Evolutionary origins of taste buds: phylogenetic analysis of purinergic neurotransmission in epithelial chemosensors

Taste buds are gustatory endorgans which use an uncommon purinergic signalling system to transmit information to afferent gustatory nerve fibres. In mammals, ATP is a crucial neurotransmitter released by the taste cells to activate the afferent nerve fibres. Taste buds in mammals display a characteristic, highly specific ecto-ATPase (NTPDase2) activity, suggesting a role in inactivation of the neurotransmitter. The purpose of this study was to test whether the presence of markers of purinergic signalling characterize taste buds in anamniote vertebrates and to test whether similar purinergic systems are employed by other exteroceptive chemosensory systems. The species examined include several teleosts, elasmobranchs, lampreys and hagfish, the last of which lacks vertebrate-type taste buds. For comparison, Schreiner organs of hagfish and solitary chemosensory cells (SCCs) of teleosts, both of which are epidermal chemosensory end organs, were also examined because they might be evolutionarily related to taste buds. Ecto-ATPase activity was evident in elongate cells in all fish taste buds, including teleosts, elasmobranchs and lampreys. Neither SCCs nor Schreiner organs show specific ecto-ATPase activity, suggesting that purinergic signalling is not crucial in those systems as it is for taste buds. These findings suggest that the taste system did not originate from SCCs but arose independently in early vertebrates.     

Variations in the presence of chloride cells in the gills of lampreys (Petromyzontiformes) and their evolutionary implications

Although confined to fresh water, non‐parasitic species of lampreys and the landlocked parasitic sea lamprey, all of which were derived relatively recently from anadromous ancestors, still develop chloride cells, whose function in their ancestors was for osmoregulation in marine waters during the adult parasitic phase. In contrast, such cells are not developed by the non‐parasitic least brook lamprey Lampetra aepyptera, which has been separated from its ancestor for >2 million years, nor by the freshwater parasitic species of the genus Ichthyomyzon. The length of time that a non‐parasitic species or landlocked parasitic form or species has spent in fresh water is thus considered the overriding factor determining whether chloride cells are developed by those lampreys.     

THE VISUAL SYSTEM AND VITAMINS A OF THE SEA LAMPREY

The porphyropsin-vitamin A₂ cycle has been found heretofore only in the retinas of bony fishes capable of existence in fresh water. Cyclostomes, due to their primitive and isolated phylogenetic position, might be expected to possess the rhodopsin-vitamin A₁ cycle common to marine elasmobranchs, almost all marine teleosts, and all terrestrial vertebrates so far examined. Yet the anadromous sea lamprey, Petromyzon marinus, possesses primarily the porphyropsin system, like an anadromous teleost. This observation greatly extends the phylogenetic association of vitamin A₂ with the capacity for freshwater existence. Compared with freshwater and anadromous teleosts, the lamprey retina contains the porphyropsin system in extremely low concentration. The remaining eye tissues, like the retina, contain both vitamins A₁ and A₂, the latter greatly predominant. The livers of larval and adult lampreys, however, appear to contain vitamin A₁ alone. This situation also is not without teleost precedent, since the carp and certain anadromous salmonids display similar reversals of vitamin A pattern in the liver and eye tissues.     

Lampreys Have a Single Gene Cluster for the Fast Skeletal Myosin Heavy Chain Gene Family

Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5′-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny.     

Secretion of endogenous lithium in teleost kidneys as a probable reflection of Na+ and water secretion in their renal proximal tubules

Unlike mammals with renal reabsorption of lithium (Li+), in freshwater and, particularly, marine teleosts net secretion of this trace element by kidneys was discovered. The ratio of Li+ natural concentration (measured by mass spectrometric isotope dilution technique) in urine to that in blood plasma--(U/P)Li--lies in the range 2-6 in the freshwater species and between 5 and 14 in marine species, i.e. as a rule it is essentially higher than the inulin concentration index (U/P)In. It is supposed that the in vivo observed lithium net secretion in whole kidney reflects and quantitatively estimates Na+ and water secretion in renal proximal tubules of teleosts.     

Effects of atrial natriuretic peptides and furosemide in conscious dogs

Effects of ANF(8-33) and Auriculin A on renal variables were investigated in conscious water-diuretic dogs. The two substances were injected intravenously (1.08 micrograms/kg in 3 min) or ANF(8-33) was infused (0.2 microgram/kg X min in 20 min). The effects were compared to those of an equinatriuretic dose of furosemide (1.0 microgram/kg X min). Injections caused increases in sodium excretion, diuresis, and osmolar clearance. No significant change in exogenous creatine clearance (CCREA) occurred. Infusion of ANF(8-33) decreased blood pressure by 14% (P less than 0.01) and increased sodium excretion by a factor of 10 (P less than 0.01). The natriuresis was a function of increases in diuresis and urinary sodium concentration, the latter by a factor of 6 (P less than 0.01). Diuresis and free-water clearance (CH2O) increased by 60% (P less than 0.01), but urine osmolality did not change significantly. After the infusion a significant decrease in PAH clearance (CPAH) (P less than 0.01) was observed. Filtration fraction (FF) did not change. The furosemide natriuresis appeared later than that of ANF without significant deviations in diuresis, CH2O, CCREA, CPAH, and FF; urine osmolality increased by 35% (P less than 0.01). The effects of ANF(8-33) differ from those of furosemide in several ways. First, the onset of natriuresis is faster, second, the natriuresis is associated by marked increases in diuresis and free-water clearance but not in urine osmolality; and third, natriuresis is followed by a reduction in renal blood flow. The rapid natriuresis of ANF can occur without changes in glomerular filtration rate.     

Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions

Although prolactin has been demonstrated to be the main hormone involved in adaptation to dilute media in several freshwater teleosts, few studies have been conducted in marine teleosts. In the Mediterranean, the sea bass Dicentrarchus labrax inhabits environments ranging from the open sea to coastal lagoons, where salinity varies greatly. We characterised the prolactin (prl) gene and analysed its expression in two organs (gill and intestine) in D. labrax acclimated to either freshwater or seawater. A 2819 bp long sequence encompassing the prl gene and a part (282 bp) of the promoter were identified, and these comprised 5 coding exons separated by 4 introns. Prolactin was similarly expressed in fresh- and seawater adapted fish, although expression in gills was significantly greater than in the intestine. Nonetheless, individuals unable to successfully regulate osmotic balance in freshwater presented overall low expression rates. Results are discussed according to the mechanism of sea bass adaptation in the wild and to their life cycle between open sea and lagoons. Finally, a phylogenetic analysis indicated that teleosts are not branched according to their life-history features (e.g. seawater vs. freshwater habitats), and no signature of positive selection was detected across the phylogeny of the prl gene in teleosts.     

Circulating angiotensins in the river lamprey, Lampetra fluviatilis, acclimated to freshwater and seawater: possible involvement in the regulation of drinking

Plasma angiotensin levels were measured for the first time in a cyclostome, the river lamprey. With the demonstration that angiotensins are present in the circulation, the possibility of a physiological role in the regulation of drinking was re-examined. Angiotensin II and III concentrations and plasma osmolalities were significantly higher in lampreys acclimated to 28 ppt seawater than in those acclimated to freshwater. No changes were found in angiotensin II and III levels 4 h after transfer from freshwater to 50% seawater, although plasma osmolality had started to rise by this time. There was a suggestion that plasma angiotensin II levels might be related to osmolality in the transfer experiment. Injection of Asp(1)Val(5)- or Asn(1)Val(5)-angiotensin II (40-169 microg/kg body wt.) did not stimulate drinking in freshwater-acclimated lampreys, even when they were still capable of drinking. The angiotensin-converting enzyme inhibitor captopril and the smooth muscle relaxant papaverine both reduced drinking rate in 50% seawater-acclimated lampreys. The data do not provide direct evidence for the involvement of the renin-angiotensin system in the control of drinking behaviour in the lamprey. Indirect evidence from the captopril effect is suggestive, but could have other explanations.     

Osmoregulation in North American eels (Anguilla rostrata LeSueur) on land and in freshwater: effects of the corpuscles of Stannius

Hypercalcemia, hypomagnesia and hypophosphatemia were observed in freshwater (FW) eels (Anguilla rostrata LeSueur) after removal of the corpuscles of Stannius. These changes did not occur if Stanniectomized (CSX) eels were removed from FW and placed on land for 12 days but did occur after the eels were returned to FW. Therefore, changes in plasma electrolyte concentrations after CSX depended upon the branchial and/or integumental influx of ions. Plasma Na⁺, Cl⁻ and osmolal concentrations decreased gradually in both sham-operated (SHM) and CSX eels on land (12 days) and in FW (12 days). Plasma K⁺almost doubled in both SHM and CSX eels after 4 days on land, remained elevated, and fell abruptly to normal within a day after the eels were returned in FW. After 2 days on land, urine flow rates in SHM and CSX eels had decreased by approximately 85%, osmolar clearance by 50% and positive free-water clearance by more than 90%. Body weights did not decrease when eels were on land so it was concluded that the reduced but continuous renal loss of water was counterbalanced by the integumental uptake of condensed water. Accepted: 21 October 1998     

A Systematic Survey of the Occurrence of the Hypothalamic Saccus Vasculosus in Teleost Fish

The saccus vasculosus (SV) was examined histologically in more than 200 species of teleosts. The Atherinomorpha (Cyprinodontiformes and Atheriniformes) completely lack the SV. Many primary freshwater teleosts (Osteoglossiformes, Cypriniformes, Characiformes, Siluriformes, Gymnotiformes, and Synbranchiformes) possess none or a reduced SV. Some secondary freshwater teleosts also possess a reduced SV, but other secondary freshwater teleosts have a well-developed SV. Some marine teleosts swimming in the surface water, for example, the Clupeiformes and Scombridae, possess a somewhat reduced SV. Bathypelagic teleosts also possess a rather reduced SV. On the other hand, most marine teleosts, benthic or nektonic in the subsurface water, have a well-developed SV. The examples are the Anguilliformes, Scorpaeniformes, Perciformes, Pleuronectiformes, and Tetraodontiformes. Since all non-teleostean actinopterygians possess a well-deveoped SV, it is assumed that the earliest teleosts also possessed a SV. In the course of adaptive radiation, some teleost groups certainly have reduced or lost the SV while others developed it to various degrees. Reduction or development of the SV might be influenced more easily by environments in certain groups and might be constrained more strongly by phylogenetic lineages in other groups.     

Contrasting population genetic structure among freshwater‐resident and anadromous lampreys: the role of demographic history,differential dispersal and anthropogenic barriers to movement

The tendency of many species to abandon migration remains a poorly understood aspect of evolutionary biology that may play an important role in promoting species radiation by both allopatric and sympatric mechanisms. Anadromy inherently offers an opportunity for the colonization of freshwater environments, and the shift from an anadromous to a wholly freshwater life history has occurred in many families of fishes. Freshwater‐resident forms have arisen repeatedly among lampreys (within the Petromyzontidae and Mordaciidae), and there has been much debate as to whether anadromous lampreys, and their derived freshwater‐resident analogues, constitute distinct species or are divergent ecotypes of polymorphic species. Samples of 543 European river lamprey Lampetra fluviatilis (mostly from anadromous populations) and freshwater European brook lamprey Lampetra planeri from across 18 sites, primarily in the British Isles, were investigated for 13 polymorphic microsatellite DNA loci, and 108 samples from six of these sites were sequenced for 829 bp of mitochondrial DNA (mtDNA). We found contrasting patterns of population structure for mtDNA and microsatellite DNA markers, such that low diversity and little structure were seen for all populations for mtDNA (consistent with a recent founder expansion event), while fine‐scale structuring was evident for nuclear markers. Strong differentiation for microsatellite DNA loci was seen among freshwater‐resident L. planeri populations and between L. fluviatilis and L. planeri in most cases, but little structure was evident among anadromous L. fluviatilis populations. We conclude that postglacial colonization founded multiple freshwater‐resident populations with strong habitat fidelity and limited dispersal tendencies that became highly differentiated, a pattern that was likely intensified by anthropogenic barriers.     

Structural changes in the zonulae occludentes of the chloride cells of young adult lampreys following acclimation to seawater

Summary Thin sections and freeze-fracture replicas have been used to study the structure of the zonulae occludentes of the branchial chloride cells in young adults of the anadromous lamprey Geotria australis, caught during their downstream migration to the sea and after acclimation to full-strength seawater (35). The chloride cells in the epithelium of the gill filaments of both freshwater- and seawater-acclimated animals form extensive multicellular complexes. In freshwater animals, the majority of chloride cells (64%) are covered by pavement cells and are thus not exposed to the external environment. Most of the other chloride cells are separated from each other by pavement cells or their processes. The zonulae occludentes between chloride cells and pavement cells and between adjacent chloride cells are extensive and characterised by a network of 4 (range 3–7) superimposed strands. In seawater-acclimated animals, the pavement cells cover only 30% of the chloride cells and their processes no longer occur between chloride cells. Whereas the zonulae occludentes between chloride cells and pavement cells are still extensive, those between chloride cells are shallow and comprise only a single strand or two parallel strands. The zonulae occludentes between the chloride cells of lampreys acclimated to seawater are similar to those in the gills of teleosts in seawater, and are thus considered to be leaky and to provide a low-resistance paracellular pathway for the passive transepithelial movement of Na⁺.     

Theory on the evolutionary history of lamprey metamorphosis: role of reproductive and thyroid axes

Metamorphosis is a developmental strategy used by only a small number of extant fishes and little is known about its phylogenetic development during the evolution history of this large group of vertebrates. The present report provides a putative evolutionary history of metamorphosis in the lamprey, an extant agnathan with direct descendancy from some of the oldest known vertebrates. The study reviews recent data on the role of the thyroid gland and its hormones in metamorphosis, summarizes some recent views on the evolution of the endostyle/follicular thyroid in lampreys, and provides new data on the content of two gonadotropin-releasing hormones (GnRH-I and -III) in brain during goitrogen-stimulated, precocious metamorphosis. These new data support an earlier viewpoint of a relationship between thyroid and reproductive axes during metamorphosis. It is proposed that the earliest lampreys were paedomorphic larvae and they lived in a marine environment; as such, they resembled in many ways the larvae from which the ancient protochordates, Larvacea, are derived. The iodide-concentrating efficiency of the endostyle was a critical factor in the evolution of metamorphosis and this gland was replaced by a follicular thyroid, for postmetamorphic animals needed to store iodine following their invasion of freshwater. Larval growth and postmetamorphic reproduction in freshwater became fixtures in the lamprey life cycle; a non-parasitic adult life-history type appeared later. The presence among extant lampreys of two different adult life-history types, and examples of the lability of the timing of sexual maturation in some species, imply that there has been a complex interplay between the thyroid and reproductive axes during the evolution of metamorphosis in lampreys. This proposal is consistent with what we know of interplay of these axes in extant adult lampreys and with the long-held viewpoint that thyroid function and sexual maturation are an association with an ancient history.     

The relative importance of the gills to ammonia and urea excretion in five seawater and one freshwater teleost species

The classic Homer Smith divided box experiment (Smith, 1929) was repeated upon five marine and one freshwater teleost species. Of the nitrogenous output (as ammonia and urea), 50–70% occurred in the head region for marine teleosts, while the freshwater tilapia studied excreted 90% from the head. Together with the data of Smith, these data indicate that nitrogen excretion by the gills is of lesser importance in marine teleosts than in freshwater species.
In the dab, Limanda limanda , the head, anus and urinary opening were all in the anterior chamber of the divided box, but 47% of nitrogen excretion still occurred in the posterior chamber, presumably across the skin. In the blenny, Blennius pholis , exposed to air, the head region accounted for 46% of urea elimination, and 26% of ammonia output.     

Binding kinetics and sequencing of hepatic alpha1-adrenergic receptors in two marine teleosts, mackerel (Scomber scombrus) and anchovy (Engraulis encrasicolus)

Liver alpha(1)-adrenoceptors (ARs) are demonstrated, or at least hypothesized, in freshwater and brackish-water teleosts, whereas no data are available for marine teleosts. This study evaluates the presence of alpha(1)-ARs in the liver of two marine teleosts, the anchovy Engraulis encrasicolus and the mackerel Scomber scombrus, and examines on a broad scale the possibility that habitats posing different challenges also influence phenotypic trait selection. Binding assays were performed also on liver membranes from the carp Cyprinus carpio as a direct comparison with a freshwater species. Scatchard analysis of [(3)H]prazosin binding to purified liver membranes from anchovy, mackerel and carp resulted in K(d) values of 1.51+/-0.085, 1.26+/-0.098, and 2.61+/-0.22 nM, and B(max) values of 87.4+/-9.12, 77+/-8.29, and 115.22+/-3.31 fmol/mg protein, respectively. Thus, alpha(1)-ARs of the two marine teleosts showed higher [(3)H]prazosin affinity compared with those of the freshwater/brackish-water fish studied thus far, whereas the number of liver binding sites did not differ significantly from that of carp, eel or trout. A preliminary phylogeny based on amino acid sequence analysis indicated the presence of at least an alpha(1A)-AR in mackerel and an alpha(1D)-AR in both anchovy and mackerel. This is the first indication of alpha(1)-AR subtypes in any marine species, but further studies are needed to ascertain the physiological role of these alpha(1)-ARs in these two marine species.     

Profiles of the female-specific serum protein in the Japanese river lamprey, Lampetra japonica (Martens), and the sand lamprey, Lampetra reissneri (Dybowski), in relation to sexual maturation

The occurrence of female-specific serum protein (FSSP) was examined in the anadromous lamprey, Lampetra japonica, and a freshwater lamprey, Lampetra reissneri, by an immunodiffusion technique using antiserum raised against the serum of sexually mature female lamprey. A considerable amount of FSSP was consistently detected in the blood of vitellogenic, but not in that of nonvitellogenic, females of both species of lampreys. The relative amount of the FSSP tended to become larger in female lampreys at the spawning period. The FSSP may correspond to the yolk precursor protein which is incorporated into ovarian oocytes during the period of exogenous vitellogenesis in the lampreys.     

Sensory structures at the surface of fish skin. II. Lateralis System

Scanning electron microscopy shows the form of the cupulae of free neuromasts in two species of teleost fish, and gives information about the organization of the free neuromasts in teleosts and lampreys. In lampreys some neuromasts were found to lack the surrounding moat and the flanking hillocks characteristic of the lateral line organs previously described in these fish. In all cases, the sensory cells had the kinocilium aligned with respect to the stereocilia on the longer axis of the neuromast surface, thus enabling the direction of effective stimulation of the free neuromasts to be deduced from their morphological arrangement.     

Categorization of the mitochondria-rich cells in the gill epithelium of the freshwater phases in the life cycle of lampreys

The distribution and ultrastructure of the mitochondria-rich (MR) cells in the gills of larval (ammocoetes) and adult lampreys (Petromyzon marinus and Geotria australis) have been studied. One type of MR cell, which is found only in ammocoetes, occurs in groups on and between gill lamellae. Freeze-fracture replicas show that the apical membrane of this ammocoete MR cell contains globular particles. The second type of MR cell, which is present in both ammocoetes and adults in freshwater, is located between lamellae and at the base of the filament. This cell usually occurs singly and is typically intercalated between ammocoete MR cells in larval lampreys and between pavement cells and pavement and chloride cells in adult lampreys. It contains rod-shaped particles in either the apical membrane (subtype A) or, far less frequently, the lateral membrane (subtype B) and in membranes of cytoplasmic vesicles and tubules. These features characterize this intercalated MR cell as a member of a group of MR cells that are also found in urinary epithelia of tetrapods and the amphibian epidermis, where they are involved in H⁺ and HCO₃ ^- secretion. Because this type of MR cell disappears when the young adult lamprey enters the sea and reappears immediately after the fully grown adult re-enters freshwater on its spawning run, it is presumably essential for osmoregulation in freshwater. On the basis of electrophysiological studies on frog skin, it is proposed that the subtype A of the branchial intercalated MR cell of lampreys provides the driving force for the Na⁺ uptake by active H⁺ secretion. By analogy with urinary epithelia, the subtype B cells may exchange Cl^- for HCO₃ ^-.     

Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.