Unique Features of River Lamprey (Lampetra fluviatilis) Myogenesis.

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates.

Sex steroids in diploid and triploid gibel carp (Carassius gibelio) of both sexes in different phases of the reproductive cycle.

The changes in concentrations of 17ß-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT) in the gonads and plasma of diploid and triploid gibel carp (Carassius gibelio) from the Siemianowka Reservoir, the Vistula River drainage were quantified and compared using an enzyme immunoassay. The phase of gonad maturity was based on histological analysis and the gonadosomatic index. All fish had properly developed gonads in the pre-spawning, spawning and late spawning phases of the reproductive cycle in the fish collected in April, June and October, respectively. Diploid and triploid females and males did not differ in mean GSI in all reproductive phases. In October, ovaries of most females contained vitellogenic oocytes, testes had large numbers of spermatozoa. The hormone concentrations in both tissues depended mainly on the reproductive phase, but not on ploidy. The patterns of changes in E2 concentrations was similar in females and males. In the pre-spawning phase, diploid and triploid females showed differences in the concentrations of 11-KT in gonads and plasma, and T in plasma. Diploid males differed in pattern of T concentrations in gonads and plasma, while all males showed a similar pattern of 11-KT plasma concentrations. Spermatozoa observed in triploid males suggest that they participate in reproduction. Gibel carps, regardless of ploidy, had an extended period of reproduction, which makes this invasive species a potentially greater threat to native ichthyofauna. The sex androgen concentrations that differed between 2 n and 3 n females could be physiological factors potentially contributing to the coexistence of gynogenetic C. gibelio females and sexual diploids.

Comparison of artificial spawning effectiveness of hCG, CPH and GnRHa in combination with dopamine inhibitors in a wild strain of ide Leuciscus idus (L.) in hatchery conditions.

Optimization of artificial reproduction, by increasing the survival rate of embryos and hatching rate, is of major importance for reducing genetic diversity, especially in fish captured from their natural habitat that subsequently spawn in hatcheries. The artificial reproduction of ide, Leuciscus idus (L.) was conducted in controlled conditions. The spawning agents included: different doses of human chorionic gonadotropin (hCG) that was compared with common carp pituitary homogenate (CPH), Ovopel - a commercial agent for induction of spawning that contains mammalian gonadotropin releasing hormone analogue (mGnRHa) with dopamine antagonists (DA): metoclopramide (MET) and Ovaprim - a commercial agent containing salmon gonadotropin releasing hormone analogue (sGnRHa) and a dopamine antagonists (DA): domperidone (DOM). There were no ovulations in females from control groups. There were no differences between the ovulation rates (90 %) or embryo survival (> 92 %) and hatching rates (> 91 %) when there was administration of hCG doses between 500 and 1000 IU/kg. When there was comparison of different spawning agents, the ovulation rate was 100 % for all treated groups. There were the shortest and longest latency times to the time of ovulation after administration of CPH (26 h) and hCG (79 h), respectively. The greatest embryo survival (> 93 %) and hatching (> 91 %) rates occurred as a result of hCG administration with these values being slightly greater than when there was treatment with Ovaprim. The association between latency time and hatching rate indicated that when there was a slower final oocyte maturation (FOM) there were greater hatching rates.

Parthenogenesis as a Solution to Hybrid Sterility: The Mechanistic Basis of Meiotic Distortions in Clonal and Sterile Hybrids.

Hybrid sterility is a hallmark of speciation, but the underlying molecular mechanisms remain poorly understood. Here, we report that speciation may regularly proceed through a stage at which gene flow is completely interrupted, but hybrid sterility occurs only in male hybrids whereas female hybrids reproduce asexually. We analyzed gametogenic pathways in hybrids between the fish species Cobitis elongatoides and C. taenia, and revealed that male hybrids were sterile owing to extensive asynapsis and crossover reduction among heterospecific chromosomal pairs in their gametes, which was subsequently followed by apoptosis. We found that polyploidization allowed pairing between homologous chromosomes and therefore partially rescued the bivalent formation and crossover rates in triploid hybrid males. However, it was not sufficient to overcome sterility. In contrast, both diploid and triploid hybrid females exhibited premeiotic genome endoreplication, thereby ensuring proper bivalent formation between identical chromosomal copies. This endoreplication ultimately restored female fertility but it simultaneously resulted in the obligate production of clonal gametes, preventing any interspecific gene flow. In conclusion, we demonstrate that the emergence of asexuality can remedy hybrid sterility in a sex-specific manner and contributes to the speciation process.

Variable occurrence of apoptosis in the testes of diploid and sterile allotetraploid Cobitis (Teleostei, Cobitidae) males during the reproductive cycle.

Cobitis species exist in both diploid and diploid-polyploid (d-p) populations, but mostly occur in the latter. They are considered an important model organism to study the biology and physiology of natural hybrid and polyploid vertebrates. Indeed, polyploidization causes a huge stress for in terms of cell physiology and alters spermatogenesis in polyploid fish. The most extensively studied mode of germ cell death during spermatogenesis in vertebrates is apoptosis. The aim of this study was to examine caspase-3 immunoexpression in the testes of Cobitis taenia from a diploid population as well as C. taenia and sterile tetraploid Cobitis from d-p populations before, during and after spawning. The obtained results suggest a different performance of apoptosis in the testes of C. taenia from the two studied populations and seems to be conditioned by their role as the only sperm donors in d-p populations. Moreover, apoptosis was an active cell death process in the testes of tetraploid Cobitis.

Chromosomal inheritance of parental rDNAs distribution pattern detected by FISH in diploid F1 hybrid progeny of Cobitis (Teleostei, Cobitidae) species has non-Mendelian character.

This study was conducted to describe the major and the minor rDNA chromosome distribution in the spined loach Cobitis taenia (2n = 48) and the Danubian loach Cobitis elongatoides (2n = 50), and their laboratory-produced diploid reciprocal F1 hybrid progeny. It was tested by fluorescence in situ hybridisation (FISH) whether the number of 28s and 5s rDNA sites in the karyotypes of diploid hybrids corresponds to the expectations resulting from Mendelian ratio and if nucleolar organiser regions (NOR)were inherited from both parents or nucleolar dominance can be observed in the induced F1 hybrid progeny. Ten (females) or twelve (males) 28s rDNA loci were located in nine uniarm chromosomes of C. taenia. Two of such loci terminally bounded on one acrocentric chromosome were unique and indicated as specific for this species. Large 5s rDNA clusters were located on two acrocentric chromosomes. In C. elongatoides of both sexes, six NOR sites in terminal regions on six meta-submetacentric chromosomes and two 5s rDNA sites on large submetacentrics were detected. The F1 hybrid progeny (2n = 49) was characterised by the intermediate karyotype with the sites of ribosome synthesis on chromosomes inherited from both parents without showing nucleolar dominance. 5s rDNA sites were detected on large submetacentric and two acrocentric chromosomes. The observed number of both 28s and 5s rDNAs signals in F1 diploid Cobitis hybrids was disproportionally inherited from the two parental species, showing inconsistency with the Mendelian ratios. The presented rDNA patterns indicate some marker chromosomes that allow the species of the parental male and female to be recognised in hybrid progeny. The 5s rDNA was found to be a particularly effective diagnostic marker of C. elongatoides to partially discern genomic composition of diploid Cobitis hybrids and presumably allopolyploids resulting from their backcrossing with one of the parental species. Thus, the current study provides insight into the extent of rDNA heredity in Cobitis chromosomes and their cytotaxonomic character.

The Legacy of Sexual Ancestors in Phenotypic Variability, Gene Expression, and Homoeolog Regulation of Asexual Hybrids and Polyploids.

Hybridization and polyploidization are important evolutionary processes whose impacts range from the alteration of gene expression and phenotypic variation to the triggering of asexual reproduction. We investigated fishes of the Cobitis taenia-elongatoides hybrid complex, which allowed us to disentangle the direct effects of both processes, due to the co-occurrence of parental species with their diploid and triploid hybrids. Employing morphological, ecological, and RNAseq approaches, we investigated the molecular determinants of hybrid and polyploid forms. In contrast with other studies, hybridization and polyploidy induced relatively very little transgressivity. Instead, Cobitis hybrids appeared intermediate with a clear effect of genomic dosing when triploids expressed higher similarity to the parent contributing two genome sets. This dosage effect was symmetric in the germline (oocyte gene expression), interestingly though, we observed an overall bias toward C. taenia in somatic tissues and traits. At the level of individual genes, expression-level dominance vastly prevailed over additivity or transgressivity. Also, trans-regulation of gene expression was less efficient in diploid hybrids than in triploids, where the expression modulation of homoeologs derived from the "haploid" parent was stronger than those derived from the "diploid" parent. Our findings suggest that the apparent intermediacy of hybrid phenotypes results from the combination of individual genes with dominant expression rather than from simple additivity. The efficiency of cross-talk between trans-regulatory elements further appears dosage dependent. Important effects of polyploidization may thus stem from changes in relative concentrations of trans-regulatory elements and their binding sites between hybridizing genomes. Links between gene regulation and asexuality are discussed.

Evidence of the sterility of allotetraploid Cobitis loaches (Teleostei, Cobitidae) using testes ultrastructure.

The diploid-polyploid populations of Cobitis distributed in Poland are usually composed of the spined loach Cobitis taenia or, less often, the Danubian loach C. elongatoides and their triploid (females) and tetraploid hybrids (females and males). The aim of this study was to determine whether tetraploid males participate in the reproduction process by analyzing their testis ultrastructure and the process of spermatogenesis in comparison with diploid males of both parental species. Tetraploid loaches were obtained from three different diploid-polyploid populations distributed in Poland. The structure of Cobitis testes are typical for most Teleostei fish with cystic-type spermatogenesis. The successive stages of developing germ cells are enclosed within cysts formed by the Sertoli cells. This paper morphologically describes the different germ cell stages of spermatogenesis (spermatogonia, spermatocytes, spermatids, and spermatozoa) of C. taenia and C. elongatoides and provides a pioneering ultrastructural analysis of tetraploid Cobitis testes which reveals their unusual structure for the first time. Thus, cysts with normal spermatogonia and spermatocytes (pachyten or leptoten stages) containing synaptonemal complexes were present and no spermatids or spermatozoa were observed. Moreover, in contrast to previously analyzed diploid species, single cells or all of the cells within the cysts displayed chromatin condensation and/or chromatin fragmentation. The obtained results clearly demonstrated that tetraploid males are sterile and diploids are fertile and are the only sperm donors in the reproduction processes of diploid-polyploid Cobitis populations.

Early Development and Post Embryonic Skeletal Morphology of the Progeny of Spined Loach Cobitis taenia L. (Teleostei, Cobitidae) and its Naturally Occurring Allotriploids.

Polyploid fishes of the genus Cobitis represent a valuable model system to study the origin and consequences of hybridization and polyploidization within vertebrates. These naturally accessible polyploids are an excellent subject to determine the advantages or disadvantages of polyploidy. We investigated the embryonic and larval development with skeletal morphology of diploid and polyploid Cobitis progeny, obtained from crosses between females and males of Cobitis taenia and between allotriploid Cobitis females and C. taenia males. Observations were made during first fourteen days post fertilization. The pattern of development of all investigated individuals was the same. However the diploids developed synchronically, achieving successive stages faster than the polyploid ones; hatching was observed at 50 and 63 hours post fertilization, respectively. Statistically significant differences in hatching success and survival rate between diploid and polyploid progeny were not observed. All newly hatched larvae were characterized by a large amount of yolk, forty myomeres, body pigmentation and four external gills. Skeletal elements of the chondrocranium in the first days post hatching consisted of the otic capsule, ethmoid plate, trabeculae cranii and Meckel's cartilage. In contrast to the diploids, the polyploid larvae were characterized by a higher number of deformities. This study gives new comparative data on the features of early development of diploid and polyploid Cobitis progeny.

Molecular cytogenetic analysis of the crucian carp, Carassius carassius (Linnaeus, 1758) (Teleostei, Cyprinidae), using chromosome staining and fluorescence in situ hybridisation with rDNA probes.

The crucian carp Carassius carassius (Linnaeus, 1758) is a species with restricted and decreasing distribution in Europe. Six males and six females of the species from the Baltic Sea basin in Poland were examined to show sequentially CMA3/AgNO3 staining pattern, DAPI staining, and, for the first time in literature, molecular cytogenetic analysis using double-colour fluorescence in situ hybridisation (FISH) with 28S and 5S rDNA probes. The karyotype consisted of 20 m, 36 sm and 44 sta chromosomes, NF=156. The AgNO3 stained NORs were most frequently located terminally in the short arms of two sm and two sta elements, and CMA3-positive sites were also observed suggesting abundant GC-rich repetitive DNA in the regions. Other CMA3-positive sites in the short arms of six to ten sm and sta chromosomes were detected. The results based on 28S rDNA FISH confirmed the location of rDNA sites. DAPI-negative staining of NORs suggested the scarcity of AT-rich DNA in the regions. FISH with 5S rDNA probe revealed 8-14 loci (ten and 12 in respectively 49 and 29% of metaphases). They were located in two sm and eight to ten sta chromosomes and six of them were larger than others. Simultaneously, mapping of the two rDNA families on the chromosomes of C. carassius revealed that both 28S and 5S rDNA probes were located in different chromosomes. Molecular cytogenetic data of C. carassius presented here for the first time give an important insight into the structure of chromosomes of this polyploid and declining species and may be useful in its systematics.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae).

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n = 48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n = 98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2 % triploids and 44.8 % tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2 days after hatching, approximately 65.1 % of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2 % on average during 2-5 and 10-15 months of life, respectively. The karyotype of tetraploid progeny (4n = 98) included 3n = 74 chromosomes of the parental female and n = 24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66 % of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Reproduction and fecundity of the golden loach, Sabanejewia baltica Witkowski, 1994 from Bug River in Poland.

In this paper we report for the first time the results of histology of the golden loach (S. baltica) gonads (25 females and 8 males) and absolute fecundity of females from the Bug River during the reproductive season. The golden loach has an asynchronous ovary and spawns in batches. The absolute fecundity of the golden loach ranged from 1507 to 7220 eggs (3050±1377). We hypothesize that the golden loach spawns twice a year.

DNA content of hepatocyte and erythrocyte nuclei of the spined loach (Cobitis taenia L.) and its polyploid forms.

We analyzed the DNA content of hepatocyte and erythrocyte nuclei of the spined loach Cobitis taenia (diploid) and its allopolyploid forms. Twenty triploid females and one tetraploid were used. At least 20,000 hepatocyte and erythrocyte nuclei were acquired and analyzed by flow cytometry. C. taenia erythrocyte nuclei contain 3.15 +/- 0.21 pg of DNA and the hepatocyte nuclei 4.45 +/- 0.46 pg of DNA. Triploid Cobitis have 5.08 +/- 0.41 pg of DNA in erythrocyte nuclei and 6.11 +/- 0.40 pg of DNA in hepatocyte nuclei, whereas the tetraploid erythrocyte and hepatocyte nuclei contained 6.60 and 7.40 pg of DNA, respectively. In general, the DNA contents correlate positively with the ploidy level of the fish investigated. The DNA content variation in the hepatocyte and erythrocyte nuclei may be due to differences in extent of chromatin condensation, which is more pronounced in the erythrocyte than hepatocyte nuclei, or to the several orders of ploidy that occur in the parenchymal liver cells.

Age, reproduction and fecundity of the spined loach Cobitis taenia L. (Pisces, Cobitidae) from Lake Klawój (Poland).

This is the first study concerning the features of the reproduction process of the karyologically identified spined loach C. taenia (2n=48). The histology of 71 ovaries, and gonadosomatic index (GSI) of karyologically identified spined loach Cobitis taenia L. from Lake Klawój (Northern Poland) were examined. The absolute and relative fecundity of 25 females was estimated by gravimetric method. The age of fish was determined according to the annual increments of otholits. The spawning of C. taenia from Lake Klawój took place from May to July, at a water temperature exceeding 18.5 degrees C. The GSI values at the beginning of the reproduction period ranged from 7 to 19%. The average absolute fecundity of females was 2078 eggs, with the number ranging from 869 to 3371 eggs. High individual variability in the gonad histology and the GSI values during the reproductive period was observed. Such variability could be the result of beginning the reproduction process in the fish at various times and, probably, due to the various numbers of batches laid and various numbers of eggs per batch.