Structural and ultrastructural characteristics of the yolk syncytial layer in Prochilodus lineatus (Valenciennes, 1836) (Teleostei; Prochilodontidae)

The yolk syncytial layer (YSL) has been regarded as one of the main obstacles for a successful cryopreservation of fish embryos. The purpose of this study was to identify and characterize the YSL in Prochilodus lineatus, a fish species found in southeastern Brazil and considered a very important fishery resource. Embryos were obtained through artificial breeding by hormonal induction. After fertilization, the eggs were incubated in vertical incubators with a controlled temperature (28 degrees C). Embryos were collected in several periods of development up to hatching and then fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Morphological analyses were carried out under either light, transmission or scanning electron microscopy. The formation of the YSL in P. lineatus embryos starts at the end of the cleavage stage (morula), mainly at the margin of the blastoderm, and develops along the embryo finally covering the entire yolk mass (late gastrula) and producing a distinct intermediate zone between the yolk and the endodermal cells. The YSL was characterized by the presence of microvilli on the contact region with the yolk endoderm. A cytoplasmic mass, full of mitochondria, vacuoles, ribosomes, endomembrane nets and euchromatic nuclei, indicated a high metabolic activity. This layer is shown as an interface between the yolk and the embryo cells that, besides sustaining and separating the yolk, acts as a structure that makes it available for the embryo. The structural analyses identified no possible barriers to cryoprotectant penetration.     

Chemical modulation on heterogeneous growth in Prochilodus lineatus (Valenciennes, 1847) (Pisces; Characiformes).

In the present study, the effect of chemical factors released by conspecifics on growth variability (heterogeneous growth - HetG) in a gregarious fish species (Prochilodus lineatus) was tested. HetG was assessed by the weight variation coefficient in two consecutive 21-day periods. The fish were grouped in tanks (4 fish in each) that received running water with constant draining. The tanks contained either conspecifics (C) or non-conspecifics (N). Four conditions were established in accordance with the tank water supply: a) water with previous contact with conspecifics throughout the experiment (CC); b) water without previous contact with conspecifics throughout the experiment (NN); c) water with previous contact with conspecifics in just the first period, 0 to 21 days (CN); and d) just in the period from 21 to 42 days (NC). At the end of the experiments, the occurrence of chemical modulation on the growth variability in P. lineatus was verified: the fish that received water with previous contact with a conspecific (C) presented exacerbation of HetG. This corroborates the notion that the predominant mechanism in the determination of intraspecific growth variation in the gregarious species is associated to chemical factors released by conspecifics.     

Origin and molecular organization of supernumerary chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae) obtained by DNA probes

In Prochilodus lineatus B-chromosomes are visualized as reduced size extra elements identified as microchromosomes and are variable in morphology and number. We describe the specific total probe (B-chromosome probe) in P. lineatus obtained by chromosome microdissection and a whole genomic probe (genomic probe) from an individual without B-chromosome. The specific B-chromosome was scraped and processed to obtain DNA with amplification by DOP-PCR, and so did the genomic probe DNA. Fluorescence in situ hybridization using the B-chromosome probe labeled with dUTP-Tetramethyl-rhodamine and the genomic probe labeled with digoxigenin-FITC permitted to establish that in this species supernumerary chromosomes with varying number and morphology had different structure of chromatin when compared to that of the regular chromosomes or A complement, since only these extra elements were labeled in the metaphases. The present findings suggest that modifications in the chromatin structure of B-chromosomes to differentiate them from the A chromosomes could occur along their dispersion in the individuals of the population.     

Utilization of endogenous reserves and effects of starvation on the health of Prochilodus lineatus (Prochilodontidae)

Prochilodus lineatus Valenciennes (curimbatá) is an important migratory Neotropical fish. It does not feed during spawning migration, and often survives after spawning. The mobilization of energy reserves and some effects of starvation (zero to eight weeks) on fish health were experimentally evaluated. Hepatic glycogen and lipids from the perivisceral fat bodies were the main reserves mobilized during the first four weeks of fasting. During this period, somatic indices and blood parameters showed that fish health was not significantly affected. However, after five weeks of food deprivation, the main energy reserves were depleted and the fish became anaemic. The loss of muscle mass indicates that protein breakdown was an important energy source after the reduction of hepatic and perivisceral reserves. Mortality was increasingly observed from seven weeks of starvation. Prior accumulation of high amounts of reserves is essential to allow movements for long distances during spawning migrations in this species.     

Intraspecific crosses resulting in the first occurrence of eight and nine B chromosomes in Prochilodus lineatus (Characiformes, Prochilodontidae)

B chromosomes are supernumerary elements present in about 15% of eukaryotic species and are most frequently heterochromatic, behave parasitically, show a transmission rate higher than standard (A) chromosomes, and can provoke harmful effects on carriers. In the current work, Prochilodus lineatus individuals carrying eight and nine B chromosomes were obtained by induced crossing performed involving breeders with different B chromosome numbers in their cells. The high B chromosome numbers found in the offspring were recorded for the first time in this species. The use of cytogenetic techniques applied in the present study revealed that regardless of the increase in number of B chromosomes in the genome of these individuals, those elements did not presented active genes, and showed their normal heterochromatic characteristic.     

Variability in the carbon isotope signature of Prochilodus lineatus (Prochilodontidae, Characiformes) a bottom-feeding fish of the Neotropical region

The spatial and seasonal variability of stable carbon isotopes in detritivorous Prochilodus lineatus , primary producers and particulate organic carbon (POC) were examined to promote a better understanding of the trophic dynamics of food webs in Neotropical ecosystems. Spatial variability in carbon isotope composition in the species showed a decreasing gradient in the Upper Paraná River system, from the Paraná River and Itaipu Reservoir subsystems to the Baía and Ivinheima subsystems. Fish δ¹³C followed the isotopic trend of POC along the Paraná River and Baía subsystem. Seasonal differences were not observed in fish. The analysis of percentile contribution of C₃ and C₄ sources in the species diet revealed the predominance of C₃ plants (phytoplanktonic and periphytic algae and riparian vegetation) in most of the analysed subsystems and seasons. Nevertheless, the considerable participation of riparian vegetation was verified, especially in the Ivinheima subsystem in the dry season. C₄ macrophytes presented a maximum contribution along the Upper Paraná River. These results demonstrate the existence of specific variability for the consumer and their food sources in different environments of the same ecosystem.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

Cardiac function of two ecologically distinct Neotropical freshwater fish: Curimbata, Prochilodus lineatus (Teleostei, Prochilodontidae), and trahira, Hoplias malabaricus (Teleostei, Erythrinidae)

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca(2+) (2.5 up to 10.5 mM) to force generation at 25 degrees C (acclimation temperature) in two ecologically distinct Neotropical teleost fish: Curimbata (active species), and trahira (sedentary species). The post-rest force was studied with and without 10 muM ryanodine in the medium. The positive inotropism observed for both species in response to increases on extracellular Ca(2+) reflected a greater Ca(2+) influx through sarcolemma, as well as an increase in Ca(2+) liberation from the SR by the Ca(2+)-induced Ca(2+) release mechanism. The significant post-rest potentiation recorded for the curimbata and trahira control preparations (3.22+/-0.24 to 6.55+/-0.77 mN mm(-2) and 0.74+/-0.07 to 2.26+/-0.26 mN mm(-2), respectively), was completely inhibited by the addition of ryanodine to the bathing medium, suggesting a potential functionality of SR for both species. Considering the differences in these species habitats, modes of life and levels of activity and the fact of a probable SR Ca(2+) cycling in a physiological temperature, we suggest that the functionality of the SR in these species is probably related to their phylogeny.     

Molecular characterization and chromosomal localization of two families of satellite DNA in Prochilodus lineatus (Pisces,Prochilodontidae), a species with B chromosomes

Prochilodus lineatus, an abundant species in the Mogi-Guaçu river basin, represents a large part of the region's fishing potential. Karyotypic analyses based on classic cytogenetic techniques have revealed the presence of 54 meta-submetacentric type chromosomes, together with the occurrence of small supernumerary chromosomes with intra and interindividual variations. This paper describes the genomic organization of two families of satellite DNA in the P. lineatus genome. The chromosomal localization these two repetitive DNA families through fluorescence in situ hybridization (FISH) demonstrated that the SATH1 satellite DNA family, composed of approximately 900 bp, was located in the pericentromeric region of a group of chromosomes of the standard complement, as well as on all the B chromosomes. The SATH2 satellite family has a monomeric unit of 441 bp and was located in the pericentromeric regions of some chromosomes of the standard complement, but was absent in the B chromosomes. Double FISH analyses showed that these two families participate jointly in the pericentromeric organization of several chromosomes of this species. The data obtained in this study support the hypothesis that the B chromosomes derive from chromosomes of the standard complement, which are carriers of the SATH1 satellite DNA.     

Shrinkage of Prochilodus lineatus (Valenciennes, 1847) larvae preserved in either ethyl‐alcohol or formalin in relation to their developmental stage and feeding condition

The effects of preservation in 95% ethyl‐alcohol and 5% formalin were analysed for 3 months on standard length of Prochilodus lineatus larvae from hatching to the end of the flexion process. Unyolked stages were raised under two feeding regimes: unfed and daily fed. All developmental stages that were preserved in formalin as well as the yolked and flexion‐postflexion larvae stored in alcohol shrank significantly (2–6%). In contrast, unyolked preflexion larvae showed a slight but significant enlargement after storage in alcohol (1%). Shrinkage of preflexion stages was 2.5% higher when stored in formalin, while both preservative agents caused similar shrinkage in flexion‐postflexion larvae (ca. 3%). Shrinkage levels after storage in alcohol were dependent on live length, decreasing or increasing with increasing length in yolked and flexion‐postflexion larvae, respectively. The feeding regime did not affect length changes after preservation in either preservative agent.     

Reproduction in the greenback grey mullet, Liza subviridis (Valenciennes, 1836)

This paper presents data on the reproductive biology of Liza subviridis , a little studied mullet species. The fish is heterosexual, exhibiting external fertilization. Six maturity stages can be macroscopically identified in the testes and seven in the ovaries. The macroscopic changes in the gonads are manifestations of histological changes occurring in the development of sperm cells and oocytes. First sexual maturity is attained in the length ranges of 9.5–11.5 cm and 10.5–11.5 cm in male and female fish respectively. The fecundity for fish measuring 10.3–13.9 cm in standard length ranged from 40 000–145 000 eggs. The relationship between fecundity ( F ) and length ( L ) can be represented as: F = 1.9044 L ^4.2998. The spawning duration in L. subviridis is restricted to a short and definite period, with all ripe ova being released within a single spawning act. A pronounced spawning season can be detected to extend from June to November. However, during off-seasons, some spawning also occurs. The correlations between spawning, rainfall and air temperature is discussed.     

Thirteen polymorphic microsatellite loci in the Neotropical fish Prochilodus argenteus (Characiformes,Prochilodontidae)

Prochilodus species inhabit the main river systems of South America and usually present commercial value to inland fishing. In the present study, we describe the isolation and characterization of 13 novel microsatellite loci in Prochilodus argenteus. The number of alleles per polymorphic locus varied from four to 22 and the observed heterozygosity ranged from 0.333 to 0.893. Additionally, cross‐species amplification was successful in two other Prochilodus species. These loci will be useful for studies of the population genetic structure in this fish group.     

Karyotypic conservatism in five species of Prochilodus (Characiformes,Prochilodontidae) disclosed by cytogenetic markers

The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number, morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns. The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in Prochilodus.     

A simple, inexpensive and successful freezing method for curimba Prochilodus lineatus (Characiformes) semen

The cryopreservation of fish sperm provides a tool by which reproduction is optimized and thereby larval production is increased. The aims of this study were to evaluate the effects of cryosolutions, motility-activation media, straw volumes and thawing temperatures on the post-thaw motility of curimba semen. Furthermore, semen cryopreserved in a simple and inexpensive cryosolution and that yielded excellent post-thaw motility was tested for fertility. Semen was diluted in each of the eight cryosolutions in a factorial of two cryoprotectants (DMSO and methylglycol) x four extenders (0.9% NaCl, 5% glucose, BTS and M III). Diluted semen was frozen in 0.5-mL straws in a nitrogen vapor vessel. Sperm motility was evaluated after thawing (60 degrees C water bath for 8s) and activation with a total of four different activation media (distilled water, 0.15% NaCl, 0.29% NaCl or 1% NaHCO(3)). To evaluate straw volume and thawing temperature, semen was diluted in 5% glucose and methylglycol and frozen in 0.5- and 4.0-mL straws. Half of the 0.5-mL straws were thawed in a water bath at 60 degrees C for 8s and the other half at 30 degrees C for 16s. The 4.0-mL straws were thawed at 60 degrees C for 24s only. In the last experiment, semen cryopreserved in 5% glucose and methylglycol, 0.5-mL straws, and thawed at 60 degrees C for 8s was tested for fertility. The results of these comparisons are presented and show that curimba semen can be successfully cryopreserved in a simple glucose solution combined with methylglycol as cryoprotectant, in 0.5-mL straws, yielding motility rates between 86% and 95% and fertilization rates between 47% and 83%.     

B-chromosome frequency stability in <Emphasis Type="Italic">Prochilodus lineatus</Emphasis> (Characiformes,Prochilodontidae)

The genus Prochilodus includes individuals ranging in size from medium to large, being highly relevant for commercial and subsistence fishing. Prochilodus species have a diploid number of 2n = 54 chromosomes and up to seven supernumerary (B) microchromosomes. Previous research has shown that B frequency increased drastically in the Mogi-Guaçu river population of Prochilodus lineatus in the early 1980s, whereas it remained about constant in the 1990s. Here we analyses B frequency in this population during the 2003–2007 period and have found that frequency has not changed significantly since 1987, and that these B chromosomes do not show the intra-individual variation in number that characterized them in the 1980s. This indicates that these B chromosomes have been neutralized, after their invasion, through their mitotic stabilization.     

Reproductive biology of Prochilodus argenteus Agassiz, 1829 (Pisces: Prochilodontidae) in São Francisco River,Brazil

Knowledge of the main reproductive parameters of a species is fundamental for comprehension of the reproductive behavior of a species within its habitat. To evaluate the reproductive biology of curimbatá, Prochilodus argenteus, an important species for commercial fisheries of the São Francisco River basin, Brazil, 153 females and 174 males were captured during 1999–2000 in the São Francisco River. The females (maximum total length 71.2 cm and maximum weight 7.8 kg) were larger than the males (65.0 cm and 4.3 kg). The weight–length ratio indicated isometric growth for females (b = 3.15) and for males (b = 3.04). Prochilodus argenteus presented total spawning from October to January. Increase in rainfall was the main triggering factor for reproduction, showing a positive correlation with the gonadosomatic index (r = 0.84 females; r = 0.95 males). Size at gonadal maturation of females and males was 31.0 and 25.5 cm, respectively. This study provides information to aid in future implementation of captive breeding and management practices that intend to reduce the ongoing depletion of natural stocks.     

Diet of labrisomid blenny Auchenionchus variolosus (Valenciennes, 1836) (Labrisomidae) during its larval development off central Chile (2012–2013)

The diet, feeding success (prey number and total volume per gut, and maximum prey width) and trophic niche width of the labrisomid blenny Auchenionchus variolosus (Valenciennes, 1836) was studied during its larval development (3.93–17.26 mm standard length). Individuals were collected in October 2012 and October 2013 in nearshore waters (<500 m offshore) from Bahía Valparaíso, central Chile with Bongo nets. When compared to the same length range, larval A. variolosus collected during 2013 showed larger upper jaw‐at‐sizes than those from 2012. This coincided with a reduction in relative importance (%IRI) of the main prey item, copepod nauplii, from ~90 to ~73%. Feeding incidence (FI) was high throughout larval development, varying from 97.3 to 100%, being similar throughout the larval development. Prey items per gut (PIPG) ranged from 0 to 35 prey, showing no differences among years. Total volume per gut (TVPG) was positively correlated with larval length, and at given larval size, TVPG ingested by larval A. variolosus was larger during 2013 (0.0607 mm³) than during 2012 (0.0301 mm³). Prey width range was 47.47–700.94 μm and was positively correlated with standard length (SL). Niche breadth was independent of larval size and did not change during larval development in 2012 or 2013. The study helps to understand the trophic interactions occurring in nearshore waters off rocky reef environments from mid‐latitudes of the Southeast Pacific coasts.     

Isolation and characterization of microsatellite DNA in the piracema fish Prochilodus lineatus (Characiformes)

We described five novel microsatellite loci for the piracema fish species Prochilodus lineatus (Characiformes), endemic to South America and of extreme importance to both commercial and artisanal fisheries across its occurrence area. A primary, unenriched genomic library was constructed and radioactively screened for repetitive motifs. Positive clones were automatically sequenced and based on the design of new primers, polymerase chain reaction assays were carried out to determine optimum reaction and electrophoretic conditions for each characterized locus. We evaluated its usefulness in population genetic studies by determining Hardy-Weinberg equilibrium, FIS and a jackknife estimate of the number of alleles for a sample of fish caught below the Funil Hydroelectric Power Plant dam (N = 95), Grande River, Brazil. The number of alleles varied from 3 to 21 and expected heterozygosities ranged from 0.58 to 0.91. Two of five loci were in Hardy-Weinberg equilibrium. Jackknife estimates of the number of alleles were higher than the observed number of alleles for three loci and could provide a measure of sampling bias. These markers should provide important tools for the determination of genetic structure, stock delimitation and reservoir fish management in the Grande River as well as to improve hatchery practices for environmental mitigation measures and to help sustain fisheries in the river.