New Tetrachromic VOF Stain (Type III-G.S) for Normal and Pathological Fish Tissues

10 páginas, 3 figuras, 1 tabla. Este trabajo esta dedicado a Sra. María Armenta.A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G<acid Fuchsin<Light green<Methyl Blue<Fast Green), which are used simultaneously, and it enables the individual tissues to be selectively differentiated and stained. Muscle fibers, collagen, reticulin and elastin fibers, erythrocytes, cartilage, bone, mucous cells, oocytes and larvae were selectively stained and differentiated. Dyes with small size and molecular weight (i.e Orange G), penetrate all tissue structures rapidly, but are only tightly retained in densely textured tissues (i.e erythrocytes). Methyl Blue is an interesting triarylmethane dye (large size and molecular weight), which is incorporated in this new VOF tetrachrome stain, and acquires histochemical significance when used at acid pH (2.8) because collagen and reticulin fibers, as well basophilic and metachromatic substances (strongly ionized sulphated glycoconjugates) can be identified. Muscle tissues show an evident green colour (Fast Green or Light Green affinities), even those isolated and/or diffuse muscle fibers present in the digestive submucosa layer. Connective tissues showed a specific and strong blue colour (Methyl Blue affinity) or mixed blue-red staining (Methyl Blue and Acid Fucshin affinities). Very noticeable is the staining of the mucous cells, as well as the hyaline capsule of the viral lymphocystic cells, which were stained blue-purple (carboxylated and/or strongly ionized sulphated groups). Cartilaginous tissues showed a blue or purple (Methyl Blue affinity) staining, and a specific red colour (Acid Fucshin affinity) was evident during calcification or in bone structures (i.e skeleton, fins, gills, teeth).This work (Spanish MCYT/AGL2003-03558).Peer reviewe

Manuel Gutiérrez-Rodríguez (1923-2009):A cytohistochemist from Cadiz

3 pages.-- Obituary.February 23, 2009 was the sad date on which Dr. Manuel Gutierrez Rodriguez passed away in his hometown of Cadiz, Spain. He was a scientist with a background of excellent, untiring research work; equally important, he was admired and warmly loved by his fellow colleagues as well as by many students at the Medical School and the Science School of the University of Cadiz, who over the years received and were influenced by his teaching.Peer reviewe

Histochemical characterisation of oocytes of the swordfish <i>Xiphias gladius</i>

This work was partially funded by ICMAN.CSIC and the Spanish projects (MCYT/AGL2005-02478 and AGL2006-13777- CO3-O2/ACU). J.B. Ortiz-Delgado is supported by the Programa Ramón y Cajal (MEC, Spain)

Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensis

Cryopreservation of germ cells would facilitate the availability of cells at any time allow ing the selection of donors and maintaining quality control for further applications such as transplan tation and germline recovery. In the present study, we analyzed the efciency of four cryopreservation protocols applied either to isolated cell suspensions or to testes fragments from Senegalese sole. In tes tes fragments, the quality of cryopreserved germ cells was analyzed in vitro in terms of cell recovery, integrity and viability, DNA integrity (fragmentation and apoptosis), and lipid peroxidation (malondialde hyde levels). Transplantation of cryopreserved germ cells was performed to check the capacity of cells to in vivo incorporate into the gonadal primordium of Senegalese sole early larval stages (6 days after hatch ing (dah), pelagic live), during metamorphosis (10 dah) and at post-metamorphic stages (16 dah and 20 dah, benthonic life). Protocols incorporating dimethyl sulfoxide (DMSO) as a cryoprotectant showed higher number of recovered spermatogonia, especially in samples cryopreserved with L-15 + DMSO (0.39 ± 0.18 × 106 cells). Lipid peroxidation and DNA frag mentation were also signifcantly lower in this treat ment compared with other treatments. An important increase in oxidation (MDA levels) was detected in samples containing glycerol as a cryoprotectant, refected also in terms of DNA damage. Transplan tation of L-15 + DMSO cryopreserved germ cells into larvae during early metamorphosis (10 dah, 5.2 mm) showed higher incorporation of cells (27.30 ± 5.27%) than other larval stages (lower than 11%). Cryopreservation of germ cells using testes fragments frozen with L-15 + DMSO was demonstrated to be a useful technique to store Senegalese sole germline.LA/P/0101/2020info:eu-repo/semantics/publishedVersio

Cuantificación y alteraciones histopatológicas producidas por concentraciones subletales de cobre en Fundulus heteroclitus

25 páginas.[EN] Fed and starved Fundulus heteroclitus specimens were exposed for 30 days to sublethal copper concentrations (800 ?g Cu2+/L). The accumulation of copper and histopathological effects on the liver and gills were studied. In both fed and starved specimens, the liver showed a progressive copper accumulation during the experimental period (from 2 to 30 days), with higher levels of copper in the liver of starved fish. Copper accumulation was also detected in the gills, and the specimens exposed to copper for 15 to 30 days (both fed and starved) showed significant differences (P < 0.005) with respect to the control group and the 2-day treated fishes. The main histopathological alterations were: vacuolization of hepatic parenchyma, desquamation of gill epithelia, lamellar telangiectasia, as well as hepatic and branchial necrosis. The results of this paper provide evidence that the nutritional status of the fish has great importance in modifying its response to sublethal copper effects.[ES]Ejemplares de Fundulus heteroclitus alimentados y mantenidos en inanición fueron expuestos a concentraciones subletales de cobre (800 µg Cu2+/L) durante un periodo de 30 días. Se estudió la acumulación de cobre, así como las alteraciones histopatológicas producidas en las branquias y en el hígado. El hígado, tanto en los organismos alimentados como en los mantenidos en ayunas, presentó acumulación progresiva hasta los 30 días, con mayores niveles en ejemplares sometidos al ayuno. Por otro lado, en las branquias se detectó también acumulación de cobre, presentando el grupo de organismos expuestos de 15 a 30 días (alimentados y sometidos a inanición) diferencias significativas (P < 0.005) con respecto al grupo control y los tratados durante 2 días. Las principales alteraciones histopatológicas fueron: vacuolización del parénquima hepático, descamación del epitelio branquial, telangiectasia lamelar, así como necrosis branquial y hepática. Los resultados obtenidos en el presente trabajo muestran la importancia del estado nutricional de peces, como modificador de la respuesta de los mismos a los efectos subletales producidos por la exposición a cobre.Peer reviewe

Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensis

[EN] Cryopreservation of germ cells would facilitate the availability of cells at any time allowing the selection of donors and maintaining quality control for further applications such as transplantation and germline recovery. In the present study, we analyzed the efficiency of four cryopreservation protocols applied either to isolated cell suspensions or to testes fragments from Senegalese sole. In testes fragments, the quality of cryopreserved germ cells was analyzed in vitro in terms of cell recovery, integrity and viability, DNA integrity (fragmentation and apoptosis), and lipid peroxidation (malondialde- hyde levels). Transplantation of cryopreserved germ cells was performed to check the capacity of cells to in vivo incorporate into the gonadal primordium of Senegalese sole early larval stages (6 days after hatching (dah), pelagic live), during metamorphosis (10 dah) and at post-metamorphic stages (16 dah and 20 dah, benthonic life). Protocols incorporating dimethyl sulfoxide (DMSO) as a cryoprotectant showed higher number of recovered spermatogonia, especially in samples cryopreserved with L-15 + DMSO (0.39 ± 0.18 × 10 6 cells). Lipid peroxidation and DNA fragmentation were also significantly lower in this treatment compared with other treatments. An important increase in oxidation (MDA levels) was detected in samples containing glycerol as a cryoprotectant, reflected also in terms of DNA damage. Transplantation of L-15 + DMSO cryopreserved germ cells into larvae during early metamorphosis (10 dah, 5.2 mm) showed higher incorporation of cells (27.30 ± 5.27%) than other larval stages (lower than 11%). Cryopreservation of germ cells using testes fragments frozen with L-15 + DMSO was demonstrated to be a useful technique to store Senegalese sole germlineSIOpen access funding provided by FCT|FCCN (b-on). This study received Portuguese national funds from Foundation for Science and Technology (FCT) through project GERMROS (EXPL/CVT-CVT/0305/2021) and European Union’s Horizon 2020 research and innovation program under grant agreement No. 871108 (AQUAEXCEL3.0). This work was also supported by CCMAR Strategic Plan-UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020. E. Fatsini was supported by FCT contract 2020.04181.CEECIN

Evidence for 5S rDNA Horizontal Transfer in the toadfish Halobatrachus didactylus (Schneider, 1801) based on the analysis of three multigene families

Abstract Background The Batrachoididae family is a group of marine teleosts that includes several species with more complicated physiological characteristics, such as their excretory, reproductive, cardiovascular and respiratory systems. Previous studies of the 5S rDNA gene family carried out in four species from the Western Atlantic showed two types of this gene in two species but only one in the other two, under processes of concerted evolution and birth-and-death evolution with purifying selection. Here we present results of the 5S rDNA and another two gene families in Halobatrachus didactylus, an Eastern Atlantic species, and draw evolutionary inferences regarding the gene families. In addition we have also mapped the genes on the chromosomes by two-colour fluorescence in situ hybridization (FISH). Results Two types of 5S rDNA were observed, named type α and type β. Molecular analysis of the 5S rDNA indicates that H. didactylus does not share the non-transcribed spacer (NTS) sequences with four other species of the family; therefore, it must have evolved in isolation. Amplification with the type β specific primers amplified a specific band in 9 specimens of H. didactylus and two of Sparus aurata. Both types showed regulatory regions and a secondary structure which mark them as functional genes. However, the U2 snRNA gene and the ITS-1 sequence showed one electrophoretic band and with one type of sequence. The U2 snRNA sequence was the most variable of the three multigene families studied. Results from two-colour FISH showed no co-localization of the gene coding from three multigene families and provided the first map of the chromosomes of the species. Conclusions A highly significant finding was observed in the analysis of the 5S rDNA, since two such distant species as H. didactylus and Sparus aurata share a 5S rDNA type. This 5S rDNA type has been detected in other species belonging to the Batrachoidiformes and Perciformes orders, but not in the Pleuronectiformes and Clupeiformes orders. Two hypotheses have been outlined: one is the possible vertical permanence of the shared type in some fish lineages, and the other is the possibility of a horizontal transference event between ancient species of the Perciformes and Batrachoidiformes orders. This finding opens a new perspective in fish evolution and in the knowledge of the dynamism of the 5S rDNA. Cytogenetic analysis allowed some evolutionary trends to be roughed out, such as the progressive change in the U2 snDNA and the organization of (GATA)n repeats, from dispersed to localized in one locus. The accumulation of (GATA)n repeats in one chromosome pair could be implicated in the evolution of a pair of proto-sex chromosomes. This possibility could situate H. didactylus as the most highly evolved of the Batrachoididae family in terms of sex chromosome biology.This work was supported by grants from the Junta de Andalucía (Spain) to the PAI BIO-219 group (LR), CACYTMAR and the “Proyecto de excelencia” RNM-03074 (CS). MUM holds a CSIC JAE fellowship.Peer Reviewe

Toxicity of malathion during Senegalese sole, Solea senegalensis larval development and metamorphosis: Histopathological disorders and effects on type B esterases and CYP1A enzymatic systems

The toxicity of malathion to Solea senegalensis was studied in a static renewal bioassay during its first month of larval life (between 4 and 30 dph). Through the use of different biomarkers and biochemical, cellular and molecular approaches (inhibition of cholinesterases [ChEs], changes in cytochrome P450-1A [CYP1A] and the study of histopathological alterations), the effects of three concentrations of malathion (1.56, 3.12, and 6.25 mu g/L) have been analyzed. In subacute exposure, malathion inhibited cholinesterase activities (AChE, BChE, CbE) in a dose- and time-dependent manner, ranging the inhibition percentage from 20% to 90%. However, the expression levels of CYP1A and AChE transcripts or proteins were not modified. Additionally, exposure to malathion provoked histopathological alterations in several organ systems of Senegalese sole in a time- and dose dependent way, namely disruption of parenchymal architecture in the liver, epithelial desquamation, pyknotic nuclei and steatosis in the intestine, disorganization of supporting cartilage, and sings of hyperplasia and hypertrophy in the gills and degeneration of the epithelial cells from the renal tubules. Malathion exposure also provoked strong disorganization of cardiac fibers from the heart. The findings provide evidence that exposure to sublethal concentrations of malathion that provoked serious injury to the fish S. senegalensis, were below the expected environmental concentrations reported in many other ecosystems and different fish species,revealing a higher sensitivity for Solea senegalensis to malathion exposure, thus reinforcing its use as sentinel species for environmental pollution in coastal and estuarine environments

Estado actual y perspectivas en acuicultura: histofisiología, histopatología y bioxicología

Actas del congreso celebrado en Puerto Real, Cádiz, los días 17-22 de febrero de 199