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We developed phyloBARCODER (https://github.com/jun-inoue/phyloBARCODER), a new web tool that can identify short DNA sequences to the species level using metabarcoding. phyloBARCODER estimates phylogenetic trees based on the uploaded anonymous DNA sequences and reference sequences from databases. Without such phylogenetic contexts, alternative, similarity-based methods independently identify species names and anonymous sequences of the same group by pairwise comparisons between queries and database sequences, with the caveat that they must match exactly or very closely. By putting metabarcoding sequences into a phylogenetic context, phyloBARCODER accurately identifies (i) species or classification of query sequences and (ii) anonymous sequences associated with the same species or even with populations of query sequences, with clear and accurate explanations. Version 1 of phyloBARCODER stores a database comprising all eukaryotic mitochondrial gene sequences. Moreover, by uploading their own databases, phyloBARCODER users can conduct species identification specialized for sequences obtained from a local geographic region or those of nonmitochondrial genes, e.g. ITS or rbcL.
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Código de Barras de DNA Taxonômico , Eucariotos , Filogenia , Eucariotos/genética , Eucariotos/classificação , Código de Barras de DNA Taxonômico/métodos , Software , Bases de Dados Genéticas , Internet , Bases de Dados de Ácidos NucleicosRESUMO
In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na+/K+-ATPase (NKA) and anti-vacuolar-type H+-ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na+/H+ exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo.
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Brânquias , Larva , Animais , Larva/metabolismo , Brânquias/metabolismo , Brânquias/citologia , Brânquias/embriologia , Tubarões/embriologia , Tubarões/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/citologiaRESUMO
The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words).
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Receptores do FSH , Receptores do LH , Animais , Receptores do LH/metabolismo , Receptores do LH/genética , Feminino , Receptores do FSH/metabolismo , Receptores do FSH/genética , Hormônio Luteinizante/metabolismo , Hormônio Foliculoestimulante/metabolismo , Peixes/metabolismo , Peixes/genética , Folículo Ovariano/metabolismoRESUMO
The diverse reproductive strategies of elasmobranchs (sharks, rays, and skates) have attracted research attention, but the endocrine control of reproduction is still incompletely known in elasmobranchs. By long-term monitoring of the egg-laying cycle in cloudy catsharks (Scyliorhinus torazame), we recently demonstrated a transient increase in plasma progesterone (P4) levels just prior to the appearance of the capsulated eggs in the oviducts. In the present study, we examined the in vivo effects of P4 administration in mature female cloudy catsharks. Although no capsulated eggs were observed following the implantation of P4-containing silicone tubing, we did find dark swollen oviducts in the abdominal cavity, in which clumps of long and coiled tendrils were observed. The tendril is an extension of the egg capsule, and the formation of the egg capsule begins with the tendril before main capsule formation. During the period of P4 implantation, the tendrils elongated, and their diameters were significantly increased on day 2 of treatment. Tendril formation was also confirmed on the day of endogenous P4 surge. Tendrils were not formed in catsharks implanted with estradiol-17ß or testosterone. Histological analysis of the oviducal gland revealed that P4 implantation induced the secretion of tendril materials from the secretory tubules in the baffle zone, while the tendril materials were stored in the cytoplasm of the secretory cells under low P4 condition. Morphometrically, the area of secreted luminal materials in the secretory tubules was highly correlated to the diameter of tendrils. Our results suggest that the P4 surge during the egg-laying cycle serves as a trigger for egg capsule formation in the oviducal gland of cloudy catshark, but the hormonal signals were incomplete as the main capsule was not formed. Further studies are required to identify the hormones required for ovulation and formation of the main egg capsule.
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For adaptation to a high salinity marine environment, cartilaginous fishes have evolved a ureosmotic strategy. They have a highly elaborate "four-loop nephron" in the kidney, which is considered to be important for reabsorption of urea from the glomerular filtrate to maintain a high concentration of urea in the body. However, the function and regulation, generally, of the "four-loop nephron" are still largely unknown due to the complicated configuration of the nephron and its many subdivided segments. Laser microdissection (LMD) followed by RNA-sequencing (RNA-seq) analysis is a powerful technique to obtain segment-dependent gene expression profiles. In the present study, using the kidney of cloudy catshark, Scyliorhinus torazame, we tested several formaldehyde-free and formaldehyde-based fixatives to optimize the fixation methods. Fixation by 1% neutral buffered formalin for 15 min resulted in sufficient RNA and structural integrities, which allowed LMD clipping of specific nephron segments and subsequent RNA-seq analysis. RNA-seq from the LMD samples of the second-loop, the fourth-loop, and the five tubular segments in the bundle zone revealed a number of specific membrane transporter genes that can characterize each segment. Among them, we examined expressions of the Na + -coupled cotransporters abundantly expressed in the second loop samples. Although the proximal II segment of the second loop is known for the elimination of excess solutes, the present results imply that the PII segment is also crucial for reabsorption of valuable solutes. Looking ahead to future studies, the segment-dependent gene expression profiling will be a powerful technique for unraveling the renal mechanisms and regulation in euryhaline elasmobranchs.
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Microdissecção , Néfrons , Animais , Peixes , Perfilação da Expressão Gênica , RNA , Ureia/metabolismoRESUMO
Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates.
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Feiticeiras (Peixe) , Hormônios Neuro-Hipofisários , Animais , Peixes , Feiticeiras (Peixe)/classificação , Feiticeiras (Peixe)/genética , Lampreias/genética , Filogenia , Vertebrados/genéticaRESUMO
As part of their osmoregulatory strategy, marine elasmobranchs retain large quantities of urea to balance the osmotic pressure of the marine environment. The main source of nitrogen used to synthesize urea comes from the digestion and absorption of food across the gastrointestinal tract. In this study we investigated possible mechanisms of nitrogen movement across the spiral valve of the cloudy catshark (Scyliorhinus torazame) through the molecular identification of two Rhesus glycoprotein ammonia transporters (Rhp2 and Rhbg) and a urea transporter (UT). We used immunohistochemistry to determine the cellular localizations of Rhp2 and UT. Within the spiral valve, Rhp2 was expressed along the apical brush border membrane, and UT was expressed along the basolateral membrane and the blood vessels. The mRNA abundance of Rhp2 was significantly higher in all regions of the spiral valve of fasted catsharks compared to fed catsharks. The mRNA abundance of UT was significantly higher in the anterior spiral valve of fasted catsharks compared to fed. The mRNA transcript of four ornithine urea cycle (OUC) enzymes were detected along the length of the spiral valve and in the renal tissue, indicating the synthesis of urea via the OUC occurs in these tissues. The presence of Rhp2, Rhbg, and UT along the length of the spiral valve highlights the importance of ammonia and urea movement across the intestinal tissues, and increases our understanding of the mechanisms involved in maintaining whole-body nitrogen homeostasis in the cloudy catshark.
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Elasmobrânquios , Nitrogênio , Animais , Amônia , RNA Mensageiro , Ureia , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de UreiaRESUMO
Clarifying the effect of the sampling protocol on the detection of environmental DNA (eDNA) is essential for appropriately designing biodiversity research. However, technical issues influencing eDNA detection in the open ocean, which consists of water masses with varying environmental conditions, have not been thoroughly investigated. This study evaluated the sampling effort for the metabarcoding-based detection of fish eDNA using replicate sampling with filters of different pore sizes (0.22 and 0.45 µm) in the subtropical and subarctic northwestern Pacific Ocean and Arctic Chukchi Sea. The asymptotic analysis predicted that the accumulation curves for detected taxa did not saturate in most cases, indicating that our sampling effort (7 or 8 replicates, corresponding to 10.5-40 L of filtration in total) was insufficient to fully assess the species diversity in the open ocean and that tens of replicates or a substantial filtration volume were required. The Jaccard dissimilarities between filtration replicates were comparable with those between the filter types at any site. In subtropical and subarctic sites, turnover dominated the dissimilarity, suggesting that the filter pore size had a negligible effect. In contrast, nestedness dominated the dissimilarity in the Chukchi Sea, implying that the 0.22 µm filter could collect a broader range of eDNA than the 0.45 µm filter. Therefore, the effect of filter selection on the collection of fish eDNA likely varies depending on the region. These findings highlight the highly stochastic nature of fish eDNA collection in the open ocean and the difficulty of standardizing the sampling protocol across various water masses.
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The Bull Shark (Carcharhinus leucas) faces varying levels of exploitation around the world due to its coastal distribution. Information regarding population connectivity is crucial to evaluate its conservation status and local fishing impacts. In this study, we sampled 922 putative Bull Sharks from 19 locations in the first global assessment of population structure of this cosmopolitan species. Using a recently developed DNA-capture approach (DArTcap), samples were genotyped for 3400 nuclear markers. Additionally, full mitochondrial genomes of 384 Indo-Pacific samples were sequenced. Reproductive isolation was found between and across ocean basins (eastern Pacific, western Atlantic, eastern Atlantic, Indo-West Pacific) with distinct island populations in Japan and Fiji. Bull Sharks appear to maintain gene flow using shallow coastal waters as dispersal corridors, whereas large oceanic distances and historical land-bridges act as barriers. Females tend to return to the same area for reproduction, making them more susceptible to local threats and an important focus for management actions. Given these behaviors, the exploitation of Bull Sharks from insular populations, such as Japan and Fiji, may instigate local decline that cannot readily be replenished by immigration, which can in turn affect ecosystem dynamics and functions. These data also supported the development of a genetic panel to ascertain the population of origin, which will be useful in monitoring the trade of fisheries products and assessing population-level impacts of this harvest.
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Reproductive modes of vertebrates are classified into two major embryonic nutritional types: yolk deposits (i.e., lecithotrophy) and maternal investment (i.e., matrotrophy). Vitellogenin (VTG), a major egg yolk protein synthesized in the female liver, is one of the molecules relevant to the lecithotrophy-to-matrotrophy shift in bony vertebrates. In mammals, all VTG genes are lost following the lecithotrophy-to-matrotrophy shift, and it remains to be elucidated whether the lecithotrophy-to-matrotrophy shift in nonmammalians is also associated with VTG repertoire modification. In this study, we focused on chondrichthyans (cartilaginous fishes)-a vertebrate clade that underwent multiple lecithotrophy-to-matrotrophy shifts. For an exhaustive search of homologs, we performed tissue-by-tissue transcriptome sequencing for two viviparous chondrichthyans, the frilled shark Chlamydoselachus anguineus and the spotless smooth-hound Mustelus griseus, and inferred the molecular phylogeny of VTG and its receptor very low-density lipoprotein receptor (VLDLR), across diverse vertebrates. As a result, we identified either three or four VTG orthologs in chondrichthyans including viviparous species. We also showed that chondrichthyans had two additional VLDLR orthologs previously unrecognized in their unique lineage (designated as VLDLRc2 and VLDLRc3). Notably, VTG gene expression patterns differed in the species studied depending on their reproductive mode; VTGs are broadly expressed in multiple tissues, including the uterus, in the two viviparous sharks, and in addition to the liver. This finding suggests that the chondrichthyans VTGs do not only function as the yolk nutrient but also as the matrotrophic factor. Altogether, our study indicates that the lecithotrophy-to-matrotrophy shift in chondrichthyans was achieved through a distinct evolutionary process from mammals.
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Tubarões , Animais , Feminino , Tubarões/genética , Tubarões/metabolismo , Vertebrados , Evolução Biológica , Mamíferos/metabolismo , Vitelogeninas/genéticaRESUMO
Homeostatic control and reproductive functions of humans are regulated at the molecular levels largely by peptide hormones secreted from endocrine and/or neuroendocrine cells in the central nervous system and peripheral organs. Homologs of those hormones and their receptors function similarly in many vertebrate species distantly related to humans, but the evolutionary history of the endocrine system involving those factors has been obscured by the scarcity of genome DNA sequence information of some taxa that potentially contain their orthologs. Focusing on non-osteichthyan vertebrates, namely jawless and cartilaginous fishes, this article illustrates how investigating genome sequence information assists our understanding of the diversification of vertebrate gene repertoires in four broad themes: (a) the presence or absence of genes, (b) multiplication and maintenance of paralogs, (c) differential fates of duplicated paralogs, and (d) the evolutionary timing of gene origins.
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Evolução Molecular , Vertebrados , Humanos , Animais , Filogenia , Vertebrados/genética , Peixes/genética , HormôniosRESUMO
Osmoregulatory behaviours should have evolutionarily modified for terrestrialisation of vertebrates. In mammals, sensations of buccal food and drying have immediate effects on postprandial thirst to prevent future systemic dehydration, and is thereby considered to be 'anticipatory thirst'. However, it remains unclear whether such an anticipatory response has been acquired in the non-tetrapod lineage. Using the mudskipper goby (Periophthalmus modestus) as a semi-terrestrial ray-finned fish, we herein investigated postprandial drinking and other unique features like full-body 'rolling' over on the back although these behaviours had not been considered to have osmoregulatory functions. In our observations on tidal flats, mudskippers migrated into water areas within a minute after terrestrial eating, and exhibited rolling behaviour with accompanying pectoral-fin movements. In aquarium experiments, frequency of migration into a water area for drinking increased within a few minutes after eating onset, without systemic dehydration. During their low humidity exposure, frequency of the rolling behaviour and pectoral-fin movements increased by more than five times to moisten the skin before systemic dehydration. These findings suggest anticipatory responses which arise from oral/gastrointestinal and cutaneous sensation in the goby. These sensation and motivation seem to have evolved in distantly related species in order to solve osmoregulatory challenges during terrestrialisation.
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ÁguaRESUMO
Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.
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Peixes , Vertebrados , Animais , Eletroporação , Proteínas de Fluorescência Verde/genética , Evolução BiológicaRESUMO
The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.
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Tubarões , Animais , Tubarões/genética , Genoma , Vertebrados , Cromatina , Disseminação de InformaçãoRESUMO
Environmental DNA (eDNA) is increasingly used to noninvasively monitor aquatic animals in freshwater and coastal areas. However, the use of eDNA in the open ocean (hereafter referred to OceanDNA) is still limited because of the sparse distribution of eDNA in the open ocean. Small pelagic fish have a large biomass and are widely distributed in the open ocean. We tested the performance of two OceanDNA analysis methods-species-specific qPCR (quantitative polymerase chain reaction) and MiFish metabarcoding using universal primers-to determine the distribution of small pelagic fish in the open ocean. We focused on six small pelagic fish species (Sardinops melanostictus, Engraulis japonicus, Scomber japonicus, Scomber australasicus, Trachurus japonicus, and Cololabis saira) and selected the Kuroshio Extension area as a testbed, because distribution of the selected species is known to be influenced by the strong frontal structure. The results from OceanDNA methods were compared to those of net sampling to test for consistency. Then, we compared the detection performance in each target fish between the using of qPCR and MiFish methods. A positive correlation was evident between the qPCR and MiFish detection results. In the ranking of the species detection rates and spatial distribution estimations, comparable similarity was observed between results derived from the qPCR and MiFish methods. In contrast, the detection rate using the qPCR method was always higher than that of the MiFish method. Amplification bias on non-target DNA and low sample DNA quantity seemed to partially result in a lower detection rate for the MiFish method; the reason is still unclear. Considering the ability of MiFish to detect large numbers of species and the quantitative nature of qPCR, the combined usage of the two methods to monitor quantitative distribution of small pelagic fish species with information of fish community structures was recommended.
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DNA Ambiental , Perciformes , Animais , Biodiversidade , DNA/análise , DNA/genética , DNA Ambiental/genética , Peixes/genética , Oceanos e Mares , Perciformes/genéticaRESUMO
Most cartilaginous fishes live in seawater (SW), but a few exceptional elasmobranchs (sharks and rays) are euryhaline and can acclimate to freshwater (FW) environments. The plasma of elasmobranchs is high in NaCl and urea concentrations, which constrains osmotic water loss. However, these euryhaline elasmobranchs maintain high levels of plasma NaCl and urea even when acclimating to low salinity, resulting in a strong osmotic gradient from external environment to body fluid. The kidney consequently produces a large volume of dilute urine to cope with the water influx. In the present study, we investigated the molecular mechanisms of dilute urine production in the kidney of Japanese red stingray, Hemitrygon akajei, transferred from SW to low-salinity environments. We showed that red stingray maintained high plasma NaCl and urea levels by reabsorbing more osmolytes in the kidney when transferred to low salinity. RNA-seq and qPCR analyses were conducted to identify genes involved in NaCl and urea reabsorption under the low-salinity conditions, and the upregulated gene expressions of Na+-K+-Cl- cotransporter 2 (nkcc2) and Na+/K+-ATPase (nka) were found in the FW-acclimated individuals. These upregulations occurred in the early distal tubule (EDT) in the bundle zone of the kidney, which coils around the proximal and collecting tubules to form the highly convoluted structure of batoid nephron. Considering the previously proposed model for urea reabsorption, the upregulation of nkcc2 and nka not only causes the reabsorption of NaCl in the EDT, but potentially also supports enhanced urea reabsorption and eventually the production of dilute urine in FW-acclimated individuals. We propose advantageous characteristics of the batoid-type nephron that facilitate acclimation to a wide range of salinities, which might have allowed the batoids to expand their habitats.
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We wanted to clone the glucocorticoid receptor (GR) from slender African lungfish (Protopterus dolloi) for comparison to the P. dolloi mineralocorticoid receptor (MR), which we had cloned and were characterizing, as well as for comparison to the GRs from humans, elephant shark and zebrafish. However, although sequencing of the genome of the Australian lungfish (Neoceratodus forsteri), as well as, that of the West African lungfish (Protopterus annectens) were reported in the first three months of 2021, we could not retrieve a GR sequence with a BLAST search of GenBank, when we submitted our research for publication in July 2021. Moreover, we were unsuccessful in cloning the GR from slender African lungfish using a cDNA from the ovary of P. dolloi and PCR primers that had successfully cloned a GR from elephant shark, Xenopus and gar GRs. On October 21, 2021 the nucleotide sequence of West African lungfish (P. annectens) GR was deposited in GenBank. We used this GR sequence to construct PCR primers that successfully cloned the GR from the slender spotted lungfish. Here, we report the sequences of nine P. dolloi GR isoforms and explain the basis for the previous failure to clone a GR from slender African lungfish using PCR primers that cloned the GR from elephant shark, Xenopus and gar. Studies are underway to determine corticosteroid activation of these slender African lungfish GRs.
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Proteínas de Peixes , Peixes , Receptores de Glucocorticoides , Animais , Clonagem Molecular , Proteínas de Peixes/genética , Peixes/genética , Isoformas de Proteínas , Receptores de Glucocorticoides/genéticaRESUMO
The many diverse reproductive strategies of elasmobranchs (sharks, skates and rays) from lecithotrophic oviparity to matrotrophic viviparity have attracted significant research attention. However, the endocrine control of elasmobranch reproduction is less well-documented largely due to their reproductive characteristics, such as a long reproductive cycle, and/or repeated internal fertilization using stored sperm in oviparous species. In the present study, for the first time, we succeeded in non-invasive monitoring of the continuing egg-laying cycle of the cloudy catshark Scyliorhinus torazame using portable ultrasound devices. Furthermore, long-term simultaneous monitoring of the egg-laying cycle and measurement of plasma sex steroids revealed cycling patterns of estradiol-17ß (E2), testosterone (T) and progesterone (P4). In particular, a decline in T followed by a reciprocal surge in plasma P4 were consistently observed prior to the appearance of the capsulated eggs, implying that P4 is likely associated with the ovulation and/or egg-case formation. While the cycling pattern of E2 was not as apparent as those of T and P4, threshold levels of E2 (>5 ng/mL) and T (>1 ng/mL) appeared to be crucial in the continuation of egg-laying cycle. The possibility to trace the dynamics of plasma sex steroids in a single individual throughout the reproductive cycles makes the catshark a useful model for regulatory and mechanistic studies of elasmobranch reproduction.
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Oviparidade , Tubarões , Animais , Estradiol , Feminino , Hormônios Esteroides Gonadais , Masculino , Progesterona , Reprodução , Sêmen , UltrassonografiaRESUMO
BACKGROUND: The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century. RESULTS: Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4, a key regulatory gene, in the pharyngeal epithelium during early developmental stages. CONCLUSIONS: Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys.
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Feiticeiras (Peixe) , Glândula Tireoide , Animais , Desenvolvimento Embrionário , Feiticeiras (Peixe)/genética , Lampreias/genética , Vertebrados/genéticaRESUMO
Na+,K+-ATPase (NKA) α-subunit 1a (α1a) and 1b (α1b) gene expressions in the gills are changeable in response to ambient salinity in a few salmonids. In this study, the expressions were compared among ambient salinities and used to infer sea entry migration of chum salmon Oncorhynchus keta fry. The expression of α1a decreased from the 2 days after seawater (SW) transfer from freshwater (FW) and was significantly lower in SW-acclimated fry than that in FW-fry. On the other hand, the expression of α1b peaked on the first to second day after SW transfer and then settled to a level 2-fold higher than in FW-fry. In fry caught in the waterfronts of the beaches, the expression levels were quite similar to those on the first and second days after SW transfer, whereas, in fry caught off beach, the expressions were identical to those of SW-acclimated fry. These suggest that fry adapt to SW with moving along the shoal in the bay, and move to off beach after completing SW adaptation. One of the physiological significances in a usage of waterfront may be to transform the gills to SW type. Only fry on the 2 days after SW transfer failed to exhibit condition factor-dependency of burst swimming, probably due to physiological perturbation, which may be related to poor predation avoidance. The physiological approach used in this study inferred sea entry migration of fry; furthermore, it shows the possible significance of adaptation to SW in the shoal is to reduce predation risk.