Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, <i>Cancer borealis</i>

SUMMARY Full-length cDNAs encoding crustacean cardioactive peptide (CCAP) were isolated from several decapod (brachyuran and astacuran) crustaceans: the blue crab Callinectes sapidus, green shore crab Carcinus maenas,European lobster Homarus gamarus and calico crayfish Orconectes immunis. The cDNAs encode open reading frames of 143 (brachyurans) and 139-140 (astacurans) amino acids. Apart from the predicted signal peptides(30-32 amino acids), the conceptually translated precursor codes for a single copy of CCAP and four other peptides that are extremely similar in terms of amino acid sequence within these species, but which clearly show divergence into brachyuran and astacuran groups. Expression patterns of CCAP mRNA and peptide were determined during embryonic development in Carcinususing quantitative RT-PCR and immunohistochemistry with whole-mount confocal microscopy, and showed that significant mRNA expression (at 50% embryonic development) preceded detectable levels of CCAP in the developing central nervous system (CNS; at 70% development). Subsequent CCAP gene expression dramatically increased during the late stages of embryogenesis (80-100%),coincident with developing immunopositive structures. In adult crabs, CCAP gene expression was detected exclusively in the eyestalk, brain and in particular the thoracic ganglia, in accord with the predominance of CCAP-containing cells in this tissue. Measurement of expression patterns of CCAP mRNA in Carcinus and Callinectes thoracic ganglia throughout the moult cycle revealed only modest changes, indicating that previously observed increases in CCAP peptide levels during premoult were not transcriptionally coupled. Severe hypoxic conditions resulted in rapid downregulation of CCAP transcription in the eyestalk, but not the thoracic ganglia in Callinectes, and thermal challenge did not change CCAP mRNA levels. These results offer the first tantalising glimpses of involvement of CCAP in environmental adaptation to extreme, yet biologically relevant stressors, and perhaps suggest that the CCAP-containing neurones in the eyestalk might be involved in adaptation to environmental stressors.

Section: Discussion Cdnas Encoding Ccap In Crustaceanssupporting

confidence: 67%

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

Chung

Wilcockson

Zmora

et al. 2006

“…the SG, PO and ACP) of C. productus (Hsu et al, 2004(Hsu et al, , 2005ade la Iglesia et al, 2005;Fu et al, 2005a,b;Messinger et al, 2005). Like those of other crustacean species (Christie et al, 1995a;Li et al, 2002aLi et al, , 2003, each of these sites has been shown to contain multiple neuroactive compounds. For example, using a combination of anatomical, mass spectrometric and molecular techniques, over 50 peptide hormones have been identified from the C. productus SG (Hsu et al, 2004(Hsu et al, , 2005ade la Iglesia et al, 2005;Fu et al, 2005a,b).…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crabCancer productus

Messinger

Kutz

et al. 2005

Self Cite

SUMMARY A club-shaped, tachykinin-immunopositive structure first described nearly two decades ago in the commissural ganglion (CoG) of three species of decapod crustaceans has remained enigmatic, as its function is unknown. Here, we use a combination of anatomical, mass spectrometric and electrophysiological techniques to address this issue in the crab Cancer productus. Immunohistochemistry using an antibody to the vertebrate tachykinin substance P shows that a homologous site exists in each CoG of this crab. Confocal microscopy reveals that its structure and organization are similar to those of known neuroendocrine organs. Based on its location in the anterior medial quadrant of the CoG, we have named this structure the anterior commissural organ (ACO). Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry shows that the ACO contains the peptide APSGFLGMRamide,commonly known as Cancer borealis tachykinin-related peptide Ia(CabTRP Ia). Using the same technique, we show that CabTRP Ia is also released into the hemolymph. As no tachykinin-like labeling is seen in any of the other known neuroendocrine sites of this species (i.e. the sinus gland, the pericardial organ and the anterior cardiac plexus), the ACO is a prime candidate to be the source of CabTRP Ia present in the circulatory system. Our electrophysiological studies indicate that one target of hemolymph-borne CabTRP Ia is the foregut musculature. Here, no direct CabTRP Ia innervation is present, yet several gastric mill and pyloric muscles are nonetheless modulated by hormonally relevant concentrations of the peptide. Collectively,our findings show that the C. productus ACO is a neuroendocrine organ providing hormonal CabTRP Ia modulation to the foregut musculature. Homologous structures in other decapods are hypothesized to function similarly.

“…C. productus was chosen for hemolymph analysis as neither of the classically ascribed Cancer neuroendocrine organs, i.e. the X-organ-sinus gland (XO-SG) system and the pericardial organ (PO), possess TRPs in this species (Fu et al, 2005), whereas in C. borealis, TRPs are present in the PO (Christie et al, 1995;Li et al, 2003) and in C. magister the distribution of TRPs is unknown. In hemolymph samples from two of the three crabs that were held without food, an abundant peak corresponding to that of CabTRP Ia (i.e.…”

Section: The Presence Of Cabtrp Ia In the Circulatory System Is Corrementioning

confidence: 99%

Midgut epithelial endocrine cells are a rich source of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and GYRKPPFNGSIFamide (Gly1-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer productus

Christie

Kutz-Naber

Stemmler

et al. 2007

Self Cite

-SIFamide) and APSGFLGMRamide [Cancer borealis tachykinin-related peptide Ia (CabTRP Ia)], respectively, both of which are known neuropeptides of Cancer species. Although the function of these midgut-derived peptides remains unknown, we found that both Gly 1 -SIFamide and CabTRP Ia were released when the midgut was exposed to highpotassium saline. In addition, CabTRP Ia was detectable in the hemolymph of crabs that had been held without food for several days, but not in that of fed animals, paralleling results that were attributed to TRP release from midgut endocrine cells in insects. Thus, one function that midgutderived CabTRP Ia may play in Cancer species is paracrine/hormonal control of feeding-related behavior, as has been postulated for TRPs released from homologous cells in insects.