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First published online June 16, 2005
Journal of Experimental Biology 208, 2467-2474 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01667

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Evolution of novel functions: cryptocyanin helps build new exoskeleton in Cancer magister

N. B. Terwilliger^1,*, M. C. Ryan¹ and D. Towle²

¹ Oregon Institute of Marine Biology, University of Oregon, Box 5389, Charleston, OR 97420, USA
² Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA

View larger version (22K): [in a new window]   Fig. 1. Cryptocyanin and hemocyanin protein levels in the hemolymph during sequential molt cycles of Cancer magister. Instar times expressed as % molt cycle, adjusted to average duration for that instar stage (2nd instar molt cycle averaged 16 days, while 6th instar averaged 30 days). Hemolymph samples were run on non-denaturing, non-dissociating pH 7.4 PAGE. Protein concentrations were determined from image analysis of gels using standard curves of purified hemocyanin and cryptocyanin of C. magister. Data expressed as means ±1 S.E.M. (2nd instar, N=13; 3rd instar, N=23; 4th instar, N=23; 5th instar, N=16; 6th instar, N=5). Arrows indicate ecdysis.

View larger version (13K): [in a new window]   Fig. 2. Cryptocyanin and hemocyanin mRNA expression in C. magister hepatopancreas during one molt cycle. Fifth-instar juvenile cohort, ecdysis on day 0 and day 28. Real-time quantitative PCR data from three target mRNAs: cryptocyanin, hemocyanin and tubulin. mRNA levels of cryptocyanin and hemocyanin were normalized to that of tubulin mRNA, which was constant throughout the molt cycle. (A) Hemocyanin mRNA/tubulin mRNA (x); (B) cryptocyanin mRNA/tubulin mRNA (x), hemocyanin mRNA/tubulin mRNA (x). Note that the y-axis scale in B is 10-fold greater than in A. Two crabs sampled per time point.

View larger version (86K): [in a new window]   Fig. 3. (A) Hepatopancreas and adnexal tissue of premolt C. magister stained with hematoxylin and eosin. Abbreviations: h, hepatopancreas tubules; b, B cells; f, F cells; r, R cells; m, muscle; rc, reserve cells in connective tissue; p, pigment granules. (B,C) In situ hybridization. Cell-specific expression of cryptocyanin and hemocyanin mRNA in hepatopancreas tissue of premolt C. magister. (B) Hemocyanin-specific antisense probes reveal that hemocyanin mRNA is expressed in R cells of hepatopancreas tubules. Hemocyanin sense probes show no reactivity. (C) Cryptocyanin-specific antisense probe shows extremely strong reactivity in R cells of hepatopancreas. Same tissue as Fig. 2B, 10 µm distant. Cryptocyanin sense probe shows no reactivity. Scale bar: A=100 µm; B,C=300 µm.

View larger version (120K): [in a new window]   Fig. 4. Immunohistology of C. magister. Tissues oriented with external surface of crab on right; counterstained with hematoxylin. (A) Late premolt crab incubated with C. magister anti-cryptocyanin monoclonal antibody shows cryptocyanin staining brown in subepidermal connective tissue (ct), elongated epidermal cells (e) and newly secreted exocuticular layer (ex). Reserve cells (rc), old exocuticle (oex), old endocuticle (oen). (B) 18 h postmolt crab shows immunoreactivity to cryptocyanin in new endocuticle. Circle indicates area enlarged in C. (C) Area of new exoskeleton enlarged 10x from B. Newly formed endocuticle (en) is immunoreactive to cryptocyanin; new exocuticle (ex) is now immunonegative. Scale bar: A=70 µm; B=100 µm; C=10 µm.

View larger version (104K): [in a new window]   Fig. 5. Reserve cells (rc) show golden to dark brown immunoreactivity to cryptocyanin antibody in 30 min postmolt crab. Hepatopancreas (h), new exocuticle (ex). New endocuticular layer has not yet formed. Counterstained with hematoxylin. Scale bar=50 µm.

View larger version (23K): [in a new window]   Fig. 6. Eyestalk ablation affects cryptocyanin patterns in hemolymph. Data expressed as in Fig. 1. Arrows indicate ecdysis. (A) Hemolymph proteins follow normal patterns in the initial post-ablate molt cycle. In the subsequent cycle, cryptocyanin does not reappear in the hemolymph, in contrast to hemocyanin. Eyestalks were ablated on day 2, 6th instar (N=7). Shortened molt cycle of ablated crabs averaged 21 days. (B) Hemolymph proteins in control crabs (N=6). Molt cycle averaged 32 days.