Sex determination studies in two species of teleost fish, Oreochromis niloticus and Leporinus elongatus

Genetic analyses of sex determination have identified sex chromosomes in many teleost fish species. However, there are several cases for which sex ratios do not fit perfectly with the expectations of heterogametic systems, suggesting the influence of either minor sex determining genes or environmental influences on the process of sex differentiation. The frequent absence of sex chromosome markers makes the identification of minor sex-determining genes very difficult. It is easier to test first the hypothesis of environmental sex determination (ESD) by studying the temperature effect, since temperature-dependent sex determination has been demonstrated to occur in several vertebrate groups including 1 fish species. To contribute to a better understanding of fish sex determination, we have tested the effects of high temperatures on sex ratios of Oreochromis niloticus, and have attempted to isolate sex chromosome molecular markers in Leporinus elongatus. Treatments of O. niloticus fry at 36 degrees C applied for 10 days and more, and starting 1 week after fertilization markedly increased the proportion of males, and progeny-testing these males confirmed that some of them are sex-reversed genetic females. Two non-coding sequences of L. elongatus Z and W chromosomes were cloned by genomic subtraction. They cross-hybridized with the genome of a close species without providing sex-specific patterns. A collection of L. elongates individuals was subjected to gonadal and chromosomal sexing, and DNA hybridization with both sequences. These analyses revealed 3 individuals having atypical W chromosomes. Interestingly, 2 of these were males having a ZW karyotype. We assume that these atypical sex chromosome arise by exchanges between Z and W chromosomes, and that a transition between female and male heterogamety is underway in this species.

Chromosome evolution in the erythrinid fish, Erythrinus erythrinus (Teleostei : Characiformes)

The genus Erythrinus belongs to the family Erythrinidae, a neotropical fish group. This genus contains only two described species, Erythrinus erythrinus being the most widely distributed in South America. Six samples of this species from five distinct Brazilian localities and one from Argentina were studied cytogenetically. Four groups were identified on the basis of their chromosomal features. Group A comprises three samples, all with 2n = 54 chromosomes, a very similar karyotypic structure, and the absence of chromosome differentiation between males and females. One sample bears up to four supernumerary microchromosomes, which look like 'double minute chromosomes' in appearance. Groups B - D comprise the three remaining samples, all sharing an X1X1X2X2/X1X2Y sex chromosome system. Group B shows 2n = 54/53 chromosomes in females and males, respectively, and also shows up to three supernumerary microchromosomes. Groups C and D show 2n = 52/51 chromosomes in females and males, respectively, but differ in the number of metacentric, subtelocentric, and acrocentric chromosomes. In these three groups ( B - D), the Y is a metacentric chromosome clearly identified as the largest in the complement. The present results offer clear evidence that local samples of E. erythrinus retain exclusive and fixed chromosomal features, indicating that this species may represent a species complex.

Chromosomal evolution in Gekkonidae. I. Chromosome painting between Gekko and Hemidactylus species reveals phylogenetic relationships within the group

Geckos are a large group of lizards characterized by a rich variety of species, different modes of sex determination and diverse karyotypes. In spite of many unresolved questions on lizards' phylogeny and taxonomy, the karyotypes of most geckos have been studied by conventional cytogenetic methods only. We used flow-sorted chromosome-specific painting probes of Japanese gecko (Gekko japonicus), Mediterranean house gecko (Hemidactylus turcicus) and flat-tailed house gecko (Hemidactylus platyurus) to reveal homologous regions and to study karyotype evolution in seven gecko species (Gekko gecko, G. japonicus, G. ulikovskii, G. vittatus, Hemidactylus frenatus, H. platyurus and H. turcicus). Generally, the karyotypes of geckos were found to be conserved, but we revealed some characteristic rearrangements including both fissions and fusions in Hemidactylus. The karyotype of H. platyurus contained a heteromorphic pair in all female individuals, where one of the homologues had a terminal DAPI-negative and C-positive heterochromatic block that might indicate a putative sex chromosome. Among two male individuals studied, only one carried such a polymorphism, and the second one had none, suggesting a possible ZZ/ZW sex determination in some populations of this species. We found that all Gekko species have retained the putative ancestral karyotype, whilst the fission of the largest ancestral chromosome occurred in the ancestor of modern Hemidactylus species. Three common fissions occurred in the ancestor of Mediterranean house and flat-tailed house geckos, suggesting their sister group relationships. PCR-assisted mapping on flow-sorted chromosome libraries with conserved DMRT1 gene primers in G. japonicus indicates the localization of DMRT1 gene on chromosome 6.

Chromosome banding patterns of four species of bats, with special reference to a case of X-autosome translocation

The karyotypes of 4 species of bats, Artibeus lituratus (Phyllostomatidae), Pipistrellus pipistrellus (Vespertilionidae), Pteropus alecto and P. giganteus (Pteropodidae), were studied after several banding techniques. For A. lituratus, in which an X-autosome translocation was observed, an analysis of the replication pattern in the rearranged chromosome was also made after BrdU incorporation.

Morphologically differentiated sex chromosomes in neotropical freshwater fish

A general survey of the occurrence of morphologically differentiated sex chromosomes in the neotropical freshwater fishes is presented. The total number of 32 occurrences involving simple XX-XY and ZZ-ZW, and multiple X1X2Y, XY1Y2 and ZW1W2 sex chromosome systems is described, with comments on the aspects of sex chromosome evolution in this fish fauna. The occurrence of different sex chromosome systems in related species of the same genus, or in different populations of the same nominal species, involving male and sometimes female heterogamety, and differences in the molecular composition of sex-linked heterochromatin, are considered as indicative of the early stage of sex chromosomes evolution in fish.

Cytogenetic analysis of A-, B-chromosomes and ZZ/ZW sex chromosomes of Characidium gomesi (Teleostei, Characiformes, Crenuchidae)

Different cytogenetic techniques were used to analyze the chromosomes of Characidium gomesi with the main objective of comparing the base composition of ZZ/ZW sex-chromosomes, B-chromosomes and the heterochromatin of A-chromosomes. The results of digestion of chromosomes with AluI restriction endonuclease (RE), silver and CMA3 staining, C-banding and fluorescence in situ hybridization (FISH) with the 18S rDNA probe suggested the existence of compositional differences between the heterochromatin of ZZ/ZW sex-chromosomes, A- and B-chromosomes, and indicated the presence of different numbers and morphology of B-chromosomes in the samples of this population.

Heteropicnotic chromatin and nucleolar activity in meiosis and spermiogenesis of Limnogonus aduncus (Heteroptera, Gerridae): a stained nucleolar organizing region that can serve as a model for studying chromosome behavior.

Males of Limnogonus aduncus were found to have the sex chromosome system X0 and chromosome number 2n = 23 (22A + X0). Testis cells were stained with lacto-acetic orcein and silver nitrate so that changes in the morphology and degree of staining of the heteropicnotic chromatin and the nucleolar material could be observed during meiosis and spermiogenesis. These structures share the same nuclear position and could be seen until almost the end of spermiogenesis. A chromosome region stained with silver nitrate was indicative of a nucleolar organizing region (NOR), which is rarely detected in Heteroptera with this technique. The NOR is located at one end of a single member of an autosome pair. The finding of this stained region enabled us to observe that the telomeric association of sister chromatids that characterizes the Heteroptera does not include the chromosome ends, where NORs are located; we also observed in anaphase that the chromosome end through which it is pulled to the pole is the one containing the NOR. Another observation was that the single nucleolar body present in the cells at anaphase never goes to the cell pole that does not receive the NOR. We conclude that L. aduncus is a good model for cytogenetic studies involving nucleolar activity and also may be useful for studying the mechanisms of activation and inactivation of kinetic activity at the chromosome ends. Although the chromosomes of Heteroptera are known to be holocentric, whether kinetic activity is restricted to one or involves both chromosome ends is still not well understood.

A new species of Endecous Saussure, 1878 (Orthoptera, Gryllidae) from northeast Brazil with the first X(1)X(2)0 chromosomal sex system in Gryllidae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Distribution of constitutive heterochromatin in species of triatomines with fragmentation of sex chromosomes X

Cytogenetic analyses of triatomines are considered to be important taxonomic tools. Thus, we analyzed the pattern of constitutive heterochromatin in 7 species of triatomine with fragmentation of the sex chromosome X, focusing on the cytotaxonomy of these triatomines. The species analyzed included Triatoma vitticeps, Triatoma melanocephala, Triatoma tibiamaculata, Triatoma protracta, Meccus pallidipennis, Panstrongylus megistus, and Panstrongylus lignarius. The seminiferous tubules of the adult males were subjected to C-banding. P. megistus and P. lignarius showed differences in chromosome number and disposition of constitutive heterochromatin, as only P. lignarius showed C-blocks in autosomes. C-banding can differentiate these species, since one of the sex chromosome (X) is heterochromatic in T. vitticeps. T. protracta showed C-blocks in both ends of all autosomes, T. tibiamaculata showed terminal C-dots in some autosomal pairs and M. pallidipennis did not show constitutive heterochromatin in autosomes. Thus, we confirmed the heterochromatic pattern of 7 species of insects and emphasized the importance of cytogenetic techniques for C-banding for taxonomy studies of the triatomines, which are important vectors of Chagas disease.

Chromosome evolution in lycosoid spiders (Araneomorphae): a scenario based on analysis of seven species of the families Lycosidae, Senoculidae and Trechaleidae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A step to the gigantic genome of the desert locust: chromosome sizes and repeated DNAs

The desert locust (Schistocerca gregaria) has been used as material for numerous cytogenetic studies. Its genome size is estimated to be 8.55 Gb of DNA comprised in 11 autosomes and the X chromosome. Its X0/XX sex chromosome determinism therefore results in females having 24 chromosomes whereas males have 23. Surprisingly, little is known about the DNA content of this locust's huge chromosomes. Here, we use the Feulgen Image Analysis Densitometry and C-banding techniques to respectively estimate the DNA quantity and heterochromatin content of each chromosome. We also identify three satellite DNAs using both restriction endonucleases and next-generation sequencing. We then use fluorescent in situ hybridization to determine the chromosomal location of these satellite DNAs as well as that of six tandem repeat DNA gene families. The combination of the results obtained in this work allows distinguishing between the different chromosomes not only by size, but also by the kind of repetitive DNAs that they contain. The recent publication of the draft genome of the migratory locust (Locusta migratoria), the largest animal genome hitherto sequenced, invites for sequencing even larger genomes. S. gregaria is a pest that causes high economic losses. It is thus among the primary candidates for genome sequencing. But this species genome is about 50 % larger than that of L. migratoria, and although next-generation sequencing currently allows sequencing large genomes, sequencing it would mean a greater challenge. The chromosome sizes and markers provided here should not only help planning the sequencing project and guide the assembly but would also facilitate assigning assembled linkage groups to actual chromosomes.

Neo-sex chromosomes of Ronderosia bergi: insight into the evolution of sex chromosomes in grasshoppers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The contrasting role of heterochromatin in the differentiation of sex chromosomes: an overview from Neotropical fishes

During the evolutionary process of the sex chromosomes, a general principle that arises is that cessation or a partial restriction of recombination between the sex chromosome pair is necessary. Data from phylogenetically distinct organisms reveal that this phenomenon is frequently associated with the accumulation of heterochromatin in the sex chromosomes. Fish species emerge as excellent models to study this phenomenon because they have much younger sex chromosomes compared to higher vertebrates and many other organisms making it possible to follow their steps of differentiation. In several Neotropical fish species, the heterochromatinization, accompanied by amplification of tandem repeats, represents an important step in the morphological differentiation of simple sex chromosome systems, especially in the ZZ/ZW sex systems. In contrast, multiple sex chromosome systems have no additional increase of heterochromatin in the chromosomes. Thus, the initial stage of differentiation of the multiple sex chromosome systems seems to be associated with proper chromosomal rearrangements, whereas the simple sex chromosome systems have an accumulation of heterochromatin. In this review, attention has been drawn to this contrasting role of heterochromatin in the differentiation of simple and multiple sex chromosomes of Neotropical fishes, highlighting their surprising evolutionary dynamism.

Development of a bovine X chromosome linkage group and painting probes to assess cattle, sheep, and goat X chromosome segment homologies.

The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We have developed bovine chromosome painting probes for Xp and Xq to assess segment homologies between the submetacentric bovine X chromosome and the acrocentric sheep and goat X chromosomes. These painting probes and their corresponding DNA libraries were developed by chromosome micromanipulation, DNA micropurification, microcloning, and PCR amplification. The bovine Xp painting probe identified an interstitially located homologous segment in the sheep and goat Xq region, most probably resulting from chromosome inversion. Ten type II (microsatellite) markers obtained from the bovine Xq library and five other X chromosome assigned, but unlinked, markers were used to generate a linkage map for Xq spanning 89.4 centimorgans. The chromosome painting probes and molecular markers generated in this study would be useful for comparative mapping and tracing of internal X chromosome rearrangements in all ruminant species and would contribute to the understanding of mammalian sex chromosome evolution.