Gonadotropin-like and adrenocorticotropin-like cells in the pituitary gland of hagfish, Paramyxine atami; immunohistochemistry in combination with lectin histochemistry

The pituitary system of the hagfish remains an enigma. The present study has aimed to detect possible adenohypophysial hormones in the pituitary gland of the brown hagfish, Paramyxine atami, by means of immunohistochemistry in combination with lectin histochemistry. Rabbit antisera raised against ovine luteinizing hormone (LH)β, proopiomelanocortin (POMC)-related peptides, and the growth hormone/prolactin family of tetrapod and fish species were used, and 25 kinds of lectins were tested. Three different types of adenohypophysial cells were revealed in the pituitary of brown hagfish. The first was stained with both anti-ovine LHβ and several D-mannose-binding lectins, such as Lens culinaris agglutinin and Pisum sativum agglutinin. This cell type predominated in the adenohypophysis in adults with developing gonads and thus appeared to be involved in the regulation of gonadal functions. The second was negative for anti-ovine LHβ but was stained with several N-acetylglucosamine-binding lectins, such as wheat germ agglutinin and Lycopersicon esculentum lectin. This cell type exhibited a weak positive reaction with anti-lamprey adrenocorticotropin (ACTH) and thus appeared to be related to POMC-like cells. The second cell type was found in the adenohypophysis regardless of the developmental state of the gonads. The third cell type was negative for both antisera and lectins. Since this cell type was numerous in juveniles and adults without developing gonads, most cells of this type were probably undifferentiated. These findings suggest that GTH and ACTH are major adenohypophysial hormones in the hagfish. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture (to M.N.).     

A germline restricted, highly repetitive DNA sequence in Paramyxineatami : an interspecifically conserved, but somatically eliminated, element

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83 bp long, has been designated “EEPa1”, for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms.The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution. Received: 7 January 1997 / Accepted: 13 May 1997     

Reproductive biology and ecology of Pacific hagfish (Eptatretus stoutii) and black hagfish (Eptatretus deani)

The reproductive biology of Pacific hagfish Eptatretus stoutii (Lockington, 1878) and black hagfish Eptatretus deani (Evermann & Goldsborough, 1907) was assessed using current and historical data. Our results found that the reproductive characteristics of both hagfish species reflect those of K-selected species, which tend to live long and exhibit slow growth rates, low fecundity (approximately 20 eggs per female) and late maturity. Additionally, females of both species commence maturation prior to males. This study provides a population profile for both species of hagfish, but further assessments are needed to effectively manage a sustainable hagfish fishery.     

Variability in the Presence of Elastic Fibre-like Structures in the Ventral Aorta of Agnathans (Hagfishes and Lampreys)

This paper investigates whether elastic fibre-like structures are present in the ventral aorta of hagfishes and lampreys. Fibres, which are morphologically similar to the elastic fibres of gnathostomatous (jawed) vertebrates, are shown to be present in the ventral aorta of the hagfish Paramyxine atami, and also, but to a lesser extent, the dorsal aorta of this species and the ventral aorta of another hagfish, Eptatretus stouti.The ‘elastic’ tissue formed irregular sheet-like aggregates, comprising well-defined amorphous material surrounded by tubular microfibrils, whose diameters ranged from 10 to 15 nm. While this tissue was most abundant in the same region of the aortae as that occupied by the elastica interna in the blood vessels of gnathostomes, it was also found deeper in the wall of these blood vessels. Although tubular microfibrils were found in the ventral aorta of the hagfish Myxine glutinosaand the lamprey Geotria australis, these were never associated with well–defined, amorphous material. This parallels the results of previous studies on Myxine glutinosaand another species of lamprey, Petromyzon marinus.Thus, the elastic fibre–like tissues found in the ventral aortae of P. atamiand E. stoutiprovide the first examples of such structures in this region in agnathan vertebrates.     

The Complete Mitochondrial Genome of the Hagfish Myxine glutinosa: Unique Features of the Control Region

The complete sequence of the mitochondrial DNA of the hagfish Myxine glutinosa has been determined. The hagfish mtDNA (18,909 bp) is the longest vertebrate mtDNA determined so far. The gene arrangement conforms to the consensus vertebrate type and differs from that of lampreys. The exceptionally long (3628-bp) control region of the hagfish contains the typical conserved elements found in other vertebrate mtDNAs but is characterized by a large number of putative hairpins, which can potentially fold into a highly compact secondary structure that appears to be unique to hagfish. The comparison of the mtDNAs of two M. glutinosa specimens, excluding the control region, shows a 0.6% divergence at the nucleotide level as a sample of intraspecies polymorphism. Received: 21 August 2000 / Accepted: 2 March 2001     

Two variable lymphocyte receptor genes of the inshore hagfish are located far apart on the same chromosome

Variable lymphocyte receptors (VLR) generate enormous diversity through assembling highly diverse leucine-rich repeat (LRR) modules and presumably function as antigen receptors in jawless vertebrates. The hagfish, which constitute major extant members of jawless vertebrates along with lampreys, have two VLR genes designated VLRA and VLRB, whereas only a single VLR gene has been identified in the lamprey. In the present study, we show by fluorescence in situ hybridization (FISH) that hagfish VLRA and VLRB are located on the same chromosome, but are far apart from each other. Analysis of available inshore hagfish complementary DNA sequences indicates that VLRA and VLRB do not share a LRR module with an identical nucleotide sequence. Physical separation of VLRA and VLRB is consistent with this observation and indicates that the two VLR genes function as separate units. The FISH protocol developed in this study should be useful for the analysis of the agnathan genome. J. K. is a research fellow of the Japan Society for the Promotion of Science.     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

FMRFamide-immunoreactive structures in the brain of the brown hagfish,Paramyxine atami: relationship with neuropeptide Y-immunoreactive structures

Summary Localization of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH₂) in the brain and hypophysis of the brown hagfish,Paramyxine atami, was examined by immuno-histochemistry specially regarding a possible relationship with neuropeptide Y (NPY). FMRFamide-immunoreactive fibers were demonstrated in many regions of the brain, with the highest density in the diencephalon. However, no immunoreactivity was found in the hypophysis. Labeled cells were chiefly located in the nucleus hypothalamicus of the diencephalon, although a few cells were recognized in the ventrolateral area of the caudal tegmentum. Examination of adjacent sections immunostained alternatively with anti-NPY antiserum and anti-FMRFamide antiserum showed overlapping of the distributional patterns of the immunoreactive structures in the brain. Moreover, the same cells in the nucleus hypothalamicus were immunostained with both antisera. Cross-blocking experiments showed that the FMRFamide-immunoreactivity is abolished by preabsorption of the antiserum with homologous antigen, but not eliminated completely by pretreatment with appropriate antigens (NPY, avian pancreatic polypeptide and methionine-enkephalin-Arg-Phe). In contrast, the NPY-immunoreactivity was blocked by pretreatment of the antiserum with NPY, pancreatic polypeptide or FMRFamide, although no blocking by enkephalin was observed. Accordingly, the present study shows that, in the brown hagfish, FMRFamide-immunoreactive structures in the brain can be recognized by anti-NPY antiserum.     

Ultrastructure of the subepidermal musculature of Xenoturbella bocki, the adelphotaxon of the Bilateria

 Xenoturbella bocki is the only species of the high-ranked taxon Xenoturbellida. The species lives on marine mud bottoms at a depth of 20–120 m and moves extremely slowly by ciliary gliding. Nevertheless it possesses a well-developed body wall musculature with outer circular muscles, a prominent layer of inner longitudinal muscles and radial muscles that extend from the outer circular myocytes to the musculature surrounding the gastrodermis. The longitudinal myocytes are not compact cells, but form fascicles of fibrils running parallel to each other. Fine cytoplasmic cords connect the fibres of a cell to each other and with its nuclear region. The muscles are embedded within a sometimes expansive extracellular matrix (ECM) that lacks any fibrillar components. All muscle cells display conspicuous and numerous cytoplasmic extensions that are intermingled with each other. Tight coupling between adjacent cell membranes is not found, but zonula adhaerens-like junctions exist. Fibrils belonging to different myocytes, but also fibrils of the same cell, are coupled by such cytoplasmic extensions. Circular, radial and at least the peripheral longitudinal myocytes display cell-matrix connections with the internal lamina, a component of the subepidermal ECM. This internal lamina projects down into the centres of the fascicles with longitudinal muscle fibrils and forms extensive attachment zones with the muscle cells, reminiscent of focal contacts. For the ingestion of food, X. bocki opens the simple mouth pore and protrudes the aciliated gastrodermis. The body wall musculature is responsible for this protrusion and also for the withdrawal of the gastrodermis. In the past, possible phylogenetic kinships with the Acoelomorpha (Plathelminthes) or the Enteropneusta and Holothuroidea were discussed, but, on the basis of all information available, X. bocki is hypothesized to be the sister taxon of the Bilateria. Accepted: 2 April 1997     

Immunohistochemical localization of neuropeptide Y-like substance in the brain and hypophysis of the brown hagfish,Paramyxine atami

The distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the brown hagfish, Paramyxine atami, was examined by use of the peroxidase-antiperoxidase method. Immunoreactive cells were found in two areas of the brain, the nucleus hypothalamicus of the diencephalon and the ventrolateral area of the caudal tegmentum, at the level of the nucleus motorius V–VII. The labeled cells of the nucleus hypothalamicus were loosely grouped and recognized as bipolar neurons. Immunolabeled fibers were widely distributed in the brain, showing the highest density in the diencephalon. They were sparse, or absent, in the olfactory bulb, habenula, primordium hippocampi, neurohypophysis, corpus interpedunculare, and dorsolateral area of the medulla oblongata. The fibers appeared to project exclusively from the ventral hypothalamus to various other portions of the brain: the anterolateral areas of the telencephalon via the basal hypothalamus, the pars dorsalis thalami, the dorsocaudal region of the mesencephalon, and the ventromedial portions of the tegmentum and anterior medulla oblongata. These findings suggest that, in the brown hagfish, NPY-like substance is involved in neuroregulation of various cerebral areas, but it may be of little significance in the control of pituitary function.     

Plant module size and dose of gall induction stimulus influence gall induction and galler performance

Several different hypotheses attribute large variations in gall abundance and galler performance to variations in plant growth rate and/or module size. The plant vigor hypothesis (PVH) predicts that galler performance will be greatest on large, fast‐growing plant modules due to their large, rapidly dividing cells that allow them to make large galls, where survival and size of survivors are usually greater. The optimal module size hypothesis (OMSH) predicts that galler performance will be greatest on intermediate‐sized modules, based on a tradeoff between ease of gall induction on small modules and increased resource availability in large modules. Here, we evaluate predictions of the PVH and OMSH during a two‐year field study by examining gall induction and full gall development success, as well as subsequent galler performance in successfully developed galls, using a shoot galling adelgid, Adelges abietis, on white spruce Picea glauca. In addition, we tested whether increased dose of gall induction stimulus on different sized modules, achieved by observing differing A. abietis densities per bud, could increase the rate of successful gall induction, as predicted by the OMSH. Galls of A. abietis appeared to be induced by a dose‐dependent stimulus from fundatrices in spring. Furthermore, the critical stimulus dose required to induce a gall appeared to increase with shoot length. These findings support the OMSH and not the PVH. Galler performance (gall volume and the number of gallicolae that emerged from each gall) in successfully developed galls was positively related to shoot length, supporting both the PVH and OMSH. We conclude that the PVH represents one component of the more inclusive OMSH, which considers both ease of gall induction on different‐sized modules and the effect of plant module size on gall size.     

Long term oviposition preference and larval performance of Schizomyia macrocapillata (Diptera: Cecidomyiidae) on larger shoots of its host plant Bauhinia brevipes (Fabaceae)

The Plant Vigor Hypothesis (PVH) proposes that natural selection on female oviposition choice results from higher fitness of larvae on more vigorous and larger plant modules. For six consecutive years we tested the PVH predictions by investigating the effect of shoot size of Bauhinia brevipes (Fabaceae) on the oviposition preference and offspring survival of the gall-midge Schizomyia macrocapillata (Diptera: Cecidomyiidae). Additionally, we analyzed the effects of bottom-up and top-down mortality forces on the system. The hypersensitive reaction (bottom-up effect) accounted for more than 90% larval mortality of S. macrocapillata, making available few galls to be found and killed by natural enemies (top-down effect). Smaller shoots were always more abundant while longer shoots were rare. Nevertheless, the percent number of galls induced by S. macrocapillata was up to 10-fold greater on the largest shoots, corroborating the preference prediction of the PVH. Schizomyia macrocapillata should use over-exploit larger shoots to maximize the preference for, and consequently increase the performance on these shoots. Our results partially support the performance prediction of the PVH: (1) the observed survival was higher than expected on longer shoots, and (2) the ratio of survival per shoot was positively related with shoot length only in 2 years. Thus, we found a link between female preference and larval performance on large-sized shoots, at least in some years. The gall-midge attack pattern in this study might be an evolved response to maximize the female preference and increased larval performance on longer shoots of the host plant.     

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

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

Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish,Eptatretus burgeri

Summary Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. These types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.We wish to express our appreciation to Professor Hideshi Kobayashi for the guidance of this study. It was aided by grants from the Ford Foundation and the Ministry of Education to Professor H. Kobayashi, and also by grants from the Population Council, New York and the Ministry of Education to Professor S. Ishii.     

Resistance of parenchyma cells in wood to degradation by brown rot fungi

Wood degradation by two basidiomycetes, Fomitopsis pinicola and Laetiporus sulphureus was studied in one conifer and four broadleaved trees: Picea abies (Norway spruce), Acer pseudoplatanus (sycamore), Betula pendula (birch), Quercus robur (common oak) and Robinia pseudoacacia (robinia). Observations of birefringence under polarized light showed that in all hosts both brown rot fungi affected cells of the early wood before those of the late wood. Degradation of cellulose, as shown by the loss of birefringence, was apparent after 6 weeks in the cell wall of fibres and fibre tracheids, but even after 12 weeks, axial parenchyma showed no signs of degradation. The results indicate that both brown rot fungi cause higher weight losses in hosts (P. abies and B. pendula) with a small amount of parenchyma cells, whereas the lowest weight losses are associated with wood containing a high amount of parenchyma cells (Q. robur and R. pseudoacacia). Resistance of parenchyma cells to degradation by brown rot fungi appears to be related to the cell wall morphology of parenchyma cells and may also reflect a low co-evolutionary adaptation of brown rot fungi to the xylem of broadleaved trees.     

Adrenergic Stimulation of Na/K Pump Current in Adult Rat Cardiac Myocytes in Short-term Culture

Passive membrane properties, steady-state Na/K pump current (I _p) and modulation of I _p by adrenergic agonists were studied with patch-clamp techniques in adult rat ventricular myocytes that were freshly isolated or maintained in culture for 1–4 days. Freshly isolated (day 0) myocytes had a 1.7–1.8 times smaller specific membrane resistance compared with that of cells on any day in culture. From day 0 to 4 there was a progressive decrease in cell capacitance (−17.6 ± 0.8 pF/day) without a parallel decline in cell dimensions. The pump current density (1.55 pA/pF) was stable over the 0–4 days in culture. In rod-shaped myocytes norepinephrine (NE) and isoproterenol (ISO) stimulated I _p in a dose-dependent manner, with an apparent affinity of 36 ± 8 and 1.5 ± 0.4 nm, and maximum stimulation of 0.65 ± 0.02 and 0.57 ± 0.02 pA/pF, respectively. Nadolol suppressed this effect, suggesting that it was mediated by β-adrenergic receptor activation. An inverse relationship was found between steady-state I _p and the stimulation of I _p by NE. In contrast to what was shown in guinea pig cardiac myocytes, in rat myocytes isoproterenol stimulation of I _p was not increased by intracellular [Ca] and it did not change the I _p -membrane potential relation. These results show that in adult rat cardiac myocytes NE and ISO are potent stimulators of Na/K pump activity, and this effect may be studied using rat myocytes maintained in short-term culture. Received: 12 November 1997/Revised: 5 March 1998     

Immunocytochemical localization of somatostatin and vasotocin in the brain of the Pacific hagfish,Eptatretus stouti

Summary The brain of the Pacific hagfish, Eptatretus stouti, was studied immunocytochemically using antisera against somatostatin (SRIH), arginine vasopressin (AVP), and adrenocorticotropic hormone (ACTH). SRIH-immunoreactive perikarya were distributed bilaterally in the postoptic nucleus and in the hypothalamic nucleus. Although several short, stained fibers were observed in the vicinity of the perikarya, SRIH-immunoreactivity was not found in the neurohypophysis, nor in other parts of the brain. On the other hand, presumed arginine vasotocin (AVT) perikarya were distributed in an arc-shaped region extending from the posterior part of the preoptic nucleus to the anterior-most end of the hypothalamic nucleus and projected their fibers to the neurohypophysis. Most presumptive AVT perikarya were located close to the paired prehypophysial arteries near the anterior end of the postoptic nucleus. In the neurohypophysis, abundant presumptive AVT-fibers terminated in the posterior dorsal wall, although some fibers terminated in the anterior dorsal wall and only a few fiber endings were found in the ventral wall. No ACTH-positive cells were detected in the hagfish brain or in the pituitary gland.Supported from a grant from the National Science Foundation PCM 8141393     

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

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

Evidence for an evolutionarily conserved interaction between cell wall biosynthesis and flowering in maize and sorghum

Background  

Factors that affect flowering vary among different plant species, and in the grasses in particular the exact mechanism behind this transition is not fully understood. The brown midrib (bm) mutants of maize (Zea mays L.), which have altered cell wall composition, have different flowering dynamics compared to their wild-type counterparts. This is indicative of a link between cell wall biogenesis and flowering. In order to test whether this relationship also exists in other grasses, the flowering dynamics in sorghum (Sorghum bicolor (L.) Moench) were investigated. Sorghum is evolutionarily closely related to maize, and a set of brown midrib (bmr) mutants similar to the maize bm mutants is available, making sorghum a suitable choice for study in this context.     

The salmonid cathelicidins: A gene family with highly varied C-terminal antimicrobial domains

The cathelicidin family of host defence peptides is regarded as an important component of the host innate immune system. Its members have been found in mammals, birds, primitive vertebrate Atlantic hagfish and, most recently, also in ray-finned fish such as rainbow trout and Atlantic salmon. By using genomic PCR amplifications and RT-PCR tissue analyses we have here investigated and characterized the cathelicidin gene family in three salmonids: brown trout (Salmo trutta fario), brook trout (Salvelinus fontinalis) and grayling (Thymallus thymallus). One or two different genes were found in each species coding for almost identical cathelin-like domains and largely varied cationic C-terminal regions. Multiple alignment of the amino acid sequences let us recognize two distinctive hallmarks of these peptides: the presence of a high number of serine and glycine residues, which may collocate them in a new class of antimicrobial peptides, and of the six-amino-acid repeated sequence RPGGGS detected in a variable number of copies among different cathelicidins. The high variation in length and sequence of this region suggests the existence of a genetically unstable region similar to that found in some mammalian cathelicidins.