Progress 10/01/00 to 09/30/05
Outputs
Blue crabs, Callinectes sapidus, make the transition from osmotic and ionic conformity to regulation at a critical salinity of 27 ppt. At that point there is an induction of carbonic anhydrase (CA) activity that can be as large as 10 fold, depending on final acclimation salinity. The crab must be exposed to the low salinity for a minimum of twelve hours for induction to occur, but once the process begins, it goes to completion. Induction of CA activity appears to be a function of increased levels of CA mRNA expression. CA mRNA increases at 6 hr after transfer to low salinity, and the initial increase in CA activity occurs at 24 hr post-transfer. The increase in CA mRNA appears to be transient, but the resulting increase in CA protein is long-lasting. Changes in CA expression appear to be under inhibitory control by a CA repressor found in the eyestalk, the major endocrine complex of the crab. This substance appears to be present in crabs at high salinity and functions
to keep CA expression, and therefore activity, at low, baseline levels. The repressor, or its effect, is removed in response to exposure to low salinity, allowing CA induction to occur. The repressor has been functionally localized to the major neurohemal storage organ of the eyestalk, the sinus gland. Furthermore, HPLC fractionation of sinus gland extract results in a series of peaks, and the fraction containing CA repressor activity is in the area of the chromatogram containing the highest density of peaks. Further work is currently in progress to isolate the single active peak and ultimately identify and characterize the repressor.
Impacts
The research has shown that changes in the expression of genes important in physiological responses to changes in the physical characteristics of the environment are a result of regulation at the transcriptional level. Specifically, the increase in carbonic anhydrase (CA) activity in the gills of the blue crab during low salinity adaptation is a result of the increase in the levels of CA mRNA. Furthermore, transcription appears to be under inhibitory regulatory control by a CA repressor localized in the major endocrine organ of the crab, the sinus gland of the eyestalk. HPLC fractionation of sinus gland extract yields a profile of peaks, with the repressor activity so far being localized to the area of the profile with the highest density of peaks.
Publications
- Henry, R.P. and D.W. Borst (2006) Effects of eyestalk ablation on carbonic anhydrase activity in the euryhaline blue crab, Callinectes sapidus: neuroendocrine control of enzyme expression. J. Exp. Zool. 305A: 23-331. Henry, R.P. (2005) Critical salinity, sensitivity, and commitment os salinity-mediated carbonic anhydrase induction in the gills of two euryhaline species of decapod crustaceans. J. Exp. Zool. 303A:45-56.
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Progress 01/01/04 to 12/31/04
Outputs
Work continuted on the characterization of the molecular response of Callinectes sapidus and Carcinus maenas to environmental low salinity. In C. sapidus, the critical salinity for the initial induction of the enzyme carbonic anhydrase (CA) was found to be 26 ppt. The induction mechanism was also found to be sensitive to changes in salinity as small as 15 mOsm (0.5 ppt). Furthermore, the crab must be exposed to low salinity for 12-18 hrs before CA induction occurs. CA induction appears to be under inhibitor control by a compound in the eyestalk. Eyestalk ablation increases CA activity, and injection of eyestalk extract inhibits this induction.
Impacts
Regulation of gene expression and physiological adaptations via inhibitory control appears to be a common theme in crustaceans. This work may also shed light on how crabs molt, which could ultimately have an impact on soft shell fisheries.
Publications
- Gannon, A.T. and R.P. Henry (2004) Oxygen and carbon dioxide sensitivity of ventilation in amphibious crabs, Cardisoma guanhumi, breathing air and water. Comp. Biochem. Physiol. 138A:111-117.
- Henry, R.P. (2004) Initial characterization of a carbonic anhydrase repressor from the eyestalks of the euryhaline green crab, Carcinus maenas. Bull. Mt. Desert Island Biol. Lab. 43:119-120.
- Smith, C.* and R.P. Henry (2004) A carbonic anhydrase repressor is found in the hemolymph of the euryhaline green crab, Carcinus maenas. Bull. Mt. Desert Island Biol. Lab. 43:108-109.
- Thomason, K.L.*, D.W. Towle, and R.P. Henry (2004) Quantitative expression of carbonic anhydrase mRNA and protein-specific activity in the gills of the euryhaline green crab, Carcinus maenas. Bull. Mt. Desert Island Biol. Lab. 43:72-73.
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Progress 01/01/03 to 12/31/03
Outputs
The expression of the gene for the enzyme carbonic anhydrase (CA) in the gills of euryhaline crustaceans is known to be increased when the crabs are exposed to low salinity, resulting in an 8-12 fold induction of the enzyme itself. Using real time, quantitative PCR (qPCR), we found that CA gene expression increases 6 fold during the first 24 hr of low salinity exposure, and peaks at an 8 fold increase by 72 hr after exposure. Protein-specific CA activity begins to increase at 48 hr after low salinity exposure, and continues to increase through four days. CA gene expression appears to be under inhibitory control by a repressor substance found in the major endocrine complex of the crab, the eyestalk. This repressor is present at high salinity, but its effects are absent in low salinity. Experiments using injections of eyestalk extract have shown that the repressor itself is most likely down-regulated as a result of low salinity exposure. Furthermore, the putative
repressor appears to be a small peptide with properties similar to peptides of the Crustacean Hypoglycemic Hormone (CHH) family. It is heat and acid stable for short periods of time (2 min), but it is heat and acid labile after 15 min of treatment.
Impacts
Regulation of gene expression and physiological adaptations via inhibitory control appears to be a common theme in crustaceans. This work may also shed light on how crabs molt, which could ultimately have an impact on soft shell fisheries.
Publications
- Henry, R.P., S. Gehnrich, D. Weihrauch, D. W. Towle. 2003. Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas. Comp. Biochem. Physiol. 136A:243-258.
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Progress 01/01/02 to 12/31/02
Outputs
The results of the past year's work were impressive. Eyestalk ablation (ESA) resulted in an increase in gill carbonic anhydrase (CA) activity even when there was no low salinity stimulus present. In Callinectes sapidus acclimated to 35 ppt, a salinity in which CA is minimally expressed, ESA alone caused a threefold increase in CA activity by 48 hr post-treatment. Additionally, if crabs were treated with ESA at 35 ppt and then transferred to 28 ppt, a salinity change of small enough proportion that it normally does not result in any CA induction, CA activity was induced by over eight fold over a 48 hr time period. And finally, if crabs were treated with ESA at 35 ppt and transferred to 15 ppt, a salinity change that normally results in an 8-10 fold increase in CA activity, this effect was potentiated by about 30%. These changes in CA induction occurred exclusively in the posterior, ion transporting gills; CA activity in the anterior, respiratory gills was unchanged for
any of the experimental treatments. These results led us to postulate the existence of a CA gene repressor, present in the eyestalk at high salinity. The action of this repressor is removed upon exposure to low salinity, allowing CA induction to occur. This idea was further supported by an experiment in which intact blue crabs, acclimated to 35 ppt, were given an injection of eyestalk extract and then transferred to 15 ppt for 48 hr. The normal salinity-mediated induction of CA was reduced by about 50%. We will now turn our attention to what controls the expression of the repressor itself.
Impacts
These are the first tangible results in the area of putative neuroendocrine control of environmentally mediated gene expression of transport-related enzymes. The results, which indicated control by a molecular "off" switch, which is removed as a result of the environmental stimulus, suggest the presence of a mechanism that may be similar to that of the control of molting and growth in crabs. If this turns out to be the case, this research will shed light not only on salinity adaptations (which determine population size through habitat range and utilization), but it may also lead to new understanding of the molt and post-molt calcification process (which may have an impact on the soft-shell crab industry).
Publications
- Skaggs, H.S. and R.P. (2002)Henry Inhibition of carbonic anhydrase in the gills of two euryhaline crabs, Callinectes sapidus and Carcinus maenas, by heavy metals. Comp. Biochem. Physiol. 133C:605-612.
- Henry, R.P., E.E. Garrelts, M.M. McCarty, and D.W. Towle (2002) Differential induction of branchial carbonic anhydrase and Na/K ATPase activity in the euryhaline crab, Carcinus maenas, in response to low salinity exposure. J. Exp. Zool. 292:595-603.
- Chadwick, M.A., H. Hunter, J.W. Feminella, and R.P. Henry (2002) Salt and water balance in Hexagenia limbata (Ephemeroptera:Ephemridae) when exposed to brackish water. Fla. Ent. 85:653-654.
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Progress 01/01/01 to 12/31/01
Outputs
The control of the expression of the enzyme carbonic anhydrase (CA), a critical enzyme in salt and water balance in euryhaline crustaceans, was examined in three species: Callinectes sapidus, Carcinus maenas, and Cancer irroratus. Exposure to low salinity induces a 10 fold increase in CA activity in the gills, the organ of salt transport. This occurs only in the posterior, ion-transporting gills of euryhaline species; no CA induction is seen in any of the gills of stenohaline, marine species. CA induction is controlled at the transcriptional level: levels of mRNA for CA increase at 24-48 hr after low salinity exposure, and levels of protein-specific CA activity increase immediately thereafter, at 48-72 hr after exposure. No increases in either CA mRNA or CA activity were seen in control tissues, the anterior gills that are specialized for respiratory gas exchange, in any species or under any experimental conditions. CA induction may be initiated by cell swelling. In
low salinity, cells take up water and increase in volume. They then initiate a volume regulatory response in which intracellular osmolytes (mostly amino acids) are released from the cell into the hemolymph. This process takes place immediately before the increase in CA mRNA and may serve as the initial signal in the pathway of CA gene expression. CA expression appears to be under negative control by a repressor substance found in the eyestalk, the major endocrine complex of crustaceans. Removal of the eyestalks (eyestalk ablation, or ESA) results in a 50-100% increase in CA activity in crabs acclimated to high salinity and given no low salinity exposure. ESA also appears to potentiate the low salinity-stimulated induction of CA. Crabs acclimated to high salinity, treated with ESA, and transferred to low salinity, showed a 28% increase in CA activity over intact crabs.
Impacts
This research will help us understand how commercially important species, such as the blue crab, can adapt to low environmental salinity and thus take advantage of the nutrient-rich habitat of the estuary. Furthermore, since many invasive species are also euryhaline, this work will provide evidence of how non-endemic species, such as the green crab, invade new environments and disrupt the existing ecological equilibrium.
Publications
- Perry, H.M., C. Trigg, K. Larsen, J. Freeman, M. Erickson, and R. Henry (2001) Calcium concentration in seawater and exoskeletal calcification in the blue crab, Callinectes sapidus. Aquaculture 34:197-208.
- Henry, R.P., M. Campoverde, and D.W. Borst (2000) Effects of eyestalk ablation on carbonic anhydrase activity in the gills of Carcinus maenas and Cancer irroratus. Bull. Mt. Desert Island Biol. Lab. 39:21-22.
- Gehnrich, S.C., D. Brooks, D. Weihrauch, D. Towle, and R. Henry (2000) Carbonic anhydrase in the hypodermis of the shore crab, Carcinus maenas, and its role in the post-molt calcification of the cuticle. Bull. Mt. Desert Island Biol. Lab. 39:26-27.
- Gehnrich, S.C., D. Brooks, D. Weihrauch, D. Towle, and R. Henry (2000) Alterations in carbonic anhydrase gene expression during low salinity adaptation in the shore crab, Carcinus maenas. Bull. Mt. Desert Island Biol. Lab. 39:28-29.
- Henry, R.P., E.E. Garrelts, M.M. McCarty, and D.W. Towle (1999) Salinity adaptations in the euryhaline green crab, Carcinus maenas. Bull. Mt. Desert Island Biol. Lab. 38:55.
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