Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis: I. Atresia of the bile ducts

The bile ducts in the liver of larval sea lamprey, Petromyzon marinus, undergo programmed degeneration during metamorphosis. The degenerative process is most dramatic in the middle metamorphic stages (3-5), and is asynchronous, occurring more rapidly in small peripheral biliary components than in larger, medial ducts. All classes of bile ducts within the biliary tree exhibit similar histological changes during regression. The initial evidence of degeneration in the epithelium is a folding of the basal lamina, and this is accompanied by cell shrinkage and disruption of cell order. "Shedding" of microvilli and cytoplasmic constituents then takes place at the apical surface resulting in the accumulation of periodic acid-Schiff positive membranous debris in the Lumen. The appearance of "hyalin bodies" in the lumen coincides with the depletion of intermediate-sized filaments from the cytoplasmic matrix. Numerous, large dense bodies, myelin figures, and autophagic vacuoles are consistently observed in necrotic cells. Following cytolysis, bile duct remnants become ensheathed within regions of fibrosis. Ultimately, these fibrous regions are replaced with cords of hepatocytes. By stage 7, all bile ducts have disappeared. The events of biliary atresia in lampreys are comparable to tissue regression which is associated with normal development and pathological conditions in other vertebrates but are particularly reminiscent of those in human biliary atresia. The unique ability of the adult lamprey to service without bile ducts enhances the value of this organism as an experimental model for studying human biliary atresia.     

Nonparenchymal liver cells and granulomas during lamprey biliary atresia

Transmission (thin sections and freeze-fracture replicas) and scanning electron microscopy were used to describe the nonparenchymal liver cells during the seven (1-7) stages of metamorphosis in the sea lamprey, Petromyzon marinus L., when bile ducts and canaliculi degenerate. The biliary atresia is accompanied by an increased diameter of fenestrae in the endothelium, an active phagocytosis by Kupffer cells in the sinusoids, and large lipid inclusions in perisinusoidal lipocytes (fat-storing or Ito cells). Plasma-like cells and foci of nonparenchymal cells (granulomas) are present in the liver interstitium during at least four stages of metamorphosis. The fenestrae in the sinusoidal wall are wider (up to 2.8-micron diameter) than normally reported for vertebrate livers but are likely a reflection of the morphogenetic and physiological events and consequences of the biliary atresia. Kupffer cells are involved in an extensive erythrophagocytosis, the storage of iron, and perhaps the incorporation of cellular components from hepatocytes. Lipocytes are the vitamin A-storing cells of the transforming liver and may be responsible for some perisinusoidal fibrosis. Granulomas are present during stages 3-6 and are focal areas where mononuclear leukocytes (lymphocytes and plasmalike cells), macrophages, and neutrophils have infiltrated the hepatic parenchyma. The function of the granulomas is not known; but their presence may be related to the porous nature of the sinusoidal wall, the tissue degeneration, and/or the physiological change (e.g., bile stasis) during biliary atresia.     

Gap junctions and zonulae occludentes of hepatocytes during biliary atresia in the lamprey

Gap junctions and zonulae occludentes of hepatocytes were examined in thin sections and freeze-fracture replicas from livers of larval and juvenile adult lampreys and during the phase of metamorphosis when bile ducts and bile canaliculi disappear (biliary atresia). Larvae possess zonulae occludentes at the canaliculi which are composed of one to five (mean = 2.81) junctional strands that provide a bile-blood barrier. Morphometry demonstrates that during biliary atresia the decreases in number of junctional strands and apico-basal depth of the zonulae occludentes are accompanied by an increase in the frequency of gaps or interruptions in the strands and in a breakdown of the bile-blood barrier. The zonulae occludentes completely disappear during metamorphosis and are not found in the adult liver. Gap junctions of the larval liver occupy 1% of the surface of the plasma membrane and have a mean area of 0.167 micron 2 but, following an initial decline in these parameters during early biliary atresia, they rise sharply in later stages of metamorphosis and in adults are 3.2% and 0.502 micron 2, respectively. The events of alteration in junctional morphology during lamprey biliary atresia is in many ways comparable to the changes in gap junctions and zonulae occludentes during experimental and pathological intra- and extrahepatic cholestasis in mammals.     

Ultrastructural features of degeneration of the gallbladder during lamprey biliary atresia

Thin sections and freeze-fracture replicas were used to examine the fine structural features of degeneration of the gallbladder during lamprey biliary atresia. The cells of the epithelium undergo a progressive accumulation of dense bodies and vacuoles, loss of glycogen, condensation of the filamentous ectoplasm, fragmentation of microvilli, and dilation of cisternae of rough endoplasmic reticulum but eventually disappear by stage 4 of metamorphosis. Zonulae occludentes in the epithelium show a progressive increase in apical-basal depth as the junctional strands fragment. The possibility of an influence of transformed, subepithelial cells on degeneration of epithelial cells is suggested by close contact of the former with the thickened, highly pleated, epithelial basal lamina. The smooth muscle cells of the larval gallbladder are believed to transform during lamprey metamorphosis into these subepithelial cells which shed their external lamina, become intimately associated with collagen and other microfibrils, and which may be capable of phagocytosis. The events of gallbladder degeneration during lamprey metamorphosis show features of apoptosis.     

Changes in the Gut Microbiome of the Sea Lamprey during Metamorphosis

Vertebrate metamorphosis is often marked by dramatic morphological and physiological changes of the alimentary tract, along with major shifts in diet following development from larva to adult. Little is known about how these developmental changes impact the gut microbiome of the host organism. The metamorphosis of the sea lamprey (Petromyzon marinus) from a sedentary filter-feeding larva to a free-swimming sanguivorous parasite is characterized by major physiological and morphological changes to all organ systems. The transformation of the alimentary canal includes closure of the larval esophagus and the physical isolation of the pharynx from the remainder of the gut, which results in a nonfeeding period that can last up to 8 months. To determine how the gut microbiome is affected by metamorphosis, the microbial communities of feeding and nonfeeding larval and parasitic sea lamprey were surveyed using both culture-dependent and -independent methods. Our results show that the gut of the filter-feeding larva contains a greater diversity of bacteria than that of the blood-feeding parasite, with the parasite gut being dominated by Aeromonas and, to a lesser extent, Citrobacter and Shewanella. Phylogenetic analysis of the culturable Aeromonas from both the larval and parasitic gut revealed that at least five distinct species were represented. Phenotypic characterization of these isolates revealed that over half were capable of sheep red blood cell hemolysis, but all were capable of trout red blood cell hemolysis. This suggests that the enrichment of Aeromonas that accompanies metamorphosis is likely related to the sanguivorous lifestyle of the parasitic sea lamprey.     

Alagille Syndrome Mimicking Biliary Atresia in Early Infancy

Alagille syndrome may mimic biliary atresia in early infancy. Since mutations in JAG1 typical for Alagille syndrome type 1 have also been found in biliary atresia, we aimed to identify JAG1 mutations in newborns with proven biliary atresia (n = 72). Five biliary atresia patients with cholestasis, one additional characteristic feature of Alagille syndrome and ambiguous liver histology were single heterozygotes for nonsense or frameshift mutations in JAG1. No mutations were found in the remaining 67 patients. All “biliary atresia” carriers of JAG1 null mutations developed typical Alagille syndrome at the age of three years. Our data do not support association of biliary atresia with JAG1 mutations, at least in Czech patients. Rapid testing for JAG1 mutations could prevent misdiagnosis of Alagille syndrome in early infancy and improve their outcome.     

Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis. II. Canalicular degeneration and transformation of the hepatocytes

Degeneration of all bile canaliculi takes place in the liver of the sea lamprey, Petromyzon marinus, during metamorphosis. Disintegration of microvilli is observed during earlier stages, and membranous debris ultimately accumulates within the canalicular lumina. Complete occlusion of the lumina and disorganization of junctional complexes is followed by a complete loss of the exocrine biliary pole of hepatocytes and a reorganization of these cells into solid cords. An increase in the size and number of acid phosphatase-containing cytoplasmic bodies coincides with the events of canalicular degeneration. These secondary lysosomes apparently participate in some manner in the isolation and disposal of iron and other bile constituents which no longer can be excreted in bile canaliculi. The loss of the exocrine biliary pole of hepatocytes is concomitant with vascular disturbances in the form of disordered arrangements of sinusoidal endothelial cells and an increase in the population of activated Kupffer cells involved in erythrophagocytosis. The significance of the shift in functional organization of the liver in adult lampreys is discussed in relation to physiological changes in this organism and to human hepatic cholestasis, for which this organism is a potentially valuable experimental model.     

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.     

A comparison of the effectiveness of sea lamprey control in Georgian Bay and the North Channel of Lake Huron

The sea lamprey control program in the Great Lakes has effectively reduced the abundance of sea lamprey, and, in conjunction with appropriate fishery management practices, has contributed to the restoration of valuable stocks of fish — particularly the salmonids. Sea lamprey producing tributaries of Lake Huron have been treated with lampricide on a continuing basis since 1966, and indications of decreasing abundance of sea lamprey have been evident since 1969. Control of sea lamprey has been more successful in Georgian Bay than in North Channel, as shown by the numbers of spawning phase animals captured in assessment devices, and by the incidence of lamprey-inflicted wounds on fish. The St. Marys River — the only known uncontrolled sources of recruitment to parasitic sea lamprey populations of Lake Huron — is suspected of being the cause of the higher levels of sea lamprey abundance observed in the northwest part of the lake.     

Iron loading in the livers of metamorphosing lampreys,Petromyzon marinus L.

Iron loading of hepatocytes was followed through the stages (1-7) of metamorphosis in lamprey (Petromyzon marinus L.) using light- and electron-microscopic histochemistry. Iron is present in ferric and ferrous forms in the hepatocytes of larval lampreys in levels that can only be detected in the electron microscope. During the initial stages (1-3) of metamorphosis iron begins to increase in the cytoplasmic matrix and in dense bodies but it is not apparent in the light microscope until stage 4. The increased accumulation of iron through the subsequent stages (5-7) of metamorphosis coincides with the advanced degeneration and ultimate disappearance of bile canaliculi and bile ducts. The absence of a bile canaliculus is concurrent with the beginning of staining of lateral cell borders for ferrous iron and with intense concentrations of ferric iron throughout the cytoplasmic matrix and within cytoplasmic dense bodies. By the end of metamorphosis the hepatocytes resemble iron-loaded hepatocytes in pathological and experimentally induced situations in other vertebrates. The iron loading of hepatocytes during metamorphosis is discussed with respect to both the concomitant atresia of the biliary tree and alteration of several aspects of blood morphology and chemistry. Since iron loading occurs synchronously in the hepatocytes of a given population of metamorphosing lampreys, this organism should prove to be a useful experimental system for investigation on cellular mechanisms of iron loading in vertebrates.     

Mitochondrial and lysosomal pathways of lamprey (Lampetra fluviatilis L.) hepatocyte death

Mechanisms of mitochondrial and lysosomal pathways of natural death of lamprey hepatocytes are described at the spring period of the prespawn migration. The mitochondrial pathway (release of cytochrome c from mitochondria into cytosol and activation of caspases) is realized by the classic scheme of apoptosis. Comparatively recently, the lysosomal pathway of cell death associated with cathepsin B activation has been revealed in cells in pathologies, specifically in obstruction of gallbladder and bile ducts. A peculiarity of lamprey hepatocytes consists in that in the adult animal liver there takes place biliary atresia (the absence of gallbladder and bile ducts. Thereby, the lamprey hepatocytes are an excellent object for study of this new pathway of cell death. We have revealed development of the mitochondrial and the lysosomal pathways of cell death of lamprey hepatocytes.     

The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae

Larval sea lamprey inhabit freshwater streams and migrate to oceans or lakes to feed after a radical metamorphosis; subsequently, mature adults return to streams to spawn. Previous observations suggested that lamprey utilize the odor of conspecific larvae to select streams for spawning. Here we report biochemical and electrophysiological evidence that this odor is comprised of two unique bile acids released by larvae. High performance liquid chromatography and mass spectrometry demonstrated that larval sea lamprey produce and release two unique bile acids, allocholic acid (ACA) and petromyzonol sulfate (PS). Electro-olfactogram (EOG) recording also demonstrated that the olfactory system of migratory adult sea lamprey is acutely and specifically sensitive to ACA and PS; detection thresholds for these compounds were approximately 10(-12) M. ACA and PS were the most potent of 38 bile acids tested and cross-adaptation experiments suggested that adult sea lamprey have specific olfactory receptor sites associated with independent signal transduction pathways for these bile acids. These receptor sites specifically recognize the key substituents of ACA and PS such as a 5 alpha-hydrogen, three axial hydroxyls, and a C-24 sulfate ester or carboxyl. In conclusion, the unique lamprey bile acids, ACA and PS, are potent and specific stimulants of the adult olfactory system, strongly supporting the hypothesis that these unique bile acids function as migratory pheromones in lamprey.     

Differences in the fatty acid composition of larvae and metamorphosing sea lampreys, Petromyzon marinus

This study was designed to evaluate biochemical changes in the fatty acid (FA) compositions of selected lipid depot (kidney and liver) and absorption (intestine) organs in larvae and metamorphosing sea lamprey, Petromyzon marinus. Palmitic or stearic acids were generally the predominant saturated fatty acids (SFA) before and during metamorphosis, but the greatest proportion of myristic acid occurred in renal triacylglycerol (TG). Monoenes, dienes, and polyenes consist mainly of 16:1, 18:1, and 20:1, 18:2 and 20:2omega6, and 18:4omega3, respectively. Alterations in these predominant fatty acids occurred during lamprey metamorphosis, but depended on tissue, lipid class, and developmental status. During metamorphosis, kidney TG and phospholipid (PL) classes tended to mobilize SFA and enhance the fatty acid unsaturation, as indicated by increased unsaturated/saturated ratio, unsaturation index (USI), and total mean chain length (MCL). There was a tendency to increase saturation in the fatty acids of liver TG and PL classes and intestine TG, FA and monoacylglycerol (MG) classes, but to increase unsaturation in the fatty acids of liver cholesteryl ester (CE), FA and MG classes and intestine PL and CE classes from larva or stage 3 to stage 7. Increased polyunsaturated fatty acids in kidney TG and PL from larvae to stage 5 transformers and intestine PL and CE from stage 3 to stage 7 transformers may reflect an osmoregulatory pre-adaptation. The presence of branched-chain SFA (BCSFA) and the odd number of fatty acids (ONFA) indicated a significant role of detritivores in the benthic larvae. Decreased abundance of BCSFA, ONFA, and 18:2 dienes occurred in the transformed intestine TG as non-trophic metamorphosis proceeded. These data suggest that sea lamprey metamorphosis may proceed in a habitat, dietary, osmoregulatory, energetic, and developmental pre-adaptation of fatty acid composition from benthic filter-feeding larvae to pelagic parasitic juveniles.     

The Pathology of Liver Cirrhosis in Patients with Cystic Fibrosis of the Pancreas

The peculiarities of the type of liver cirrhosis that occurs in patients with cystic fibrosis of the pancreas depend on a number of factors. Two such factors, which have received little attention in the past, became apparent during a study of the livers of patients dying of this disease at the Henry Ford Hospital, Detroit. Firstly, the onset of the disease in fetal life may disturb the development of the bile duct system whose normal development is essential for normal structural relationships to be maintained in the liver. Focal lesions of intrahepatic biliary atresia will then complicate the histologic picture of “multi-lobular biliary cirrhosis”. Secondly, scars formed in an infantile liver will considerably distort the subsequent growth of the liver, resulting in bizarre nodularity. Despite massive deformation large portions of the liver will still be composed of primary parenchyma that will enable normal liver functions as revealed by laboratory tests.     

Multi-Sample Pooling and Illumina Genome Analyzer Sequencing Methods to Determine Gene Sequence Variation for Database Development

Determination of sequence variation within a genetic locus to develop clinically relevant databases is critical for molecular assay design and clinical test interpretation, so multisample pooling for Illumina genome analyzer (GA) sequencing was investigated using the RET proto-oncogene as a model. Samples were Sanger-sequenced for RET exons 10, 11, and 13–16. Ten samples with 13 known unique variants (“singleton variants” within the pool) and seven common changes were amplified and then equimolar-pooled before sequencing on a single flow cell lane, generating 36 base reads. For comparison, a single “control” sample was run in a different lane. After alignment, a 24-base quality score-screening threshold and 3` read end trimming of three bases yielded low background error rates with a 27% decrease in aligned read coverage. Sequencing data were evaluated using an established variant detection method (percent variant reads), by the presented subtractive correction method, and with SNPSeeker software. In total, 41 variants (of which 23 were singleton variants) were detected in the 10 pool data, which included all Sanger-identified variants. The 23 singleton variants were detected near the expected 5% allele frequency (average 5.17%±0.90% variant reads), well above the highest background error (1.25%). Based on background error rates, read coverage, simulated 30, 40, and 50 sample pool data, expected singleton allele frequencies within pools, and variant detection methods; ≥30 samples (which demonstrated a minimum 1% variant reads for singletons) could be pooled to reliably detect singleton variants by GA sequencing.     

Relocalization of membrane enzymes accompanies biliary atresia in lamprey liver

Summary Light- and electron-microscopic histochemical procedures were used to show the distribution of the membrane-bound enzymes alkaline phosphatase (Alp), adenosine triphosphatase (ATPase), and 5-nucleotidase (5-nuc) in the livers of lamprey, Petromyzon marinus, throughout the life cycle. In larvae, the three enzymes are located at the biliary pole on the canalicular membranes of microvilli. At metamorphosis the enzymes become localized at all lateral cell surfaces of hepatocytes as bile canaliculi degenerate in the programmed regression of the entire biliary tree. This latter pattern of enzyme distribution persists during the parasitic adult phase but no activity is evident in individuals in the spawning migration. As the timing of the relocalization of enzymatic activity correlates well with a build-up of bile products and iron during metamorphosis, it is suggested that the lateral surface may be the new site for transport of these products.Supported by NSERC of Canada grant no. A5945 to J.H.Y.     

Molecular defense responses in roots and the rhizosphere against Fusarium oxysporum

Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting “soldier” rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.     

A new Liopropoma sea bass (Serranidae,Epinephelinae, Liopropomini) from deep reefs off Cura?ao,southern Caribbean,with comments on depth distributions of western Atlantic liopropomins

Collecting reef-fish specimens using a manned submersible diving to 300 m off Curaçao, southern Caribbean, is resulting in the discovery of numerous new fish species. The new Liopropoma sea bass described here differs from other western Atlantic members of the genus in having VIII, 13 dorsal-fin rays; a moderately indented dorsal-fin margin; a yellow-orange stripe along the entire upper lip; a series of approximately 13 white, chevron-shaped markings on the ventral portion of the trunk; and a reddish-black blotch on the tip of the lower caudal-fin lobe. The new species, with predominantly yellow body and fins, closely resembles the other two “golden basses” found together with it at Curaçao: L. aberrans and L. olneyi. It also shares morphological features with the other western Atlantic liopropomin genus, Bathyanthias. Preliminary phylogenetic data suggest that western Atlantic liopropomins, including Bathyanthias, are monophyletic with respect to Indo-Pacific Liopropoma, and that Bathyanthias is nested within Liopropoma, indicating a need for further study of the generic limits of Liopropoma. The phylogenetic data also suggest that western Atlantic liopropomins comprise three monophyletic clades that have overlapping depth distributions but different depth maxima (3–135 m, 30–150 m, 133–411 m). The new species has the deepest depth range (182–241 m) of any known western Atlantic Liopropoma species. Both allopatric and depth-mediated ecological speciation may have contributed to the evolution of western Atlantic Liopropomini.