RESUMEN
CD4 and LAG-3 are related molecules that are receptors for MHC class II molecules. Their major functional differences are situated in their cytoplasmic tails, in which CD4 has an activation motif and LAG-3 an inhibitory motif. Here, we identify shark LAG-3 and show that a previously identified shark CD4-like gene has a genomic location, expression pattern, and motifs similar to CD4 in other vertebrates. In nurse shark (Ginglymostoma cirratum) and cloudy catshark (Scyliorhinus torazame), the highest CD4 expression was consistently found in the thymus whereas such was not the case for LAG-3. Throughout jawed vertebrates, the CD4 cytoplasmic tail possesses a Cx(C/H) motif for binding kinase LCK, and the LAG-3 cytoplasmic tail possesses (F/Y)xxL(D/E) including the previously determined FxxL inhibitory motif resembling an immunoreceptor tyrosine-based inhibition motif (ITIM). On the other hand, the acidic end of the mammalian LAG-3 cytoplasmic tail, which is believed to have an inhibitory function as well, was acquired later in evolution. The present study also identified CD4-1, CD4-2, and LAG-3 in the primitive ray-finned fishes bichirs, sturgeons, and gars, and experimentally determined these sequences for sterlet sturgeon (Acipenser ruthenus). Therefore, with CD4-1 and CD4-2 already known in teleosts (modern ray-finned fish), these two CD4 lineages have now been found within all major clades of ray-finned fish. Although different from each other, the cytoplasmic tails of ray-finned fish CD4-1 and chondrichthyan CD4 not only contain the Cx(C/H) motif but also an additional highly conserved motif which we expect to confer a function. Thus, although restricted to some species and gene copies, in evolution both CD4 and LAG-3 molecules appear to have acquired functional motifs besides their canonical Cx(C/H) and ITIM-like motifs, respectively. The presence of CD4 and LAG-3 molecules with seemingly opposing functions from the level of sharks, the oldest living vertebrates with a human-like adaptive immune system, underlines their importance for the jawed vertebrate immune system. It also emphasizes the general need of the immune system to always find a balance, leading to trade-offs, between activating and inhibiting processes.
Asunto(s)
Tiburones , Animales , Humanos , Genómica , Antígenos de Histocompatibilidad Clase II/genética , MamíferosRESUMEN
Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947T, and produced antagonistic compounds of < 3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.
Asunto(s)
Enfermedades de los Peces/terapia , Flavobacterium/efectos de los fármacos , Oncorhynchus mykiss/microbiología , Animales , Acuicultura/métodos , Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/microbiología , Infecciones por Flavobacteriaceae/terapia , Flavobacterium/genética , Flavobacterium/patogenicidad , Oncorhynchus mykiss/genética , Piel/microbiologíaRESUMEN
Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.
Asunto(s)
Infestaciones Ectoparasitarias/veterinaria , Enfermedades de los Peces/parasitología , Inmunoglobulina M/fisiología , Manosa/inmunología , Platelmintos/inmunología , Takifugu/parasitología , Secuencia de Aminoácidos , Animales , Western Blotting , Cilios/inmunología , Clonación Molecular , ADN Complementario/genética , ADN Complementario/inmunología , Infestaciones Ectoparasitarias/inmunología , Infestaciones Ectoparasitarias/parasitología , Electroforesis en Gel de Poliacrilamida , Enfermedades de los Peces/inmunología , Expresión Génica , Branquias/parasitología , Especificidad del Huésped , Concentración de Iones de Hidrógeno , Inmunoglobulina M/sangre , Inmunoglobulina M/genética , Inmunoglobulina M/aislamiento & purificación , Membrana Mucosa/química , Membrana Mucosa/inmunología , Membrana Mucosa/parasitología , Platelmintos/patogenicidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Takifugu/inmunología , Infecciones por Trematodos/inmunología , Infecciones por Trematodos/parasitología , Infecciones por Trematodos/veterinariaRESUMEN
Rapid radiation associated with phenotypic divergence and convergence provides an opportunity to study the genetic mechanisms of evolution. Here we investigate the genus Takifugu that has undergone explosive radiation relatively recently and contains a subset of closely-related species with a scale-loss phenotype. By using observations during development and genetic mapping approaches, we show that the scale-loss phenotype of two Takifugu species, T. pardalis Temminck & Schlegel and T. snyderi Abe, is largely controlled by an overlapping genomic segment (QTL). A search for candidate genes underlying the scale-loss phenotype revealed that the QTL region contains no known genes responsible for the evolution of scale-loss phenotype in other fishes. These results suggest that the genes used for the scale-loss phenotypes in the two Takifugu are likely the same, but the genes used for the similar phenotype in Takifugu and distantly related fishes are not the same. Meanwhile, Fgfrl1, a gene predicted to function in a pathway known to regulate bone/scale development was identified in the QTL region. Since Fgfr1a1, another memebr of the Fgf signaling pathway, has been implicated in scale loss/scale shape in fish distantly related to Takifugu, our results suggest that the convergence of the scale-loss phenotype may be constrained by signaling modules with conserved roles in scale development.
Asunto(s)
Escamas de Animales/fisiología , Escamas de Animales/efectos de la radiación , Takifugu/genética , Adaptación Biológica/genética , Animales , Mapeo Cromosómico , Peces/genética , Fenotipo , Filogenia , Sitios de Carácter Cuantitativo/genética , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
The lymphotoxin (LT)/LTß receptor (LTßR) axis is crucial for the regulation of immune responses and development of lymphoid tissues in mammals. Despite the importance of this pathway, the existence and function of LT and LTßR remain obscure for nonmammalian species. In this study, we report a nonmammalian LTßR and its ligand. We demonstrate that TNF-New (TNFN), which has been considered orthologous to mammalian LT, was expressed on the cell surface as a homomer in vitro. This different protein structure indicates that TNFN is not orthologous to mammalian LTα and LTß. Additionally, we found that LTßR was conserved in teleosts, but the soluble form of recombinant fugu LTßR did not bind to membrane TNFN under the circumstance tested. Conversely, the LTßR recombinant bound to another ligand, LIGHT, similar to that of mammals. These findings indicate that teleost LTßR is originally a LIGHT receptor. In the cytoplasmic region of fugu LTßR, recombinant fugu LTßR bound to the adaptor protein TNFR-associated factor (TRAF) 2, but little to TRAF3. This difference suggests that teleost LTßR could potentially activate the classical NF-κB pathway with a novel binding domain, but would have little ability to activate an alternative one. Collectively, our results suggested that LIGHT was the original ligand for LTßR, and that the teleost immune system lacked the LT/LTßR pathway. Acquisition of the LT ligand and TRAF binding domain after lobe-finned fish may have facilitated the sophistication of the immune system and lymphoid tissues.
Asunto(s)
Receptor beta de Linfotoxina/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Evolución Molecular , Células HEK293 , Humanos , Insectos , FN-kappa B/metabolismo , Alineación de Secuencia , Factor 3 Asociado a Receptor de TNF/metabolismo , TakifuguRESUMEN
Recently, mammalian basophils have been highlighted as having roles in allergy and antiparasitic immunity; however, there is little information about the functions and evolutionary origin of basophils, because they are the least abundant leukocyte in most vertebrates. In this study, we characterized the teleost basophils that are abundant in the peripheral blood of fugu (Takifugu rubripes). Fugu basophils have two distinct granules: reddish-purple and dark violet ones. Teleost fish do not have IgG and IgE, but we found that fugu IgM bound on the surface of the basophils, and the cross-linked IgM induced degranulation of both types of granules. This indicates that teleost basophils can be activated in an Ab-dependent manner. Furthermore, papain induced the degranulation of the reddish-purple granules, which contain histamine, and the released granules stimulated the migration of various leukocytes. In contrast, chitin elicited the degranulation of the dark violet granules, which resulted in CD4+ T cell-specific migration. Thus, fugu basophils control immune responses via two distinct Ab-independent mechanisms. In addition, fugu basophils endocytosed soluble Ag and expressed MHC class II and B7-H1/DC. These findings suggested that fugu basophils can interact with T cells as APCs. Thus, the Ab-dependent basophil activation predates the emergence of IgG and IgE, and fish basophils exhibit different dynamics and features of degranulation to distinct stimuli compared with mammalian basophils. Some features of teleost basophils are more similar to those of mammalian mast cells than to those of mammalian basophils.
Asunto(s)
Basófilos/inmunología , Takifugu/inmunología , Animales , Degranulación de la Célula/inmunología , Inmunoglobulina M/inmunologíaRESUMEN
Nocardiosis causes serious economic damage in the fish farming of Japanese yellowtail fish. Nocardia seriolae identified as pathogenic bacterium is an intracellular-pathogen. In general, induction of cell-mediated immunity (CMI) is effective in infection defense against intracellular-pathogen. However, the conventional formalin-killed N. seriolae (FKC) vaccine induces humoral immunity. Interleukin-12 (IL-12) is Th1 type heterodimeric cytokine and induces cell differentiation in mammals. Our previous study showed that recombinant amberjack IL-12 has a role in CMI induction in vitro and could be a possible CMI inducing adjuvant. However, its adjuvant effect of fish IL-12 was not studied. In the present study, six types of amberjack recombinant IL-12 (rIL-12) were mixed and injected into amberjack with FKC. Firstly, we analyzed Th1- and Th2- related gene expression and monitored Th1/Th2 status followed by investigation of antibody titer. As a result, Th1-type immunity was induced in FKC + rIL-12 vaccinated fish. Secondly, we checked Th1/Th2 status of vaccinated fish after 10 days of N. seriolae infection using the expression of related genes. High T-bet/GATA-3 ratio was observed in FKC + rIL-12 vaccinated fish, suggesting that Th1 cells possesing antigen memory were induced against N. seriolae infection. Finally, the survival rate in challenge test showed that 88% of FKC + rIL-12 vaccinated fish was survived at 34 days after N. seriolae injection whereas PBS (control) and FKC only were exterminated. These result suggest that i) rIL-12 is viable CMI inducible adjuvant and ii) production of Th1 cells having antigen memory resulting from activation of IL-12 signaling pathway is important for defense against N. seriolae infection.
Asunto(s)
Vacunas Bacterianas/inmunología , Proteínas de Peces/genética , Interleucina-12/genética , Nocardiosis/veterinaria , Nocardia/inmunología , Perciformes , Adyuvantes Inmunológicos/farmacología , Animales , Formaldehído/farmacología , Nocardiosis/prevención & control , Proteínas Recombinantes/genética , Vacunas de Productos Inactivados/inmunologíaRESUMEN
How parasites recognize their definitive hosts is a mystery; however, parasitism is reportedly initiated by recognition of certain molecules on host surfaces. Fish ectoparasites make initial contact with their hosts at body surfaces, such as skin and gills, which are covered with mucosa that are similar to those of mammalian guts. Fish are among the most primitive vertebrates with immune systems that are equivalent to those in mammals, and they produce and secrete IgM into mucus. In this study, we showed that the monogenean parasite Heterobothrium okamotoi utilizes IgM to recognize its host, fugu Takifugu rubripes Oncomiracidia are infective larvae of H. okamotoi that shed their cilia and metamorphose into juveniles when exposed to purified d-mannose-binding fractions from fugu mucus. Using liquid chromatography-tandem mass spectrometry analysis, proteins contained in the fraction were identified as d-mannose-specific IgM with two d-mannose-binding lectins. However, although deciliation was significantly induced by IgM and was inhibited by d-mannose or a specific Ab against fugu IgM, other lectins had no effect, and IgM without d-mannose affinity induced deciliation to a limited degree. Subsequent immunofluorescent staining experiments showed that fugu d-mannose-specific IgM binds ciliated epidermal cells of oncomiracidium. These observations suggest that deciliation is triggered by binding of fugu IgM to cell surface Ags via Ag binding sites. Moreover, concentrations of d-mannose-binding IgM in gill mucus were sufficient to induce deciliation in vitro, indicating that H. okamotoi parasites initially use host Abs to colonize host gills.
Asunto(s)
Inmunoglobulina M/inmunología , Manosa/metabolismo , Membrana Mucosa/inmunología , Takifugu/inmunología , Takifugu/parasitología , Trematodos/fisiología , Animales , Afinidad de Anticuerpos , Sitios de Unión de Anticuerpos , Cromatografía Liquida , Cilios/fisiología , Branquias/parasitología , Inmunidad Mucosa , Inmunoglobulina M/metabolismo , Larva/inmunología , Larva/fisiología , Manosa/inmunología , Membrana Mucosa/parasitología , Espectrometría de Masas en TándemRESUMEN
Interleukin (IL) -12 is a heterodimeric cytokine mainly produced by monocytes, macrophages, and dendritic cells in mammals. IL-12p70 composed of IL-12p35 and IL-12p40, is known to play a crucial role in promoting cell-mediated immunity (CMI) through Th1 differentiation and IFN-γ production. Although two types of IL-12p35 (p35a, p35b) and three types of IL-12p40 (p40a, p40b and p40c) have been identified in several fish species, the knowledge on functional characteristics of teleost IL-12 is still limited. In the present study, we cloned two types of IL-12p35 and three types of IL-12p40 genes in amberjack and yellowtail, and analyzed their expressions in response to stimulation with Nocardia seriolae in amberjack. As a result, four types of IL-12 (IL-12p35a, p35b, p40a and p40b) and IFN-γ mRNA were increased by live-N. seriolae stimulation but not by formalin-killed N. seriolae, suggesting that four types of IL-12 (p35, p35b, p40a and p40c) participate in promoting CMI. Subsequently, we produced six types of recombinant IL-12p70 (rIL12p70) protein in insect cells. Head kidney leukocytes were cultured with formalin-killed N. seriolae and six types of rIL-12p70 to elucidate the role of amberjack IL-12p70 in induction of CMI. After stimulation, IFN-γ expression was elevated whereas IL-10 expression was suppressed in Head kidney leukocytes stimulated with four types of rIL-12 (p40a/p35a, p40c/p35a, p40a/p35b, p40a/p35b). On the other hand, two types of rIL-12 (p40b/p35a, p40b/p35b) only elicited down regulation of IL-10 expression. These results indicate that all amberjack IL-12p70 isoforms are involved in Th1 -differentiation and promotion of CMI with different manners. Fish IL-12 has a potential for the promising vaccine adjuvant.
Asunto(s)
Infecciones por Actinomycetales/veterinaria , Vacunas Bacterianas/inmunología , Enfermedades de los Peces/terapia , Proteínas de Peces/genética , Interleucina-12/genética , Nocardiaceae/inmunología , Perciformes , Infecciones por Actinomycetales/microbiología , Infecciones por Actinomycetales/terapia , Secuencia de Aminoácidos , Animales , Enfermedades de los Peces/microbiología , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Interleucina-12/química , Interleucina-12/metabolismo , Subunidad p35 de la Interleucina-12/genética , Subunidad p35 de la Interleucina-12/metabolismo , Subunidad p40 de la Interleucina-12/genética , Subunidad p40 de la Interleucina-12/metabolismo , Filogenia , Alineación de Secuencia/veterinaria , Vacunas Atenuadas/inmunología , Vacunas de Productos Inactivados/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Correlated suits of behaviors (behavioral syndrome) are commonly observed in both inter- and intraspecific studies. In order to understand the genetic basis of such a correlation between species, we compared ten behaviors classified into five categories (acclimation, feeding, normal swimming, reaction to a novel object and activity in a novel environment) between two pufferfish species, Takifugu rubripes and T. niphobles. The two species showed consistent differences in nine behaviors with a significant correlation among behaviors. Quantitative trait locus (QTL) analysis using second generation hybrids revealed that different sets of small effect QTL are associated with the observed interspecific behavioral disparity. This indicates that correlations in temperament traits between them are governed by many genes with small effects, and each behavior has been selected to form particular combination patterns. One of the QTL showing small pleiotropic effect includes the Drd4 gene known for its association with behavioral traits in some animal taxa including mammals.
Asunto(s)
Conducta Animal/fisiología , Takifugu/genética , Animales , Genotipo , Reacción en Cadena de la Polimerasa , Sitios de Carácter Cuantitativo/genética , Especificidad de la EspecieRESUMEN
The genetic mechanisms underlying host specificity of parasitic infections are largely unknown. After hatching, the larvae of the monogenean parasite, Heterobothrium okamotoi, attach to the gill filaments of hosts and the post-larval worms develop there by consuming nutrients from the host. The susceptibility to H. okamotoi infection differs markedly among fish species. While this parasite can grow on tiger pufferfish (also called fugu), Takifugu rubripes, it appears to be rejected by a close congener, grass pufferfish, Takifugu niphobles, after initial attachment to the gills. To determine the genetic architecture of the pufferfish responsible for this host specificity, we performed genome-wide quantitative trait loci analysis. We raised second generation (F2) hybrids of the two pufferfish species and experimentally infected them with the monogenean in vivo. To assess possible differences in host mechanisms between early and later periods of infection, we sampled fish three h and 21days after exposure. Genome scanning of fish from the 3h infection trial revealed suggestive quantitative trait loci on linkage groups 2 and 14, which affected the number of parasites on the gill. However, analysis of fish 21days p.i. detected a significant quantitative trait locus on linkage group 9 and three other suggestive quantitative trait loci on linkage groups 7, 18 and 22. These results indicated the polygenic nature of the host mechanisms involved in the infection/rejection of H. okamotoi. Moreover the analyses suggested that host factors may play a more important role during the growth period of the parasite than during initial host recognition at the time of attachment. Within the 95% confidence interval of the linkage group 9 quantitative trait locus in the fugu genome, there were 214 annotated protein-coding genes, including immunity-related genes such as Irak4, Muc2 and Muc5ac.
Asunto(s)
Especificidad del Huésped , Interacciones Huésped-Patógeno , Platelmintos/fisiología , Tetraodontiformes/genética , Tetraodontiformes/parasitología , Animales , Ligamiento Genético , Branquias/parasitología , Sitios de Carácter CuantitativoRESUMEN
N-acetyl-d-glucosamine (GlcNAc) is one of the components of peptidoglycan, a biopolymer in the bacterial cell wall. We purified a novel GlcNAc-binding protein, designated as fGBP-78, from sera of fugu (Takifugu rubripes). The fGBP-78 is a heteromer of 78- and 25-kDa subunits. Moreover, fGBP-78 exerted remarkable inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria, including ones virulent for marine fish species as well as non-pathogenic Escherichia coli. These results suggest that fGBP-78 contributes to bacterial clearance in fugu. Furthermore, the nanoLC-MS/MS and Western blotting analyses reveal that the 78-kDa subunit is the fugu IgM heavy chain. In addition, the molecular mass of the other subunit (25 kDa) was equal to that of the Ig light chain. Overall, results indicate that fGBP-78 is an IgM molecule presumably acts as a natural antibody. This paper reports a novel function of teleost IgM as a significant suppresser against bacterial growth.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas Portadoras/inmunología , Proteínas de Peces/inmunología , Inmunoglobulina M/inmunología , Subunidades de Proteína/inmunología , Takifugu/inmunología , Acetilglucosamina/química , Acetilglucosamina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/farmacología , Proteínas Portadoras/sangre , Proteínas Portadoras/farmacología , Proteínas de Peces/sangre , Proteínas de Peces/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/crecimiento & desarrollo , Inmunidad Innata , Inmunoglobulina M/sangre , Inmunoglobulina M/farmacología , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/sangre , Subunidades de Proteína/farmacología , Takifugu/sangre , Takifugu/genética , Takifugu/microbiologíaRESUMEN
Closely related species have often evolved dramatic differences in body size. Takifugu rubripes (fugu) is a large marine pufferfish whose genome has been sequenced, whereas T. niphobles is the smallest species among Takifugu. We show that, unsurprisingly, the juvenile growth rate of T. rubripes is higher than that of T. niphobles in a laboratory setting. We produced F(2) progenies of their F(1) hybrids and found one quantitative trait locus (QTL) significantly associated with variation in juvenile body size. This QTL region (3.5 Mb) contains no known genes directly related to growth phenotype (such as IGFs) except Fgf21, which inhibits growth hormone signaling in mouse. The QTL in Takifugu spp. is distinct from the region previously known to control body size variations in stickleback or tilapia. Our results suggest that in the fish tested herein, genomic regions underlying body size evolution might have different genetic origins. They also suggest that many diverse traits in Takifugu spp. are amenable to genetic mapping.
Asunto(s)
Tamaño Corporal/genética , Takifugu/genética , Animales , Cruzamientos Genéticos , Evolución Molecular , Factores de Crecimiento de Fibroblastos/genética , Variación Genética , Fenotipo , Sitios de Carácter Cuantitativo , Takifugu/crecimiento & desarrolloRESUMEN
For understanding the functions of the growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family of hormones, we examined pituitary mRNA expression of these hormones in anguillid eels in relation to salinity difference, silvering, and seasonal change. Female Japanese eels (Anguilla japonica) were collected in the brackish Hamana Lake and its freshwater rivers from July to December. To clarify the effect of salinity, the habitat use history of the eels were determined using otolith microchemistry. Expression levels of mRNA of each hormone were determined using real time PCR. Although GH and PRL have been known to be osmoregulatory hormones, there were no consistent differences in expression levels of these hormones between different salinity habitats. In contrast, SL mRNA expression was higher in eels from freshwater rivers than from the brackish lake. GH mRNA expression clearly decreased during silvering, whereas PRL and SL mRNA expression did not change. We also showed that PRL mRNA and SL mRNA decreased in the brackish lake and PRL mRNA increased in freshwater rivers from autumn to early winter. These findings provide basic knowledge for a further understanding of the role of these hormones.
Asunto(s)
Anguilla/genética , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Glicoproteínas/metabolismo , Hormonas Hipofisarias/metabolismo , Prolactina/metabolismo , Salinidad , Animales , Biometría/métodos , Peso Corporal , Ecosistema , Femenino , Proteínas de Peces/genética , Glicoproteínas/genética , Lagos , Pigmentación , Hormonas Hipofisarias/genética , Prolactina/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Ríos , Estaciones del Año , Especificidad de la EspecieRESUMEN
Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.
Asunto(s)
Sustitución de Aminoácidos/genética , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Procesos de Determinación del Sexo/genética , Takifugu , Animales , Evolución Biológica , Femenino , Estudios de Asociación Genética , Ligamiento Genético , Heterocigoto , Homocigoto , Masculino , Mutación Missense/genética , Receptores de Péptidos/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Cromosomas Sexuales/genética , Takifugu/genética , Takifugu/fisiologíaRESUMEN
Teleost IL-6 is upregulated after antigen stimulation; therefore, we hypothesized that fish IL-6 contributes to antibody production during immune responses against infections. To verify this hypothesis, we first cloned IL-6R and gp130 in fugu (Takifugu rubripes) in the present study. The membrane and soluble forms of IL-6R were identified by the identification of cDNA clones of IL-6R homologues. Three STAT3-docking sites were found in the intracellular region of fugu gp130. Expression analysis showed that fugu IL-6R and gp130 were expressed in mIgM(+) B cells, suggesting that fugu B cells are stimulated by IL-6. Recombinant fugu IL-6 (rfIL-6) increased the gene expression of secretory antibodies by mIgM(+) B cells in vitro. The rfIL-6 and soluble form of rfIL-6R activated STAT3 phosphorylation in the B cells and a cultured cell line transfected with fugu gp130. These results indicate that fugu IL-6 enhances antibody production in the B-cell lineage via gp130 and STAT3 signaling.
Asunto(s)
Formación de Anticuerpos , Receptor gp130 de Citocinas/metabolismo , Proteínas de Peces/metabolismo , Interleucina-6/inmunología , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Takifugu/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Filogenia , Alineación de SecuenciaRESUMEN
The compact genome of fugu (Takifugu rubripes) has been used widely as a reference genome for understanding the evolution of vertebrate genomes. However, the fragmented nature of the fugu genome assembly has restricted its use for comparisons of genome architecture in vertebrates. To extend the contiguity of the assembly to the chromosomal level, we have generated a comprehensive genetic map of fugu and anchored the scaffolds of the assembly to the 22 chromosomes of fugu. The map consists of 1,220 microsatellite markers that provide anchor points to 697 scaffolds covering 86% of the genome assembly (http://www.fugu-sg.org/). The integrated genome map revealed a higher recombination rate in fugu compared with other vertebrates and a wide variation in the recombination rate between sexes and across chromosomes of fugu. We used the extended assembly to explore recent rearrangement events in the lineages of fugu, Tetraodon, and medaka and compared them with rearrangements in three mammalian (human, mouse, and opossum) lineages. Between the two pufferfishes, fugu has experienced fewer chromosomal rearrangements than Tetraodon. The gene order is more highly conserved in the three teleosts than in mammals largely due to a lower rate of interchromosomal rearrangements in the teleosts. These results provide new insights into the distinct patterns of genome evolution between teleosts and mammals. The consolidated genome map and the genetic map of fugu are valuable resources for comparative genomics of vertebrates and for elucidating the genetic basis of the phenotypic diversity of ~25 species of Takifugu that evolved within the last 5 My.
Asunto(s)
Mapeo Cromosómico , Evolución Molecular , Genoma , Mamíferos/genética , Takifugu/genética , Animales , Cromosomas , Secuencia Conservada/genética , Orden Génico , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Ratones , Repeticiones de Microsatélite , Zarigüeyas/genética , Oryzias/genética , Filogenia , Análisis de Secuencia de ADN , Tetraodontiformes/genéticaRESUMEN
A skin mucus lectin exhibiting a homodimeric structure and an S-S bond between subunits of ~40 kDa was purified from flathead Platycephalus indicus (Scorpaeniformes). This lectin, named FHL (FlatHead Lectin), exhibited mannose-specific activity in a Ca(2+)-dependent manner. Although FHL showed no homology to any previously reported lectins, it did exhibit ~20% identity to previously discovered plasma kallikreins and coagulation factor XIs of mammals and Xenopus laevis. These known proteins are serine proteases and play pivotal roles in the kinin-generating system or the blood coagulation pathway. However, alignment analysis revealed that while FHL lacked a serine protease domain, it was homologous to the heavy-chain domain of plasma kallikreins and coagulation factor XI therefore suggesting that FHL is not an enzyme but rather a novel animal lectin. On the basis of this finding, we investigated the lectin activity of human plasma kallikrein and revealed that it could indeed act as a lectin. Other genes homologous to FHL were also found in the genome databases of some fish species, but not in mammals. In contrast, plasma kallikreins and coagulation factor XI have yet to be identified in fish. The present findings suggest that these mammalian enzymes may have originally emerged as a lectin and may have evolved into molecules with protease activity after separation from common ancestors.
Asunto(s)
Peces , Calicreínas/metabolismo , Lectinas/metabolismo , Secuencia de Aminoácidos , Animales , Factor XI/química , Factor XI/metabolismo , Perfilación de la Expresión Génica , Humanos , Calicreínas/sangre , Calicreínas/química , Lectinas/análisis , Lectinas/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The profiles of sex steroids (estradiol-17ß, testosterone and 11-ketotestosterone) and the mRNA levels of gonadotropins (luteinizing hormone and follicle-stimulating hormone) were investigated before and after downstream migration in females of the Japanese eel species Anguilla japonica, which were collected in the brackish Hamana Lake and its inlet freshwater rivers. Eels were separated into three groups using otolith microchemistry: 'migrants' that grew in the inlet rivers and then made a downstream migration to Hamana Lake mainly in October and November; 'non-migrant' yellow eels caught in rivers during the same season; and 'residents,' which were yellow eels caught in rivers in August. Sex steroid levels, especially those of testosterone and 11-ketotestosterone, were higher in migrants than in non-migrants and residents. Real-time quantitative PCR analysis indicated that mRNA levels of luteinizing hormone (LH) ß-subunits were significantly higher in migrants than in other groups, whereas those of follicle-stimulating hormone ß-subunits did not show significant changes during downstream migration. The high levels of these hormones during downstream migration raise the question about if they also play a role in motivating the migratory behavior of eels.
Asunto(s)
Anguilla/fisiología , Migración Animal/fisiología , Estradiol/sangre , Gonadotropinas/metabolismo , Testosterona/análogos & derivados , Testosterona/sangre , Animales , Ecosistema , Estradiol/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ríos , Testosterona/metabolismoRESUMEN
The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.