabs acclimated to 10 ppt. CA activity, mRNA expression, and CA protein concentrations did not change in anterior gills. These results indicate that low salinity-stimulated CA induction is under transcriptional regulation, and that the increase in CA activity is a result of the increase in gene expression and synthesis of new enzyme. Changes in mRNA appear to be transient, but once synthesized, the CA protein appears to persist in the gill for an extended time. In a separate set of experiments, green crabs acclimated to 32 ppt were transferred directly to salinities of 25, 20, 15, and 10 ppt. CA activity and mRNA concentrations increased with decreasing salinity, peaking at 15 ppt but decreasing between 15 and 10 ppt. The decrease may represent a breakdown in the mechanism of transport-related protein induction near the lower salinity limit of this species.