Immunocytochemical study of fibronectin in the sea lamprey,Petromyzon marinus,and the Atlantic hagfish,Myxine glutinosa

Summary Immunoreactive fibronectin-like material was localized within tissues of agnathans (hagfishes and lampreys) by an immunoperoxidase technique. Fibronectin was detected in basement membranes and in loose and dense connective tissues throughout the agnathan body. A fibronectin-like component was also identified in the plasma of both lampreys and hagfishes. The results indicate that fibronectin or a fibronectin-like material is a major component of agnathan connective tissues. Although there were some variations in the localization of fibronectin both between the lamprey and the hagfish and between agnathan and other vertebrate tissues, the generalized pattern of distribution of fibronectin in the agnathans supports the view that this protein, like that in higher vertebrates, plays a role in cellmatrix adhesion and tissue organization.     

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.     

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.     

Furunculosis in cultured American eel Anguilla rostrata (Le Sueur)

Diseased American eels, Anguilla rostrata , were collected from an eel culture facility in South Carolina and examined. The disease was characterized by skin lesions located on the body. A bacterium identified as Aeromonas salmonicida was isolated from the lesions and shown to be the causative agent. The bacterium was also isolated from the kidney during later stages of the disease.     

Temperature behavioral responses of the American eel,Anguilla rostrata (Lesueur), from Maryland

Acute temperature preference tests were conducted with American eels, Anguilla rostrata, collected from Maryland's eastern shore. Eels were acclimated to temperatures of 6, 12, 18, 24 and 30°C. Final temperature preferendum was 16.7°C. Data differ from the temperature responses of the majority of fishes tested to date in that acclimation temperature did not influence selected temperatures. Similar results were obtained for various other fishes (Oncorhynchus, Salmo, Salvelinus) by other investigators. Behavioral responses at various acclimation temperatures were observed.     

Intercellular junctions in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary The intramembrane organization of the occluding junctions in the gill epithelium of the Atlantic hagfish, Myxine glutinosa, was studied by means of freeze-fracture electron microscopy. Mitochondria-rich cells, characterized by assemblies of rod-shaped particles in the luminal plasma membrane and by an extensive intracellular amplification of the basolateral plasma membrane, are singly distributed between the pavement cells in the gill epithelium of this marine and stenohaline cyclostome. The occluding junctions between mitochondria-rich cells and pavement cells do not differ from those between adjacent pavement cells, concerning the number of superimposed strands (median 6, range 4–9) and their geometrical organization. These observations suggest that, in contrast to marine teleosts, the paracellular pathway plays a minor role in transepithelial ion movements in the hagfish gill epithelium. The findings are in agreement with the absence of hypoosmoregulatory mechanisms in hagfish, as have been evolved in various marine vertebrates. In addition, small communicating junctions are demonstrated between pavement cells; they possibly serve for a coordinated synthesis and secretion of mucus by the pavement cells.Dedicated to Prof. H. Leonhardt on the occasion of his 70th birthday     

Binding sites for atrial natriuretic peptide in the kidney and aorta of the hagfish (Myxine glutinosa)

1. In the atlantic hagfish (Myxine glutinosa) binding sites for atrial natriuretic peptide (ANP) were visualized by autoradiography in contractile structures of the renal system (glomeruli, neck segment, and archinephric duct) and in the aorta. 2. Since the location of binding sites is comparable to that in higher vertebrates including man, these results suggest that ANP may act as a hormone already in cylcostomata.     

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.     

Glomerular bypass shunts in the kidney of the Atlantic hagfish,Myxine glutinosa

Summary The vascular pathways associated with the glomerulus of the Atlantic hagfish, Myxine glutinosa have been studied by scanning electron microscopy of corroded resin casts of the vasculature. Although the overall pattern of the renal vasculature did not differ from earlier reports, a previously unreported vascular pathway which arose from the renal artery and bypassed the glomerular capillaries in 28% of glomeruli was clearly demonstrated. Glomerular bypass shunts either ran to join the loose capillary network around Bowman's capsule and thereby drain into the network of vessels associated with the mesonephric duct (ureter), or ran directly into the ureteral system of vessels and subsequently into the posterior cardinal veins. Glomerular bypass shunts which theoretically permit renal arterial blood to bypass the process of filtration may play a role in the regulation of body fluid volume.     

Primary structure of the minor haemoglobins from the sea lamprey (Petromyzon marinus, Cyclostomata)

Erythrocytes of the adult Sea Lamprey Petromyzon marinus contain several haemoglobin species, but only the main constituent has hitherto been sequenced. The present paper describes the determination of the primary structures of the two minor species, whose electrophoretic mobilities are higher and lower than that of the main component. Tryptic peptides from both chains were purified by high-performance liquid chromatography, then sequenced and aligned by homology with the main haemoglobin. The fast and the major components appeared to be very similar, differing in only four positions (pos. 5: Ser----Thr; pos. 33: Thr----Ser; pos. 86: Val----Ala; pos. 99: Gly----Arg). The slow haemoglobin component, however, differed from the main component with respect to 27 amino-acid residues. The position of the three globins of Petromyzon marinus in the phylogenetic tree of haemoglobins is discussed and a relationship with primitive alpha-chains is postulated.     

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.     

Volume and pH regulation in agnathan erythrocytes: comparisons between the hagfish,Myxine glutinosa,and the lampreys,Petromyzon marinus and Lampetra fluviatilis

The responses of hagfish (Myxine glutinosa) and lamprey (Lampetra fluviatilis and Petromyzon marinus) erythrocytes to osmotic swelling in hypoosmotic medium and to acid-base disturbances induced by ammonium chloride prepulse were studied. The erythrocytes of hagfish regulated neither cell volume after osmotic swelling nor intracellular pH after acidification. In contrast, the erythrocytes of lamprey lost potassium and chloride after osmotic swelling, whereby their volume recovered. Furthermore, the red cell pH of lamprey recovered from experimental acidification in a nominally bicarbonate-free medium in the presence of sodium, confirming that the pathway involved is sodium/proton exchange.Abbreviation DMO 5,5-dimethyloxazolidine-2,4-dione     

Immunoreactive endozepine-like peptides in the brain and pituitary of the Atlantic hagfish, Myxine glutinosa

Endozepines are a family of peptides capable of displacing benzodiazepines from their specific binding sites, to which belong the diazepam-binding inhibitor and the octadecaneuropeptide (ODN). This paper reports the distribution of ODN-related peptides, investigated for the first time by immunocytochemistry, in different brain and pituitary regions of the Atlantic hagfish, Myxine glutinosa. Immunoreactive ODN-like material was found in the telencephalon at the level of bundles of different olfactory nerve fibres. Moreover, at the level of the pallium, immunoreactive multipolar neurons were observed in the pars parvocellularis of the stratum griseum superficialis. Similar immunopositive nerve cell bodies were found in the nucleus medialis of the central prosencephalic complex. In the mesencephalon, few immunoreactive neurons lining and contacting the mesencephalic ventricle were detected; such nerve cells could be involved in the regulation of cerebrospinal fluid homeostasis. Dorsally in the mesencephalon, numerous ODN-containing cell bodies were present in the area praetectalis. The rhomboencephalon was immunostained only in the octavolateral area and in the nucleus motorius magnocellularis of the trigeminal nerve. Furthermore, ODN immunoreactivity was also present in the nerve cells of ganglia of the ophthalmic division of the trigeminal nerve complex. The immunocytochemical patterns described here in the brain of M. glutinosa suggest an involvement of ODN-like peptides as neuromodulators in sensory pathways, such as olfactory and visual. Finally, ODN-like substances were localized in discrete populations of adenohypophysial cells and in tanycytes lining the neurohypophyseal walls, suggesting for endozepines a paracrine and/or endocrine control of pituitary hormones release and a neurohormone role respectively. These results could give new insights into the chemioarchitecture of the brain of myxinoids.     

Seasonal concentrations of reproductive steroids in the gonads of the Atlantic hagfish, Myxine glutinosa

Changes in gonadal morphology, gonadal estradiol, and progesterone were examined in Atlantic hagfish, Myxine glutinosa, during a period of 17 months, beginning in April, 2001. Atlantic hagfish were captured from the ocean on a monthly basis. A total of 60 hagfish were divided into three different size classes of twenty hagfish each (small 20-35 cm, medium 35-45 cm, large 45-55+cm) and transported to the University of New Hampshire for sampling. Overall, in the medium and large size hagfish, estradiol and progesterone had significantly elevated peaks in January, 2001. There were significant increases in estradiol concentrations in January, with relatively low fluctuations in levels for the rest of the sampling period. Progesterone concentrations increased significantly in January, 2002, in medium and large hagfish, and remained elevated until June and April, 2002, for the two size classes respectively. The majority of hagfish sampled were females or hermaphrodites; few true males were identified in any of the samples. The number of females with large eggs increased following the estradiol peak in January and hermaphrodites with mature sperm were identified in the July, 2002, sample. These data represent the first evidence for a seasonal reproductive cycle in M. glutinosa and only the second seasonal reproductive cycle identified in any hagfish species.     

Photokinetic responses and diel activity of sea lamprey Petromyzon marinus ammocoete larvae

Twenty-four ammocoete larvae of the sea lamprey Petromyzon marinus were tested individually for 3-day periods in electronic shuttleboxes (Ichthyotrons) with natural lighting through windows, to determine their diel activity pattern in terms of hourly percentages of the 24-h total. Mean hourly activity was slightly greater during the day (4–7 % h⁻¹) than at night (3·7% h⁻¹). Peak hourly activity (7·2% h⁻¹) occurred during the dawn crepuscular period as light levels were increasing, while the minimum hourly activity (1·6 % h^-l) occurred during the dusk crepuscular period while light levels were decreasing. The observed activity pattern is atypical of other aquatic animals similarly tested, and might be explained as an extraptically mediated photoinetic response.     

Angiotensin I-converting enzyme-like activity in tissues from the Atlantic hagfish (Myxine glutinosa) and detection of immunoreactive plasma angiotensins

Using a highly sensitive fluorimetric assay, significant levels of angiotensin I -converting enzyme-like activity (ACELA) were detected in a range of tissues (branchial heart, gill, kidney with associated vasculature and archinephric duct, liver, whole brain and gut) from the Atlantic hagfish (Myxine glutinosa). The highest ACELA occurred in heart and gill (1.8 and 1.5 nmol His–Leu min⁻¹ mg protein⁻¹, respectively). The mammalian angiotensin I-converting enzyme (ACE) inhibitor, captopril, at 10⁻⁵ M was a potent inhibitor of the ACELA found in all hagfish tissues. Radioimmunoassay showed that immunoreactive angiotensins (251.8±11.8 pM) were detectable in hagfish plasma. The validity of the assay for measurement of hagfish angiotensins was indicated by the parallelism of the angiotensin II standard curve against serially diluted hagfish plasma. Measurement of immunoreactive plasma angiotensins and detection of significant levels of ACELA in a wide range of tissues gives indirect evidence for the presence of a renin–angiotensin system in hagfishes, the earliest evolved group of craniates.