Molecular forms of acetylcholinesterase from the skeletal muscle of the ammocoete of the lamprey Petromyzon marinus

1. We have studied the cholinesterase activity from the skeletal muscle of the ammocoete of the lamprey Petromyzon marinus. 2. On the basis of pharmacologic and kinetic criteria, we conclude that the enzyme is true acetylcholinesterase, not pseudocholinesterase. 3. The acetylcholinesterase was found to be present in both globular and asymmetric forms. 4. In contrast to muscle of adult spawning lamprey, where globular esterase is almost exclusively G4, we found that muscle from ammocoete also contains significant amounts of G1 and G2. 5. This difference may be related to the physiological states of the lamprey during the various stages of its life cycle.     

An electrophoretic and immunoelectrophoretic study of serum proteins during the life cycle of the lamprey, Petromyzon marinus L

Serum proteins of upstream migrants, parasitic adults, early and late metamorphosing animals, and ammocoetes of Petromyzon marinus L. were examined by polyacrylamide gel electrophoresis and crossed-immunoelectrophoresis. The electrophoretic patterns for two proteins, SDS-1 and CB-III, were found to change during the life cycle. quantitative measurements of these two proteins showed that they continued to increase until in the adult they together constituted approx. 85% of the total serum protein. Evidence was obtained for a protein present in ammocoetes and metamorphosing animals, but not in upstream migrants.     

Refinement of a molecular model for lamprey hemoglobin from Petromyzon marinus

A molecular model for the protein and ambient solvent of the complex of cyanide with methemoglobin V from the sea lamprey Petromyzon marinus yields an R-factor of 0.142 against X-ray diffraction data to 2.0 A resolution. The root-mean-square discrepancies from ideal bond length and angle are, respectively, 0.014 A and 1.5 degrees. Atoms that belong to planar groups deviate by 0.012 A from planes determined by a least-squares procedure. The average standard deviation for chiral volumes, peptide torsion angle and torsion angles of side-chains are 0.150 A3, 2.0 degrees and 19.4 degrees, respectively. The root-mean-square variation in the thermal parameters of bonded atoms of the polypeptide backbone is 1.21 A2; the variation in thermal parameters for side-chain atoms is 2.13 A2. The model includes multiple conformations for 11 side-chains of the 149 amino acid residues of the protein. We identify 231 locations as sites of water molecules in full or partial occupancy. The sum of occupancy factors for these sites is approximately 154, representing 28% of the 550 molecules of water within the crystallographic asymmetric unit. The environment of the heme in the cyanide complex of lamprey methemoglobin resembles the deoxy state of the mammalian tetramer. In particular, the bond between atom NE2 of the proximal histidine and the Fe lies 5.1 degrees from the normal of the heme plane. In deoxy- and carbonmonoxyhemoglobins, the deviations from the normal to the heme plane are 7 to 8 degrees and 1 degree, respectively. Furthermore, the inequality in the distance of atom CD2 of the proximal histidine from the pyrrole nitrogen of ring-C of the heme (distance = 3.29 A) and CE1 from the pyrrole nitrogen of ring-A (distance = 3.06 A) is characteristic of deoxyhemoglobin, not carbonmonoxyhemoglobin, where these distances are equal. Finally, a hydrogen bond exists between carbonyl 111 and the hydroxyl of tyrosine 149. The corresponding hydrogen link in the mammalian tetramer is central to the T to R state transition and is present in deoxyhemoglobin but absent in carbonmonoxyhemoglobin. We suggest that the low affinity of oxygen for lamprey hemoglobin may be a consequence of these T-state geometries.     

Shifting patterns of nitrogen excretion and amino acid catabolism capacity during the life cycle of the sea lamprey (Petromyzon marinus)

The jawless fish, the sea lamprey (Petromyzon marinus), spends part of its life as a burrow-dwelling, suspension-feeding larva (ammocoete) before undergoing a metamorphosis into a free swimming, parasitic juvenile that feeds on the blood of fishes. We predicted that animals in this juvenile, parasitic stage have a great capacity for catabolizing amino acids when large quantities of protein-rich blood are ingested. The sixfold to 20-fold greater ammonia excretion rates (J(Amm)) in postmetamorphic (nonfeeding) and parasitic lampreys compared with ammocoetes suggested that basal rates of amino acid catabolism increased following metamorphosis. This was likely due to a greater basal amino acid catabolizing capacity in which there was a sixfold higher hepatic glutamate dehydrogenase (GDH) activity in parasitic lampreys compared with ammocoetes. Immunoblotting also revealed that GDH quantity was 10-fold and threefold greater in parasitic lampreys than in ammocoetes and upstream migrant lampreys, respectively. Higher hepatic alanine and aspartate aminotransferase activities in the parasitic lampreys also suggested an enhanced amino acid catabolizing capacity in this life stage. In contrast to parasitic lampreys, the twofold larger free amino acid pool in the muscle of upstream migrant lampreys confirmed that this period of natural starvation is accompanied by a prominent proteolysis. Carbamoyl phosphate synthetase III was detected at low levels in the liver of parasitic and upstream migrant lampreys, but there was no evidence of extrahepatic (muscle, intestine) urea production via the ornithine urea cycle. However, detection of arginase activity and high concentrations of arginine in the liver at all life stages examined infers that arginine hydrolysis is an important source of urea. We conclude that metamorphosis is accompanied by a metabolic reorganization that increases the capacity of parasitic sea lampreys to catabolize intermittently large amino acid loads arising from the ingestion of protein rich blood from their prey/hosts. The subsequent generation of energy-rich carbon skeletons can then be oxidized or retained for glycogen and fatty acid synthesis, which are essential fuels for the upstream migratory and spawning phases of the sea lamprey's life cycle.     

The hemoglobin of the sea lamprey,Petromyzon marinus

The blood hemoglobin of the sea lamprey presents a curious mixture of primitive and highly specialized properties. Like muscle hemoglobin, it has a molecular weight of about 17,000, and apparently contains a single heme. Its isoelectric point is like that of a typical invertebrate hemoglobin. Its amino acid composition is partly characteristic of invertebrate) partly of vertebrate hemoglobins (Pedersen; Roche and Fontaine). In the present experiments, the oxygen equilibrium curve of this pigment was measured at several pH's. As expected, it is a rectangular hyperbola, the first such function to be observed in a vertebrate blood hemoglobin. Other hemoglobins known to possess this type of oxygen dissociation curve—those of vertebrate muscle, the worm Nippostrongylus, and the bot-fly larva—appear to serve primarily the function of oxygen storage rather than transport. Lamprey hemoglobin on the contrary is an efficient oxygen-transporting agent. It achieves this status by having, unlike muscle hemoglobin, a relatively low oxygen affinity, and a very large Bohr effect. In these properties it rivals the most effective vertebrate blood hemoglobins.     

Developmental transformations in a normal series of embryos of the sea lamprey Petromyzon marinus (Linnaeus)

Lamprey development is of interest to evolutionary biologists because it can inform our understanding of primitive vertebrate developmental patterns. In this study, we describe and illustrate some of the principle landmarks of organogenesis in the embryonic sea lamprey Petromyzon marinus L. at different chronological ages. We examined 63 fixed embryos spanning Piavis developmental stages 11-18+ (5-70 days postfertilization) by gross observation and histology. This period begins at late neurulation stages and ends with the formation of the larva (ammocoete). A significant difference with some previous accounts is that the anus develops not from a persistent blastopore, but by secondary canalization and proctodeum formation at the former site of the blastopore. Further, we show that the ciliated bands of the pharyngeal roof originate in the esophagus, distinguishing it from the intestine. We clarify the epithelialization of the gut, showing that the secondary gut cavity is progressively epithelialized from each end. We identify possible germ cells in the coelomic and cloacal walls. Balfour's "subnotochordal rod" is lacking in our specimens; we suggest that he may have misinterpreted the corpus adiposum. Our study is of potential value to the growing number of biologists interested in lamprey development and provides a character set that will be used : 1) in a phylogenetic study of vertebrate development, and 2) to prepare a staging series for the lamprey based on parsimony analysis.     

Genomic analysis of Hox clusters in the sea lamprey Petromyzon marinus

The sea lamprey Petromyzon marinus is among the most primitive of extant vertebrates. We are interested in the organization of its Hox gene clusters, because, as a close relative of the gnathostomes, this information would help to infer Hox cluster organization at the base of the gnathostome radiation. We have partially mapped the P. marinus Hox clusters using phage, cosmid, and P1 artificial chromosome libraries. Complete homeobox sequences were obtained for the 22 Hox genes recovered in the genomic library screens and analyzed for cognate group identity. We estimate that the clusters are somewhat larger than those of mammals (roughly 140 kbp vs. 105 kbp) but much smaller than the single Hox cluster of the cephalochordate amphioxus (at more than 260 kb). We never obtained more than three genes from any single cognate group from the genomic library screens, although it is unlikely that our screen was exhaustive, and therefore conclude that P. marinus has a total of either three or four Hox clusters. We also identify four highly conserved non-coding sequence motifs shared with higher vertebrates in a genomic comparison of Hox 10 genes.     

High efficiency of meiotic gynogenesis in sea lamprey Petromyzon marinus

Induction of androgenesis and gynogenesis by applying a pressure (PS) or heat shock (HS) to double the haploid chromosomal set results in progenies possessing only chromosomes from a single parent. This has never been accomplished in representatives of Agnatha. The objective of this study was to induce gynogenesis and androgenesis in sea lamprey Petromyzon marinus. For gynogenesis experiments, ultraviolet (UV)-irradiated sperm was used to activate sea lamprey eggs and HS or PS were applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation of immobilized sperm was performed for 1 min at 1,719 J m(-2). HS of 35+/-1 degrees C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the induction time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of pre-hatched embryos in comparison to 16 and 24 min after activation. In control groups, without shock treatment (no diploidization), there were no survivors. All deformed, gynogenetic embryos were confirmed to be haploids and died prior to burying themselves in the sand. We confirmed by flow cytometry that progenies produced using both shock methods surviving to the next stage, burying in the substrate, were diploid gynogenetic. For the androgenesis experiments, UV-irradiated eggs (1,719 J m(-2) for 1 min) were fertilized with non-treated sperm and HS was applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. HS of 35+/-1 degrees C was applied 110, 140, 170, 200, and 230 min after activation for 2 min. Low yields of androgens were obtained and all animals died within a week after hatching. These techniques will allow to establish meiotic gynogenetic lines of sea lamprey for determining sex differentiation in this species and to analyze its hormonal and environmental regulation.     

Changes in the proximate body composition of the landlocked sea lamprey Petromyzon marinus (L.) during larval life and metamorphosis

Estimates were made of the growth rates and proximate body composition of larval and metamorphosing P. marinus (L.) collected at various times of the year from Shelter Valley Creek, Lake Ontario. Analysis of length-frequency data indicates that the average duration of larval life was 6 years, with metamorphosis occurring predominantly in the length range above 13 cm. Increases in length were almost entirely restricted to the warmest months and did not take place during the final year of larval life. Three categories were thus recognized for the proximate analysis: ammocoetes <13 cm, ammocoetes > 13 cm and metamorphosing individuals. In ammocoetes <13 cm, seasonal differences were observed in the regression coefficients in the logarithmic relationships between wet weight and length and between each of water, lipid and ash and the wet weight. No such difference was found for the regressions between protein and wet weight. For a fixed length (9 cm), the wet weight varied only slightly during the year, although a small peak was seen in May. When considered on the basis of fixed weight (1 g), the relative amount of lipid deposited was greatest in May/July, coincident with a high diatom density. The water content followed an inverse pattern to that of lipid, while the protein and ash contents showed little seasonal variation and exhibited values lower than those normally found in teleosts. Throughout the last year of larval life, the animal stored a greater proportion of lipid, presumably to facilitate the energy demands of metamorphosis during which this food store underwent a marked reduction.     

Somatostatin concentrations in the pancreatic-intestinal tissues of the sea lamprey, Petromyzon marinus L., at various periods of its life cycle.

1. Somatostatin concentrations were measured in homogenates of the pancreas-intestinal tissues from each period of the life cycle of Petromyzon marinus using radioimmunoassay. 2. Levels were very low in larva (4.0 pg/mg wet weight) and in the first three stages of metamorphosis, but increased from stage 4 onwards and reached a high in upstream-migrating adults (210.0 ng/mg). 3. These data correlate well with our previous morphological and immunohistochemical observations on the morphogenesis of somatostatin-containing D-cells during the life cycle and indicate that the increased concentration of hormone accompanies the development of the endocrine pancreas in lampreys.     

Molecular evolution of the rhodopsin gene of marine lamprey,Petromyzon marinus

Visual pigment genes have been isolated from a marine lamprey, Petromyzon marinus. We report here the rhodopsin gene, spanning 21.2 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Southern analysis suggests that the lamprey genome contains a single rhodopsin gene. The amino acid (aa) sequence deduced from this gene has 92% sequence similarity with that of the river lamprey rhodopsin. The data reveal that aa substitutions occurred more often in the transmembrane region than in the non-transmembrane region, possibly reflecting functional adaptation of the rhodopsin during the last 500 million years of the jawless fish evolution.     

Multiple functions of a multi-component mating pheromone in sea lamprey Petromyzon marinus

The role of the C24 sulphate in the mating pheromone component, 7α,12α,24-trihydroxy-5α-cholan-3-one 24-sulphate (3kPZS), to specifically induce upstream movement in ovulated female sea lampreys Petromyzon marinus was investigated. 7α,12α-dihydroxy-5α-cholan-3-one 24-oic acid (3kACA), a structurally similar bile acid released by spermiated males, but lacking the C24 sulphate ester, was tested in bioassays at concentrations between 10(-11) and 10(-14) molar (M). 3kACA did not induce upstream movement in females or additional reproductive behaviours. In contrast, spermiated male washings induced upstream movement, prolonged retention on a nest and induced an array of nesting behaviours. Differential extraction and elution by solid-phase extraction resins showed that components other than 3kPZS + 3kACA are necessary to retain females on nests and induce nest cleaning behaviours. All pheromone components, including components in addition to 3kPZS + 3kACA that retain females and induce nest cleaning behaviours were released from the anterior region of the males, as had been reported for 3kPZS. It is concluded that the sea lamprey male mating pheromone has multiple functions and is composed of multiple components.     

Migration and spawning energetics of the anadromous sea lamprey,Petromyzon marinus

Synopsis Energy expended in migration and reproduction was determined from measurements of caloric concentration and body and gonodal weight for nontrophic sea lampreys collected from different sites along the St. John River, New Brunswick. The estimated cost of locomotion in swimming the 140 km which separates the estuary from the spawning redds was 300 and 260 kcal for males and females respectively. Acutal distance which lampreys swam as well as mean swimming speed were estimated from a linear regression equation relating energy expenditure for locomotion and body weight. Energy expenditure for breeding was considerably greater than that catabolized throughout the upstream migration.     

Immunocytochemical localization of insulin and somatostatin in the endocrine pancreas of the sea lamprey Petromyzon marinus L., at various stages of its life cycle

Summary Light-microscopic immunocytochemistry and routine staining techniques were used to localize insulin and somatostatin-immunoreactive cells within the endocrine pancreatic tissue of the lamprey, Petromyzon marinus, during various stages of the life cycle. The endocrine pancreas of larvae consists solely of follicles of insulin-immunoreactive cells surrounding the junction of oesophagus, intestine and bile duct. Somatostatin-immunoreactive cells are restricted to the intestinal epithelium. In both parasitic and upstream-migrating adults the endocrine pancreas consists of cranial and caudal portions, both containing separate populations of insulin and somatostatin-immunoreactive cells.Supported by NSERC of Canada grant no. A5945 to JHY