Sexual maturation in female triploid plaice, Pleuronectes platessa, and plaice × flounder, Platichthys flesus, hybrids

The gonads of immature female triploid plaice ( Pleuronectes plafcssa ) and plaice × flounder ( Platichthys flesus ) hybrids produced from cold-shocked eggs were examined. They were less than half the size of those in diploids of similar age and contained only small numbers of developing oocytes. These were similar both in size and cytological appearance to those in the controls. The bulk of the ovarian tissue was composed of nests of small undifferentiated cells resembling the oogonia in the ovaries of diploid fish. Mature, 5 year old triploid hybrids which had not produced eggs over the previous two spawning seasons were killed in January when the diploid controls were nearing spawning condition. The ovaries of these fish were smaller than those of the controls and contained degenerating oocytes at maturation stage V. The advantage of sterile female triploid fish for fish-cultivation is discussed.     

Temperature-dependent development rates of plaice (Pleuronectes platessa L.) eggs from the Irish Sea

Experimentally derived temperature-dependent development ratesfor planktonic fish eggs are required for developing IndividualBased Models (IBMs) of early life history stages and for estimatingdaily egg production in ichthyoplankton surveys. Developmentrates of Irish Sea plaice eggs were experimentally determinedfor temperatures between 4.5 and 12° C. Differences in developmentrates were apparent when these results were compared with literaturedata for eggs from North Sea parents. On average, plaice eggsfrom the Irish Sea developed more rapidly and hatched up to2 days earlier compared with North Sea eggs incubated at thesame temperature. In terms of parental effects, larger femalestended to produce larger eggs but egg size decreased over successivebatches from the same female within a year. Temperature-dependentegg development rates were influenced by egg size in a non-linearmanner for eggs incubated at 6° C, but egg size had no effectat higher temperatures. Since most plaice eggs sampled fromthe Irish Sea during ichthyoplankton surveys in 1995 and 2000were found in waters cooler than 8° C, such effects maymodify egg development rates in the wild.     

Predation by sprat and herring on pelagic fish eggs in a plaice spawning area in the Irish Sea

Predation on planktonic fish eggs was examined by stomach content analysis of sprat and herring sampled in a plaice spawning area to the east of the Isle of Man in March 1993. Plankton samples were taken to examine prey selection. The clupeids showed a selection for the later developmental stages of plaice eggs. Plaice eggs had a refuge in size from predation by sprat <80 mm total length which selected smaller non-plaice eggs. However, herring and sprat >80 mm total length showed a strong selection for plaice eggs, presumably due to their large size.     

Genetic and reproductive potential of spermatozoa of diploid and triploid males obtained from interspecific hybridization of Misgurnus anguillicaudatus female with M. mizolepis male

Diploid and triploid interspecific hybrid male progeny obtained from mating Misgurnus anguillicaudatus with M. mizoleis were reported to have histologically fertile and sterile testes, respectively. However, their reproductive capacity is still unclear because mating tests have not been examined using mature hybrids. Here, we examined physiological and genetic characteristics of spermatozoa of diploid and triploid hybrids. In diploid hybrid males, 1n, 2n and 4n spermatozoa showing low motility were detected. However, spermatozoa of three diploid hybrid males could generate 2n larvae. Therefore, only 1n spermatozoa of diploid hybrid males was fertile to produce larva. The chromosomes of diploid hybrid males were transmitted to spermatozoa by random segregation between the homologous chromosomes because most larvae had one allele derived from both M. anguillicaudatus and M. mizolepis at all loci examined. In triploid hybrid males, spermatozoa could be categorized to three different types based on their ploidy status. Type 1: In the first and second males, sperm samples mainly comprised 6n spermatozoa. Motility and fertility were not recorded. Type 2: The third male gave a large proportion of 6n spermatozoa as well as a small proportion of 1n spermatozoa. Although no motility was observed, larvae arose from eggs inseminated with such spermatozoa. Type 3: In the fourth male, only 1n spermatozoa were detected and their motility was vigorous. When eggs were fertilized with such 1n spermatozoa, normal larvae hatched. 1n spermatozoa of the triploid hybrid male only included the M. anguillicaudatus genome. In Misgurnus fishes, diploid hybrid males exhibited semi-sterility or slight fertility. On the contrary, triploid hybrid males were sometime fertile due to the production of 1n spermatozoa by a kind of transformation of meiosis like meiotic hybridogenesis.     

Does triploidization produce functional sterility of triploid males of tench <Emphasis Type="Italic">Tinca tinca</Emphasis> (L.)?

Triploidy interferes with gametogenesis in all fish species tested so far. In fish it results in complete female sterility however, males are still able to develop testis. The reason why sterility levels in triploid fishes differ among species and between sexes is unclear. In the present study the reproductive capacity of triploid males of tench was studied. Flow cytometry revealed sperm cells of triploids to be largely aneuploid with high mosaic DNA, oscillating from haploid DNA to diploid DNA content. Analysis of variance showed an insignificant influence of ploidy level on the percentage of motile spermatozoa, as well as on spermatozoa velocity. Experimental crosses between normal diploid female and triploid males resulted in the appearance of triploid progeny, which exhibited genotypes composed of microsatellite alleles inherited from the founder female and additional allele derived from the donor male. We can conclude that the triploid males analysed in the present study were capable to fertilize eggs derived from diploid females.     

Studies on the skin of plaice (Pleuronectes platessa L.)

A histological, histochemical and ultrastructural examination of the skin of wild and cultured plaice was carried out, using fish from each year class from 0+ to 4+. The skin was shown to be similar in general structure to that of other teleosts but a previously undescribed cell, designated the Eosinophilic Granular Cell, a dendritic secretory cell found throughout the basal layers of the epidermis, is described. It was fixed only by formalin or dichromate, and contained numerous acidophilic granules. Melanin-bearing macrophages were observed migrating through the epithelium, but no DOPA or tyrosinase positive cells were observed by the methods used. Mast cells were very common in the dermis but were only demonstrable by special techniques. The melanophore and guano-phore systems are described and although no melanophores or melanocytes were found in the unpigmented areas of partially pigmented hatchery-reared fish, the integrity of the guanophore system was complete in such fish.     

Comparative histology of the testes of the spined loach Cobitis taenia L. and natural allotetraploids of Cobitis (Pisces, Cobitidae)

The spined loach Cobitis taenia L. creates exclusively diploid and mixed diploid–polyploid populations. Allotriploid females, which co-exist with C. taenia or C. elongatoides and a few tetraploid males and females dominate in most Cobitis mixed populations. They reproduce gynogenetically and produce triploid eggs that are stimulated to development by sperm from Cobitis males. Some of these eggs are fertilized, which leads to the production of bisexual tetraploids. Males of C. taenia (2n = 48) from a diploid population in Lake Klawój, Northern Poland (46 individuals) and from a mixed Cobitis population in the Bug River, Eastern Poland (7 individuals), and three tetraploid males (4n = 98) from the same mixed population were examined. All the fish were analyzed karyologically and histologically. Tubules with cysts of the testes of C. taenia from both populations were filled with germ cells at various developmental stages. Among fishes from Lake Klawój sperm maturation in batches simultaneous with the batch spawning of C. taenia females was found. The testes of the loach C. taenia, from a mixed population in the Bug River, were filled with spermatozoa over the entire reproductive season. Sperm maturation in batches was not observed. Sperm maturation in batches seems to be only connected with a few diploid males in this population. So, a continuous process of spermatogenesis in their testes is required. Only in the testes of all tetraploid Cobitis males were cells characteristic of the early stages of spermatogenesis observed, i.e. without spermatids and spermatozoa. Furthermore, the histological sections of the testis of a male captured in August, revealed fragments with connective tissue between the germ cells. However the participation of tetraploid, infertile Cobitis males in the process of reproduction in the investigated mixed population remains controversial. The results obtained so far, reveal that even the infertile sperm of tetraploid males may induce gynogenesis in Cobitis triploid females.     

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

The host specificity of Lepeophtheirus pectoralis (Müller, 1776) (Copepoda: Caligidae)

The host specificity of Lepeophtheirus pectoralis (Müller) was examined experimentally by exposing different fish species to infection by artificially reared copepodid larvae. Copepodids which were hatched from eggs of adults parasitic on plaice ( platessae copepodids) preferred plaice to all other fishes tested, whereas copepodids which were hatched from eggs of adults parasitic on flounder ( flesi copepodids) preferred flounder to all other fish species. These behavioural differences suggest that two strains of L. pectoralis exist, one ( platessae ) adapted to plaice as its host and the other ( flesi ) to flounder. Comparison of an experimentally derived order of host preference with a table of occurrence obtained from the literature, suggests that a third strain of L. pectoralis , adapted to dab as its host, might also occur.
The process of infection by L. pectoralis copepodids is also described. It comprises a host location phase, during which the copepodid enters the habitat of its flatfish hosts and locates a host individual, and an attachment phase. The host location phase appears to be governed by changes in the activity of the copepodid and by its positively rheotactic response to water currents produced by the host. The attachment phase is probably based on the response of the copepodid to chemical factors produced by the host.     

Isolation of plaice (Pleuronectes platessa) alpha1-microglobulin: conservation of structure and chromophore

A cDNA coding for plaice (Pleuronectes platessa) alpha1-microglobulin (Leaver et al., 1994, Comp. Biochem. Physiol. 108B, 275-281) was expressed and purified from baculovirus-infected insect cells. Specific monoclonal antibodies were then prepared and used to isolate the protein from plaice liver and serum. Mature 28.5 kDa alpha1-microglobulin was found in both liver and serum. The protein consisted of an 184 amino acid peptide with a complex N-glycan in position Asn123, one intrachain disulfide bridge and a yellow-brown chromophore. Physicochemical characterization indicated a globular shape with a frictional ratio of 1.37, electrophoretic charge-heterogeneity and antiparallel beta-sheet structure. A smaller, incompletely glycosylated, yellow-brown alpha1-microglobulin as well as a 45 kDa precursor protein were also found in liver. The chromophore was found to be linked to alpha1-microglobulin intracellularly. Recombinant plaice alpha1-microglobulin isolated from insect cells had the same N-terminal sequence, globular shape and yellow-brown color as mature alpha1-microglobulin, but carried a smaller, fucosylated, non-sialylated N-glycan in the Asn123 position. The concentration of alpha1-microglobulin in plaice serum was 20 mg/l and it was found both as a 28.5 kDa component and as high molecular weight components. Thus, the size, shape, charge and color of plaice alpha1-microglobulin were similar to mammalian alpha1-microglobulin, indicating a high degree of structural conservation between fish and human alpha1-microglobulin. The monoclonal antibodies against plaice alpha1-microglobulin cross-reacted with human alpha1-microglobulin, emphasizing the structural similarity.     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

Genes for three different isoforms of transforming growth factor-beta are present in plaice (Pleuronectes platessa) DNA

Although transforming growth factor-beta (TGF-beta) genes have been described in several species of fish, whether an individual fish possesses more than one member of this multigene family has yet to be established. During this study, three DNA fragments were isolated from the plaice (Pleuronectes platessa) by homology cloning. Sequence analysis revealed that each fragment closely resembled a distinct member of the TGF-beta family. Each putative plaice TGF-beta clustered individually with a different TGF-beta subgroup during phylogenetic analysis suggesting that these may be the plaice homologues of vertebrate TGF-beta 1/4/5, -beta 2 or -beta 3. The first direct evidence for the presence of multiple TGF-beta genes in a single fish species is presented.     

优雅蝈螽精子形成过程中核的形态结构变化观察（英文）

【目的】螽斯精子结构复杂,具有特征性的箭头状顶体,是研究昆虫精子形成的理想材料。为了研究螽斯精子形成过程中的动态变化机制,特别是细胞核的凝集机制和箭头状顶体的发生机制,本研究对优雅蝈螽Gampsocleis gratiosa精细胞和精子的细胞核进行了观察。【方法】选择发育良好的优雅蝈螽成虫精巢为研究材料,利用透射电镜技术、普通光学显微镜和荧光显微镜技术,制作光镜切片和电镜切片进行观察。【结果】根据其形态结构变化特征,将优雅蝈螽精子形成过程中的细胞核分为4个阶段：圆形核、叶形核、柱状核和成熟阶段。我们还通过常规HE染色,结合DNA特异性荧光探针DAPI,证明了圆形核时期,精细胞内具有两个明显的球状结构,一个为细胞核,另一个是原顶体;精子成熟阶段,精子尾部排出的细胞质微滴中含有DNA。【结论】优雅蝈螽精子形成过程中,精细胞的细胞核经历了显著的形态变化,精细胞核的形态变化与细胞骨架微管相关,细胞核塑形伴随着染色质的重组。本研究为进一步阐明直翅目昆虫精子形成的分子机制奠定了基础。     

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.     

Survival of discarded plaice (Pleuronectes platessa) from Norway lobster (Nephrops norvegicus) otter‐trawl fishery

The introduction of landing obligations in the European Union common fisheries policy, has increased the focus on discard survival. Since species with scientifically proven high survival rates may be exempted from the landing obligations, the economic significance for the fishing industry is large. Landing obligations include individuals below the minimum conservation reference size (MCRS), which are not allowed to be sold for human consumption. The Norway lobster (Nephrops norvegicus) fishery is a high value fishery, but with a substantial amount of bycatch of plaice (Pleuronectes platessa) below the MCRS. In the present study, observations were made on the short‐term survival of plaice at or below the MCRS, discarded from a small otter board trawler targeting Nephrops in Skagerrak (ICES 3a). The average short‐term survival of plaice was 15% at haul level, ranging from 0% to 39%, after 10 days of captive observation. Survival significantly decreased with time on deck and the retention of debris in the codend (p < 0.0001), while survival was not correlated to size. Vitality assessments, using a combined reflex impairment and injury score, were tested as predictor of survival and were significantly associated to survival (p < 0.0001) both for individual fish and when grouped by hauls. Seabird predation behaviour was assessed and it was estimated that seabirds predated or pursued 85% of discarded plaice below the MCRS.     

Shock-induced triploidy and its effect on growth and gonad development of the European catfish, Silurus glanis L.

Triloidy was induced in European catfish, Silurus glanis L., by cold-shocking eggs at 4°C for 30 and 40 min respectively, starting 5 min after fertilization. The hatching success of cold-shocked eggs was 25–30%. Cold shocks longer than 1 h caused total mortality. The triploid character of the cold-shocked European catfish was proved by karyological and red blood cell size analyses. The gonads of the triploid fish were significantly smaller than those of the diploids, while the growth rate values of the triploids were significantly higher than those of the diploids.     

Otolith oxygen and carbon stable isotopes in wild and laboratory-reared plaice (Pleuronectes platessa)

The relationship between water temperature, growth rate, and otolith isotopic ratios was measured for juvenile plaice (Pleuronectes platessa) reared at two temperatures (11 and 17°C) and two feeding regimes (1 and 3 prey items·ml^?1). The otolith isotope ratios in individual fish ranged from ?2 to ?4 for carbon isotope ratios (δ¹³C) and from 0.2 to 1.9 for oxygen isotope ratios (δ¹⁸O). The otolith oxygen isotope ratios were significantly affected by water temperature, but not by feeding level, and there were no significant synergistic effects. The fractionation of oxygen isotopes during otolith growth was independent of individual growth rate. Carbon isotope ratios were not significantly affected by food ration or water temperature, but were related to fish growth rate. The carbon isotope ratios were negatively correlated with fish length in the colder water treatments, and tended to increase with fish length in the warm water treatments. The laboratory-determined relationship between otolith oxygen isotope ratio and water temperature was applied to individuals of five species (plaice, cod, whiting, haddock, gurnard) collected in a single trawl sample. The otolith derived temperatures often overestimated measured water temperatures. The difference between real and estimated water temperatures varied between species, and the closest fit was for field-caught plaice.     

Effect of ploidy on scale-cover pattern in linear ornamental (koi) common carp Cyprinus carpio

The effect of ploidy on scale-cover pattern in linear ornamental (koi) common carp Cyprinus carpio was investigated. To obtain diploid and triploid linear fish, eggs taken from a leather C. carpio female (genotype ssNn) and sperm taken from a scaled C. carpio male (genotype SSnn) were used for the production of control (no shock) and heat-shocked progeny. In heat-shocked progeny, the 2 min heat shock (40° C) was applied 6 min after insemination. Diploid linear fish (genotype SsNn) demonstrated a scale-cover pattern typical for this category with one even row of scales along lateral line and few scales located near operculum and at bases of fins. The majority (97%) of triploid linear fish (genotype SssNnn) exhibited non-typical scale patterns which were characterized by the appearance of additional scales on the body. The extent of additional scales in triploid linear fish was variable; some fish had large scales, which covered almost the entire body. Apparently, the observed difference in scale-cover pattern between triploid and diploid linear fish was caused by different phenotypic expression of gene N/n. Due to incomplete dominance of allele N, triploids Nnn demonstrate less profound reduction of scale cover compared with diploids Nn.     

Transfer of passive immunity from mother to young in a teleost fish: haemagglutinating activity in the serum and eggs of plaice, Pleuronectes platessa L

Plaice were immunized against rabbit red blood cells (rbc) and the haemagglutinating activity in their sera and eggs was isolated by gel filtration in Sephacryl S300 SF. A "heavy" (greater than 6.69 X 10(5) Daltons) and a "light" (less than 2.32 X 10(5) Daltons) component of haemagglutinating activity were present in plaice immune serum and eggs. The "heavy" haemagglutinating component had the characteristics of immunoglobulin M (IgM) antibody. By contrast the "light" component was suppressed greatly by monosaccharides but not by 2-mercaptoethanol (2-ME) and it was not precipitable against heterologous rabbit anti-plaice Ig immune serum. Since this "light" component was also identified in the serum and eggs of non-immunized plaice the activity was attributed to non-specific lectin-like activity. The presence of an inducible, high molecular weight, haemagglutinating Ig-like protein in plaice serum and eggs suggests the possibility of transfer of passive adaptive immunity from mother to young in this fish.