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Abstract: The population genetic structure of the Peruvian scallop Argopecten purpuratus from three different wild populations along the Peruvian coast was analyzed using nine microsatellite loci and a partial region (530bp) of the mitochondrial 16S rRNA gene. A total of 19 polymorphic sites in the 16S rRNA gene defined 18 unique haplotypes. High genetic diversity was presented in all populations. Statistical analysis of mitochondrial DNA revealed no significant genetic structure (Φ ST =0.00511, P =0.32149) among the three localities. However, microsatellite analysis showed low (2.86%) but highly significant (P =0.0001) genetic differentiation among populations, most of the variation was found in Independencia Bay population, which is located in the Peruvian National Reserve of Paracas. Neutrality tests based on mitochondrial haplotypes were performed to assess signatures of recent historical demographic events. Overall results from Tajima''s D and Fu''s F S tests indicated significant deviations from neutrality. To our knowledge, this study constitutes the first investigation based on mitochondrial and microsatellite markers on the genetic structure of A. purpuratus. [Copyright &y& Elsevier]

Scallops (Bivalvia: Pectinidae) comprise more than 350 extant taxa including species of high economic importance. However, its phylogenetic classification remains unclear and so far only 7 scallop mitogenomes have been determined. In this study, the mitochondrial genomes of two congeneric scallop species Pecten albicans and Pecten maximus were determined. Both mitogenomes contain 12 protein-coding genes ( atp8 is missing), two ribosomal genes, 20–22 transfer RNA genes, all encoded in the same strand. Overall, both Pecten mitogenomes are highly similar with high nucleotide (93%) and amino acid (98.7%) sequence identity. Both mitogenomes also contain the same gene order arrangement, which is a novel contribution within Pectinidae. The highly similar mitochondrial organization between both Pecten species suggested a recent speciation event. Phylogenetic analysis based on complete protein-coding gene information as well as large synteny gene blocks confirmed the sister group relationship between the genera Pecten (subfamily Pectininae) and Argopecten (tribe Aequipectini). [ABSTRACT FROM AUTHOR]

Abstract: Food control policies regarding to seafood label authenticity have become a global issue due to increased incidence of species substitution or mislabelling. Proper species-level identification in processed scallop products is hindered by the lack of morphological characters such as their valves. In order to identify four commercially important scallop species (Argopecten purpuratus, Argopecten irradians, Mizuhopecten yessoensis, Pecten albicans) a species-specific multiplex PCR reaction is described herein. Novel reverse species-specific primers in combination with one universal forward primer designed to amplify a partial region of the mitochondrial 16S rRNA gene were assayed in fresh as well as in manufactured scallop samples. All PCR reactions showed a high specificity allowing an unambiguous species authentication. [Copyright &y& Elsevier]

Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratus auratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types. • Two types of sperm, diploid and aneuploid, are found among tetraploid ginbuna. • Incorporation of the paternal genome into triploid eggs produced tetraploids. • Aneuploid sperm was less motile than haploid and diploid sperm. • Spermatocytes with multivalent configurations generate diploid sperm. • The occurrence of synapsis at meiosis may cause ploidy differences in sperm. [ABSTRACT FROM AUTHOR]

The effect of replacement of glucose in glucose-methanol extender (GM) with either sucrose and trehalose on the sperm motility characteristics of cryopreserved semen and the post-thaw storage of salmon, brown trout, brook trout, sex-reversed female rainbow trout and whitefish was investigated. Semen samples were diluted 1:5 or 1:9 (for sex-reversed females) in 9% methanol containing 0.18 M glucose, sucrose or trehalose. After thawing (40 °C, 5 s), sperm was stored for 0, 30, 60, 120 and 240 min at 4 °C and assessed for sperm motility. The type of sugar had no effect on the percentage of sperm motility of brown trout (65–73%), brook trout (51–58%) or sex-reversed female rainbow trout (38–40%). Glucose and trehalose secured the highest post-thaw sperm motility after thawing (0 min) in salmon (64 ± 7%) and whitefish (52 ± 8%), respectively. The longest period (determined as the last time point when the percentage of motility did not significantly change) was recorded for cryopreserved semen of brook trout (60–120 min). The shortest was recorded for whitefish semen (30 min). Our results suggest that glucose, sucrose or trehalose can be used interchangeably for the cryopreservation of brown trout, brook trout and sex-reversed semen. However, the best results in terms of post-thaw sperm motility were achieved for salmon with glucose and sucrose and for whitefish with trehalose and sucrose. Cryopreserved sperm can be stored for prolonged periods of at least 30 min without a loss in sperm motility, which can facilitate more organised hatchery work. Statement of relevance The results of this study confirmed the high level of utility of cryopreservation employing a 0.18 M glucose and 9% methanol extender for freezing, among Salmonidae species. We have demonstrated for the first time the utility of this method for freezing Atlantic salmon semen. Furthermore it was shown that disaccharides are more efficient than glucose for preserving of whitefish post-thaw motility. [ABSTRACT FROM AUTHOR]

Abstract: Spontaneous polyploids and mosaics have often been observed in artificially propagated larvae of the Japanese eel, Anguilla japonica. However, the mechanisms responsible for such unusual cytotypes are unclear. In this study, we examined the relationship of such polyploidization and mosaicisms in larvae resulting from artificial propagation to egg quality (fertilization rate and hatching rate) and viability of larvae, and then clarified the inducing factors and the mechanism for occurrence of such phenomena. Eggs stripped from females after induced maturation were artificially inseminated with sperm pre-cultured with artificial seminal plasma. Ploidy was determined by measuring the relative DNA content of the nuclei with flow cytometry. Of 968 embryos from 32 full-sib families, 9.1% were determined to be abnormal, most of which were triploids (86.5% of abnormal embryos); others were haploids (1.1%), aneuploids (2.3%), and mosaics (10.1%). The percentage of normal diploids from each family varied between 56.3% and 100% (90.9±11.7%, n =32). A significant positive correlation was found between the fertilization rate (P <0.001) or the hatching rate (P <0.001) and the percentage of diploids. Survival rate of triploid eels was similar to diploid eels at 10days after fertilization whereas aneuploids were inviable. When eggs were left in the body cavity of the female for four hours after ovulation and subsequently fertilized, the percentage of diploids decreased. We tried to elucidate the cause for the occurrence of spontaneous triploids by genetic analysis using 26 microsatellite DNA markers, which have been developed and mapped in relation to the centromere. These results suggest that the occurrence of cytogenetically unusual progeny is associated with over-ripening or aging of ova caused by the lapse of time from ovulation until fertilization, and spontaneous triploid larvae are derived from the duplication of the maternal chromosome set by inhibition of the second polar body release after normal meiosis I (crossing over) in oocyte and fertilization with normal sperm. [Copyright &y& Elsevier]

Diploid androgenotes were produced without egg irradiation in the loach, Misgurnus anguillicaudatus. Eggs of wild-type diploid females were fertilized with diploid sperm of a neo-tetraploid male and then cold-shock treated at 3 °C (range, ±0.5 °C) for 30 minutes just after fertilization to eliminate the female nucleus. After hatching, ploidy status of the hatched larvae was analyzed by flow cytometry, which revealed putative diploid androgenotes as well as larvae possessing other ploidies. Five independent microsatellite DNA markers were genotyped to confirm all-male inheritance of the resultant diploid larvae. The mean ± SD yield rate of diploid androgenetic larvae to total eggs used was 12.29 ± 3.25% in the cold-shock group and 22.23 ± 13.42% in the UV-irradiated group (P > 0.05). No diploid androgenetic larvae were detected in the intact control group. To our knowledge, this is the first report demonstrating successful induction of diploid androgenotes without egg irradiation in fish. [ABSTRACT FROM AUTHOR]

Abstract: The solutions commonly used to dilute or cryopreserve sperm are commonly composed of salts, buffers and cryoprotectants, which may affect gametes and subsequent fertilization success. Here, we have evaluated the effects of several cryoprotectants (methanol; MeOH, dimethyl sulfoxide; DMSO and dimethyl acetamide; DMA at concentrations of 0.25, 0.5 and 1%) and different ions (potassium, calcium and magnesium at concentrations of 1.25, 2.5, 5.0 and 10mM) as sperm diluents upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus sperm. Our results demonstrated that DMSO (at 1%) decreased sperm motility while calcium and magnesium ions (from 2.5mM) induced sperm aggregation and reduced sperm motility. Reduced fertilization rates were observed with potassium (from 1.25mM), calcium (at 10mM), magnesium (at 10mM), DMA (at 1%), and DMSO (at 1%). We conclude that specific ions and cryoprotectants, and their relative concentrations caused effect upon loach gametes. These data are important to consider for the preparation of sperm diluents and activating solutions in order to manage gamete quality for artificial propagation. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to establish a protocol of sperm cryopreservation in Misgurnus anguillicaudatus and verify the applicability of the obtained protocol in other loach species. We evaluated the following parameters: inseminating dose, thawing temperatures (20, 25 and 30°C for 10s), extenders (loach or cyprinid extenders), internal cryoprotectants (dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), glycerol (Gly), ethylene glycol (EG), and methanol (MeOH) at 0, 5, 10 and 15%), external cryoprotectants (bovine serum albumin 1 and 2%; sucrose 0.5 and 1%; glucose 0.5 and 1%; glycine 0.5 and 1%), activating solutions (distilled water, dechlorinated tap water, 25mM NaCl and 50mM NaCl), and hatchability of the eggs when fertilized with fresh or cryopreserved sperm. After the evaluation of these parameters, we optimized the cryopreservation using the following procedure: thawing temperature at 25°C for 10s; loach or cyprinid extenders; methanol at 10 or 15% as internal cryoprotectants; glycine 0.5% or bovine serum albumin 1% as external cryoprotectants and 50mM NaCl for sperm activation. Using this procedure, the fertilizability of the post-thawed sperm was 47% in comparison to the fresh sperm, at the minimum inseminating dose (687.65spermatozoaegg−1 mL−1). Based on this protocol, sperm from other loach species Lefua nikkonis, Misgurnus mizolepis and Barbatula toni were cryopreserved successfully. [Copyright &y& Elsevier]

Abstract: Although tetraploidy is considered to be important and useful for the production of sterile triploids and fertile allo-tetraploids in aquaculture, in most cases the resultant embryos exhibit extremely low survival. In this study, we aimed to clarify the cause of the inviability of tetraploids in masu salmon. Firstly, we compared developmental rates of the first cell cycle among 9 single pairs produced by all possible matings between 3 females and 3 males. The results showed that the fertilized eggs developed almost synchronized among individuals from each pair but asynchronous among pairs and this asynchronous development was more apparent among pairs from different female than those from different male. Secondly, we induced both tetraploidy (4N) and gynogenetic diploidy (G2N) by the same hydrostatic pressure shock (PS) conditions (700 kg/cm2, 7 min duration) for the first cleavage inhibition using single-pair mating. This experiment was designed to verify whether the cause of mortality of induced tetraploidy was the elevated ploidy itself. Eggs from one female were divided into 2 groups and fertilized with normal or UV-irradiated sperm from one male, respectively. Then each group of eggs was subdivided into 6 groups: one was an intact control and the other 5 groups were treated with PS at every 30 min from 5 to 7 hpf at 10 °C. As a result, the treated eggs of 4N and G2N showed the highest survival (90.8% and 60.6%, respectively) and embryogenesis rate (both 100% relative to surviving eggs) at 33 dpf, only when PS treatment was performed at late prometaphase (6 hpf and 6.5 hpf, respectively). The other PS groups showed extremely low survival (1.5–52.8% and 3.1–14.8%), ceased morphogenetic development and developed into only undifferentiated cell masses. This experiment was repeated 2 more times using other single-pair mating and the results showed the same tendency. The embryonic bodies were confirmed by flow cytometry to have approximately objective ploidy, tetraploid, hypo- or hypertetraploid in 4N and diploid or hypodiploid in G2N. However, all tetraploid embryos, even those with normal morphology, began to die simultaneously around the hatching period (34 dpf), while G2N embryos showed normal morphology and survived beyond 50 dpf (55.6%). Most tetraploid embryos showed malformation and had very poor vascular systems even in normal-looking ones. These results suggest that the mortality of induced tetraploids depends not only on the side effects of the PS treatment but also strongly depends on the induced tetraploidy itself. [Copyright &y& Elsevier]

Abstract: We have investigated the relationship between maturational competence and hatching rate of eggs ovulated following injection of lutenizing hormone releasing hormone ethylamide (LH-RHa) in the cultured hybrid (bester) sturgeon (Huso huso female×Acipenser ruthenus male). Among 10 steroids tested (including progesterone) in a previous study, 17α-hydroxyprogesterone (17αOHP) was the most potent inducer of final maturation in the bester, with results similar to 17α,20β,21-trihydroxy-4-pregnen-3-one and 17α,20β-dihydroxy-4-pregnen-3-one. We therefore used 17αOHP to estimate maturational competence in the present study. In vitro oocyte maturation response to 17αOHP at a concentration of 0.1 μg/ml was correlated with ovulatory response to hormone treatment and quality of ovulated eggs. Two females exhibited higher in vitro oocyte maturation rate (100% germinal vesicle breakdown), ovulated sooner after hormone injection, and also had a higher hatching rate (77, 92%). In the F2 bester broodstock with a lower hatching rate (10–21%), a higher incidence (15–64%) of spontaneous triploidy was found. These results indicate that maturational competence can be estimated by in vitro oocyte maturation response to 17αOHP at 0.1 μg/ml and that this is a useful technique for selecting the proper female for breeding. Ovulatory success, hatching rate and occurrence of triploidy are thus all predictable in the cultured bester. [Copyright &y& Elsevier]

Abstract: The sex ratios of triploids and gynogenetic diploids in the hybrid sturgeon, the bester (Huso huso female×Acipenser ruthenus male), have been investigated to confirm the possibilities of sex control by chromosome manipulation in sturgeon. Viable gynogenetic diploids were produced by activating eggs with ultraviolet-irradiated bester sperm followed by heatshock treatment. Triploids were induced by the same method after fertilization of eggs with normal bester sperm. A total of 380 progeny individuals were analyzed by flow cytometry, and classified into 217 diploids (2n) and 163 triploids (3n). The percentages of females are 40–55% in control diploid, 60.0–73.3% in triploid and 70.0–80.0% in gynogenetic groups. Since these progeny reared under same environmental conditions, the presence of males in gynogenetic progeny groups derived from five different pairs suggested the involvement of a female heterogametic {ZW (female)/ZZ (male)} sex-determination system in bester. The percentage of males in each groups most closely approximated the percentages expected when the sex-determining element on the W chromosome segregates independently of the centromere during meiosis in females. If the WW “super”-female is viable in sturgeon, such females would be produce all female progeny when crossed to normal ZZ males. Moreover, inducing gynogenetic diploid in endangered sturgeon population with genetically inactivated “donor” sperm from other sturgeon species is likely to offer a useful method for restoration, since both sexes and not just an all-female population would be predicted. [Copyright &y& Elsevier]

In the loach Misgurnus anguillicaudatus, triploid (diploid female×tetraploid male) females laid both large-sized triploid and small-sized haploid eggs. First-generation triploid gynogens were produced by activating the large-sized eggs with UV-irradiated spermatozoa. Mature 4-year-old gynogens were all females and laid one, two or three types of gametes, simultaneously: large-sized, small-sized and intermediate-sized eggs. Frequencies of such eggs differed among individuals of the first-generation triploid gynogens. When the large-sized eggs were triggered to induce gynogenetic development by UV-irradiated spermatozoa, the resultant second-generation gynogens had triploid DNA content. However, the gynogens induced from the intermediate-sized eggs exhibited aneuploid (1.4–1.5n) chromosome numbers and karyotypes. Gynogens from the small-sized eggs died before feeding, probably due to the expression of haploid syndrome. Among the second-generation triploid gynogens, some individuals revealed DNA fingerprints identical to the mother, but others gave patterns slightly different from the mother when the samples were digested with HinfI and then hybridized with oligonucleotide probes (GACA)4, (GGAT)4 and 33.15. These results showed that both clonal and aclonal unreduced eggs might be formed in the triploid gynogens. [Copyright &y& Elsevier]

Androgenetic doubled haploids (DHs) were induced in the loach Misgurnus anguillicaudatus (Cobitidae) without irradiation of the eggs. The eggs of wild-type females were activated with the intact sperm of an orange-phenotype male, and treated (within 10 s of activation) at 3 ± 0.5 °C for 30 min, to eliminate the female nucleus. The eggs were then incubated in a water bath at 20 ± 0.5 °C for 35 min. Finally, diploidy was restored (65 min after activation) by heat-shock treatment at 42 ± 0.5 °C for 2 min. Under these conditions, the yield rate (mean ± SD) of putative DHs relative to the total number of eggs used was 10.43 ± 1.69%, which was significantly higher ( P < 0.05) than the yield rates obtained under the remaining heat-shock initiation conditions (55 min, 60 min, and 70 min after activation). We analyzed the ploidy status of the putative DH by using flow cytometry. All-male inheritance was confirmed by the expression of the recessive orange body color trait and microsatellite genotypes. We detected no maternally derived alleles or heterozygous genotypes at any of the 28 loci (covering 27 linkage groups) of loach, indicating the exclusively paternal inheritance and homozygosity of the obtained androgenetic DHs. [ABSTRACT FROM AUTHOR]