Dasyatis Akajei Research Articles

Characteristics of H and L Subunits with Mass Spectrometry, Electrophoresis and Transmission Electron Microscopy in Liver Ferritin of Dasyatis Akajei

Liver ferritin of Dasyatis akajei (DALF) with mass spectrum purity was prepared in batches. Transmission electron microscopy (TEM) was used to determine the molecular sizes of DALF, protein shell, and iron core, respectively. SDS-PAGE analysis reveals that DALF consists of two different types of subunits, H and L subunits. Moreover, the two types of both H and L subunits and their homology were further identified by peptide mass fingerprinting. Redox reagents such as natural red, thionine, and methyl viologen and the acidity at pH 1.5 does not have abilities for weakening the interaction intensity of both H-L and L-L subunit polymers to form L subunit ions for mass spectrum (MS) analysis with MALDI-TOF MS, respectively. However, using a combined approach of both increasing the laser intensity and decreasing the matrix pH synchronously, an MALDI-TOF mass spectrometer can directly determine molecular weights of both H and L subunits in DALF, indicating that the interaction intensity both H-L and L-L subunit polymers is higher than that of H-H subunit type in DALF.

Characteristics of a self-assembled fibrillar gel prepared from red stingray collagen

A translucent collagen gel was formed from a transparent acidic solution of red stingray collagen by adjusting to physiological ionic strength and pH in phosphate buffer and then incubating at 25–37°C. During fibril formation from red stingray collagen, the turbidity increased when the NaCl concentration was increased at constant pH and the rate of fibril formation was accelerated by higher pH or lower NaCl concentration. The T m of red stingray collagen fibrillar gel was estimated as 44.3 ± 3.5°C, which was higher than that of the collagen solution, 33.2°C. In addition, red stingray collagen gel maintained its shape without melting and was suitable for culture of mouse stromal cells at 37°C.

Ghrelin-like peptide with fatty acid modification and O-glycosylation in the red stingray, Dasyatis akajei

BackgroundGhrelin (GRLN) is now known to be an appetite-stimulating and growth hormone (GH)-releasing peptide that is predominantly synthesized and secreted from the stomachs of various vertebrate species from fish to mammals. Here, we report a GRLN-like peptide (GRLN-LP) in a cartilaginous fish, the red stingray, Dasyatis akajei.ResultsThe purified peptide contains 16 amino acids (GVSFHPQPRS10TSKPSA), and the serine residue at position 3 is modified by n-octanoic acid. The modification is the characteristic of GRLN. The six N-terminal amino acid residues (GVSFHP) were identical to another elasmobranch shark GRLN-LP that was recently identified although it had low identity with other GRLN peptides. Therefore, we designated this peptide stingray GRLN-LP. Uniquely, stingray GRLN-LP was O-glycosylated with mucin-type glycan chains [N-acetyl hexosamine (HexNAc)3 hexose(Hex)2] at threonine at position 11 (Thr-11) or both serine at position 10 (Ser-10) and Thr-11. Removal of the glycan structure by O-glycanase made the in vitro activity of stingray GRLN-LP decreased when it was evaluated by the increase in intracellular Ca2+ concentrations using a rat GHS-R1a-expressing cell line, suggesting that the glycan structure plays an important role for maintaining the activity of stingray GRLN-LP.ConclusionsThis study reveals the structural diversity of GRLN and GRLN-LP in vertebrates.

Identification of angiotensinogen genes with unique and variable angiotensin sequences in chondrichthyans

The renin–angiotensin system is an enzyme-linked hormonal cascade that plays an important role in body fluid and cardiovascular regulation. The system is initiated by the action of renin on the precursor protein, angiotensinogen (AGT), whose sequence information is scarce because of its high variability among species. In the present study, we cloned AGT in chondrichthyans (elasmobranchs: Triakis scyllium, Dasyatis akajei, Leucoraja erinacea and a holocephalan: Callorhinchus milii). Homology was low among AGTs thus far identified; 25–28% between elasmobranchs and tetrapods and 33–61% even within chondrichthyans. All chondrichthyan angiotensin (ANG) II’s have a unique Pro 3 instead of Val 3 as seen in all other species. In addition, holocephalan ANG II has an unusual His 4 instead of Tyr 4. In addition, and the N-terminal amino acid, which is usually Asp 1 in tetrapods and Asn 1 in fishes, was highly variable (Asp, Asn or Tyr) in chondrichthyans. Molecular phylogenetic analysis showed that chondrichthyan AGT precursors are clustered into a group separated from those of tetrapods and teleosts. The AGT gene was most abundantly expressed in the liver, followed by the kidney, interrenal tissue and rectal gland of Triakis where biological actions of ANG II have been demonstrated. Collectively, we identified diversified AGT genes for the first time in chondrichthyes and showed that their ANG II’s have unique amino acid residues at positions 1, 3 and 4. High variability of ANG II sequences in chondrichthyans is discussed in relation to their unique regulatory mechanisms such as urea-based osmoregulation.

한국 연안산 어류의 미네랄 함량 및 영양평가

To measure the mineral content of fishes, we collected 177 individuals from 53 fish species from the east (Pohang), west (Gunsan), and south (Tongyeong) coasts of Korea. The mean content of the macro mineral in a 100 g sample taken from the edible portion of each fish was (in descending order): P(207.4 mg), K(169.7 mg), Na(101.6 mg), Ca(44.4 mg), and Mg(30.4 mg). In comparison, the mean content of micro mineral in the fishes was (in descending order): Zn (<TEX>$8.98{\mu}g/g$</TEX>), Fe (<TEX>$5.03{\mu}g/g$</TEX>), Cu (<TEX>$0.76{\mu}g/g$</TEX>), Mn (<TEX>$0.43{\mu}g/g$</TEX>), and Ni (<TEX>$0.08{\mu}g/g$</TEX>). We could not observe clear regional variation in the mineral content of fish species. With regard to minerals considered essential for humans, the amount of Ca per sample (100 g) was the highest in the Scyliorhinus torazame (465.4 mg), and than followed by Ammodytes personatus (338.0 mg), Dasyatis akajei (267.4 mg), Raja kenojei (248.1 mg), Conger myriaster (174.1 mg), Clupanodon punctatus (86.8 mg), Muraenesox cinereus (81.2 mg), and Engraulis japonica (70.4 mg). The daily average intake of the mineral through the fishes consisted of P (100.6 mg), K (82.3 mg), Na (49.3 mg), Mg (14.8 mg), Zn (0.44 mg), Fe (0.24 mg), Cu (0.04 mg) and Mn (0.02 mg). The respective intakes of macro mineral (P, Mg, Na, Ca, K) from the fishes were about 14.4, 4.2, 3.3, 3.1 and 1.8% of the dietary reference intakes for Koreans (KDRIs) set by the Korean Nutrition Society. In comparison, the respective intakes of micro mineral (Cu, Zn, Fe, Mn) from the fishes were about 4.6, 4.4, 2.4, and 0.6% of the KDRIs.

Use of red stingray,brushtooth lizardfish and chub mackerel in Nara Prefecture

Use of red stingray,brushtooth lizardfish and chub mackerel in Nara Prefecture

Biochemical properties of acid-soluble collagens extracted from the skins of underutilised fishes

Acid-soluble collagens (ASCs) were extracted from the skins of several underutilised fishes, namely dusky spinefoot ( Siganus fuscescens), sea chub ( Kyphosus bigibbus), eagle ray ( Myliobatis tobijei), red stingray ( Dasyatis akajei) and yantai stingray ( Dasyatis laevigata). The yields of the ASCs from skins of dusky spinefoot and sea chub were about 3.4–3.9%, and from ray species were about 5.3–5.7%, on a dry weight basis. According to the electrophoretic pattern, ASCs consisted of two different α-chains (α1 and α2) and were classified as type I collagen. However, the molecular weights of α2-chain for ray species were lower than those of bony fishes. ASC from ray species contained a higher content of imino acids than those from dusky spinefoot and sea chub. The denaturation temperatures ( T d) of ray species were about 33 °C, which was about 5 °C higher than those of dusky spinefoot and sea chub. The high T d of ray species suggested the possibility of its utilisation as a substitute for mammalian collagen.

Isolation and characterization of cDNA sequences of l-gulono-gamma-lactone oxidase, a key enzyme for biosynthesis of ascorbic acid, from extant primitive fish groups

Most advanced teleosts lack l-gulono-gamma-lactone oxidase (GULO), a key enzyme required for the biosynthesis of ascorbic acid. However, extant representatives of primitive species including sturgeon and many cartilaginous fishes, are exceptional in their ability to synthesize ascorbic acid de novo. In the present study, full-length GULO cDNAs were isolated from white sturgeon ( Acipenser transmontanus) and two shark species belonging to the Triakidae ( Triakis scyllium and Mustelus manazo). The open reading frames from all three species contained 440 amino acids and the deduced polypeptides had similar hydropathy profiles, predicted molecular masses and theoretical pI values. These GULO sequences exhibited high amino acid identity (67–97%) with each other, and also shared 61–71% identity with mammalian GULOs. Based on the GULO sequences obtained from these species, we developed degenerate primers for the isolation of partial GULO sequences by RT-PCR from other primitive species including another shark ( Mustelus griseus, Triakidae), a spiny dogfish ( Squalus acanthias, Squalidae), two ray species ( Raja kenojei, Rajidae and Dasyatis akajei, Dasyatidae) and four sturgeons ( Acipenser baeri, A. gueldenstaedtii, A. naccarii and A. ruthenus, Acipenseridae). Overall, sequence identities of these amplified GULO segments among primitive species were 63–99% at the nucleotide level and 67–100% at the amino acid level. Considerable numbers of amino acid residues were unique to either fish or mammals, and Acipenseriform species occupied an intermediate position, sharing several residues with either fish or mammalian GULOs. Phylogenetic analyses based on parsimony, distance and likelihood methods of both nucleotide and amino acid sequences resulted in trees that were in agreement with known taxonomy. The transcription and enzyme activity of GULO were kidney-specific when measured by biochemical assay and reverse transcription-PCR.

DESCRIPTION OF A NEW ASCARID SPECIES IN ELASMOBRANCHS FROM TAIWAN STRAIT

A new ascarid of elasmobranchs, Terranova amoyensis sp. n., is described from the intestine of the red stringray, Dasyatis akajei (Müller and Henle), from Taiwan Strait. This is the second report from this genus with a gubernaculum in fish. The new species differs from its congeners mainly in the following combination of characters: the 3 lips are unequal in size and different in shape, i.e., the subventral lips are asymmetric bilaterally and larger than the dorsal lip; the latter lip is bilaterally symmetrical. The ventriculus is spherical, and the cecum is short relative to the esophagus. Spicules are unequal in size, 1.48 mm long for the right spicule and 1.34 mm for the left spicule, or 4.3 and 3.9% of body length, respectively. Nineteen pairs of caudal sessile papillae (excluding phasmids) are present, of which 13 pairs are precloacal; pairs 14 and 15 lie close together on either side of the cloaca, composing ad-cloacal papillae (the pair near the tail end are twins). The remaining 4 pairs of postcloacal papillae form a group near the tail tip (pair 18 also twins and near the phasmids). The gubernaculum is I-shaped. The excretory pore is located on top of a coniform prominence whose location is just behind or between the bases of the 2 subventral lips. The vulva is prominent at one-third of the anterior part of the body; the distance from the vulva opening to the anterior body end is 17.4 mm (13.61-21.41), 26.8% (25.14-28.93%) of the body length.

A REDESCRIPTION OF THE FISH LEECH PTEROBDELLA AMARA (= RHOPALOBDELLA JAPONICA) (HIRUDINIDA: PISCICOLIDAE) BASED ON SPECIMENS FROM THE TYPE LOCALITY IN INDIA AND FROM AUSTRALIA

Pterobdella amara is redescribed from specimens from the type locality in India, and from the type host in Queensland, Australia. A neotype is designated from the type locality. The presence of conspicuous finlike bodies along the lateral margins of P. amara was greatly exaggerated in the original description. The trachelosome and anterior portion of the urosome are wide and flat, but there are no finlike bodies present. The urosome tapers abruptly posterior to the flattened portion, becomes cylindrical, and remains of constant width to the caudal sucker. The oral sucker is small and the caudal sucker is terminal. The body is smooth and lacks pigmentation, except for 1 pair of eyes on the oral sucker. Important internal characters are 2 pairs of mycetomes, 5 pairs of testisacs, confluent gonopores, extensive accessory gland cells on the male atrium and ejaculatory ducts, a spacious coelom consisting of dorsal and ventral sinuses with expansive connections between each at urosome ganglia, and interganglionic expansions of the dorsal sinus. Rhopalobdella japonica from stingrays in Japan is synonymized with P. amara and becomes a subjective junior synonym. This leech is a parasite in the oral cavity of the stingrays Pastinachus sephen and Himantura uarnak in India and Australia, and Dasyatis akajei in Japan.

Molecular cloning and characterization of CD9 cDNA from cartilaginous fish, red stingray, Dasyatis akajei

CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. In this study, we describe the isolation of the full-length cDNA encoding for CD9 molecule (daCD9) of red stingray, Dasyatis akajei. This 1252 bp cDNA was isolated from leukocyte cDNA library and contains 681 bp open reading frame encoding 226 amino acid residues. Amino acid sequences analysis and structure prediction display approximately 50% identity to higher vertebrates with the presence of conserved structures, including the four transmembrane domains and certain characteristic residues. Southern blot analysis shows that daCD9 exists as a single copy gene. Northern blot analysis reveals that daCD9 is highly expressed in gill and spleen although its expression can be found in other tissues suggesting daCD9 might play an important role in immune defense in this fish.

Effect of pH and Phosphate on Trapping Capacity of Various Heavy Metal Ions With Ferritin Reactor in Flowing Seawater

We describe a protein reactor consisting of native liver ferritin of Dasyatis akajei (DALF) and a dialysis bag. Our goal was to study a ferritin reactor for its capacity to trap various heavy metal ions (M2+) in flowing seawater. The reactor is sensitive and inexpensive and can be operated by nonprofessional technicians. A positive relationship between the number of trapped M2+ with the DALF reactor and its concentration in the flowing seawater was observed. Both the pH in the medium and the phosphate content within the ferritin cavity strongly affected trapping capacity. It was found that the ferritin released its phosphate compound directly with a shift in pH without the need for releasing reagent, which differs from the phosphate release characteristics of horse spleen ferritin, as previously described. This behavior evidently makes the trapping capacity with the ferritin reactor weaken, indicating that this trapping capacity is tightly connected to its phosphate compound. Our study shows that a self-regulation ability of the ferritin shell rather than its phosphate compound plays an important role in controlling the rate and capacity of trapping M2+. The ferritin reactor was constructed to monitor the contamination level of M2+ in flowing seawater. Our preliminary data along with fieldwork indicate that the DALF reactor is an analytical means for effectively monitoring the contamination level of M2+ in flowing seawater.

Characteristics of trapping copper ions with scrolled ferritin reactor in the flowing seawater.

Native liver ferritin of Dasyatis akajei (DALF), apoDALF, and reconstituted DALF were employed to construct a ferritin reactor, respectively. An apparatus consisting of a mixer, a ferritin reactor, and a magnetic stirrer was constructed to study capacity and feasibility of trapping Cu2+ in the flowing seawater. The experimental results showed that the numbers of trapping Cu2+ with DALF reactor were higher than these with the reactors of apoDALF and reconstituted DALF, respectively, giving the maximal numbers of 98 +/- 5 Cu2+ per molecular DALF in 120 h. We found that the iron layer with a high ratio of phosphate to ion on the surface of the ferritin core played an important role in increasing numbers of trapping Cu2+. In addition, we found two positive relations of dependence of trapping Cu2+ numbers with the reactor on the incubation time and on the Cu2+ concentration in the flowing seawater. Another apparatus consisting of a buoyage, an isolation basket equipped with griddling, and a scrolled ferritin reactor was constructed to study the feasibility of trapping Cu2+ in the sea area. Moreover, the present studies indicated that this apparatus had been used to not only analyze and evaluate the concentration variety of various heavy metal ions such as Cu2+ and Pb2+ diluting by the seawater but also monitor the formation of pollution degree by various small organic molecules during the climax and the neap.

Cloning, sequence analysis and evolutionary conservation of a full-length cDNA encoding cyclophilin A from red stingray Dasyatis akajei

Cloning, sequence analysis and evolutionary conservation of a full-length cDNA encoding cyclophilin A from red stingray Dasyatis akajei

트롤 어선에서 어획후 투기된 어류의 생존율

끌그물로부터 탈출한 소형어류의 생존율을 향상시킬 수 있는 자원관리형 어구어법을 개발하기 위한 기초 조사로서, 트롤망에서 양망 후 투기되는 어류의 생존율을 조사하기 위한 시험조업이 한국 남해안 및 제주도 근해에서 이루어졌다. 트롤망의 끝자루를 탈출한 소형어류를 선내에 비치된 사육수조에 수용하여 시간대별로 생존지속 시간을 조사하였다. 그 결과를 요약하면 다음과 같다. 1. 트롤 어구로부터 어획된 어류를 선상에 비치된 사육수조에 넣어 생존율을 조사한 결과 살오징어, 한치, 갈치, 달고기, 삼치, 참조기 및 눈볼대등 대부분의 어종은 양망후 즉시 사망하였다. 2 두툽상어, 붕장, 가오리, 말쥐치, 부채새우 등은 수조내에서 장시간 생존하였으므로 이들 어종은 투기된 후에 생존할 확률이 높을 것으로 생각된다. 3. 탈출장치를 빠져나온 등가시치는 수조에 수용된지 6 시간 이내에 전부 사망하였으나,97% 의 두툽상어와 72% 의 붕장어는 72시간 이상 생존하였다. 4. 끝자루를 빠져나온 가오리는 수조에 수용된지 60시간 이내에 모두 사망하였으나, 25% 의 말쥐치와 문어는 72시간 이상 생존하였다. 5. 홍감팽의 경우 탈출장치와 끝자루를 빠져나온 개체간의 생존율을 비교해보면, 끝자루를 빠져나온 홍감팽은 72 시간 이내에 전부 사망하였으나, 탈출장치를 빠져나온 것의 8.3%는 72시간 이상 생존하는 것을 보여줌으로서, 탈출장치를 빠져나온 개체의 생존율이 다소 높은 것을 보여주었다. 6. 그러나 부채새우의 경우를 비교해 보면, 끝자루를 탈출한 새우의 생존율이 75%로서 탈출장치를 빠져나온 부채새우의 생존율 33% 보다 훨씬 높게 나타났다. 따라서, 탈출후의 어종별 생존 특성에 대해서 보다 많은 연구가 필요할 것으로 본다. Experimental fishing was carried out to investigate the survival rate of fishes discarded after hauling from the trawl and to develop the fishing gear and method for fisheries management which can improve the survival rate of young fishes escaped from the trawl codend in offshore korean southern sea and off Cheju Island of Korea. The young fishes were bred in fish cage on the board to measure the sustainable survival time for fishes escaped from grid panel and codend. The obtained results are summarized as follows' 1. Japanese flying squid(Todarodes pacjficus), mitra squid(Loligo chinesis), hair-tail( Trichiurus lepturus), john dory(Zeus japonicus), spanish mackerel(Scomberomorus niphonius), redlip croaker (Pseudosciaena polyactis) and blackthroat seaperch(Doederliinia berycoides) were dead instantly after hauling. 2. Survival rate of tiger shark(Galeocerdo cuvier), conger eel (Conger myriaster), red skate (Dasyatis akajei), black scraper(Navodon modestus) and japanese fan lobster(Ibacus ciliatus) might be high after discarding because they survived for long hours in fish cage. 3. Blotched eelpout(Zoarces gili) escaped from the escapement device(grid) was dead within 6 hours in the water tank installed on the board but 97% of tiger shark and 72% of conger eel survived over 72 hours. 4. Red skate escaped from trawl codend was dead within 60 hours in the water tank installed on the board but sustainable survival time of 25% of octopus(Paroctopus dofleini) and 100% of black scraper was over 72 hours. 5. Compared with the survival rate of rosefish(Helicolenus higendorfi) escaped from the escapement device(grid) and trawl codend, all of the rosefish escaped from the grid was dead within 7.2 hours but 8.3% of the rosefish escaped from the codend survived over 72 hours. 6. In comparison with the survival rate of japanese fan lobster survived over 72 hours, survival rate of japanese fan lobster escaped the codend was much higher as 75% than 33% of japanese fan lobster escaped from the grid.

A naturally enhanced green fluorescent protein from magnificent sea anemone ( Heteractis magnifica) and its functional analysis

A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR. The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 β-strands of hmGFP were similar to those of GFP from other species. The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification. The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized. The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm. The expression of hmGFP and the chimera IPL∼hmGFP in CHO cells has shown that the fusion protein IPL∼hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein.

Purification, electrophoretic behavior, and kinetics of iron release of liver ferritin of Dasyatis akajei.

From the liver of fish Dasyatis akajei, ferritin has been isolated by thermal denaturation and ammonium sulfate fractionation and then further purified by anion exchange chromatography and gel exclusion chromatography. The molecular weight of the liver ferritin of D. akajei (DALF) was measured to be 400 kDa by PAGE. Moreover, SDS-PAGE experimentation indicates that protein shell of DALF consists of the H and L subunits with molecular weight of 18 and 13 kDa, respectively. Using isoelectric focusing with pH ranging from 5.0 to 6.0, the ferritin purified by the PAGE exhibited three bands with different pI values in the gel slab. Diameters of the protein shell and iron core were also investigated by transmission electron microscope and determined to be 10-12 nm and 5-8 nm, respectively. A kinetic study of DALF reveals that the rate of self-regulation of the protein shell rather than the complex surface of the iron core plays an important role in forming a process for iron release with mixed orders.

The functional similarity and structural diversity of human and cartilaginous fish hemoglobins

Although many descriptions of adaptive molecular evolution of vertebrate hemoglobins (Hb) can be found in physiological text books, they are based mainly on changes of the primary structure and place more emphasis on conservation than alterations at the functional site. Sequence analysis alone, however, does not reveal much about the evolution of new functions in proteins. It was found recently that there are many functionally important structural differences between human and a ray ( Dasyatis akajei) Hb even where sequence is conserved between the two. We have solved the structures of the deoxy and CO forms of a second cartilaginous fish (a shark, Mustelus griseus) Hb, and compared it with structures of human Hb, two bony fish Hbs and the ray Hb in order to understand more about how vertebrate Hbs have functionally evolved by the selection of random amino acid substitutions. The sequence identity of cartilaginous fish Hb and human Hb is a little less than 40 %, with many functionally important amino acid replacements. Wider substitutions than usually considered as neutral have been accepted in the course of molecular evolution of Hb. As with the ray Hb, the shark Hb shows functionally important structural differences from human Hb that involve amino acid substitutions and shifts of preserved amino acid residues induced by substitutions in other parts of the molecule. Most importantly, βE11Val in deoxy human Hb, which overlaps the ligand binding site and is considered to play a key role in controlling the oxygen affinity, moves away about 1 Å in both the shark and ray Hbs. Thus adaptive molecular evolution is feasible as a result of both functionally significant mutations and deviations of preserved amino acid residues induced by other amino acid substitutions.

Molecular Cloning of Proopiomelanocortin cDNA from an Elasmobranch, the Stingray, Dasyatis akajei

Recently, we have characterized a new MSH (named δ-MSH) which joins the group of MSHs (α, β, γ) in dogfish proopiomelanocortin (POMC). The present study has confirmed the presence of δ-MSH in POMC of another member of the elasmobranchian order, the stingray, Dasyatis akajei, by cDNA cloning from pituitary mRNAs. Overlapping partial cDNA clones corresponding to stingray POMC were amplified by PCR from single-strand cDNA prepared from pituitary poly (A)+ RNA. Excluding the poly A tail, stingray POMC cDNA consists of 1077 base pairs (bp). It contains a 912-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a POMC of 280 aa. γ-MSH, α-MSH, ACTH, δ-MSH, β-MSH, and β-endorphin are located at POMC (50–61), (115–127), (115–153), (182–193), (226–242), and (245–280), respectively. The stingray POMC is smaller than that of the dogfish POMC (294 aa) mainly due to the absence of a sequence of 11 consecutive aa between δ-MSH and β-MSH. δ-MSH has been found only in the elasmobranchs and, therefore, δ-MSH might have evolved after the divergence of chondrichthians from the ancestral vertebrate lineage and before divergence of sharks and rays.

Rhopalobdella japonica n. gen., n. sp. (Hirudinea, Piscicolidae) from Dasyatis akajei (Chondrichthyes: Dasyatididae) in the northwestern Pacific.

A new genus and species of piscicolid leech in the Platybdellinae inhabits the oral cavity of Dasyatis akajei in the northwestern Pacific Ocean near Tanabe, Japan. The genus Rhopalobdella n. gen. is characterized externally by very small oral and caudal suckers and a smooth body that is widest just posterior to the clitellum. Eyespots and ocelli are lacking. The coelom is spacious with large segmental connecting sinuses between dorsal and ventral sinuses. There are 5 pairs of testisacs, an unusually extensive epididymis, and a very large bursa. Conducting tissue is absent. There are 2 pairs of esophageal diverticula and very well developed nephridia. Rhopalobdella japonica n. gen. n. sp. is characterized by a urosome that tapers strongly to the caudal sucker and by a single gonopore; the common oviduct opens into the posterior portion of the bursa. The coelomic and excretory systems resemble Aestabdella, but in other respects the genera are quite different. This is the first marine leech reported from rays in the northwestern Pacific.