Bombesin-like immunoreactive material in the gut, and the effect of bombesin on the stomach circulatory system of an elasmobranch fish, Squalus acanthias

The distribution, nature and amount of bombesin-like immunoreactivity (IR) in the gastrointestinal canal and its afferent vessels was investigated in the spiny dogfish (Squalus acanthias) together with the in vitro effect of synthetic bombesin on perfusion flow through the vascularly perfused dogfish stomach. Nerve fibres showing bombesin-like IR frequently occurred in the walls of the anterior mesenteric and coeliac arteries and the intrinsic vessels of the gut. Chromatographic studies revealed that multiple peaks of bombesin-like IR material were present in extracts of the spiny dogfish gastrointestinal vessels. Bombesin-like IR was also present in muscle and mucosal layers of the gut with higher levels in muscle compared with mucosa, and higher levels in the stomach than in the intestine and the rectum. Exogenous bombesin increased the flow through the vasculary perfused spiny dogfish stomach in a dose-dependent manner. Studies with tetrodotoxin and atropine showed that bombesin probably exerts its effect directly on the vascular musculature. It is concluded from this study that bombesin-like material is present in nerves innervating the gut circulatory system of the spiny dogfish. Bombesin may affect the blood-flow to the gastrointestinal canal, possibly via a direct effect on vascular smooth muscle.     

Rapid miocene-pliocene dispersal and evolution of Mediterranean rajid fauna as inferred by mitochondrial gene variation

Rajidae (colloquially known as skates and rays) experienced multiple and parallel adaptive radiations allowing high species diversity and great differences of species composition between regional faunas. Nevertheless, they show considerable conservation of bio-ecological, morphological and reproductive traits. The evolutionary history and dispersal of North-east Atlantic and Mediterranean rajid fauna were investigated throughout the sequence analysis of the control region and 16S rDNA mitochondrial genes. Molecular estimates of divergence times indicated recent origin and rapid dispersal of the present species. Compared with the ancient origin of the family (Late Cretaceous), the present species diversity arose in a relatively narrow time-window (12 Myr) from Middle Miocene to Early Pleistocene, likely by speciation processes related to dramatic geological and climatic events in the Mediterranean. Nucleotide substitution rates and phylogenetic relationships indicated Mediterranean endemic skates derived from sister species with wider distribution during Late Pliocene-Pleistocene. Skate phylogeny and systematics obtained using mitochondrial gene variation were largely consistent with those based on morpho-anatomical data.     

Behaviour of fish by-catch in the mouth of a crustacean trawl

The behaviour of fish by-catch was recorded and characterized by in situ observations in the mouth of a crustacean trawl using an underwater camera system with artificial light, at depths between 106 and 461 m, along the central coast of Chile. The groups or species studied were rattails (family Macrouridae), Chilean hake Merluccius gayi gayi, sharks (orders Carcharhiniformes and Squaliformes), skates (family Rajidae), flatfishes (genus Hippoglossina) and small benthopelagic and demersal fishes (orders Osmeriformes, Stomiiformes, Gadiformes, Ophidiiformes and Perciformes). The fish behaviour was categorized in terms of (1) position in the water column, (2) initial orientation with respect to the trawl, (3) locomotion and (4) swimming speed with respect to the trawl. Rattails, sharks, skates and flatfishes were passive in response to the trawl and showed similar behavioural patterns, with most fishes observed sitting or touching the bottom with no swimming or other activity. Merluccius gayi gayi was the most active species, displaying a wide combination of behavioural responses when the trawl approached. This species showed several behavioural patterns, mainly characterized by swimming forward at variable speed. A fraction of small bentho-pelagic and demersal fishes also showed an active behaviour but always at lower speed than the trawl. The species-specific differences in behaviour in the mouth of the trawl suggest that improvements at the level of the footrope can be made to reduce by-catch, especially of passive species.     

The synarcual cartilage of batoids with emphasis on the synarcual of Rajidae

Comparison of embryonic specimens with juvenile and mature specimens of other skates indicates that the relative developmental sequence of events is maintained among several taxa within larger clades. However, there is a fundamental difference between the pattern of chondrification and the pattern of calcification in skates. Early in ontogeny a short synarcual surrounds the first free vertebral centrum. Additional neural arch segments are incorporated from anterior to posterior and the relative length of the synarcual cartilage to total length of the body normalizes early. A secondary direction of chondrification, from ventral to dorsal, is also present. Juveniles and subadults show that synarcual calcification is relatively late compared to the calcification of other regions of the skeleton and proceeds from lateral to medial. Comparison with extinct taxa also indicates that there is a decrease in vertebral centrum involvement with the synarcual cartilage over the evolutionary history of the clade. Results from exploratory analyses of morphospace and taxonomy reveal that phylogeny explains part, but not all, of the data on the synarcual in Rajidae. There is evidence of individual and ontogenetic variation among all species of skates examined, however, phylogenetically informative variation prevails. Comparison with other batoids demonstrates a trend where the number of vertebral centra flanked by the synarcual cartilage decreases among more derived taxa indicating a high degree of convergent morphology among batoids with potential functional significance. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Species composition of skates (Rajidae) in commercial fisheries around the British Isles and their discarding patterns

Recent regulations have required European nations to report commercial landings of Rajidae (skates) to species level since 2008. Morphological similarities between some species, variability in colouration and regional differences in common names may compromise the accuracy of some of these data. An increased proportion of rajid landings reported by the U.K. (England, Wales and Northern Ireland) are now reported to species level (42% in 2008, rising to 92% in 2010). Recent landings (2007-2010) of Rajidae by the U.K. indicated that the majority of reported landings were made by otter trawl (55·9%), tangle and gillnet (18·7%) and beam trawl (15·5%). Approximately 70% of recent landings originated from four ICES Divisions: the Irish Sea (VIIa), western English Channel (VIIe), Bristol Channel (VIIf) and southern North Sea (IVc). Recent species-specific landings of Rajidae are appraised in terms of the species reported and the overall composition, and potential problems identified. Data from observer trips have been used to estimate the species composition of Rajidae taken in some of the main commercial fisheries operating around the British Isles, and these data are compared to landings. Although there was typically broad agreement between these data sets in terms of the main species landed, misidentification issues were apparent and Rajidae with highly patchy distributions may be under-represented in observer data. Data from observer trips were also used to examine the discard and retention pattern. Most rajid species were first retained from total lengths, L(T) , of 27-34 cm, with 50% retention occurring at between 49 and 51 cm and near-full retention at L(T) of 60-67 cm. Beam trawls captured a higher proportion of smaller individuals, whilst gillnets (>150 mm mesh size) caught proportionally more larger rajids.     

Multiple paternity analysis in the thornback ray Raja clavata L

Skates (Rajidae) are characterized by slow growth rate, low fecundity, and late maturity and are thus considered to be vulnerable to exploitation. Although understanding mating systems and behavior are important for long-term conservation and fisheries management, this aspect of life history is poorly understood in skates. Using 5 highly polymorphic microsatellite loci, we analyzed egg clutches collected from 4 female Raja clavata captured in the wild to test for multiple paternity. Using the reconstructed multilocus genotypes method to explain the progeny genotype array, we showed that all 4 clutches were sired by a minimum of 4-6 fathers and, thus, female thornback rays are polyandrous. Whether polyandry in R. clavata is natural or a consequence of overexploitation remains uncertain. This is the first report of multiple paternity in a rajiform species and any oviparous elasmobranch.     

Immunohistochemical localization of bombesin-like peptides in the digestive tract of the bowfin,Amia calva

1. The distribution of bombesin-like immunoreactivity was determined in the gastrointestinal tract of the primitive holostean fish, the bowfin (Amia calva) using immunocytochemistry.2. Immunostaining using two different antisera raised against frog skin bombesin revealed a population of apparent endocrine cells containing bombesin-like immunoreactivity in the epithelium of the antral mucosa in the stomach.3. No bombesin-containing endocrine cells were present in any other segment of the gut. Bombesinergic nerves were not observed anywhere in the bowfin gastrointestinal tract.4. The antral bombesin endocrine cells were of the open type and were distributed diffusely from the base to the tips of antral glands, with some tendency to cluster near the base of the glands.5. These results suggest that a bombesin-like peptide may play an endocrine role in control of gastric functions such as regulation of acid secretion and gastric motility. These results support the hypothesis that bombesin serves a role in bony fish analogous to the role of antral cholecystokinin in amphibia and antral gastrin in amniotes.     

Molecular barcoding of skates (Chondrichthyes: Rajidae) from the southern Northeast Atlantic

Serra‐Pereira, B., Moura, T., Griffiths, A. M., Gordo, L. S. & Figueiredo, I. (2010). Molecular barcoding of skates (Chondrichthyes: Rajidae) from the southern Northeast Atlantic. —Zoologica Scripta, 40, 76–84. Due to their vulnerability to fishing pressure, many species of skate (Rajidae) in the Northeast Atlantic are undergoing declines in abundance. The assessment of stock status and subsequent proposal of management measures are often complicated by high levels of species diversity and endemism, coupled with morphological and ecological conservatism, which makes distinguishing between species difficult. To improve the identification of skates and investigate the phylogenetic position of endemic species the cytochrome c oxidase subunit I (COI) was sequenced in 12 species (Dipturus oxyrinchus, Leucoraja naevus, Leucoraja circularis, Neoraja iberica, Raja brachyura, Raja clavata, Raja maderensis, Raja microocellata, Raja miraletus, Raja montagui, Raja undulata, Rostroraja alba) inhabiting the Portuguese waters. Based on sequence divergence R. maderensis and R. clavata only differ by 1% of the 652 bp COI sequence, questioning the recognition of R. maderensis (considered to be endemic to Madeira and the Azores), as a reproductively isolated species. Otherwise, there was clear phylogenetic support for the different genera and all the remaining species, although the genetic divergence was low compared to other chordates. In particular, COI analysis allowed clear identification of the morphologically similar species R. brachyura and R. montagui.     

Distribution of bombesin-like peptides in the alimentary canal of several vertebrate species

The objective of this study was to quantitate and characterize the variants of bombesin-like immunoreactivity in the alimentary canal of the rat, rabbit, hawk, owl, dog, monkey and human. Bombesin-like immunoreactivity was found throughout the entire gastrointestinal tract of all species studied. In the rat, the highest concentration of bombesin-like immunoreactivity was found in the colon. Gel chromatography showed that bombesin-like immunoreactivity corresponded to gastrin-releasing peptide (GRP-27) and GRP-10. In the dog, the greatest concentration of bombesin-like immunoreactivity was observed in the mucosal layer of the fundus, whereas the concentration of bombesin-like immunoreactivity in the muscle layer of the dog did not vary significantly from region to region. Gel chromatography showed that bombesin-like immunoreactivity in the dog corresponded to GRP-27, bombesin, GRP-10, and a smaller fragment. In the human, the concentration of bombesin-like immunoreactivity did not vary significantly from region to region in the mucosal and muscular layers. Gel chromatography of human fundal mucosa showed that bombesin-like immunoreactivity peaks occur in the regions of GRP-27, bombesin and GRP-10. These findings substantiate the observation that bombesin-like peptides play a variety of roles in the regulation of gut function.     

A comparison of the central gastrointestinal antitransit effects of morphine and bombesin in the mouse

The gastrointestinal motor effects of centrally-given morphine and bombesin were compared in mice. Both compounds produced a dose-related decrease in the propulsion of a marker along the gut when given by the intracerebroventricular (i.c.v.) or intrathecal (i.th.) routes. Co-administration of the same compound by both routes was found to produce a marked increase in potency for morphine, but only a slight increase in potency for bombesin. Isobolographic analysis of the gut effects of these compounds revealed a multiplicative brain-spinal cord interaction for morphine, but an additive interaction for bombesin. These results are consistent with the interpretation that morphine can act at either the level of the brain or the spinal cord, activating independent pathways which ultimately converge to alter gut propulsion. In contrast, spinal bombesin requires communication to supraspinal sites in order for its gut effects to occur, suggesting activation of a common outflow pathway from the central nervous system.     

Molecular Organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural Features and Evolution of Piscine 5S rRNA Genes and Nontranscribed Intergenic Spacers

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Centrally-mediated bombesin effects on gastrointestinal motility

Administration of bombesin into the lateral cerebral ventricle (i.c.v.) of rats results in a dose-related delay in gastric emptying and small intestinal transit. Recordings of intestinal intraluminal pressure in this species show that the i.c.v. peptide produces a dose-related increase in the frequency of duodenal contractions, and a complex inhibitory/excitatory jejunal effect at low and high doses, respectively. Intrathecal (i.th.) or i.c., but not intraperitoneal (i.p.), bombesin produces a dose-related slowing of gastrointestinal and colonic transit in mice. I.c.v. bombesin is 13.5 and 3406 times more potent in inhibition of gastrointestinal transit than when given by the i.th. or i.p. routes, respectively. Similarly, the i.c.v. peptide is 1.54 and over 11000 times more potent in slowing mouse colonic transit than when given by the i.th. or i.p. routes, respectively. The substance P analogue, D-Arg1, D-Pro2, D-Trp7,9, Leu11-Substance P (DAPTL-SP)(a reported bombesin antagonist in vitro) was not effective in blocking the gastrointestinal transit effects of the peptide in vivo. Transection of the spinal cord at the level of the second thoracic vertebra (T2) eliminates the gastrointestinal and colonic effects of i.th., but not i.c.v. bombesin. Thus, bombesin can affect motor function of the gut via activity within the brain or spinal cord of rats and mice; the activity of the peptide when given at the supraspinal level depends on an intact vagus nerve and adrenal-pituitary axis, while the activity of the peptide given at the spinal level appears to depend on the integrity of ascending spinal-supraspinal pathways.     

Histological and immunohistochemical studies of the endocrine cells of the gastrointestinal mucosa of the toad (Bufo regularis)

Using histological and immunhistochemical techniques, nine endocrine cell types were observed in the mucosa of the gastrointestinal tract of the toad, Bufo regularis, viz. enterochromaffin, somatostatin, glucagon, pancreatic polypeptide (PP), secretin, gastric inhibitory peptide (GIP), gastrin-C-terminal pentapeptide (GTPP), neurotensin and bombesin cells. The enterochromaffin cells were distributed throughout the gastrointestinal tract except the rectum. Somatostatin, glucagon, PP, secretin, GIP and GTPP cells were observed both in the stomach and in the small intestine. Neurotensin cells were seen only in the ileum and bombesin cells only in the pyloric and antral parts of the stomach. Immunostaining of consecutive sections did not reveal more than one polypeptide hormone in any of these cell types. It is concluded from the present results that the toad gastrointestinal mucosa contains endocrine cell types that are more or less homologous to those in the mammal alimentary tract, though some of them exhibit a different topographic distribution.     

Histological and immunohistochemical studies of the endocrine cells of the gastrointestinal mucosa of the toad (Bufo regularis)

Summary Using histological and immunhistochemical techniques, nine endocrine cell types were observed in the mucosa of the gastrointestinal tract of the toad,Bufo regularis, viz. enterochromaffin, somatostatin, glucagon, pancreatic polypeptide (PP), secretin, gastric inhibitory peptide (GIP), gastrin-C-terminal pentapeptide (GTPP), neurotensin and bombesin cells. The enterochromaffin cells were distributed throughout the gastrointestinal tract except the rectum. Somatostatin, glucagon, PP, secretin, GIP and GTPP cells were observed both in the ileum and bombesin cells only in the pyloric and antral parts of the stomach. Immunostaining of consecutive sections did not reveal more than one polypeptide hormone in any of these cell types. It is concluded from the present results that the toad gastrointestinal mucosa contains endocrine cell types that are more or less homologous to those in the mammal alimentary tract, though some of them exhibit a different topographic distribution.     

The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge

Urea synthesis via the hepatic ornithine urea cycle (OUC) has been well described in elasmobranchs, but it is unknown whether OUC enzymes are also present in extrahepatic tissues. Muscle and liver urea, trimethylamine oxide (TMAO), and other organic osmolytes, as well as selected OUC enzymes (carbamoyl phosphate synthetase III, ornithine transcarbamoylase, arginase, and the accessory enzyme glutamine synthetase), were measured in adult little skates (Raja erinacea) exposed to 100% or 75% seawater for 5 d. Activities of all four OUC enzymes were detected in the muscle. There were no changes in muscle OUC activities in skates exposed to 75% seawater; however, arginase activity was significantly lower in the liver, compared to controls. Urea, TMAO, and several other osmolytes were significantly lower in the muscle of little skates exposed to 75% seawater, whereas only glycerophosphorylcholine was significantly lower in the liver. Urea excretion rates were twofold higher in skates exposed to 75% seawater. Taken together, these data suggest that a functional OUC may be present in the skeletal muscle tissues of R. erinacea. As well, enhanced urea excretion rates and the downregulation of the anchor OUC enzyme, arginase, in the liver may be critical in regulating tissue urea content under dilute-seawater stress.     

Bombesin evoked acetylcholine release from the guinea pig antrum

Bombesin induced contraction and acetylcholine (ACh) release of the longitudinal muscle strip of the guinea pig antrum were examined using the standard organ bath technique and the superfusion system. Bombesin increased frequency and tonus of rhythmic contraction in a dose dependent manner (10(-10)M - 10(-7)M). The effects of bombesin on frequency of contraction were not affected by atropine, propranolol, phentolamine, hexamethonium and tetrodotoxin. The effects on tonus, on the other hand, were significantly reduced by atropine, and the dose response curve to bombesin was shifted to the right. There was a remarkable increase of 3H-ACh release by the superfusion of bombesin (10(-8)M), which was almost completely abolished in Ca-free medium, but not affected by hexamethonium and tetrodotoxin. These results suggest that mechanism of bombesin effects on frequency is different from that on tonus; frequency response to bombesin is not dependent on autonomic nervous system but due to a direct effect on smooth muscle cells, whereas tonic response to the peptide is partly mediated by ACh release via a mechanism independent of sodium spike.     

Evidence that bombesin releases extragastric gastrin in man

Bombesin-induced gastrin release from extragastric sources has been investigated in two groups of patients without gastric antrum: 11 patients with total gastrectomy and 11 patients with subtotal (Billroth II) gastrectomy. A 30-min bombesin infusion (5 ng . kg-1 . min-1) caused a prompt significant gastrin increase (P less than 0.05) in both groups of patients. The gastrin response to bombesin was significantly (P less than 0.005) lower in patients without antral tissue than in the control group (n = 7). The individual peak gastrin responses, in totally (TG) and subtotally (SG) gastrectomized patients, were significantly over basal levels (TG: peak 100.3 +/- 12 vs. basal 62.8 +/- 9.1, P less than 0.005; SG: peak 96.9 +/- 9.4 vs. basal 72.4 +/- 6.8, P less than 0.001; pg/ml, mean +/- S.E.M.). These data indicate that bombesin acts not only on antral G cells, but on all gastrin cells in the gastrointestinal tract.     

Bombesin-induced stimulation of cardiac parasympathetic innervation

The central nervous system effects of bombesin on cardiovascular function were examined in conscious, freely-moving rats. Intracerebroventricular administration of bombesin elevated mean arterial pressure, reduced heart rate and inhibited cold-induced tachycardia. Adrenalectomy prevented bombesin-induced elevations of mean arterial pressure. In contrast, bombesin-induced bradycardia was neither adrenal-dependent nor a baroreceptor-mediated reflex response to increased arterial pressure. Systemic atropine methyl nitrate treatment attenuated bombesin-induced bradycardia, suggesting that bombesin acts within the central nervous system to stimulate cardiac vagal activity.     

Evolutionary transitions among egg-laying,live-bearing and maternal inputs in sharks and rays

Sharks and rays are thought to have a large number of independent origins of live-bearing. We examined evolutionary transitions to live-bearing and maternal input to embryos in this subclass by optimizing reproductive characters onto a composite phylogeny. Egg-laying (40 per cent of all species) is the likely ancestral reproductive mode for this clade, and there is evidence that live-bearing has evolved independently 9–10 times and maternal input 4–5 times. Most transitions (12–15) have been toward live-bearing with provisioning limited to yolk. These have occurred from egg-laying ancestors or live-bearing taxa that provide maternal input to embryos. Only 2–3 transitions have occurred in the other direction, i.e. away from yolk-only bearing. Egg-laying has evolved from live-bearing ancestors in skates, Rajidae (25 per cent of all species) and possibly in the zebra shark, Stegostoma fasciata. Thus, although there has been an overall trend toward the evolution of live-bearing in elesmobranchs, the evolution of additional maternal input has been extremely labile.