Effects of temperature on free‐embryonic diapause in the autumn‐spawning bitterling Acheilognathus rhombeus(Teleostei: Cyprinidae)

In bitterling Acheilognathus rhombeus , developmental arrest always occurred at stage D of the free‐embryonic phase, regardless of incubation temperature. Developmental arrest was terminated only by a cold treatment at 4° C for 60–90 days, initiated 10 days post‐hatching. After the termination of developmental arrest, free‐embryos became larvae c . 6 months after hatching, regardless of the time of initiation and duration of the cold treatment. In hybridization experiments between A. rhombeus and several species of spring‐spawning bitterlings, free‐embryos became free‐swimming larvae within 60 days after hatching in all experiments. Developmental arrest was not observed in any of the hybrids, regardless of parental sex. These results suggest that free‐embryonic diapause in A. rhombeus is not induced by environmental factors, such as cold, but by genetic factors, which are recessive to those in spring‐spawning bitterlings. Free‐embryonic diapause in A. rhombeus appears to be an adaptation to winter, which might have evolved with reproduction in autumn among autumn‐spawning bitterling species. This is the only report of free‐embryonic diapause after hatching in fishes, and only the second example of diapause in fishes, along with annual killifishes (Rivulidae).     

Comparative phylogeography of two bitterlings, Tanakia lanceolata and T. limbata (Teleostei, Cyprinidae), in Kyushu and adjacent districts of western Japan, based on mitochondrial DNA analysis

To understand the geographical patterns of genetic variation in freshwater fishes in western Japan, the genetic structures of populations of Tanakia lanceolata and T. limbata (Teleostei, Cyprinidae) in this area were investigated using mitochondrial DNA (mtDNA) cytb sequences. Neighbor-joining trees of mtDNA haplotypes revealed four and three genetically divergent groups in T. lanceolata and T. limbata, respectively. Each group was restricted to one or the other of the geographical regions in the area studied. The patterns of geographical divergence in the two species showed some similarities, which seem to reflect common historical events experienced by freshwater fishes distributed in western Japan. On the other hand, dissimilarities were also found in the patterns, indicating that species-specific historical processes also occurred. Within one region, T. lanceolata was less differentiated than T. limbata, suggesting a difference in the dispersal abilities of the two bitterlings. In addition, several individuals in Kunichika River on Shikoku island were morphologically identified as T. lanceolata but had mtDNA haplotypes of T. limbata. We tentatively suggest that these individuals are hybrids of the two species, but further studies employing nuclear markers are necessary to validate this hypothesis.     

Reproductive Ecology and Host Utilization of Four Sympatric Bitterling (Acheilognathinae,Cyprinidae) in a Lowland Reach of the Harai River in Mie,Japan

Bitterling are fishes that use freshwater mussels for oviposition. The reproductive ecology and pattern of mussel utilization of four sympatric species of bitterling, Acheilognathus rhombeus, A. tabira tabira, Tanakia lanceolata, and T. limbata, were investigated in a lowland river with seven sympatric mussel species. Three bitterling species are spring spawners with overlapping spawning seasons. A. rhombeus is an autumn-spawning species and is temporally isolated in its reproduction from the other species. Ovipositor length during oviposition of T. limbata and T. lanceolata was short, while those of A. tabira tabira and A. rhombeus were long. Most T. limbata inhabited near-shore areas, whereas the two other spring-spawning species were distributed across the entire river. All bitterling species used Inversidens brandti, Obovalis omiensis and Inversiunio jokohamensis as spawning hosts, but not the other mussel species available. T. lanceolata, A. tabira tabira and A. rhombeus showed spawning preferences for O. omiensis and I. brandti. However, T. limbata did not show clear preferences for any of the mussel species they used. A. t. tabira showed a significant preference for large I. brandti in offshore areas, while the other spring-spawning bitterling showed a preference for mussels inshore. These results are discussed in the context of reproductive resource partitioning.     

Intestinal morphology in oily bitterlings (Cyprinidae: Acheilognathinae)

We evaluated inter- and intraspecific variations of the intestinal coiling patterns and intestinal length of the oily bitterlings, Tanakia limbata, Tanakia koreensis, Tanakia somjinensis, and Tanakia signifer. All the oily bitterling species shared a unique intestinal coiling pattern, expressed as an oval shaped loop. The ratio of the intestinal length to standard length varied among species, viz., both T. koreensis and T. signifer have significantly longer intestines than T. limbata and T. somjinensis. The intestinal length may be a useful diagnostic character between two sibling species, T. koreensis and T. somjinensis, although we observed intraspecific variation in the intestinal length of T. limbata and T. koreensis among collection sites.     

Cytogenetics off interpopulation Cuphea lanceolata hybrids.

Cuphea lanceolata Ait. (Lythraceae) is an annual diploid (x = 6) with medium-chain fatty acid rich seed oils. Wild C. lanceolata populations are classified as C. lanceolata f. silenoides or C. lanceolata f. lanceolata on the basis of flower pigment differences. Although these taxa are taxonomically close, their interfertility has not been demonstrated. We describe meiotic phenomena underlying the sterility of hybrids between C. lanceolata f. silenoides (LNS-43) and C. lanceolata f. lanceolata (LNC-78) populations. We assayed metaphase and anaphase I microsporocytes of the parent and hybrid populations. The hybrids were female and male sterile. The mean percentage of stainable pollen was 94.9% for the parents and 1.1% for the hybrids. Chromosomes paired and disjoined normally in the parents (LNS-43 and LNC-78) and abnormally in the hybrids (LNS-43 x LNC-78 and LNC-78 x LNS-43). Univalents, unequal chromosome distributions, and laggards were observed in the hybrids. The mean number of univalents per cell was 0.00 for the parents and 5.95 for the hybrids, the mean number of bivalents per cell was 6.00 for the parents and 1.51 for the hybrids, and the mean number of chiasmata per cell was 9.19 for the parents and 4.04 for the hybrids. The most frequently observed (75%) anaphase I chromosome distribution for the hybrids was 7:5:0 (pole-pole-laggards). The genome affinities of the hybrids were half those of the parents (a mean of 0.5 for the hybrids as opposed to 1.0 for the parents). Although C. lanceolata f. silenoides and C. lanceolata f. lanceolata freely hybridize, their progeny are sterile, and the genetic diversity of LNC-78, and perhaps of C. lanceolata f. lanceolata as a whole, cannot be accessed through hybrids with C. lanceolata f. silenoides or C. viscosissima.     

Predicting distributions of seven bitterling fishes in northern Kyushu,Japan

The distributions of seven bitterling species and subspecies—Tanakia lanceolata, T. limbata, Acheilognathus tabira nakamurae, A. rhombeus, Rhodeus ocellatus kurumeus, R. ocellatus ocellatus, and R. atremius atremius—in northern Kyushu were predicted using generalized linear models (GLMs) in order to provide information helpful for conserving native bitterlings and preventing the expansion of alien bitterling species. Predictions were made according to the following procedure: (1) a set of GLMs for each species was formulated using environmental data from 710 sites that were derived using digital maps and GIS software, from which the best fit model for each species was selected using the Akaike information criterion for predicting the fish occurrence, (2) model performance was evaluated based on the receiver-operating characteristics (ROC) analysis using occurrence and environmental data from 362 sites, and (3) potential distributions of the bitterling were analyzed using the best fit models and environmental data for 1,272 sites, of which 200 data points without occurrence data were prepared. The best fit models revealed that 4–6 environmental factors were important in predicting seven bitterling distributions, which was supported by the area under the ROC curve (AUC) values of these fishes ranging from 0.753 to 0.927. The AUC values in model evaluation were significantly greater than 0.5 for six fishes, suggesting the moderate accuracies of these best fit models for predicting the fish distributions. These predictive models can be used for evaluating potential native bitterling richness and the potential distribution expansion of an alien subspecies.     

H-Y antigen as a tool for the determination of the heterogametic sex in Amphibia

H-Y antigen was investigated in three amphibian species with different degrees of sex-chromosome differentiation: Bufo bufo, Triturus vulgaris, and Pyxicephalus adspersus. No heteromorphic sex chromosomes were found in B. bufo, but an examination of the progeny of hermaphrodites (Ponse, 1942) indicated that the female of this species was heterogametic (ZW). Sex chromosomes differing only by a very small heterochromatic region at their telomeres were found in the male of T. vulgaris (XY). Pyxicephalus adspersus revealed high differentiated ZW sex chromosomes. The results of the H-Y antigen studies on these three species indicate that H-Y antigen is expressed only in the heterogametic sex, irrespective of differences in morphological differentiation of the sex chromosomes. Therefore, H-Y antigen could be a valuable tool in determining the heterogametic sex, not only in Amphibia but possibly also in other vertebrate species that have either evolved no heteromorphic sex chromosomes or where sex-reversal experiments are not possible.     

Meiosis in gray voles of the subgenus microtus (rodentia, arvicolinae) and in their hybrids

The results of light and electron microscopic (EM) studies of meiosis in Microtus arvalis males of the karyoform "arvalis" (2n = 46, NFa = 80), in hybrids between the chromosomal forms arvalis and obscurus (2n = 46, NFa = 68), in M. rossiaemeridionalis voles (2n = 54, NFa = 54), and in a hybrid between the species M. rossiaemeridionalis and M. kermanensis (2n = 54, NFa = 54) are presented. SC (synaptonemal complex) karyotypes of the parental forms and the hybrids were constructed on the basis of measurements of the length ofautosomal SCs revealed by the EM analysis in spermatocytes at the stage of middle pachytene. The SC karyotypes of M. arvalis and the hybrids female obscurus x male arvalis consist of 22 synaptonemal complexes of autosomal bivalents and the axial elements of the synaptonemal complexes of the sex chromosomes X and Y. The SC karyotypes of M. rossiaemeridionalis and the hybrid M. rossiaemeridionalis x M. kermanensis consist of 26 synaptonemal complexes of autosomal bivalents and a sex bivalent; they differ only in the length of the Y chromosome axis (Y chromosome in the hybrid was inherited from M. kermanensis). Asynaptic configurations of the autosomal SCs were not observed in the hybrids. The SC axial elements of the X and Y chromosomes in the parental forms and in the hybrids were located close to each other throughout pachytene, but they did not form a synaptic region. The normal synapsis in sterile hybrids (M. rossiaemeridionalis x M. kermanensis) and the behavior of the sex chromosomes in meiosis in fertile and sterile hybrids are discussed in the context of specific features of meiosis and reproductive isolation.     

Phylogenetic relationships of bitterlings based on mitochondrial 12S ribosomal DNA sequences

Analysis of the nucleotide sequence of the mitochondrial 12S ribosomal RNA gene of 27 species or sub-species of bitterlings showed that bitterlings comprise an Acheilognathus clade and a Tanakia-Rhodeus clade, partially supporting an earlier classification based on morphology and karyology. The monophyly of Acheilognathus is confirmed, but that of Tanakia and Rhodeus remains poorly resolved. Within the Tanakia–Rhodeus clade, all species or sub-species having a diploid chromosome number of 46 form a monophyletic group. Results support the hypothesis that evolutionary trends of bitterling karyotypes involve reduction from 2 n =48 to either 2 n =44 (by Robertsonian translocation) or 2 n =46 (by non-Robertsonian translocation).     

白豆杉的核型和性染色体的研究

白豆杉pseudotaxus chienii(Cheng)Cheng是我国裸子植物特有属之一,雌雄异常,根尖 细胞染色体分析表明：雌株有一对异形性染色体,异配性别,属ZW型;雄株是同配性别,属ZZ型,雌株的型为2n=2x=24=22m(2SAT ZW) 2T,雄株的核型为2n=2x=24=22m(2SAT ZZ) 2T。Giemsa C-带,显示,Z染色体长短臂均具端带,W染色体不显带。     

Steroids, aromatase and sex differentiation of the newt Pleurodeles waltl

In the newt Pleurodeles waltl, genetic sex determination obeys female heterogamety (female ZW, male ZZ). In this species as in most of non-mammalian vertebrates, steroid hormones play a key role in sexual differentiation of gonads. In that context, male to female sex reversal can be obtained by treatment of ZZ larvae with estradiol. Male to female sex reversal has also been observed following treatment of ZZ larvae with testosterone, a phenomenon that was called the "paradoxical effect". Female to male sex reversal occurs when ZW larvae are reared at 32 degrees C during a thermosensitive period (TSP) that takes place from stage 42 to stage 54 of development. Since steroids play an important part in sex differentiation, we focussed our studies on the estrogen-producing enzyme aromatase during normal sex differentiation as well as in experimentally induced sex reversal situations. Our results based on treatment with non-aromatizable androgens, aromatase activity measurements and aromatase expression studies demonstrate that aromatase (i) is differentially active in ZZ and ZW larvae, (ii) is involved in the paradoxical effect and (iii) might be a target of temperature. Thus, the gene encoding aromatase might be one of the master genes in the process leading to the differentiation of the gonad in Pleurodeles waltl.     

Effects of androgens on sex differentiation of the urodele Pleurodeles waltl

In nonmammalian vertebrates, steroids have been hypothesized to induce somatic sex differentiation, since manipulations of the steroidal environment of gonads have led to various degrees of sex reversal. Whereas the critical role of estrogens in ovarian differentiation is well documented, studies on androgens have produced a perplexing variety of results depending upon species variations and nature of androgens used. In this way, testosterone induces masculinization of females in some species but provokes paradoxical feminization of males in many other species such as the urodelan Pleurodeles waltl. In reptiles this phenomenon could be interpreted by conversion of exogenous testosterone to estradiol by aromatase. Treatments of Pleurodeles larvae with nonaromatizable androgens bring support to this hypothesis and suggest a role of androgens in sex differentiation. Dihydrotestosterone (DHT) could not induce the paradoxical feminization of ZZ larvae. In addition, DHT as well as 11beta-hydroxy-androstenedione could drive a functional male differentiation of ZW larvae. Moreover, other 5alpha reduced androgens also induced sex reversal of female larvae. Yet, the 5alpha reductase inhibitor CGP 53133 and antiandrogens such as flutamide or cyproterone acetate did not exert any effect on male sex differentiation of ZZ larvae. Though the precise role of androgens is still unknown, especially for 11-oxygenated androgens, our results suggest an implication in male sex differentiation. In this way, testosterone could play a pivotal role in being metabolized either into other androgens during testis differentiation or into estradiol during ovarian differentiation.     

Fertility in first-generation hybrids of roach, Rutilus rutilus (L.), and silver bream, Blicca bjoerkna (L.)

Fertility in first‐generation hybrids of roach, Rutilus rutilus, and silver bream, Blicca bjoerkna, was investigated. Sperm and egg production of hybrids at first sexual maturity were examined. Eggs from female hybrids were artificially fertilized with the sperm of a corresponding hybrid male; a hybrid male from the reciprocal crossbreeding; a parental species male R. rutilus; and a parental species male B. bjoerkna. The results revealed that gametogenesis was normal in female hybrids. However, in male hybrids, a low efficiency of gametogenesis was observed. The semen of male hybrids was extremely dilute, with spermatozoa concentration lower than that in parental species. Nevertheless, these F1 hybrids (males and females) from reciprocal crossbreeding were fertile. F2 and backcross generations were produced, but F2 crosses from the female hybrid and corresponding hybrid male displayed a drastically slower hatching rate. Also higher proportions of deformed embryos were hatched than in other post‐F1‐generation crosses.     

A comparative study on morphology, growth rate and reproduction of Clarias gariepinus (Burchell, 1822), Heterobranchus longifilis Valenciennes, 1840, and their reciprocal hybrids (Pisces, Clariidae)

A detailed study on reproduction indicated that female C. gariepinus mature earlier (5–7 months) than female H. longifilis (12–14 months). In the reciprocal hybrids, female first sexual maturity was attained at 20–21 months. Both reciprocal hybrids and parental species displayed an equilibrated sex ratio. Macroscopical and microscopical observations revealed numerous abnormalities in gonadal development of the hybrids. GSI and fecundity in female hybrids were considerably lower than those found in the parental species. Moreover, intra-ovarian tumors occurred in 20% of the hybrids. In hybrid males, GSIs were generally higher than those found in the parental species, but the spermatozoa concentration in the semen was about 100 times less. Despite these abnormalities, viable larvae, resulting from F2 and backcross fertilizations, were obtained.     

Sex determination system of the rosy bitterling, Rhodeus ocellatus ocellatus

The rosy bitterling, Rhodeus ocellatus ocellatus, is the only fish species known in which artificial triploids are always male, regardless of the kind of polyploidization technique used. In order to elucidate the genetic sex determination system of the rosy bitterling, two kinds of gynogenesis were carried out: retention of the second polar body (GRSPB) and suppression of the first cleavage (GSFC). The sex ratio of progeny was nearly 7:1 (:) for both GRSPB and GSFC, while those of control and parental fish were almost 1:1. In backcrosses of female progeny by GRSPB and normal diploid males, male progeny were observed at low frequency (one or two individuals in each experiment), except in one experiment where the appearance rate of males was about 50%. From results of gynogenesis and backcrosses, the following conclusions can be made. The genetic sex determination system of the rosy bitterling is a heterogametic female system (ZW). Survival rate of superfemales (WW), produced by gynogenesis, is much lower than that of males (ZW).There is a possibility that crossovers between sex determining genes and a centromere occur in the first meiosis. With repect to the mechanism of unisexuality (male) of artificial triploids of the rosy bitterling, only males (ZZZ and ZZW) are presumed viable, while females (ZWW) are probably inviable.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

H-Y Antigen Expression in Temperature Sex-Reversed Turtles (Emys orbicularis)

H-Y antigen has been used as a marker for the heterogametic sex and is assumed to be an organizing factor for the heterogametic gonad. In the turtle Emys orbicularis , H-Y antigen is restricted to the female cells, indicating a female heterogamety (ZZ/ZW) sex-determining mechanism. Moreover, the sexual differentiation of the gonads is temperature sensitive, and complete sex reversal can be obtained at will. In this framework the relationships between H-Y antigen, temperature, and gonadal phenotype were studied. Mouse H-Y antiserum was absorbed with blood and gonadal cells of control wild male and female adults, and with blood and gonadal cells from three lots of young turtles from eggs incubated at 25–26°C (100% phenotypic males), at 30–30.5°C (100% phenotypic females), or at 28.5–29°C (majority of females with some males and intersexes). The residual activity of H-Y antiserum was then estimated using an immunobacterial rosette technique. In adults, both blood cells and gonadal cells were typed as H-Y negative in males and as H-Y positive in females. In each of the three lots of young, blood cells were H-Y negative in some individuals and H-Y positive in others. The proposed interpretation is that the H-Y negative individuals were genotypic males (ZZ) and the H-Y positive were genotypic females (ZW). The gonads of these animals were then pooled in different sets according to their sexual phenotype and to the presumed genotypic sex (i.e., blood H-Y phenotype). Testicular cells were typed as H-Y negative in genotypic males as well as in the presumed sex-reversed genotypic females; likewise, ovarian cells were typed as H-Y positive in genotypic females as well as in the presumed sex-reversed genotypic males. These results provide additional evidence that H-Y antigen expression is closely associated with ovarian structure in vertebrates displaying a ZZ/ZW sex-determining mechanism.     

Production of intersexes and the evolution of androdioecy in the clam shrimp Eulimnadia texana (Crustacea,Branchiopoda, Spinicaudata)

Summary

The production of low numbers of offspring that exhibit a mixture of male and female traits (termed “intersexes”) is commonly reported for crustaceans. The production of intersexes has been ascribed to both genetic and non-genetic (e.g., parasitic infections and environmental pollutants) causes. Herein we report on two observed types of intersexes in the clam shrimp Eulimnadia texana: (1) a “morphological” intersex, possessing secondary male characteristics (e.g., claspers) and an eggproducing gonad, and (2) a “gonadal” intersex, possessing primarily male traits (e.g., male secondary sexual characters and male gamete production) but also producing low levels of abortive female gametes. We propose that these intersexes are likely the products of low frequencies of crossing over between the sex determining chromosomes that result in the array of observed mixed sexual phenotypes. Additionally, we suggest that the low-level production of intersexes, combined with the ephemeral nature of the habitats occupied by these shrimp, may explain the preponderance of androdioecy (mixtures of males and hermaphrodites) found in these clam shrimp, and possibly branchiopods more generally.     

Reproductive behavior of intersexes of an intertidal amphipod Corophium volutator

Abstract. Intersexes are common in crustaceans. Typically, these intersexes are sterile or function as females, but prior evidence from laboratory experiments suggests that intersexes of a key species of gammaridean amphipod, Corophium volutator , might function as males. We observed that intersexes of C. volutator behaved as males by crawling (mate-searching) on a mudflat during ebb tides and pairing in burrows with female amphipods. In the laboratory, intersexes and males did not differ in aspects of crawling such as movement rate and measures of burrow investigation. I`ntersexuality was costly in that intersexes crawled less often than males on a mudflat, formed fewer pairs with females than males, and remained in tandem less often with receptive females than males. The use of PCR-based identification methods failed to identify the presence of transovarial, feminizing, microsporidian parasites as a major cause of intersexuality in this species in that infected females did not produce broods that contained more intersexes than broods produced by uninfected females. Because intersexes may be mistaken as females, the percentage of functional males in amphipod populations may be underestimated: an important consideration given male limitation in populations of C. volutator. The occurrence of intersexes has significant implications for studies on the evolution and ecology of sex ratios, and the use of crustaceans as indicators of environmental quality.