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First published online September 9, 2005
Journal of Experimental Biology 208, 3543-3551 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01794

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Environmental hypoxia influences hemoglobin subunit composition in the branchiopod crustacean Triops longicaudatus

J. A. Guadagnoli^1,3, A. M. Braun^2,3, S. P. Roberts³ and C. L. Reiber^3,*

¹ College of Osteopathic Medicine, Touro University, Nevada, Henderson, NV 89014, USA
² Biology Department, Dominican University, Chicago, IL 60305, USA
³ Department of Biological Sciences, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA

View larger version (14K): [in a new window]   Fig. 1. Spectral analysis of Triops hemolymph fractions. The fraction with the greatest protein absorbance peak (280 nm) coincides with the fraction having the greatest Hb absorbance peak (415 nm). AU, arbitrary units.

View larger version (17K): [in a new window]   Fig. 2. Differences in hemolymph protein concentration between normoxic- and hypoxic-reared adult females. Hypoxic-reared females have significantly more Hb than normoxic-reared individuals (*P<0.001).

View larger version (17K): [in a new window]   Fig. 3. Changes in hemolymph protein concentration when Triops were transferred to different environmental partial oxygen pressures. (A) Normoxic-reared females transferred to hypoxia. Pooled samples from 1-3 and 4-7 days after hypoxic transfer. (B) Hypoxic-reared females transferred to normoxia. Pooled samples from 1-6 and 7-14 days after normoxic transfer (*P<0.05).

View larger version (82K): [in a new window]   Fig. 4. Representative one-dimensional gels of changes in Hb subunit expression with time spent in either a normoxic or hypoxic environment. (A) Variation in Hb isoforms in normoxic-reared control (NC), hypoxic-reared control (HC) and hypoxic-transferred animals [HTD = hypoxic transfer day (n)]. (B) Variation in Hb isoforms in NC, HC and normoxic-transferred animals [NTD = normoxic transfer day (n)].

View larger version (27K): [in a new window]   Fig. 5. ImageQuant Analysis of changes in Hb subunit isoforms in animals reared in normoxia and transferred to hypoxia: (A) Hb; (B) Hbß; (C) Hb and (D) Hb. The intensity of each band (Hb, Hbß, Hb and Hb) is expressed as a percentage of the total intensity of all the bands in each lane. Asterisks indicate significantly different from normoxic-reared control (NC) and hypoxic transfer day 1 (HTD1) (P<0.001).

View larger version (92K): [in a new window]   Fig. 6. Two-dimensional gel electrophoresis of the hypoxic induction of Triops Hb. Normoxic-reared (NR), hypoxic transfer day 2 and 4 (HTD2, HTD4) and hypoxic-reared (HR). A train of spots corresponded with the molecular masses of Hb and Hb; therefore, more than one of the spots was removed and sequenced for comparison. The lettered circled spots were selected for NH2-terminal amino acid sequencing.

View larger version (53K): [in a new window]   Fig. 7. (A) An example of a 2D-DIGE gel obtained by combining samples of normoxic and hypoxic Hb on the same gel. The gel was scanned at the appropriate wavelength for each fluor. Top panel, combined scan; lower two panels are the separated scans. (B) An example of DeCyder analysis of spots. Areas outlined in blue represent an increase of at least 2.5 times normoxic values. The spot enclosed in pink is the specific area being analyzed by DeCyder software. The lower panels demonstrate the difference in volume between the spot on the normoxic vs the hypoxic gel.