Light and electron microscopic immunocytochemical localization of thyroglobulin in the thyroid gland of the anadromous sea lamprey,Petromyzon marinus L., during its upstream migration

Summary Antibodies made against thyroglobulin (TG) were used in an immunocytochemical study for the light and electron microscopic localization of TG in the thyroid gland of the anadromous sea lamprey, Petromyzon marinus, during its upstream migration. TG was found in the follicular lumen and in some colloid droplets within the follicular cells. Except for an immunoreactive product observed in a small portion of the interstitial connective tissue, the location of TG in the lamprey was similar to that in the thyroid of the rat.Supported by National Research Council of Canada Grant no. A5945 to J.H.Y. We thank Dr. F.W.W. Beamish and Mr. R. Robinson who helped in the capture of the lamprey     

Immunohistochemical observations of the endocrine pancreas during metamorphosis of the sea lamprey,Petromyzon marinus L.

Summary Light-microscopic immunohistochemistry was used to localize insulin- and somatostatin-immunoreactive cells within developing endocrine pancreatic tissue of metamorphosing lampreys, Petromyzon marinus. The extrahepatic common bile duct and a portion of the intrahepatic bile duct develop into the caudal portion of the endocrine pancreas. The cranial pancreas is composed of follicles originating in the intestinal and diverticular epithelia, thus following the method of formation of pancreatic follicles from gut epithelium in larvae. In both the cranial and caudal portions, and in an intermediate cord of isolated follicles which connect these two major masses, insulin-immunoreactive cells appear first and are followed by cells showing somatostatin-immunoreactivity. In all stages of metamorphosis individual endocrine cells demonstrate immunoreactivity to a single hormone. Biliary atresia in lamprey may have some adaptive significance in providing cells that produce a caudal endocrine pancreas.Supported by NSERC of Canada grant No. A5945 and MRC of Canada grant No. MA8629 to JHY     

Morphological analysis of geographic variation of sea lamprey ammocoetes in Portuguese river basins

The main objective of this study was to compare the morphological variability of sea lamprey (Petromyzon marinus L.) larvae from the main Portuguese river basins. Samples were collected in rivers Minho, Lima, Cávado, Vouga, Mondego, Tejo and Guadiana. Specimens were analysed in terms of morphometric (linear body measures) and meristic (number of myomeres) characters to investigate the hypothesis of population fragmentation between river basins caused by some degree of homing behaviour. The discriminant analysis showed a morphological segregation of the studied populations based on the characters head, tail and branchial length. The discriminatory power of the meristic characters was comparatively weaker, with the number of trunk myomeres, and to some extent the head myomeres, being responsible for the reduced separation between groups. Both analyses were consistent in identifying the cephalic region as the most important morphological feature to discriminate populations of sea lamprey larvae in the Portuguese territory. The largest cephalic region of the ammocoetes sampled in the northern river basins may be responsible for a better feeding efficiency and, consequently, higher values of condition factor. Guest editors: S. Dufour, E. Prévost, E. Rochard & P. Williot Fish and diadromy in Europe (ecology, management, conservation)     

Ultrastructural study of the retinal pigment epithelium during metamorphosis in the sea lamprey (Petromyzon marinus L.)

Summary The sequence of morphological changes in the retinal pigment epithelium during the metamorphic period of the sea lamprey Petromyzon marinus L. has been investigated using electron microscopy. At early metamorphic stages (stages I and II), photoreceptors are present in a small zone of the retina. During these stages, the lateral surface of the epithelial cells shows zonulae occludentes and adhaerentes. The degree of cell differentiation varies throughout the retinal pigment epithelium. Cells covering the differentiated photoreceptors in the central retina have phagosomes, whereas pigment granules appear only in the retinal pigment epithelium dorsal to the optic nerve head. Most epithelial cells have myeloid bodies; their morphology is more complex around the optic nerve head. At stage III, when photoreceptors develop over the whole retina, the distribution of cytoplasmic organelles is almost homogeneous in the retinal pigment epithelium. Subsequently, the basal plasma membrane of the epithelial cells becomes progressively folded and their apical processes enlarged. In addition, extensive gap junctions develop between retinal pigment cells. In late metamorphic stages, noticeable growth of myeloid bodies occurs and consequently the retinal pigment epithelium resembles that of the adult. This study also describes, for the first time, the presence of wandering phagocytes in the retinal pigment epithelium of lampreys; their role in melanosome degradation is discussed.     

Behavioral thermoregulation by Ammocoete larvae of the sea lamprey (Petromyzon marinus) in an electronic shuttlebox

Ammocoete larvae of the sea lamprey Petromyzon marinus, a member of the primitive vertebrate class Agnatha, were tested for thermoregulatory behavior in an electronic shuttlebox (ichthyotron). The final preferendum derived from pooled data for 24 individually tested ammocoetes was characterized by a mean of 13.6 ± 0.17 (s.e.m.)°C, a mode and median of 140°C, and a range of voluntarily occupied temperatures from 10–19°C over a 3-day period.     

Distribution of lamprey gonadotropin-releasing hormone-III (GnRH-III) in brains of larval lampreys (Petromyzon marinus)

Two immunoreactive forms of gonadotropinreleasing hormone (GnRH), lamprey GnRH-I and lamprey GnRH-III, were found in neurons in larval sea lampreys (Petromyzon marinus). Using antisera preferentially directed against either lamprey GnRH-I or-III, dense reaction product was seen in cell bodies in the rostral hypothalamus and preoptic area. Reaction product was also dense in fibers to and within the neurohypophysis, in addition to numerous fibers which projected caudally, beyond the neurohypophysis through the mesencephalon. The majority of immunoreactive GnRH was lamprey GnRH-III, and when lamprey GnRH-I was seen, it was in cells that appeared to contain both forms of GnRH. A small number of cells found in the caudal hypothalamus contained only immunoreactive lamprey GnRH-III, and these may constitute a functional subgroup within the population of GnRH neurons. In animals undergoing metamorphosis there was a large increase in reaction product in all GnRH-containing cells and fibers. A striking change within the distribution of GnRH cells was localized to a distinct group of GnRH-immunoreactive cells (GnRH-I and-III) in the ventral anterior hypothalamic area. These cells were minimally detectable in larvae, but during metamorphosis became densely filled with immunoreactive product in perikarya and distal processes. The results are consistent with the hypothesis that lamprey GnRH-III is an important form of GnRH during the maturation of GnRH cells and fibers, and further indicates that these cells have attained their normal positions in the preoptic area and hypothalamus before metamorphosis.     

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     

Changes in the ultrastructure of the gills of the river lamprey,Lampetra fluviatilis (L.), during the anadromous spawning migration

Summary Two types of mitochondria-rich cells were found in the interplatelet areas of the gills of the migrating river lamprey. Both cell types are thought to be responsible for ion-transport across the gills. In the fresh-run migrant the gills are dominated by large, flask-shaped cells which show some ultra-structural similarities with the teleost chloride cell and have been tentatively referred to as ion-excretory cells. During the spawning migration the ion-excretory cells are replaced by smaller, mitochondria-rich cells which are similar in structure to the presumed ion-transporting cells in the ammocoete gill. They lack the tubular, smooth-membraned endoplasmic reticulum so characteristic of the lamprey ion-excretory cell and the teleost chloride cell and have been referred to as ion-uptake cells. The ion-uptake cells are found during the stenohaline, freshwater phases of the lamprey's life history. Ion-excretory cells are present during the periods of the life cycle when the animal is euryhaline. The possibility that the ion-excretory cells are also responsible for ion-uptake in fresh water is discussed.     

Alterations to the subepithelial layers of the larval alimentary tract during metamorphosis in the sea lamprey Petromyzon marinus,with emphasis on tissue interactions

Light-microscopic histochemistry and conventional electron microscopy were used to study the changes to the subepithelial layers in the larval esophagus of the sea lamprey Petromyzon marinus during metamorphosis. During early stages of metamorphosis, smooth muscle cells of the muscularis mucosae and tunica muscularis dedifferentiate into myofibroblast-like cells, which make contact with the basal lamina of the overlying mucosal epithelium. During later stages, these myofibroblast-like cells redifferentiate into smooth muscle cells, reforming the muscularis mucosae and tunica muscularis. Alterations to the extracellular matrix occur concomitantly.     

Lamins of the sea lamprey (Petromyzon marinus) and the evolution of the vertebrate lamin protein family

Lamin proteins are found in all metazoans. Most non-vertebrate genomes including those of the closest relatives of vertebrates, the cephalochordates and tunicates, encode only a single lamin. In teleosts and tetrapods the number of lamin genes has quadrupled. They can be divided into four sub-types, lmnb1, lmnb2, LIII, and lmna, each characterized by particular features and functional differentiations. Little is known when during vertebrate evolution these features have emerged. Lampreys belong to the Agnatha, the sister group of the Gnathostomata. They split off first within the vertebrate lineage. Analysis of the sea lamprey (Petromyzon marinus) lamin complement presented here, identified three functional lamin genes, one encoding a lamin LIII, indicating that the characteristic gene structure of this subtype had been established prior to the agnathan/gnathostome split. Two other genes encode lamins for which orthology to gnathostome lamins cannot be designated. Search for lamin gene sequences in all vertebrate taxa for which sufficient sequence data are available reveals the evolutionary time frame in which specific features of the vertebrate lamins were established. Structural features characteristic for A-type lamins are not found in the lamprey genome. In contrast, lmna genes are present in all gnathostome lineages suggesting that this gene evolved with the emergence of the gnathostomes. The analysis of lamin gene neighborhoods reveals noticeable similarities between the different vertebrate lamin genes supporting the hypothesis that they emerged due to two rounds of whole genome duplication and makes clear that an orthologous relationship between a particular vertebrate paralog and lamins outside the vertebrate lineage cannot be established.     

Locomotor activity of river lamprey Lampetra fluviatilis (L.) during the spawning season

At high latitudes (62–64°N) the river lamprey Lampetra fluviatilis (L.) is shown to have a 24-hr locomotor activity during its spawning period just before midsummer. By keeping lampreys in cold water, the spawning was delayed about one month. Analysis of event recorder graphs indicate that the 24-hr activity is caused by addition of diurnal spawning activity to the basic nocturnal activity and is not caused by weak Zeitgeber conditions during midsummer.Water temperature might indirectly determine time of spawning of the river lamprey.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.     

Movement of Sacramento Sucker, Catostomus occidentalis, and Hitch, Lavinia exilicauda, during a Spring Release of Water from Camanche Dam in the Mokelumne River, California

Synopsis We recorded the movement of Sacramento suckers, Catostomus occidentalis, and hitch, Lavinia exilicauda, before and during a controlled flood release on a regulated California Central Valley River, using radio telemetry. Both species made small, local movements (<550 m) during pre-flood flows. During flood releases, some individual suckers made significantly larger movements (>8100 m) both up and downstream of pre-flood flow locations within the main channel while others did not. In contrast, increased flows did not significantly influence hitch movement from a side-channel pool. Sacramento suckers tended to move upstream during flow increases and downstream during flow reductions while no strong relationship for hitch was apparent. These data show that native Central Valley fishes may exhibit a variety of responses to flow change, including schooling and spawning activity, movements to refugia from higher velocities and no marked change. Managers must take into account life history, age and timing associated with specific species when implementing controlled flow strategies.     

Neuroendocrine and behavioral responses to weak electric fields in adult sea lampreys (Petromyzon marinus)

We characterized the behavioral and neuroendocrine responses of adult sea lampreys (Petromyzon marinus) to weak electric fields. Adult sea lampreys, captured during upstream spawning migration, exhibited limited active behaviors during exposure to weak electric fields and spent the most time attached to the wall of the testing arena near the cathode (−). For adult male sea lampreys, exposure to weak electric fields resulted in increased lamprey (l) GnRH-I mRNA expression but decreased lGnRH-I immunoreactivities in the forebrain, and decreased Jun (a neuronal activation marker) mRNA levels in the brain stem. Similar effects were not observed in the brains of female sea lampreys after weak electric field stimulation. The influence of electroreception on forebrain lGnRH suggests that electroreception may modulate the reproductive systems in adult male sea lampreys. The changes in Jun expression may be associated with swimming inhibition during weak electric field stimulation. The results for adult sea lampreys are the opposite of those obtained using parasitic-stage sea lampreys, which displayed increased activity during and after cathodal stimulation. Our results demonstrate that adult sea lampreys are sensitive to weak electric fields, which may play a role in reproduction. They also suggest that electrical stimuli mediate different behaviors in feeding-stage and spawning-stage sea lampreys.     

Growth and intermediary metabolism of larval and metamorphosing stages of the landlocked sea lamprey,Petromyzon marinus L

Synopsis Seasonal changes in blood, liver and muscle substrate (glucose, glycogen and lipid) concentrations and enzyme (pyruvate kinase (PyK), fructose diphosphatase (FDPase), NADP-isocitrate dehydrogenase (ICDH), malic enzyme (ME) and the hexose monophosphate shunt dehydrogenases (HMSD)) activities were assessed in ammocoete and metamorphosing stages of a stream stock of the landlocked sea lamprey, Petromyzon marinus L. In all developmental stages studied, muscle rather than liver tissue served as the main site of carbohydrate and fat storage. Blood glucose and muscle lipid exhibited a positive relationship while liver HMSD and muscle ME activity, a negative relationship, with ammocoete weight. These responses were attributed to a proliferation of red fibers and adipocytes in the ammocoete muscle as the time of metamorphosis approched. Muscle lipid stores of ammocoetes in their last year of larval life increased dramatically during the fall and winter preceding metamorphosis. Changes in tissue enzyme activity of ammocoetes in their last year of larval life indicated that the liver was the site of amino acid incorporation into fat while muscle was the site of lipogenesis from glucose. During the non-trophic period of metamorphosis, stored material was catabolized to provide energy for protein synthesis.     

Fungal biomass and decomposition in Spartina maritima leaves in the Mondego salt marsh (Portugal)

Spartina maritima (Curtis) Fernald is a dominant species in the Mondego salt marsh on the western coast of Portugal, and it plays a significant role in estuarine productivity. In this work, leaf litter production dynamics and fungal importance for leaf decomposition processes in Spartina maritima were studied. Leaf fall was highly seasonal, being significantly higher during dry months. It ranged from 42 g m^-2 in June to less than 6 g m^-2 during the winter. Fungal biomass, measured as ergosterol content, did not differ significantly between standing-decaying leaves and naturally detached leaves. Fungal biomass increased in wet months, with a maximum of 614 g g^-1 of ergosterol in January in standing-decaying leaves, and 1077 g g^-1 in December, in naturally detached leaves, decreasing greatly in summer. Seasonal pattern of fungal colonization was similar in leaves placed in litterbags on the marsh-sediment surface. However, ergosterol concentrations associated with standing-decaying and naturally detached leaves were always much higher than in litterbagged leaves, suggesting that fungal activity was more important before leaf fall. Dry mass of litterbagged leaves declined rapidly after 1 month (about 50%), mostly due to leaching of soluble organic compounds. After 13 months, Spartina leaves had lost 88% of their original dry weight. The decomposition rate constant (k) for Spartina maritima leaves was 0.151 month^-1.     

Combining limnological and palaeolimnological approaches in assessing degradation of Lake Pskov

Historical spawning grounds for sea lampreys (Petromyzon marinus L.) in most Portuguese river basins are becoming inaccessible due to the construction of impassable dams and/or weirs. Studies like the one described in this paper are particularly important in areas like the Vouga river basin, where there is a considerable fishing effort from both professional fishermen and poachers. In fact, for management and conservation purposes, it is important to clarify several aspects of the sea lamprey spawning run in this particular watershed. Therefore, a total of 30 radio tagged, migrating sea lampreys were released in the River Vouga and some of its main tributaries during 2004 and 2005. Results from the tracking sessions were used to determine the effect of poaching on the spawners’ population and the characteristics of the resting sites used during the upstream movement. The rivers’ stretches were also characterized according to the type of substrate present in the riverbed and flow type, in order to determine its aptitude to constitute appropriate spawning habitats for sea lampreys. We have identified in the upstream stretches of River Vouga, and in the tributary River Caima, characteristics that are particularly suitable for the construction of nests by the spawners. Presence of larvae provided evidence that spawners migrated into the River Vouga’s upper reaches. However, abundance and age class diversity appeared to be higher downstream of Sernada and Carvoeiro weirs, corroborating the telemetry data which suggested difficulty in passing these obstacles during low precipitation years. In the River Caima, migration was most predictable, which might be related to daily water releases from a small hydropower dam at dusk, that stimulated the lampreys to resume migration. Poaching has had a considerable negative effect on the success of the tagged lampreys’ migration: 76% of the animals released during 2005 were captured. Electronic supplementary material  Supplementary material is available for this article at and accessible for authorized user     

Nutritional status of adult sea lamprey (Petromyzon marinus Linnaeus, 1758) during spawning migration in the Minho River,NW Iberian Peninsula

The anadromous sea lamprey (Petromyzon marinus) stop feeding during spawning migration; following the migration effort, the energy reserves of their tissues are expected to be mobilised and depleted. The present work aimed at testing if mobilisation of nutrients occurred in sea lampreys during the 2011 spawning run in the Minho River. Professional fishermen at three sampling sites captured the individuals used: two at the beginning of the freshwater spawning migration (one at the river mouth and one upstream the estuary) and one near the spawning grounds (35 km upstream the estuary limit). The total lipids (Folch extraction), protein (nitrogen conversion), energy content (direct calorimetry) and fatty acids (GC/EI‐MS‐SIM) were determined in the carcass (n = 19) and female liver (n = 8) and gonads (n = 8). The results indicated that a reduction of carcass dry matter during migration was mainly caused by a lipid decrease (51.0 ± 2.46% downstream and 38.0 ± 2.92% upstream, LSmean ± SE). In females, a decrease in the measured amounts of liver lipids was also observed (4.28 ± 2.637 g downstream and 0.64 ± 2.206 g upstream, LSmean ± SE) and the monounsaturated fatty acids consistently composed the larger fraction of lipids in all tissues (ranging between 42.9 ± 4.20% and 54.9 ± 3.43% of total fatty acids, LSMean ± SE). The results suggest a dynamic nutrient mobilisation and the major importance of liver fatty acids (such as 14:1n‐5 and 16:1n‐7) as possible energy sources for embryonic development or combustion during spawning migration. The P. marinus individuals captured at the intermediate sampling site (approximately 35 km upstream river mouth) in the middle of the season seemed as ready to spawn as the upstream sea lampreys.     

Demineralization of the vertebral skeleton in Atlantic salmon Salmo salar L. during spawning migration

In Atlantic salmon, Salmo salar, the mineral rate of vertebrae in a given fish varies according to the position of the vertebra along the rachidian axis. Indeed, the mean rate goes from 49% in the anterior vertebrae and raises to 51% in post-truncal vertebrae. Although no significant difference in the mineral rate was noticed between males and females either in the lower river basin or after spawning, the mineral rate of vertebral bone decreased significantly (1–2%) during spawning migration. Vertebrae, like scales, are an important reservoir of calcium from which fasting salmon draws the minerals and organic materials necessary for the substantial remodeling of cranial bones in males and for sexual maturation. We hypothesize that mineral decrease in vertebrae may be the result of a halastasic demineralization of the vertebral tissues.