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First published online September 19, 2006
Journal of Experimental Biology 209, 3862-3872 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02425

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Identification and developmental expression of mRNAs encoding crustacean cardioactive peptide (CCAP) in decapod crustaceans

J. S. Chung¹, D. C. Wilcockson², N. Zmora¹, Y. Zohar¹, H. Dircksen³ and S. G. Webster^2,*

¹ Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA,
² School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, Wales, UK
³ Department of Zoology, Stockholm University, Svante Arrhenius väg 14, S-106 Stockholm, Sweden

View larger version (48K): [in a new window]   Fig. 1. Alignments and comparisons of deduced amino acid sequences of CCAP precursor peptides from Callinectes sapidus, Carcinus maenas, Homarus gammarus and Orconectes immunis. Identical and similar amino acids are in black and grey shaded boxes, respectively. SP, signal peptide; CCAP AP1, 2, crustacean cardioactive peptide associated peptides 1, 2; CCAP, crustacean cardioactive peptide, CCAP AP3, 4, crustacean cardioactive peptide associated peptides 3, 4. Gaps (-) have been introduced to maximise sequence identities.

View larger version (18K): [in a new window]   Fig. 2. Expression of crustacean cardioactive peptide (CCAP) mRNA during embryonic development of Carcinus maenas. Three to 11 independent measurements of different embryo batches were made at each developmental stage. Error bars indicate +1 s.e.m. Developmental stages were defined according to (Chung and Webster, 2004); additionally, the percentage development is indicated in brackets. During early developmental stages (yolk-limb buds), very low, but significant numbers of transcripts were observed, which are shown in brackets.

View larger version (93K): [in a new window]   Fig. 3. Developmental profiles of crustacean cardioactive peptide (CCAP)-expressing neural structures during embryonic development of Carcinus maenas. All confocal images have been stacked (30-32 slices; distance between slices: 0.8-2 µm) and flattened. (A) Lateral view of embryo at 70% development. Arrow points to the first immunoreactive (IR) structures seen on the dorsal side of the embryo corresponding to the position of the heart, which by this stage of development, has a regular beat (y, yolk sac; e, eye) (B) Ventral view of embryo at 70% development, showing bilaterally symmetrical IR structures, probably corresponding to segmental nerves (upper arrow) and projections adjacent to the heart (lower arrow). (C) Dorsal view of embryo at 80% development. Small arrow points to anteriorly projecting IR structure which is probably the developing anterior ramification. At this time IR structures corresponding to the segmental nerves (sn) and thoracic ganglion (tg) become visible. (D) Dorsal view of embryo at 85% development. Small arrows point to a series of prominent varicosities in the axons forming part of the developing thoracic ganglion. At this time the anterior ramification (ar) becomes digitate. (E) Dorsal view of embryo at 100% development showing IR structures associated with the segmental nerves adjacent to the anterior ramifications. (F) Dorsal view of IR structures associated with the thoracic ganglion at 100% development. Contralaterally projecting axons (small arrows) can be observed and weakly IR structures probably corresponding to perikarya (large arrows) can be seen. The developing pericardial organs (po) become prominent at this stage. (G) Dorsolateral view of thoracic ganglion at 100% development. This image has been rotated to show three pairs of weakly IR perikarya (arrows, asterisks). (H) Lateral view of embryo at 85-90% development, showing a single IR axon within a segmental nerve (sn) arising from the thoracic ganglion (tg), and a second, anteriorly projecting axon within a segmental nerve. The ring-like structure of the developing pericardial organ (po) is prominent in this orientation. Scale bars, 50 µm.

View larger version (24K): [in a new window]   Fig. 4. Expression of crustacean cardioactive peptide (CCAP) mRNA in Carcinus maenas tissues. Inset panel shows 430 bp PCR product (using CCAP F1, R1 gene-specific primers) amplified from cDNA derived from reverse transcription of 100 ng total RNA from eyestalk (Es), cerebral ganglion (Cg), circum-oesophageal commissure (Co), thoracic ganglion (Tg), heart (H), hepatopancreas (He), testis (Te) and ovary (Ov) tissues. Numbers to right of panel are sizes of ladder markers (bp). Histogram shows levels of CCAP mRNA from quantitative RT-PCR expressed as copies per µg RNA for Es (N=14), Cg (N=8) and Tg (N=8). Error bars indicate +1 s.e.m. Eyestalk tissues contain significantly more CCAP mRNA than cerebral ganglia (*asterisk, P<0.05, Dunn's multiple comparison).

View larger version (31K): [in a new window]   Fig. 5. Transcription profiles for crustacean cardioactive peptide (CCAP) mRNA during the moult cycle of Carcinus (A,C,E) and Callinectes (B,D,F). Data are shown as un-normalized (copy no. per thoracic ganglion; A,B), normalized against total RNA (copy no. µg-1 RNA x106; C,D), and normalized against a housekeeping gene, arginine kinase (AK; copy no. CCAP mRNAx103/106 copies of AK mRNA; E,F). For Carcinus, N=5-11 independent samples were taken at each moult stage. For Callinectes, N=8-10. Error bars indicate +1 s.e.m.

View larger version (18K): [in a new window]   Fig. 6. The effect of 1 h exposure of Callinectes to severe hypoxia (0.5% O2) upon crustacean cardioactive peptide (CCAP) and arginine kinase (AK) expression in eyestalk tissues (A) and the thoracic ganglion (B), expressed as copy no. per eyestalk and per ganglion, respectively. Open bars indicate expression levels at t=0, filled bars at t=1 h (N=6). Error bars indicate +1 s.e.m. Asterisk indicates significant (P<0.05, Students t-test) reduction of mean CCAP levels after exposure to hypoxic conditions.

View larger version (14K): [in a new window]   Fig. 7. Scaled schematic of crustacean cardioactive peptide (CCAP) precursor peptide structures of crustaceans (Callinectes sapidus, Carcinus maenas, Homarus gammarus, Orconectes immunis) compared with those of insects (Periplaneta americana, Manduca sexta, Drosophila melanogaster). Black bar represents CCAP; pale grey bar, amidation site; dark grey bars, potential dibasic, tribasic and tetrabasic cleavage sites. Numbers on left refer to unprocessed sizes of precursors, those within boxes, to the various peptides that may be potentially generated following cleavage.