A program for the identification of the pelagic eggs of Black Sea fishes

A computer program was developed for the identification of the teleost fish eggs that may be found in the pelagic zone of the Black Sea. The program identifies eggs of 70 species, using up to 28 descriptive characters, and may be adapted for use outside of the Black Sea.

Diets of thornback ray (Raja clavata) and tope shark (Galeorhinus galeus) in the bottom longline fishery of the Azores, northeastern Atlantic

Tope shark (Galeorhinus galeus) and thornback ray (Raja clavata) are the two most captured elasmobranch species by the Azorean bottom longline fishery. In order to better understand the trophic dynamics of these species in the Azores, the diets of thornback ray and tope shark caught in this area during 1996 and 1997 were analyzed to describe feeding patterns and to investigate the effect of sex, size, and depth and area of capture on diet. Thornback rays fed mainly upon fishes and reptants, but also upon polychaetes, mysids, natant crustaceans, isopods, and cephalopods. In the Azores, this species preyed more heavily upon fish compared with the predation patterns described in other areas. Differences in the diet may be due to differences in the environments (e.g. in the Azores, seamounts and oceanic islands are the major topographic features, whereas in all other studies, continental shelves have been the major topographic feature). No differences were observed in the major prey consumed between the sexes or between size classes (49−60, 61−70, 71−80, and 81−93 cm TL). Our study indicates that rays inhabiting different depths and areas (coastal or offshore banks) prey upon different resources. This appears to be related to the relative abundance of prey with habitat. Tope sharks were found to prey almost exclusively upon teleost fish: small shoaling fish, mainly boarfish (Capros aper) and snipefish (Macroramphosus scolopax), were the most frequent prey. This study illustrates that thornback rays and tope sharks are top predators in waters off the Azores.

Responses of plasma transaminase activity in Cyprinus carpio var. communis to mercury toxicity

The present study reports the behavioural and enzymological responses in a freshwater teleost fish, Cyprinus carpio var. communis, exposed to acute and sublethal toxicities of mercuric chloride. During acute treatment, significant behavioural changes like erratic swimming, excess mucus secretion and increased opercular movements were noticed. During acute and sublethal treatments, both aspartate amino transferase and alanine amino transferase activity increased throughout the study period. Comparing the treatments, the changes in enzyme activities were found high in acute treatment and all the values were significant at 5% level. The above findings can be used as non-specific biomarkers of environmental pollutants.

Effect of butachlor on spermatogenesis, testosterone amount and testes tissue Rutilus frisii kutum Kamenskii 1901

Morphological assessment of sexually mature Rutilus frisii kutum Kamenskii 1901 caught from the rivers (Shirud, Khoshkrud, Sepidrud and Chelavand Rivers) flowing in the southwest Caspian Sea region was conducted and sperm volume, total sperm count and sperm concentration of abnormal sperms were determined after exposing the spawners to 60% herbicide butachlor (machete). Spawners under study were maintained in tanks (1000 l) at the Shahid Ansari Teleost Fish Hatchery and exposed to two different concentrations (25% and 75% of its LC50 value) of butachlor. Results obtained indicate that exposure to high butachlor toxicity (75% of its LC50 value) decreased sperm volume to 0.61 ± 0.42 cc in 2-3 year old fishes and to 0.55 ± 0.42 cc in fishes above 3 years of age, while that in fish exposed to low butachlor toxicity (25% of its LC50 value) decreased to 1.55 ± 0.42 cc in 2-3 year old fishes and to 1.28 ± 0.42 cc in fishes above 3 years of age. The sperm volume under normal conditions in R. frisii kutum is 4.6 ± 0.42 cc in 2-3 year olds and 4.58 ± 0.42 cc in fishes above 3 years of age. The total sperm count in R. frisii kutum is 39.74 ± 2.5 billion spermatozoa/cc in 2-3 year olds and 42.99 ± 2.5 billion spermatozoa/cc in fishes above 3 years of age. When exposed to high butachlor toxicity, total sperm count dropped to 16.92 ± 2.5 billion spermatozoa/cc in 2-3 year olds and to 15.98 ± 2.5 billion spermatozoa/cc in fishes above 3 years of age. Similarly total sperm count in R. frisii kutum exposed to low butachlor toxicity was recorded as 23.6 ± 2.5 billion spermatozoa/cc in 2-3 year olds and 29.4 ± 2.5 billion spermatozoa/cc in fishes above 3 years of age. Under normal conditions, on the basis of morphology, spermatozoa showed only 10 ± 1.92% of abnormal sperms. The number of abnormal sperms increased by 28.6 ± 1.92% in fishes exposed to high butachlor toxicity, while that in fishes exposed to low butachlor toxicity increased by 19.7 ± 1.92% in 2-3 year olds and 16.6 ± 19.2% in fishes above 3 years of age. It is evident from the results obtained that increase in level of pollution caused a decrease in sperm volume but an increase in the percentage of abnormal sperms. Results obtained indicate that exposure to high butachlor toxicity (75% of its LC50 value) decreased testostron hormone to 0.31 ± 0.22 ng/ml in high butachlor toxicity, and to 0.45 ± 0.22 ng/ml in low butachlor toxicity (25% of its LC50 value). Testostron hormone dropped to 0.53 ± 0.22 ng/ml in 2-3 year olds and to 0.79 ± 0.22ng/ in fishes above 3 years of age. The testostron hormone under normal conditions in R. frisii kutum is 2.7 ± 0.22 ng/ml. It is evident from the results obtained that increase in level of pollution caused a decrease in testostron hormone

真骨鱼类DMRT基因家族的连锁结构及其系统发生

利用已经报道的多个物种基因组序列资源,我们通过比较真骨鱼类(Teleost fish)共有的DMRT1-5基因所在染色体位置的基因组结构特征,从基因组水平证明了真骨鱼类和四足动物的相应DMRT基因的直系同源关系,揭示了DMRT4和DMRT5是因为真骨鱼类和四足动物的共同祖先发生了染色体重复事件而由同一基因分歧演变形成的。同时通过基因连锁分析,探测到包括SNF2和elavL家族基因在内的多个可能与DMRT基因功能密切相关的基因,为进一步研究DMRT基因和它们之间可能存在的调控机制提供了新的线索。

Gene transfer into genomes of human cells by the sleeping beauty transposon system

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

Purificação e caracterização de proteínas Gla nos modelos peixe e mamífero

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2010

Structure, tissue distribution and estrogen regulation of splice variants of the sea bream estrogen receptor alpha gene

Estrogen actions are mainly mediated by specific nuclear estrogen receptors (ERs), for which different genes and a diversity of transcript variants have been identified, mainly in mammals. In this study, we investigated the presence of ER splice variants in the teleost fish gilthead sea bream (Sparus auratus), by comparison with the genomic organization of the related species Takifugu rubripes. Two exon2-deleted ERα transcript variants were isolated from liver cDNA of estradiol-treated fish. The ΔE2 variant lacks ERα exon 2, generating a premature termination codon and a putative C-terminal truncated receptor, while the ΔE2,3* variant contains an in-frame deletion of exon 2 and part of exon 3 and codes for a putative ERα protein variant lacking most of the DNA-binding domain. Both variants were expressed at very low levels in several female and male sea bream tissues, and their expression was highly inducible in liver by estradiol-17β treatment with a strong positive correlation with the typical wild-type (wt) ERα response in this tissue. These findings identify novel estrogen responsive splice variants of fish ERα, and provide the basis for future studies to investigate possible modulation of wt-ER actions by splice variants.

Evolution of the extracellular matrix in deuterostomes and the influence of calcitropic hormones

Tese de dout., Biologia (Biologia Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes

Whole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1–35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1–34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2–34)PTHrP, (3–34)PTHrP and (7–34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1–34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

Three rounds (1R/2R/3R) of genome duplications and the evolution of the glycolytic pathway in vertebrates

Affiliation: Henner Brinkmann : Département de biochimie, Faculté de médecine, Université de Montreal

Étude du polymorphisme du gène majeur d’histocompatibilité de classe IIb (MHIIb) chez l’omble de fontaine (Salvelinus fontinalis)

Les molécules classiques du complexe majeur d’histocompatibilité de classe II (CMHII) sont des glycoprotéines de surface spécialisées dans la présentation de peptides, principalement dérivés de pathogènes extracellulaires, aux récepteurs des lymphocytes T CD4+ afin d’initier la réponse immunitaire adaptative. Elles sont encodées, avec celles du CMH de classe I, par les gènes les plus polymorphiques identifiés jusqu’à maintenant, avec plusieurs loci et une grande diversité allélique à chacun d’eux. De plus, le polymorphisme des gènes du CMHII n’est pas limité qu’aux séquences codantes. Il est également observé dans les promoteurs où on a démontré ses effets sur le niveau d’expression des gènes. La variation de la régulation d’un gène est considérée comme un facteur important et pour laquelle des modifications morphologiques, physiologiques et comportementales sont observées chez tous les organismes. Des séquences d’ADN répétées impliquées dans cette régulation ont été identifiées dans les régions non-codantes des génomes. D’un autre côté, la sélection par les pathogènes permettrait l’évolution et le maintien du polymorphisme des gènes du CMH chez les vertébrés. À ce sujet, plusieurs études ont montré l’implication de différents allèles du CMH dans la résistance ou la susceptibilité aux maladies. Cette étude avait pour objectifs de caractériser le polymorphisme du gène MHIIb chez l’omble de fontaine (Salvelinus fontinalis) et de documenter ses effets au niveau de la survie conférée par des allèles et/ou génotypes particuliers lors d’une infection, ainsi que sur la variation du niveau d’expression du gène dans différentes conditions. Dans une première partie, nous avons identifié un total de 6 allèles du gène MHIIb, désignés Safo-DAB*0101 à Safo-DAB*0601, qui montrent une grande similarité avec les séquences codantes provenant de poissons téléostéens et de l’humain. L’analyse des séquences du domaine b1 a permis de détecter l’effet d’une pression sélective positive pour maintenir le polymorphisme dans cette région de la molécule. Quatre de ces allèles ont été testés lors d’une expérience d’infection avec le pathogène Aeromonas salmonicida afin d’évaluer l’effet qu’ils pouvaient avoir sur la survie des poissons. Nous avons trouvé que l’allèle DAB*0101 était significativement associé à la résistance à la furonculose. En plus d’avoir été identifié chez les individus homozygotes pour cet allèle, l’effet a également été remarqué au niveau de la survie les poissons de génotype DAB*0101/*0201. À l’opposé, les facteurs de risque élevé obtenus pour les génotypes DAB*0201/*0301 et DAB*0301/*0401 suggèrent plutôt une association à la susceptibilité. Étant donné la faible fréquence à laquelle l’allèle DAB*0101 a été retrouvé dans la population, le modèle de la sélection dépendante de la fréquence pourrait expliquer l’avantage conféré par ce dernier et souligne l’importance de ce mécanisme pour le maintien du polymorphisme du gène MHIIb chez l’omble de fontaine. Dans une seconde partie, nous avons rapporté la présence d’un minisatellite polymorphique formé d’un motif de 32 nucléotides dans le second intron du gène MHIIb, et pour lequel un nombre exclusif de répétitions du motif a été associé à chaque allèle (69, 27, 20, 40, 19 et 25 répétitions pour les allèles DAB*0101 à DAB*0601 respectivement). L’expression relative de quatre allèles a été évaluée dans des poissons hétérozygotes aux températures de 6 ºC et 18 ºC. Les résultats indiquent que les allèles possédant un long minisatellite montrent une réduction de l’expression du gène d’un facteur 1,67 à 2,56 par rapport aux allèles qui en contiennent un court. De même, des allèles qui incluent des minisatellites de tailles similaires n’affichent pas de différence significative au niveau de l’abondance du transcrit aux deux températures. De plus, l’effet répressif associé aux longs minisatellites est amplifié à la température de 18 ºC dans des poissons de trois génotypes différents. Nous avons finalement observé une augmentation significative par un facteur 2,08 de l’expression totale du gène MHIIb à la température de 6 ºC. Ces résultats appuient l’implication des séquences d’ADN répétées dans la régulation de l’activité transcriptionnelle d’un gène et suggèrent qu’un minisatellite sensible aux différences de températures pourrait être soumis aux forces sélectives et jouer un rôle important dans l’expression de gènes et l’évolution des organismes poïkilothermes.

Bioecologia da ictiofauna marinha descartada pelo arrasto camaroeiro em praias da Bacia Potiguar, Brasil

The composition of ichthyofauna discarded by trawling shrimping, their reproductive status and feeding ecology were studied on the beaches of Basin Rio Grande do Norte, Brazil. Fish were collected monthly in the year of 2012. During biometrics, portions of the digestive tract and of gonads were removed, fixed in formalin 10% and Bouin, respectively, for be submitted to histological processing by the techniques of hematoxylin-eosin. Stomach content analyzes were performed using the methods of Frequency of Occurrence and Volumetric and was calculated the repletion index. Throughout the study period were recorded a total of 49 species. The fish assemblages differed between sections monitoring, with the highest abundance, biomass and indices of richness and diversity in sections B, D and C. Already the excerpt A, showed higher values for dominance and equitability. In the cluster analysis according to the faunal similarity was observed the formation of three groups: group I formed by excerpts B and D, group II by excerpt C and group III formed by excerpt A. The assessment of reproductive stage revealed that the fish assemblages discarded by trawling are composed mainly of juveniles. Regarding the feeding ecology, the species Larimus breviceps, Menticirrhus littoralis and Pomadasys corvinaeformis characterized as carnivorous with tendency to carcinofagia. Already Conodon nobilis characterized as carnivorous with tendency to piscivory, but all proved generalist-opportunistic with increase of feeding activity during drought. The dendrogram of grouping of the species developed based on the food items used shows the formation of four groups: Group I consists of species that feed mainly of "gastropod" and "sediment"; group II of "teleost fish"; the group III of "crustacea" and group IV of "echinodermata" and "bivalve". The anatomical and histological characteristics of the digestive tract were consistent with the dietary habits of the analyzed species. In this context, the Costa Branca of Rio Grande do Norte can be considered a feeding site and recruitment for juveniles, which, opportunistically, utilize resources associated with the background

Transport stress in matrinxa, Brycon cephalus (Teleostei : Characidae), at different densities

Matrinxa, Brycon cephalus, is a native teleost fish from the Amazon Basin, and is of economic importance for cultivation for food and sport in Brazil. Mortality losses due to handling and transport of this stenohaline freshwater species are common. The effects of transportation at different densities on the biochemical stress responses of matrinxa (mean weight 1 kg) were examined. Fish were subjected to three different transport densities (100, 200, and 300 kg m(-3)) for four h in water with added salt (0.6%). The fish were bled at departure (baseline level), arrival (immediately after transportation) and at 24 and 96 h after arrival (recovery period). Blood glucose, cortisol, sodium, chloride, potassium and ammonia were used as stress bioindicators. No mortality was observed and no alterations in plasma cortisol were registered. However, blood glucose and ammonia levels increased and serum sodium and plasma chloride decreased on arrival for the fish transported at the highest densities. These stress responses were transient and the concentrations returned to baseline levels within 24 h. This study showed that matrinxa can be transported at densities as high as those tested in the present study, at least under the conditions employed in this study. A recovery period of at least 24 h is strongly recommended.