Sex Difference in the Cloacal Gland in the Hagfish,Eptatretus burgeri,and its Possible Significance in Reproduction

The cloacal gland of the hagfish, Eptatretus burgeri, was studied histologically in four seasons. Before the breeding season (spring and summer) the male cloacal gland was larger than the female cloacal gland. The gland was largest in the males with a more mature testis. After the breeding season (autumn and winter) there was no sex difference in the size of the cloacal gland. The cloacal gland consists of mucus and thread cells, as do the lateral slime glands. The sperm may be entangled within slime in the cloacal gland and be shed outside as a slimy sperm mass. Such a slimy sperm mass may play an important role in reproduction of the hagfish.     

The Preparation of Poly(A)±mRNA from the Hagfish Slime Gland

The isolation of translatable poly(A)^±mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37°C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.     

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.     

Eptatretus eos: A new species of hagfish (Myxinidae) from the Tasman Sea

A new species of hagfish (Myxinidae),Eptatretus eos, is described on the basis of one specimen from the Tasman Sea, west of New Zealand. This five-gilled, deep-sea species differs from previously described hagfish in having the unpaired nostril prolonged into a tube-like snout and in being uniformly pink.     

The preparation of poly(A)+mRNA from the hagfish slime gland

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37 degrees C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.     

Structural forms and possible roles of aligned cytoskeletal biopolymers in hagfish (slime eel) mucus

The hagfish, marine benthic dwellers, are primitive vertebrates which, upon stress, secrete massive quantities of mucus from slime glands of the integument by the holocrine mode. Lysis of each gland thread cell releases a single, intermediate filament-rich thread ( 60 cm × 1.5 μm), which collectively modulates the hydration and cohesiveness of mucus. In this report, with the Pacific hagfish (Eptatretus stouti), we showed that the threads exhibit a tendency for self-assembly into novel, aligned and intertwined cytoskeletal aggregates (“cables”). Large cables (15–40 cm in length, 50–800 μm in width) can be physically prepared from the whole mucous exudate, but similar smaller cables (0.1–10 cm in length, 50–200 μm in width) were found occurring naturally, particularly at the ends of laid eggs. Threads and cables may facilitate adhesion of mucus to the integument of the fish, restrict eggs to an optimum spawning site, and abet defense against invasive organisms.     

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     

Plasma cells in adult Atlantic hagfish,Myxine glutinosa

Summary Hagfishes, the most primitive vertebrates, are of special interest for the evolution of immune responses. Eptatretus stoutii, the Pacific hagfish, is able to mount cellular and humoral immune responses but all attempts to demonstrate in them the presence of plasma cells have failed. In the present study we demonstrate for the first time plasma cells identifiable by ultrastructural criteria in the pronephros, a primitive lymphohaemopoietic organ, of Myxine glutinosa, the Atlantic hagfish.     

Myxine formosana, a new species of hagfish (Myxiniformes: Myxinidae) from the southwestern waters of Taiwan

Myxine formosana, a new hagfish species, is described on the basis of the specimens from the Pacific Ocean southwest of Taiwan at depths 588–1500 m. It is five-gilled and white-headed with a three-cusp multicusp on the anterior set of cusps and a two-cusp multicusp on the posterior set. Myxine formosana, M. circifrons, M. mccoskeri, and M. robinsi are superficially rather similar, but M. formosana has low to vestigal caudal finfolds. It is the first record of the genus Myxine from Taiwan. Received: October 25, 2000 / Revised: March 17, 2001 / Accepted: April 21, 2001     

The chromosomes of some lower chordates

The chromosomes of three species of lower chordates were examined using a squash technique on small pieces of testis. Ciona intestinalis, a tunicate of the order Enterogona, has fourteen pairs of minute chromosomes. Styela plicata, a tunicate of the order Pleurogona, has sixteen pairs of chromosomes whose total size is approximately twice that of the Ciona chromosomes and about 10% of that of a typical mammalian complement. The hagfish, Eptatretus stoutii, of the suborder Myxinoidea, order Cyclostomata, has twenty-four pairs of chromosomes and what appear to be one to four small supernumeraries in some animals. The hagfish chromosomes are large, approaching the size of a typical mammalian complement. These size relationships agree in general with a concept of a small ancestral vertebrate genome which evolved into the larger present day genomes through a series of duplications of genetic material.This work was supported in part by Grant No. 247-701 from the San Diego State College Foundation.     

Cyclical Changes in Weight and Fat Content of the Liver and their Relationship to Reproduction in the Hagfish Eptatretus burgeri (Cyclostomata)

Patzner, R. A. 1980. Cyclical changes in weight and fat content of the liver and their relationship to reproduction in the hagfish Eptatretus burgeri (Cyclostomata). (Zoological Institute, University of Salzburg, Austria and Misaki Marine Biological Station, University of Tokyo, Japan.) — Acta zool. (Stockh.) 61(3): 157–160. The changes in weight and fat content of the liver in the West Pacific hagfish Eptatretus burgeri were studied over a period of one year. The liver somatic index shows a high increase from the beginning of December until migration in deeper water in July. From the beginning of April to the end of August the difference between male and female liver somatic index is statistically significant. This fact is brought in connection with the production of vitellogenin in the female liver.     

Effects of dark-light step transitions on circadian locomotor activity rhythms of the hagfish,Eptatretus burgeri

Captive hagfish,Eptatretus burgeri, were subjected to step transitions from continuous dark (DD) to continuous light (LL) and their locomotor activity patterns recorded. Free-running activity rhythms occurred in both the DD and LL regimes. The timing of the transition influenced the circadian period (τ), the relationship between the period response (δτ, or τ{inLL}–τ{inDD}, change in the circadian period) and the former being represented by a cosine curve δτ became most positive and most negative when hagfish underwent a DD-LL transition shortly before the beginning of the duration of activity (α) and shortly after the end of such, respectively. The phase response (δ-phase, change in the relative timing of the activity phase) was characterized by a delay in the activity phase after the DD-LL transition, although its magnitude bore no relation to the timing of the transition.     

Complement-like protein from the phylogenetically primitive vertebrate,Eptatretus Stouti,is a Humoral Opsonin

• 1.1. Serum from the Pacific hagfish,Eptatretus stouti,contains a complement-like protein (CLP).
• 2.2. CLP from unfractionated hagfish serum and from affinity-purified preparations binds to yeast cell surfaces.
• 3.3. Incubation with CLP enhances the phagocytosis of yeast by hagfish leukocytes.
• 4.4. CLP-mediated opsonization can be inhibited by anti-CLP antibody, EDTA, d(+)mannose and l(+)rhamnose.
• 5.5. Additional opsomic factors are also in hagfish serum.

     

Panbiogeographical analysis of distribution patterns in hagfishes (Craniata: Myxinidae)

Aim To analyse the worldwide distribution patterns of hagfishes using panbiogeographical track analysis, and to attempt to correlate these patterns with the tectonic history of the ocean basins. Location Atlantic and Pacific oceans. Method The distributions of 47 out of 70 species of hagfish (in the genera Eptatretus, Myxine, Nemamyxine, Neomyxine, and Paramyxine) were studied by the panbiogeographical method of track analysis. The analysis was performed using distributional data obtained from the collections included in the Ocean Biogeographic Information System (OBIS, http://www.iobis.org ) and FishBase ( http://www.fishbase.org ), with additional records from the literature. Individual tracks were obtained for each species by plotting localities and connecting them by minimum‐spanning trees. Generalized tracks were determined from the spatial overlap between individual tracks. Results Six generalized tracks were found: in the Gulf of Mexico, Caribbean Sea, South‐eastern Atlantic, Western Pacific, North‐eastern Pacific and South‐eastern Pacific. Main conclusions The distribution patterns of myxinids are marked by a high degree of endemism and vicariance, and are correlated with the tectonic features involved in many of the events that led to the development of oceanic basins. The main massing of the group is around the Pacific Basin. In the Atlantic Ocean, the distribution of Myxine glutinosa seems to correspond to a classic trans‐oceanic track and vicariance resulting from the opening of the Atlantic Ocean during the Cretaceous. In the Pacific Ocean, the distribution of the Eptatretus and Paramyxine species is clearly associated with the margins of the Pacific tectonic plate. The generalized tracks of hagfishes are shared by several other groups of marine organisms, including many from shallow tropical waters, implying a common history for this marine biota. Overall, vicariance is a major feature of hagfish distribution, suggesting vicariant differentiation of widespread ancestors as a result of sea‐floor spreading between continents in connection with ocean formation.     

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.     

Different Pressure Resistance of Lactate Dehydrogenases from Hagfish is Dependent on Habitat Depth and Caused by Tetrameric Structure Dissociation

The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined. LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure of LDH-A₄ from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures.     

The partial sequence of the first 30 residues from the amino-terminus of hemoglobin B in the hagfish,Eptatretus stoutii: Homology with lamprey hemoglobin

Summary The partial sequence of the first 30 residues from the amino-terminus of hemoglobin B in the hagfish,Eptatretus stoutii, has been determined. Considerable homology was found between this sequence and the corresponding sequence of lamprey hemoglobin. Both hagfish and lamprey hemoglobins have an additional segment of 9 residues at the amino-terminus as compared with mammalian hemoglobins.This work was initiated at the University of Texas at Austin, and continued in Dr. Charles Yanofskey's laboratory at Stanford University.     

FMRF -amide-like immunoreactivity in brain and pituitary of the hagfish Eptatretus burgeri (Cyclostomata)

Summary Paraffin sections of brain and pituitary of the hagfish Eptatretus burgeri were immunostained with an antiserum to FMRF-amide. Immunoreactivity was visible in a large number of neurons in the posterior part of the ventromedial hypothalamus and in long neuronal processes extending cranially from the hypothalamus to the olfactory system and caudally to the medulla oblongata. FMRF-amide-like immunoreactivity was also found in cells of the adenohypophysis. These observations suggest that the hagfish possesses a brain FMRF-amide-like transmitter system and pituitary cells containing FMRF-amide-like material.Antisera to ACTH, -MSH and pancreatic polypeptide gave no immunoreaction in hagfish brain or pituitary. D aspartic acid - F phenylalanine - L leucine - M methionine - R arginine; - W tryptophan - Y tyrosine     

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: ██     

Morphological analysis of the hagfish heart. I. The ventricle,the arterial connection and the ventral aorta

We have studied the heart in three species of hagfish: Myxine glutinosa, Eptatretus stoutii, and Eptatretus cirrhatus and report about the morphology of the ventricle, the arterial connection and the ventral aorta. On the whole, the hagfish heart lacks outflow tract components, the ventricle and atrium adopt a dorso‐caudal rather than a ventro‐dorsal relationship, and the sinus venosus opens into the left side of the atrium. This may indicate a “defective” cardiac looping during embryogenesis. The ventral aorta is elongated in M. glutinosa and E. stoutii but sac‐like in E. cirrhatus. The ventricles are entirely trabeculated. The myocytes show a low myofibrillar content and junctional complexes formed by fascia adherens and desmosomes. Gap junctions could not be demonstrated. Myocardial cells in M. glutinosa contain numerous lipid droplets. These droplets are less numerous in E. stoutii and practically absent in E. cirrhatus, suggesting different metabolic requirements. Other cell types present in the ventricle are chromaffin cells and granular leukocytes that contain rod‐shaped granules. The ventricle‐aorta connection is guarded by a bicuspid valve with left and right, pocket‐like leaflets. The leaflets extend from the cranial end of the ventricle into the aorta but the junction is asymmetrical. This junction contains a ganglion‐like structure in E. cirrhatus. The ventral aorta shows endothelial, media, and adventitial layers. The media contains smooth muscle cells surrounded by dense bands formed by tightly‐packed extracellular filaments. In addition, a short number of elastic fibers are observed in M. glutinosa and E. stoutii. Cellular and extracellular elements are more loosely organized in the aorta of E. cirrhatus. The collagenous adventitia contains ganglion‐like cells in the three species. In the absence of nerves, chromaffin and ganglion‐like cells may control the activity of the myocardium and that of the aortic smooth muscle cells, respectively. J. Morphol. 277:326–340, 2016. © 2015 Wiley Periodicals, Inc.