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.     

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.     

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.     

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.     

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.     

Sea lamprey Petromyzon marinus: an exception to the rule of homing in anadromous fishes

Anadromous fishes are believed to make regular circuits of migration in the sea before homing to their natal rivers. Sea lamprey Petromyzon marinus is an anadromous fish that is an exception to this life-history pattern. It also differs from other anadromous fishes in that its adult phase is parasitic, a feeding strategy that should make homing problematic for lamprey cohorts that become widely dispersed through transport by the diverse hosts they parasitize. We sequenced a portion of the mitochondrial DNA control region from sea lampreys collected from 11 North American east coast rivers to test for genetic evidence of homing. There were no significant differences (chi2=235.1, p=0.401) in haplotype frequencies among them, with almost 99 per cent of haplotypic diversity occurring within populations. These findings, together with concordant genetic results from other geographical regions and ancillary information on pheromonal communication, suggest that sea lamprey does not home but rather exhibits regional panmixia while using a novel 'suitable river' strategy to complete its life cycle.     

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.     

Surface morphology and functions of pharyngeal structures in the larval lamprey Petromyzon marinus

To gain insight into the multiple functions of a complex biological structure, the morphology of the pharynx of the larva (ammocoete) of the lamprey Petromyzon marinus was investigated with scanning electron microscopy and histochemistry (PAS and Alcian blue). Features studied include the gills, the parabranchial chambers external to the gills, intrapharyngeal ciliary tracts, the ridged pharyngeal roof, the floor, and the intrapharyngeal taste buds. Significant findings are: (1) All (nonciliated) cells lining these structures are covered with microvilli or microridges. The pattern and packing density of these membrane features vary among different pharyngeal structures. The lumenar membranes of pharyngeal lining cells overlie a mucous prosecretion in the apical cytoplasm, suggesting that the microvilli/ridges on these membranes function to anchor mucus. (2) Patterns of microvilli/ridges on the gill respiratory lamellae differ among ammocoetes of different species. (3) Pharyngeal osmo-regulatory cells (“chloride cells”) could not be identified on the basis of the microvillus/ridge pattern. (4) Two types of ciliary tracts are present within the pharynx. One has tall (x = 13 μm) and densely packed cilia, whereas the other has shorter (x = 7 μm) and less densely packed ones. Because mucus covers both types of tracts their function appears to involve the transport of mucus. (5) Food particles were found on the lateral surfaces of the gill filaments and on the surfaces of the parabranchial chambers. It appears that goblet cells in the epithelia of these regions secrete mucus in which the particles are trapped.     

First record of Renibacterium salmoninarum in the sea lamprey (Petromyzon marinus)

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a widespread problem with major implications for salmonid fish species. The mechanisms by which the bacterium has reached high levels of infection previously unrecorded in the Laurentian Great Lakes are presently unknown. Research involving reservoirs and mechanisms of R. salmoninarum transmission in fish is lacking because of the ecologic complexity of heterogeneous habitats and the lack of adequate funding. Herein, we report on the isolation of R. salmoninarum from the kidneys of the sea lamprey (Petromyzon marinus). The bacterium was cultured from kidneys of 16% and 4% of lampreys collected from two locations within the Lake Ontario watershed in 2003 and 2004, respectively. The identity of bacterial colonies was verified with the nested polymerase chain reaction and quantitative enzyme-linked immunosorbent assay.     

Effects of proteinase inhibitors on fertilization in sea lamprey (Petromyzon marinus)

A search for alternative sterilants in parasitic fish encouraged us to explore the usefulness of proteinase inhibitors for this purpose. Fertilization in sea lamprey species (Petromyzon marinus L.) was inhibited by chymotrypsin and trypsin inhibitors 4'-acetamidophenyl 4-guanidinobenzoate (AGB), chymostatin, tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) when these substances were added into a fertilization medium at the time of fertilization. Preincubation of eggs before fertilization with 100 microM TPCK, but not TLCK, resulted in inhibition of fertilization. Conversely, preincubation of spermatozoa with TLCK, but not TPCK, produced inhibition of fertilization. These data suggest the involvement of the chymotrypsin-like activity of eggs and trypsin-like activity of spermatozoa in fertilization. However, enzymes present in sperm suspensions were able to hydrolyze a chymotrypsin substrate N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) but not trypsin substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The nature of this activity can be characterized as serine protease and our results indicate the involvement of serine proteinases in the fertilization of sea lamprey.     

Retinal development in the lamprey (Petromyzon marinus L.): premetamorphic ammocoete eye.

Development of the retina of the ammocoete begins early in embryogenesis, with the formation of the optic vesicle, but development of the rudimentary eye is suspended and remains arrested during larval life. Prior to the onset of metamorphosis, the retina of the ammocoete is completely undifferentiated, with the exception of a small area (Zone II) surrounding the optic nerve head, where all of the adult retinal layers are found. The photoreceptors in this area have developed to include synaptic contacts as well as inner and outer segments. The pigment epithelium in this area, too, has differentiated to include well-formed melanin granules, myeloid bodies and endoplasmic reticulum and is closely associated with the receptor cell outer segments. With the approach of metamorphosis, differentiation of the remainder of the retina (Zone I) begins, taking place in a radial fashion from the optic nerve head. Differentiating pigment epithelial cells adjacent to the differentiated retinal zone begin to accumulate melanin granules. In the neural retina, junctional complexes are established in the form of an external limiting membrane, and connecting cilia project into the optic ventricle. Photoreceptor differentiation begins with the formation of a mitochondria-filled ellipsoid within the inner segment. Development and differentiation of the ammocoete retina is unique to vertebrates in that only a small area of differentiated retina is present during the larval stage. The remainder of the retina differentiates and becomes functional during metamorphosis.