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First published online February 29, 2008
Journal of Experimental Biology 211, 978-988 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014423
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Ionoregulatory changes during metamorphosis and salinity exposure of juvenile sea lamprey (Petromyzon marinus L.)

Patrick Reis-Santos1,*, Stephen D. McCormick2,3 and Jonathan M. Wilson1,{dagger}

1 Laboratório de Ecofisiologia, Centro Interdiscplinar de Investigação Marinha e Ambiental (CIIMAR), Rua dos Bragas 289, 4050-123 Porto, Portugal
2 USGS, Conte Anadromous Fish Research Center, Turners Falls, MA 01376, USA
3 Department of Biology, University of Massachusetts, Amherst, MA 01003, USA


Figure 1
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  		Fig. 1. Plasma sodium (Na+) (mEq l–1) (A,B) and plasma chloride (Cl–) concentrations (µEq l–1) (C,D) in ammocoetes (white bars) and transformers (grey bars) of P. marinus. (A,C) Ammocoetes and transformers (stages 3–5) acclimated to freshwater (FW) and 25{per thousand} salinity and sampled from the Fort River, MA, USA (hatched bars). (B,D) Ammocoetes and transformers (stage >=6) acclimated to deionized water (DW), freshwater (FW) or saline water of 10, 20, 30 and 35{per thousand}. Bars with like characters are not significantly different (ANOVA; P<0.05). Upper and lower case characters are used for transformers and ammocoetes, respectively, in B and D. Asterisks indicate a significant difference from the ammocoetes at a given salinity.

 

Figure 2
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  		Fig. 2. Branchial Na+/K+-ATPase activity (µmol ADP mg–1 protein h–1) (A,B) and {alpha} subunit expression determined by immunoblotting (C,D) in ammocoetes (white bars) and transformers (grey bars) of P. marinus. (A,C) Ammocoetes and transformers (stages 3–5) acclimated to freshwater (FW) and 25{per thousand} salinity and sampled from the Fort River, MA, USA (hatched bars). (B,D) Ammocoetes and transformers (stage >=6) acclimated to deionized water (DW), FW or saline water of 10, 20, 30 and 35{per thousand}. Representative immunoblots of sea lamprey ammocoete and transformer gill probed with the {alpha} subunit antibody {alpha}RbNKA are shown above their respective graphs. Bars with like characters are not significantly different (ANOVA; P<0.05). Upper and lower case characters are used for transformers and ammocoetes, respectively, in B and D. Asterisks indicate a significant difference from the ammocoetes at a given salinity.

 

Figure 3
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  		Fig. 3. Branchial residual (ouabain-insensitive) activity (µmol ADP mg–1 protein h–1) in ammocoetes (white bars) and transformers (grey bars) of P. marinus. (A) Ammocoetes and transformers (stages 3–5) acclimated to freshwater (FW) and 25{per thousand} salinity and sampled from the Fort River, MA, USA (hatched bars). (B) Ammocoetes and transformers (stage >=6) acclimated to deionized water (DW), FW or saline water of 10, 20, 30 and 35{per thousand}. Bars with like characters are not significantly different (ANOVA; P<0.05). Asterisks indicate a significant difference from the ammocoetes at a given salinity.

 

Figure 4
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  		Fig. 4. H+-ATPase B subunit expression determined by immunoblotting in ammocoetes (white bars) and transformers (grey bars) of P. marinus. (A) Ammocoetes and transformers (stages 3–5) acclimated to freshwater (FW) and 25{per thousand} salinity and sampled from the Fort River, MA, USA (hatched bars). (B) Ammocoetes and transformers (stage >=6) acclimated to deionized water (DW), FW or a salinity of 10, 20, 30 and 35{per thousand}. Bars with like characters are not significantly different (ANOVA; P<0.05). Upper and lower case characters are used for transformers (TR) and ammocoetes (AM), respectively, in B. Representative immunoblots of sea lamprey ammocoete and transformer gill probed with the H+-ATPase B subunit antibody B2/BvA1 are shown above their respective bars.

 

Figure 5
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  		Fig. 5. Double immunofluorescence localization of H+-ATPase (green; A,A') and Na+/K+-ATPase (red; B,B') in the gills of a freshwater ammocoete of P. marinus with the corresponding merged image overlaid with DAPI nuclear staining (blue) and differential interference contrast (DIC) for orientation (C,C'). The area within the box has been enlarged 3x. Arrows indicate apical H+-ATPase labelling and arrowheads indicate basolateral Na+/K+-ATPase labelling. Scale bar, 50 µm.

 

Figure 6
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  		Fig. 6. Immunolocalization of Na+/K+-ATPase (red; A,B,C) and H+-ATPase (green; A',B',C') in gill filament sagital sections of transformers of P. marinus acclimated to freshwater (FW; A,A',A''), 10{per thousand} (B,B',B'') and 25 {per thousand} (C,C',C'') salinity. Images were merged with DAPI nuclear staining (blue) and differential interference contrast (DIC) images for orientation (A'',B'',C''). Scale bar, 50 µm.

 

Figure 7
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  		Fig. 7. Localization of Na+/K+-ATPase immunoreactive (IR) cells on the afferent edge of the gill filament of a freshwater transformer. Image capture conditions were optimized for (A) the cluster of strongly IR chloride cells (CCs) and (B) weakly IR cells indicated by arrowheads and arrows, respectively. (C) The corresponding merged image overlaid with DAPI nuclear staining (blue) and differential interference contrast (DIC) is given for orientation. Scale bar, 25 µm.

 

Figure 8
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  		Fig. 8. Indirect immunofluorescence double labelling of a gill filament sagittal sections of a Fort River P. marinus transformer for (A) carbonic anhydrase (green) or (D) H+-ATPase B subunit (green) double labelled for Na+/K+-ATPase {alpha} subunit (red; B and E, respectively) with the corresponding merged images with the overlaid with DAPI nuclear staining (blue) and differential interference contrast (DIC) images for orientation (C and F, respectively). Scale bar, 50 µm.

 




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