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First published online March 14, 2008
Journal of Experimental Biology 211, 1114-1119 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016758
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Aquaporins play a role in desiccation and freeze tolerance in larvae of the goldenrod gall fly, Eurosta solidaginis

Benjamin N. Philip*, Shu-Xia Yi, Michael A. Elnitsky and Richard E. Lee, Jr

Department of Zoology, Miami University, Oxford, OH 45056, USA


Figure 1
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  		Fig. 1. Overall protein staining with Ponceau S demonstrates that there is no difference in banding pattern between samples prepared at different temperatures. Protein samples were treated at either 95°C for 3 min or 60°C for 10 min before SDS-PAGE electrophoresis. Markers are in kDa.

 

Figure 2
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  		Fig. 2. Immunoidentification of (A) AQP2 (N=2), (B) AQP3 (N=3) and (C) AQP4 (N=2) in protein extracts of E. solidaginis larvae using mammalian antisera. Fifty micrograms of protein were loaded onto each lane. Larvae were acclimated for ~4 months at 4°C or –20°C. Desiccated larvae were kept at 0% relative humidity for 4 days. Below each band on these representative gels is the corresponding average relative abundance (RA), with the RA of 4°C treated samples set to 1.0.

 

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  		Fig. 3. Fluorescence micrographs of fat body (A–F), midgut (G–L) and salivary gland (M–P) treatments used to determine the role of aquaporins in freeze tolerance. The glycerol (A,G,M) and mercuric chloride control tissues (E,K) were kept at 4°C. The treated tissues were cooled from 4°C to –20°C over 2 h and left at –20°C for another 2 h. All tissues were frozen in Coast's solution (C,I,O), Coast's solution + 0.25 mol l–1 glycerol (B,H,N), Coast's solution + 0.25 mol l–1 glycerol + 0.2 mmol l–1 mercuric chloride (D,J,P) or Coast's solution + 0.25 mol l–1 glycerol + 0.2 mmol l–1 mercuric chloride + 2 mmol l–1 β-mercaptoethanol (F,L). The scale bar is 100 µm for J, M, N and O and 200 µm for all other micrographs.

 

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  		Fig. 4. The survival of (A) fat body and (B) midgut tissue samples decreased when they were frozen in the presence of an aquaporin inhibitor, mercuric chloride. Controls (held at 4°C for 4 h) were incubated in Coast's solution + 0.25 mol l–1 glycerol with or without 0.2 mmol l–1 HgCl2. The remaining groups, labelled Coast's, Coast's + glycerol (Coast's solution + 0.25 mol l–1 glycerol), mercuric chloride (Coast's solution + 0.25 mol l–1 glycerol + 0.2 mmol l–1 HgCl2) and β-mercaptoethanol (Coast's solution + 0.25 mol l–1 glycerol + 0.2 mmol l–1 HgCl2 + 2 mmol l–1 β-mercaptoethanol), were cooled from 4°C to –20°C over 2 h and held at –20°C for 2 h before cell viability was assessed (N=4 per treatment). Different letters signify a significant difference between mean cell survival among treatments (P<0.05).

 

Figure 5
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  		Fig. 5. The survival of frozen salivary glands was unaffected by the presence of an aquaporin inhibitor, mercuric chloride. The control (held at 4°C for 4 h) was incubated in Coast's solution + 0.25 mol l–1 glycerol. The remaining groups, labelled Coast's, Coast's + glycerol (Coast's solution + 0.25 mol l–1 glycerol) and mercuric chloride (Coast's solution + 0.25 mol l–1 glycerol + 0.2 mmol l–1 HgCl2), were cooled from 4°C to –20°C over 2 h and held at –20°C for 2 h before cell viability was assessed (N=4 per treatment). Different letters signify a significant difference between mean cell survival among treatments (P<0.05).

 

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© The Company of Biologists Ltd 2008