Divergent Pathways of Ammonia and Urea Production and Excretion during the Life Cycle of the Sea Lamprey.

Little is known about nitrogenous waste (N waste) handling and excretion (JN waste) during the complex life cycle of the sea lamprey (Petromyzon marinus), an extant jawless fish that undergoes a complete metamorphosis from a filter-feeding larva (ammocoete) into a parasitic juvenile that feeds on the blood of larger, jawed fishes. Here, we investigate the ammonia- and urea-handling profiles of sea lampreys before, during, and after metamorphosis. The rates of ammonia excretion (Jamm) and urea excretion (Jurea) significantly decreased after the onset of metamorphosis, with the lowest rates observed during midmetamorphosis. Near the completion of metamorphosis, rates of JN waste (JN waste=Jamm+Jurea) significantly increased as sea lampreys entered the juvenile period. Feeding juvenile lampreys had greater than 10- to 15-fold higher Jamm and fivefold higher Jurea compared to nonfed juveniles, which corresponded to higher postprandial (postfeeding) concentrations of plasma ammonia and urea. The routes of Jamm and Jurea completely diverged following metamorphosis. In larvae, Jamm was equally split between branchial (gills) and extrabranchial (skin plus renal) pathways, but following metamorphosis, >80% of ammonia was excreted via the gills in nonfeeding juvenile lampreys, and >95% of ammonia was excreted via the gills in adult sea lampreys. Urea, on the other hand, was predominantly excreted via extrabranchial routes and, to a lesser extent, the gills in larvae and in nonfeeding juveniles. In adults, however, virtually all urea was excreted via urine. Reverse transcription polymerase chain reaction and in silico analyses also indicated that a urea transporter encoded by a slc4a2-like gene is present in lampreys. The branchial expression of this transporter is modulated throughout sea lamprey life history, as it is higher in the larvae and steadily decreases until the adult stage. We conclude that the divergent pathways of Jamm and Jurea during the sea lamprey life cycle reflect changes in their habitat, lifestyle, and diet. Further, the near-complete reliance on renal routes for Jurea in adult sea lampreys is unique among fishes and may reflect the ancestral condition of how this N waste product was handled and excreted by the earliest vertebrates.

Thyroid hormone and retinoid X receptor function and expression during sea lamprey (Petromyzon marinus) metamorphosis.

Sea lampreys (Petromyzon marinus) are members of the ancient class Agnatha and undergo a metamorphosis that transforms blind, sedentary, filter-feeding larvae into free-swimming, parasitic juveniles. Thyroid hormones (THs) appear to be important for lamprey metamorphosis, however, serum TH concentrations are elevated in the larval phase, decline rapidly during early metamorphosis and remain low until metamorphosis is complete; these TH fluctuations are contrary to those of other metamorphosing vertebrates. Moreover, thyroid hormone synthesis inhibitors (goitrogens) induce precocious metamorphosis and exogenous TH treatments disrupt natural metamorphosis in P. marinus. Given that THs exert their effects by binding to TH nuclear receptors (TRs) that often act as heterodimers with retinoid X receptors (RXRs), we cloned and characterized these receptors from P. marinus and examined their expression during metamorphosis. Two TRs (PmTR1 and PmTR2) and three RXRs (PmRXRs) were isolated from P. marinus cDNA. Phylogenetic analyses group the PmTRs together on a branch prior to the gnathostome TRα/ß split. The three RXRs also group together, but our data indicated that these transcripts are most likely either allelic variants of the same gene locus, or the products of a lamprey-specific duplication event. Importantly, these P. marinus receptors more closely resemble vertebrate as opposed to invertebrate chordate receptors. Functional analysis revealed that PmTR1 and PmTR2 can activate transcription of TH-responsive genes when treated with nanomolar concentrations of TH and they have distinct pharmacological profiles reminiscent of vertebrate TRß and TRα, respectively. Also similar to other metamorphosing vertebrates, expression patterns of the PmTRs during lamprey metamorphosis suggest that PmTR1 has a dynamic, tissue-specific expression pattern that correlates with tissue morphogenesis and biochemical changes and PmTR2 has a more uniform expression pattern. This TR expression data suggests that THs, either directly or via a metabolite, may function to positively modulate changes at the tissue or organ levels during lamprey metamorphosis. Collectively the results presented herein support the hypothesis that THs have a dual functional role in the lamprey life cycle whereby high levels promote larval feeding, growth and lipogenesis and low levels promote metamorphosis.

The influence of acclimation temperature on the lipid composition of the larval lamprey, Petromyzon marinus, depends on tissue and lipid class.

This study was designed to examine the effect of thermal acclimation on the lipid composition of fat depot organs the liver and kidneys of larval sea lamprey, Petromyzon marinus. We found that 21 °C-acclimated larvae possessed lower total lipid amounts in the liver (39% lower) and kidneys (30% lower) than 13 °C-acclimated larvae. Relatively lower lipid contents in the liver and kidneys of 21 °C-acclimated lamprey primarily resulted from a reduction in stored lipid reserve, triacylglycerol, but not the structural lipid, phospholipid. Compared to 21 °C-acclimated larvae, 13 °C-acclimated larvae were found to possess fewer saturated fatty acids (SFAs) and more unsaturated fatty acids (USFAs) in renal triacylglycerol and phospholipid classes, while there were no significant differences in the SFAs and USFAs of hepatic triacylglycerol, phospholipid, cholesteryl ester, fatty acid, and monoacylglycerol classes. Fewer SFAs, found in the kidney triacylglycerol of 13 °C-acclimated lamprey, were due to lower 12:0 and 14:0 fatty acids, but those in the renal phospholipid class were characterized by fewer 14:0, 15:0, and 16:0 fatty acids. More USFAs in renal triacylglycerol, as indicated by a higher unsaturation index, primarily resulted from higher polyunsaturated fatty acids (18:2ω6, 18:3ω3, and 18:4ω3); whereas, in the renal phospholipid class, this was a result of higher monoenes (18:1, 20:1, and 22:1ω9) and ω3 polyunsaturated fatty acids (18:4ω3). These data suggest that the influence of thermal acclimation on the lipid composition of lamprey fat depot organs depends on tissue and lipid class.

Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L.

BACKGROUND: Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis. MATERIALS AND METHODS: Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined. RESULTS: The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis. CONCLUSIONS: DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model.

Identity and distribution of immunoreactive adenohypophysial cells in the pituitary during the life cycle of sea lampreys, Petromyzon marinus.

Adrenocorticotropin (ACTH), melanotropins (MSHs), growth hormone (GH) and gonadotropin (GTH) have been identified or cloned from the pituitary gland of sea lampreys (Petromyzon marinus). The present study was designed to gain insights into the functional significance of these hormones through a description of changes in the occurrence and distribution of cells immunoreactive to their antibodies at several different stages of the sea lamprey life cycle. ACTH-like cells and MSH-like cells were distributed in the rostral pars distalis and the pars intermedia, respectively, throughout the life cycle from ammocoetes (larvae) to pre-spawning adults. A large number of ACTH-like cells were observed during the pre-spawning period when animals may experience the highest stressful conditions. On the other hand, the number of MSH-like cells increased markedly during metamorphosis, in accordance with the completion of eye development. A small number of GH-like cells were present in the proximal pars distalis during the larval and metamorphic phases, but the number of cells increased markedly during the parasitic period, which corresponded well with the rapid somatic growth. GTH-like cells were not observed in the pituitary during the larval and metamorphic phases, but were present in the proximal pars distalis of immediately post-metamorphosed animals. Since there was a high accumulation of GTH-like cells in pre-spawning adults, these cells appeared to be involved in gonadotropic functions. The results of changing immunoreactivity during the lamprey life cycle suggest that lamprey adenohypophysial hormones, ACTH, MSH, GH and GTH, may possess biological functions similar to those of more advanced gnathostome vertebrates. Given that lampreys represent the most ancient group of vertebrates, it is most likely that these hormones have been conserved for their functions throughout vertebrate evolution.

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.

Cloning of stanniocalcin (STC) cDNAs of divergent teleost species: Monomeric STC supports monophyly of the ancient teleosts, the osteoglossomorphs.

Molecular cloning of teleost stanniocalcin (STC) cDNAs was undertaken in two species of order Osteoglossiformes of subdivision Osteoglossomorpha and one species of each of orders Cypriniformes and Perciformes within the subdivision Euteleostei. The elephantnose (Gnathonemus petersii) and the butterflyfish (Pantadon buchholzi) are basal teleosts in different osteoglossiforme suborders yet their 218 amino acid (aa) mature hormones, from prehormones of 249 and 251aa, respectively, have only 10 cysteine residues. A substitution for cysteine at the intermonomeric disulfide linkage site, implies that their STCs exist as monomeric peptides, as is the case with STC from another osteoglossormorph, arawana [Amemiya, Y., Marra, L.E., Reyhani, N., Youson, J.H., 2002. Stanniocalcin from an ancient teleost: a monomeric form of the hormone and a possible extracorpuscular distribution. Mol. Cell. Endocrinol. 188, 141-150]. The STC cDNA of the generalized teleost and cyprinid, the white sucker (Catostomus commersoni), encodes a prehormone of 249aa with a signal peptide of 31aa and a mature protein of 218aa that possesses 11 cysteine residues. The latter feature is consistent with a previous analysis that white sucker mature STC is a glycosylated, homodimeric peptide [Amemiya, Y., Marra, L.E., Reyhani, N., Youson, J.H., 2002. Stanniocalcin from an ancient teleost: a monomeric form of the hormone and a possible extracorpuscular distribution. Mol. Cell. Endocrinol. 188, 141-150]. An open reading frame of the STC cDNA of the derived teleost and perciforme, the smallmouth bass (Micropterus dolomieui), encodes a prehormone of 255aa with a signal peptide of 33aa and a mature protein of 222aa. The position of the 11 cysteines in smallmouth bass STC suggests that it exists as a homodimeric peptide. A phylogenetic analysis, using the new STC-1 amino acid sequences and those in the gene data base provided strong support for monophyly of the Osteoglossomorpha and indicated, with positioning of white sucker and smallmouth bass, that this molecule has some utility as a taxonomic marker. This analysis also suggested that two STC-1 gene sequences exist in multiple fish genomes, and that they may be a product of the fish-specific genome duplication. The mutation in the osteoglossomorph STC likely occurred after the appearance of the first teleosts and before movement of the tectonic plates.

Expression of neuropeptide Y family peptides in the brain and gut during stages of the life cycle of a parasitic lamprey (Petromyzon marinus) and a nonparasitic lamprey (Ichthyomyzon gagei).

The long-term objective of our research is to show that internal factors may be key to triggering metamorphosis and directing the life history types in lampreys (parasitism versus nonparasitism). Since neuropeptide Y family peptides are key players in the endocrine-mediated feeding and reproductive events in mammals, a role for these peptides in the control of feeding behavior and development can be predicted for lampreys. We have investigated the expression pattern of these peptides in the brain and in the gut during different stages of the life cycle of the parasitic lamprey, Petromyzon marinus, and the nonparasitic lamprey, Ichthyomyzon gagei. We provide a description of the cloning and sequencing of P. marinus and I. gagei cDNA for neuropeptide Y (NPY), peptide tyrosine-tyrosine (PYY), and peptide methionine-tyrosine (PMY). Using sequence-specific primers, the mRNA expression patterns for these peptides in brain and gut of larval (ammocoete) and adult (juvenile, prespawning) lampreys were examined by semiquantitative RT-PCR. The observations extend a potential role of neuropeptide Y family peptides in the modulation of feeding behavior and reproductive maturity in lampreys.

Lamprey parasitism of sharks and teleosts: high capacity urea excretion in an extant vertebrate relic.

We observed 10 sea lampreys (Petromyzon marinus) parasitizing basking sharks (Cetorhinus maximus), the world's second largest fish, in the Bay of Fundy. Due to the high concentrations of urea in the blood and tissues of ureosmotic elasmobranchs, we hypothesized that sea lampreys would have mechanisms to eliminate co-ingested urea while feeding on basking sharks. Post-removal urea excretion rates (J(Urea)) in two lampreys, removed from separate sharks by divers, were initially 450 ( approximately 9000 micromol N kg-1 h-1) and 75 times ( approximately 1500 micromol N kg-1 h-1) greater than basal (non-feeding) rates ( approximately 20 micromol N kg-1 h-1). In contrast, J(Urea) increased by 15-fold after parasitic lampreys were removed from non-ureosmotic rainbow trout (Oncorhynchus mykiss). Since activities of the ornithine urea cycle (OUC) enzymes, carbamoyl phosphate synthetase III (CPSase III) and ornithine carbamoyl transferase (OCT) were relatively low in liver and below detection in intestine and muscle, it is unlikely that the excreted urea arose from de novo urea synthesis. Measurements of arginase activity suggested that hydrolysis of dietary arginine made a minor contribution to J(Urea.). Post-feeding ammonia excretion rates (J(Amm)) were 15- to 25-fold greater than basal rates in lampreys removed from both basking sharks and rainbow trout, suggesting that parasitic lampreys have a high capacity to deaminate amino acids. We conclude that the sea lamprey's ability to penetrate the dermal denticle armor of sharks, to rapidly excrete large volumes of urea and a high capacity to deaminate amino acids, represent adaptations that have contributed to the evolutionary success of these phylogenetically ancient vertebrates.

Primary structure of stanniocalcin in two basal Actinopterygii.

The primary structure of stanniocalcin (STC), the principal product of the corpuscles of Stannius (CS) in ray-finned fishes, was deduced from STC cDNA clones for two species of holostean, the gar, Lepisosteus osseus and the bowfin, Amia calva. Overlapping partial cDNA clones were amplified by polymerase chain reaction (PCR) from single-strand cDNA of the CS. Excluding the poly(A) tail, the cDNAs of 1863 base pairs [bp] (gar) and 914 bp (bowfin) contained the 5' untranslated region followed by the coding region and the 3' untranslated region. Both the gar and bowfin STC cDNA encode a prehormone of 252 amino acids (aa) with a signal peptide of 32 aa and a mature protein of 220 aa. The deduced aa sequence of gar STC shows 87% identity with bowfin STC, 60-72% identity with most vertebrate STCs and 26% identity with mouse STC2. Phylogenetic analysis of the sequences support a view that the gar and bowfin form a monophyletic holostean clade. RT-PCR revealed in the gar and bowfin that, just as in mammals and rainbow trout, the expression of STC mRNA is widely spread in many tissues and organs. Since the gar and bowfin are representatives of the most ancient fishes known to possess CS, the corpuscular-derived STC molecule in fish has had a conserved evolution.

Effect of alloxan and insulin immunoneutralization on circulating thyroid hormone levels in larval landlocked sea lampreys, petromyzon marinus.

The effects of alloxan, an insulin (INS)-secreting cell toxin, and INS immunoneutralization on circulating levels of thyroid hormones (thyroxine, T(4); triiodothyronine, T(3)) were examined in larval landlocked sea lampreys, Petromyzon marinus. Animals were injected intraperitoneally with either (Experiment 1) saline (0.6%) or alloxan (20 or 200 microg/g body weight), or with (Experiment 2) normal rabbit serum or anti-lamprey INS. Alloxan (200 microg/g) decreased plasma T(3), but not T(4), in larvae by about 45-80%. Three, six, or nine hr after acute immunoneutralization of lamprey INS with anti-lamprey INS, plasma T(3) levels decreased by 13-30%, relative to controls. These data indicate that INS deficiency can regulate the thyroid system of larval lampreys. There is some suggestion that INS may mediate the metamorphic processes by modulating thyroid hormone concentrations.

Cloning of a functional vitamin D receptor from the lamprey (Petromyzon marinus), an ancient vertebrate lacking a calcified skeleton and teeth.

The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.

KClO(4) inhibits thyroidal activity in the larval lamprey endostyle in vitro.

An in vitro experimental system was devised to assess the direct effects of the goitrogen, potassium perchlorate (KClO(4)), on radioiodide uptake and organification by the larval lamprey endostyle. Organification refers to the incorporation of iodide into lamprey thyroglobulin (Tg). Histological and biochemical evidence indicated that endostyles were viable at the termination of a 4h in vitro incubation. A single iodoprotein, designated as lamprey Tg, was identified in the endostylar homogenates by polyacrylamide gel electrophoresis and Western blotting. Lamprey Tg was immunoreactive with rabbit anti-human Tg serum and had an electrophoretic mobility similar to that of reduced porcine Tg. When KClO(4) was added to the incubation medium, both iodide uptake and organification by the endostyle were significantly reduced relative to controls as determined by gamma counting, and gel-autoradiography and densitometry, respectively. Western blotting showed that KClO(4) significantly lowered the total amount of lamprey Tg in the endostyle. Based on the results of this in vitro investigation, we conclude that KClO(4) acts directly on the larval lamprey endostyle to inhibit thyroidal activity. These data support a previous supposition from in vivo experimentation that KClO(4) acts directly on the endostyle to suppress the synthesis of thyroxine and triiodothyronine, resulting in a decrease in the serum levels of these two hormones.

Stanniocalcin from an ancient teleost: a monomeric form of the hormone and a possible extracorpuscular distribution.

Stanniocalcin (STC) is a homodimeric glycoprotein hormone implicated in calcium and phosphate regulation in both teleost fish and mammals. In the present study, immunostaining with salmon STC antiserum demonstrated that STC cells were localized in both the corpuscles of Stannius (CS) and in specific cells of the distal renal tubules of the silver arawana, Osteoglossum bicirrhosum, an ancient ray-finned fish (actinoptergian) and basal teleost (Order Osteoglossiforme). The morphology of these STC-immunoreactive kidney cells was similar to renal 'chloride' (mitochondrial-rich) cells. The immunoreactive renal cells were present in two of three other osteoglossiformes and absent in the gar, a nonteleost actinopterygian and the eel, another basal teleost. The arawana STC cDNA encodes a prehormone of 249 amino acids (aa) with a signal peptide of 31 aa and a mature protein of 218 aa. The deduced aa sequence of arawana STC shows 54-66% identity with other teleost STCs and 51-52% identity with mammalian STC-1. The deduced aa sequence of arawana STC contains ten cysteines, compared with 11 in teleost STC and in mammalian STC-1. The cysteine substitution occurs at the site of inter-monomeric disulfide linkage. Western blot analysis revealed a single 21 kDa band under non-reducing conditions, and a single band of 25 kDa under reducing conditions. These data indicate that arawana STC exists as a monomeric peptide. Northern blot analysis detected a 3.3 kb STC mRNA confined to the CS, with no hybridization signal in either the remaining kidney or in gut, muscle, brain and heart. The significance of the STC signal in cells of the renal tubules of arawana and two other Osteoglossiforme species requires further investigation. This is the first report of a monomeric form of STC in any vertebrate and the first evidence of STC in renal tubules of adult fish.

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.

Macroscopic structure of the fin-rays and their annuli in pectoral and pelvic fins of Chinook Salmon, Oncorhynchus tshawytscha.

Light microscopy, enzyme clearing, and staining techniques were used to describe the structure of fin-rays in pectoral and dorsal fins of adult and juvenile chinook salmon,Oncorhynchus tshawytscha. In addition, several decalcification agents, fixatives, and staining methods were employed to demonstrate and determine the nature of the fin-ray annulus (yearly growth ring). Etched, transverse sections of fin-rays were examined by scanning electron microscopy (SEM). A description is provided of specific morphological regions along the length of fin-rays in both fins. Each ray consists of parallel halves, medial and lateral hemisegments, which diverge proximally and embrace underlying radial cartilages and are segmented distally. Fibrocartilage masses are associated with bone and hyaline cartilage in the proximal regions. There is evidence of periosteal, and perhaps endochondral, osteogenesis in the fin-rays. A novel fixation-staining method is described to depict the fin ray annulus in adult chinook salmon. Transverse sections of rays of adults, but not juveniles, demonstrate alternating narrow-dark and wide-light purple bands following prolonged decalcification/fixation in Bouin's fluid only with subsequent hematoxylin-eosin staining. The dark and light bands correspond to translucent (annuli) and opaque zones, respectively, in dried, unstained sections in transmitted light and in backscatter SEM. In conjunction with different light microscopic staining procedures, etched, transverse sections of fin-ray bone in SEM suggest that annuli are a manifestation of an ordered regionalization of the extracellular matrix of the bone in the fin-rays. J. Morphol. 239:297-320, 1999. © 1999 Wiley-Liss, Inc.

Metamorphosis of the olfactory organ of the sea lamprey (Petromyzon marinus L.): Morphological changes and morphometric analysis.

In larval sea lampreys (Petromyzon marinus), a small, relatively inconspicuous olfactory organ sac contains small, densely packed olfactory receptor neurons and sustentacular cells. During metamorphosis, the larval organ transforms into a prominent lamellar structure with large distinct olfactory epithelial cells that is characteristic of the adult lamprey. In the present study, scanning electron microscopy and light microscopy are used to examine changes during the seven stages (1-7) of metamorphosis. The magnitude of growth over the course of metamorphosis is evident from the doubling of the relative weight of the nasal sac. During early metamorphosis (stages 1 and 2), the larval olfactory organ enlarges, and by stage 3 specific adult structures begin to form, namely a nasal valve between the nasal tube and the organ, lamellar folds, and diverticuli of the accessory olfactory organ. Subsequent development involves widening of the cells lining the lamellar folds to the form characteristic of postmetamorphic lampreys. Although the cells in the troughs initially retain numerical density values that are significantly higher than those on the lamellar surfaces, by stage 7 values decline both in troughs and along lamellar surfaces to those observed in adults. These results show that although expansion of the olfactory organ is ongoing throughout metamorphosis, remodeling occurs early (by stage 3). This timing provides space for extensive olfactory receptor neuron neurogenesis and differentiation and correlates with the transformation of some organs that were previously examined. This is the first report in any species of olfactory receptor neuron zonation based on morphometric characteristics. J. Morphol. 231:41-52, 1997. © 1997 Wiley-Liss, Inc.

Morphology and ultrastructure of possible integumentary sense organs in the estuarine crocodile (Crocodylus porosus).

The skins of crocodylids and gavialids can be distinguished from those of alligatorids by the presence of darkly pigmented pits, known as integumentary sense organs (ISOs), on the postcranial scales. The structure of ISOs, in Crocodylus porosus, was studied using light microscopy and scanning and transmission electron microscopy. The stratum corneum of the epidermis in the area of the ISO is thinner, while the stratum germinativum is thicker, relative to other regions of the integument. Beneath the epidermal layer the ISO region has a paucity of collagen fibers relative to the rest of the dermis. Widely dispersed fibrocytes, nerve terminals, and chromatophores occur throughout the ISO region of the dermis, but these elements are concentrated in the area immediately beneath the stratum germinativum in the ISO region. The morphology of the ISOs suggests that they are sensory organs. It has traditionally been assumed that sensory organs on the amniote integument have a mechanosensory function. However, alternate functional interpretations of this structure are possible, and a resolution awaits further work. © 1996 Wiley-Liss, Inc.

Configuration of the medial and lateral segments of duck (Anas platyrhynchos) salt glands.

Salt glands of the domestic duck Anas platyrhynchos differ from those of the herring gull Larus argentatus and other birds. In ducks, each salt gland consists of distinct medial and lateral segments. Centrally located drainage ducts that extend along the entire length of these medial and lateral segments collect hypertonic fluid secreted by an array of lobules. Each lobule is formed by a single mass of branched tubules in which the direction of capillary blood flow is opposite to that of the secreted fluid. This fluid drains from the medial segment through an external duct that opens into the nasal cavity at the base of the vestibular fold. A duct from the lateral segment loops and opens onto the surface of the nasal septum. The structure and function of the secretory cells is reviewed briefly within the context of our study of the configuration of duck nasal salt glands.

Morphology of the intestine of prefeeding and feeding adult lampreys, Petromyzon marinus (L): The mucosa of the posterior intestine and hindgut.

The epithelium of the posterior intestine and hindgut of recently metamorphosed adult lampreys (Petromyzon marinus L.) prior to and during spontaneous feeding was examined using light and electron microscopy. These two regions differ slightly in their general morphology but possess the same mucosal cell types. Included are caveolated absorptive and mucous cells, which are not present in more cephalic regions of the intestine, and ciliated and enteroendocrine cells. During feeding, the caveolated cells undergo dramatic transformation in their structure, namely, through the acquisition of numerous heterophagic vacuoles. Due to their morphology and to the fact that there are low amounts of lipid, it is suspected that caveolated cells are primarily involved in the absorption of protein components from the ingested host blood and body fluids. Iron in caveolated cells may result from the degradation of ingested heme or reflect the excretion of bile products at this location in the intestine. Mucous cells are likely responsible for lubrication of the luminal surface and may be important as a stem cell for the mucosal epithelium.