Ultrastructural characterization of the sex chromosomes during spermatogenesis of spiders having holocentric chromosomes and a long diffuse stage

An ultrastructural study has been made of spermatogenesis in two species of primitive spiders having holocentric chromosomes (Dysdera crocata, XO and Segestria florentia X₁X₂O). Analysis of the meiotic prophase shows a scarcity or absence of typical leptotene to pachytene stages. Only in D. crocata have synaptonemal complex (SC) remnants been seen, and these occurred in nuclei with an extreme chromatin decondensation. In both species typical early prophase stages have been replaced by nuclei lacking SC and with their chromatin almost completely decondensed, constituting a long and well-defined diffuse stage. Only nucleoli and the condensed sex chromosomes can be identified. — In S. florentina paired non-homologous sex chromosomes lack a junction lamina and thus clearly differ from the sex chromosomes of more evolved spiders with an X₁X₂O male sex determination mechanism. In the same species, sex chromosomes can be recognized during metaphase I due to their special structural details, while in D. crocata the X chromosome is not distinguishable from the autosomes at this stage. — The diffuse stage and particularly the structural characteristics of the sex chromosomes during meiotic prophase are reviewed and discussed in relation to the meiotic process in other arachnid groups.     

First evidence of achiasmatic male meiosis in the water bears Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae)

Chromosome behaviour during male meioses has been studied in two bisexual amphimictic populations of two tardigrade species, namely Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae). Both bisexual populations exhibit a diploid chromosome number 2n=12 and no sex chromosomes were identified. DAPI staining and C-banding data indicate that all chromosomes of the bisexual population of R. coronifer are acrocentric. In both species, at male meiotic prophase, all six bivalent homologous chromosomes are aligned side by side along their length and show no evidence of chiasmata. However, in the oocytes of both species a chiasma is generally present in each bivalent at diplotene stage. Lack of recombination is previously unknown in tardigrades, but is a well known phenomenon in many other metazoans where it is always restricted to the heterogametic sex. In tardigrades there is no evidence of heterochromosomes, but it does not mean that in tardigrades, the heterogametic sex does not exist. The adaptive and evolutionary significance of achiasmatic meiosis is discussed.     

“Nodules” in the achiasmatic meiosis of Bithynia (mollusca,prosobranchia)

In spermatocytes of Bithynia leachi and B. tentaculata, synaptonemal complexes with nodules in an achiasmatic meiosis were demonstrated for the first time by means of the spreading technique. The number and position of the nodules are different in each species and are independent of the length of the chromosomes. Although no crossing over occurs in Bithynia chiasma-like terminal connections of the chromosomes are preserved in diakinesis by the presence of the nodules. — Enzymatic treatments revealed information regarding their composition of alkaline proteins, RNA and DNA. In all proteolytic digestions the nodules more of less retain their position in the central region, while they disappear immediately if SCs are exposed to DNase. On the basis of the results a model of the composition and the function of nodules can be developed considering the concept of a lampbrush-organization of the chromosomes in early meiotic prophase.     

Chromosome 1 in crested and marbled newts (Triturus)

The heteromorphic chromosomes 1 of Triturus cristatus carnifex and T. marmoratus were studied in mitotic metaphase after staining with the Giemsa C-banding technique and with the fluorochromes, DAPI (AT-specific) and mithramycin (GC-specific). They were also examined in the lampbrush form under phase-contrast before fixation and after fixation and staining with Giemsa. Chromosomes 1 of T.c. carnifex are asynaptic and achiasmatic throughout most of their long arms. They are also heteromorphic in most of their long arms for the patterns of Giemsa and fluorochrome staining and the distribution of distinctive lampbrush loops. The heteromorphic regions correspond to the regions that are asynaptic and achiasmatic. They stain more strongly with mithramycin and more weakly with DAPI than the remainder of the chromosomes, signifying that their DNA is relatively rich in GC. The patterns of staining with Giemsa and fluorochromes and the distributions of distinctive lateral loops vary from one animal to another in the same species and even in the same population. The asynaptic and achiasmatic regions of chromosomes 1 in T. marmoratus extend throughout the whole of the long arms and well beyond the heterochromatic region. Chiasmata form only in the short arm and occasionally in the short euchromatic segment at the tip of the long arms. The staining patterns of chromosomes 1 in T. marmoratus differ from those in T.c. carnifex although, like carnifex, their DNA is relatively GC-rich. The chromosomes 1 of T. marmoratus are more submetacentric than those of T.c. carnifex. In T. marmoratus chromosome 1B is about 12% shorter than 1A. There is a short paracentric inversion heterozygosity in the long arm of chromosome 1B in T. marmoratus which probably accounts for the lack of chiasmata in the euchromatin that separates the centromere from the start of the heterochromatin. In both carnifex and marmoratus, embryos that are homomorphic for chromosome 1 arrest and die at the late tailbud stage of development. The same applies to F₁ hybrid embryos T.c. carnifex x T. marmoratus, and this has permitted identification of chromosomes 1A and 1B in both species. There is no correspondence between patterns of Giemsa or fluorochrome staining of the heteromorphic regions of chromosome 1 and any feature of the lampbrush chromosomes. However, the short euchromatic ends of the long arms of chromosomes 1 in both species are distinguished in the lampbrush form by a series of uniformly small loops of fine texture associated with very small chromomeres. The Giemsa C-staining patterns of both chromosomes 1A and 1B are different in each of the four subspecies of T. cristatus. T.c. karelinii stands out by having unusually large masses of Giemsa C-staining centromeric heterochromatin on all but 1 of its 12 chromosomes. A scheme is proposed for the evolution of chromosome 1 in T. cristatus and T. marmoratus, based on all available cytological and molecular data.     

Comparative studies on sex chromosomes in different species

InLocusta migratoria (XO),Mus musculus, Rattus norvegicus, Mesocricetus auratus, Cricetulus griseus andHomo sapiens typical sex vesicle structures are visible in early meiotic prophase stages up to pachynema. The structures include whole sex chromosomes or parts thereof. The heterologous parts and the solitary X chromosome ofLocusta pass diplonema, diakinesis and first metaphase nearly in mitotic shape. Entirely heterologous sex chromosomes are kept together by a unilateral and achiasmatic end connection. The sex vesicle is interpreted as a special structure of allocyclic sex chromosomes or parts of them, corresponding in early meiotic stages to the chromocenters of mitotic interphase nuclei. The formation of the sex vesicle is independent of the orthoploidy of nuclei and of the DNA ratio between autosomes and sex chromosomes. Heteropycnotic behaviour of sex chromosomes in spermatids is interpreted as a condition capable of blocking genetic activity, like in the Barr bodies of female somatic nuclei, giving equal chances of fertilization to both types of gametes.Based on a paper read at the XI International Congress of Genetics, of which an abstract has appeared in the congress proceedings, Genetics Today, Vol. 1, p. 299 (1963).     

Evolution of karyotype,sex chromosomes,and meiosis in mygalomorph spiders (Araneae: Mygalomorphae)

Spider diversity is partitioned into three primary clades, namely Mesothelae, Mygalomorphae, and Araneomorphae. Mygalomorph cytogenetics is largely unknown. Our study revealed a remarkable karyotype diversity of mygalomorphs. Unlike araneomorphs, they show no general trend towards a decrease of 2n, as the chromosome number was reduced in some lineages and increased in others. A biarmed karyotype is a symplesiomorphy of mygalomorphs and araneomorphs. Male meiosis of some mygalomorphs is achiasmatic, or includes the diffuse stage. The sex chromosome system X₁X₂0, which is supposedly ancestral in spiders, is uncommon in mygalomorphs. Many mygalomorphs exhibit more than two (and up to 13) X chromosomes in males. The evolution of X chromosomes proceeded via the duplication of chromosomes, fissions, X–X, and X‐autosome fusions. Spiders also exhibit a homomorphic sex chromosome pair. In the germline of mygalomorph males these chromosomes are often deactivated; their deactivation and pairing is initiated already at spermatogonia. Remarkably, pairing of sex chromosomes in mygalomorph females is also initiated at gonial cells. Some mygalomorphs have two sex chromosome pairs. The second pair presumably arose in early‐diverging mygalomorphs, probably via genome duplication. The unique behaviour of spider sex chromosomes in the germline may promote meiotic pairing of homologous sex chromosomes and structural differentiation of their duplicates, as well as the establishment of polyploid genomes. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 377–408.     

Structural and numerical chromosomal polymorphism in natural populations ofAlopecurus (Poaceae)

Considerable structural and numerical chromosomal variation has been found in natural populations ofAlopecurus. Interchange heterozygotes, identified by multivalent formation during meiosis, have been recovered in four out of six species studied and are reported for the first time in the diploidsA. bulbosus, andA. myosuroides, and the tetraploidA. pratensis. B chromosomes have been found in two species,A. pratensis andA. myosuroides and also in inter-specific hybrids betweenA. pratensis andA. arundinaceus. The characteristics, distribution and meiotic behaviour of both interchange heterozygotes and B chromosomes are described.     

The Origin of the Achiasmatic XY Sex Chromosome System in Cacopsylla peregrina (Frst.) (Psylloidea, Homoptera)

The status of an extra univalent, if it is a B chromosome or an achiasmatic Y chromosome, associating with the X chromosome in male meiosis of Cacopsylla peregrina (Frst.) (Homoptera, Psylloidea) was analysed. One extra univalent was present in all males collected from three geographically well separated populations, it was mitotically stable, and showed precise segregation from the X chromosome. These findings led us to propose that the univalent represents in fact a Y chromosome. The behaviour of the X and Y chromosomes during meiotic prophase suggested that their regular segregation was based on an achiasmatic segregation mechanism characterised by a 'touch and go' pairing of segregating chromosomes at metaphase I. To explain the formation of the achiasmatic Y within an insect group with X0 sex chromosome system, it was suggested that the Y chromosome has evolved from a mitotically stable B chromosome that was first integrated into an achiasmatic segregation system with the X chromosome, and has later become fixed in the karyotype as a Y chromosome.     

First evidence of sex chromosome pre-reduction in male meiosis in the Miridae bugs (Heteroptera)

The karyotype and male meiosis of Macrolophus costalis Fieber (Insecta, Heteroptera, Miridae) were studied using C-banding, AgNOR-banding and DNA sequence specific fluorochrome staining. The chromosome formula of the species is 2n = 28(24+X1X2X3Y). Male meiotic prophase is characterized by a prominent condensation stage. At this stage, two sex chromosomes, "X" and Y are positively heteropycnotic and always appeared together, while in autosomal bivalents homologous chromosomes were aligned side by side along their entire length, that is, meiosis is achiasmatic. At metaphase I, "X" and Y form a pseudobivalent and orient to the opposite poles. At early anaphase I, the "X" chromosome disintegrates into three separate small chromosomes, X1, X2, and X3. Hence both the autosomes and sex chromosomes segregate reductionally in the first anaphase, and separate equationally in the second anaphase. This is the first evidence of sex chromosome pre-reduction in the family Miridae. Data on C-heterochromatin distribution and its composition in the chromosomes of this species are discussed.     

Aberrant spermatogenesis and sex determination in Bourletiellidae (Hexapoda, Collembola), and their evolutionary significance

Light and electron microscopic evidence is provided to describe a new example of a postzygotic sex-determination system in two collembolan species, Bourletiella arvalis and B. hortensis. In B. arvalis, where chromosome number could be assessed, both sexes are homogametic (n=6) and all zygotes have an identical chromosome composition (2n=12). However, male embryos develop after the loss of two sex chromosomes, making the male genotype 2n=10 (4AAX₁0X₂0). On the other hand, female embryos develop if the zygote retains all chromosomes and the female genetic system is, therefore, 4AAX₁X₁X₂X₂ (2n=12). As an apparent consequence of the lack of two chromosomes in the male germ cells, spermatogenesis is aberrant. At the first meiotic division, in fact, the two resulting secondary spermatocytes receive a different number of chromosomes: six and four. The cells which receive six chromosomes (one haploid set of four autosomes and two sex chromosomes) proceed through the meiotic process and the two spermatids generated produce two spermatozoa by a normal spermiogenesis. The cells receiving only four chromosomes do not undergo the second meiotic division and soon degenerate. The degenerating cells can be considered a morphological marker for this process, as they are easily recognizable at the electron microscope from the functional secondary spermatocytes by the appearance of the nucleus (totally condensed), the reduction of the cytoplasm (limited to a thin layer surrounding the nucleus), and the lack of most cytoplasmic organelles (with the exception of a couple of centrioles). Electron microscopic evidence has been collected for both species, allowing to extend the same process to B. hortensis, even if chromosomes could not be counted in this species. Therefore, as a result of the spermatocyte elimination, the efficiency of spermatogenesis is reduced to 50%. This process is identical to that observed in other collembolan species of the suborder Symphypleona, and it is suggested that it represents a synapomorphic feature uniting the families Dicyrtomidae, Sminthuridae and Bourletiellidae (Sminthuriformia). It is also suggested that the process is related with the finding of a distorted sex ratio in natural populations and, possibly, with the evolution of parthenogenesis. This hypothesis is supported by the fact that chromosome pairing and genetic recombination occurs only during female meiosis, while chromosomes do not pair during male meiosis. Accepted: 27 December 2000     

B chromosomes in Stylidium crossocephalum (Angiospermae:Stylidiaceae)

Populations of Stylidium crossocephalum contain two common types of B chromosomes, macro B chromosomes and micro B chromosomes. The macro B chromosomes are telocentric, slightly smaller than the smallest A chromosomes and mitotically unstable. They have so far been found associated with 6 of the 16 stable genomes known in S. crossocephalum, occurring in populations covering a substantial portion of the species range. Micro B chromosomes are about one third the length of the smallest A chromosome, acrocentric and show some mitotic instability. They occur associated with 3 stable genomes in populations found in the more medial regions of the species range. Both types of B chromosomes generally show regular behaviour during meiosis, although when two or more are present their pairing efficiency is reduced when compared to the A chromosomes. In addition a single very large mega chromosome was found in a single cell of one heterokaryotypic plant. Its origin although conjectural at this stage may be of relevance in understanding the origin of macro and micro B chromosomes in this species.     

The cytology of Brachycome line ariloba

Hybrids between B. campylocarpa (2n=8) and B. lineariloba Race A (2n=4) have been made and studied in both meiosis and mitosis. In B. campylocarpa A × B. lineariloba A₃, meiotic pairing is limited, and suggests that there are segmental homologies between the two lineariloba chromosomes and three of the four campylocarpa chromosomes. The chromosomal relationship between the two species is not close. In hybrids there is a marked difference or asynchrony in the condensation patterns of the chromosomes from the two parental sources. Condensation behaviour is not determined by the genome and must be under local control within each chromosome.     

Males vs. female meiotic prophase in a grasshopper,Arcyptera microptera (Orthoptera: Acridiae)

Female and male first meiotic prophase are analyzed in the grasshopper Arcyptera microptera with special reference to the heteropycnotic chromatin. Differences in the behaviour of the megameric and X-chromosomes, in nuclear size and in the response to C-banding treatment are described. The evidence for a distinctive meiotic prophase in both sexes is compared with results obtained in other species of grasshoppers by other authors using more advanced stages. The discussion centers around the role of the diffuse stage and the differential behaviour of the X-chromosome in the two sexes.     

Interaction of B chromosomes with A or B chromosomes in segregation in insects

Additional or B chromosomes not belonging to the regular karyotype of a species are found in many animal and plant groups. They form a highly heterogeneous group with respect to their morphology and behaviour both in mitosis and meiosis. Achiasmatic mechanisms that ensure the segregation of a B chromosome from another B chromosome or from an A chromosome are reviewed. An achiasmatic mechanism characterized by the "distance pairing" of segregating univalents at metaphase I was found to be responsible for the preferential segregation of B chromosome univalents in Hemerobius marginatus L. (Neuroptera), and a mechanism characterized by the "touch and go pairing" of segregating univalents was responsible for the highly regular segregation of a B chromosome and the X chromosome in Rhinocola aceris (L.) (Psylloidea, Homoptera). The latter mechanism resulted in the integration of a B chromosome to the A chromosome set as a Y chromosome in a psyllid species Cacopsylla peregrina (Frst.). Furthermore, B chromosomes can disturb the regular segregation of the achiasmatic X and Y chromosomes resulting in the formation of X0/XY polymorphism in a population, which might precede the loss of the Y chromosome. The absence of observations on accurately functioning achiasmatic segregation mechanisms in grasshoppers (Orthoptera) was attributed to the X and B chromosomes, which re-orient one or several times during metaphase I. Apparently, these re-orientations mask any achiasmatic segregation mechanism that might operate during meiotic prophase in these insects.     

Chromosome numbers, sex determining systems, and patterns of the C-heterochromatin distribution in 13 species of lace bugs (Heteroptera, Tingidae).

The karyotypes, sex chromosome systems, and male meiotic patterns in 13 species belonging to 10 genera of the family Tingidae were studied. Data on eleven species, one subgenus, and 5 genera are presented for the first time, and the chromosome formula of Acalypta parvula is revised. Karyotypes of all species included six pairs of autosomes. Most of the species displayed an XY sex chromosome system, in four species, belonging to genera of Acalypta and Kalama, the X0 system was found. Male meiosis is chiasmatic for autosomes. Sex chromosomes are achiasmatic and undergo pre-reductional meiosis. Using C-banding technique, for the first time constitutive heterochromatin was localized on chromosomes in all the species studied. The heterochromatin was found either in telomeres or in some species in interstitial locations, evidencing that a quite substantial redistribution of chromosome material within chromosomes might occur without fragmentations or fusions. In two species, a supernumerary (B) chromosome was found. In addition, the male reproductive system of four species was examined and the number of testicular follicles was determined as two per testis.     

Cytogenetic analysis in the spermatogenesis of Triatoma melanosoma (Reduviidae; Heteroptera)

Triatomines are of great concern in public health because they are vectors of Chagas' disease. This study presents an analysis of the species Triatoma melanosoma. The cytogenetic characteristics of triatomines include holocentric chromosomes, post-reductional meiosis in the sex chromosomes and nucleolar fragmentation in the meiotic cycle. The methodology utilized consisted of the techniques of lacto-acetic orcein staining and silver ion impregnation. The organs analyzed were adult testicles. The results enabled to classify the chromosomes by number and size, being three large, eight medium and one small heterochromosome. The three largest chromosomes and the heterochromosomes showed heteropyknotic chromatin in meiosis. The heterochromosomes in 8.05% of the cells in metaphase I behaved as pseudobivalents, contrasting with 91.95% of the cells with individualized sex chromosomes, confirming the achiasmatic nature of these chromosomes. However, the pseudobivalents occurred prominently in metaphase II (78.38%), this fact probably is related to the post-reductional nature of the sex chromosomes. The nucleolus in T. melanosoma persisted until the diplotene phase after which it began to fragment. Nucleolar corpuscles were observed in metaphases I and II and during anaphases I and II, these characteristics being related to the phenomenon of nucleolar persistence. In the initial spermatids, peripheral silver ion impregnation occurred, which could be analogous to the pre-nucleolar corpuscles observed after fragmentation. Thus, this study extends our knowledge of the characteristics of triatomines, in particular, heteropyknotic degree, kinetic activity, formation of sex chromosome achiasmatic pseudobivalency, confirmation of the fragmentation phenomenon, and post-meiotic nucleolar reactivation.     

The meiotic behaviour of the XY pair in Lutreolina crassicaudata (Marsupialia: Didelphoidea)

The meiotic behaviour of the XY pair of the didelphid Lutreolina crassicaudata is analyzed by microspreading of spermatocytes for visualization of chromosomal axes and by three-dimensional reconstruction of spermatocyte nuclei from EM thin sections. The delay in pairing of sex chromosomes compared to autosomes and the absence of a synaptonemal complex between the axes of the X and Y chromosomes, already described for South American marsupials by three-dimensional reconstruction and for Australian species with synaptonemal complex microspreadings, is confirmed for this species. Sections demonstrate that at the diffuse stage and diplotene the dense plate occupies the region of the inner face of the nuclear envelope in contact with the XY body. Spreads show an structure similar in staining to the axes that becomes apparent simultaneously with the dense plate, called a balloon. The mechanism of XY pairing during meiotic prophase appears to be common to American and Australian marsupials as the same morphological pattern is found in all the species described. This mechanism is different from the way of pairing and segregation known for eutherian sex chromosomes.     

Genotypes affecting the condensation and transmission of heterochromtic B chromosomes in the mealybug Pseudococcus affinis

In male mealybugs (Pseudococcide: Homoptera) the set of chromosomes of paternal origin becomes heterochromatic (H) and genetically inactive in early embryogenesis. During spermatogenesis the two sets segregate and only the meiotic products with the euchromatic (E) set form sperm. Individuals of Pseudococcus affinis (Maskell) may carry a B chromosome (B) which is usually heterochromatic. During prophase I of spermatogenesis, however, the B becomes even less condensed than the E set and usually segregates with the E set. We have previously shown that natural populations of P. affinis contain genotypes that can reduce the rate of transmission (k) of Bs from more than 0.9 to less than 0.1. We now demonstrate that these genotypes suppress k either by enhancing or by preventing the decondensation of the B, which in turn affects the position of the B on the metaphase plate and its segregation. We also demonstrate that radiation-induced fragment of the B, and a piece of a B which has been translocated onto a piece of an H chromosome, retain their characteristic pattern of condensation and thus that the condensation of the B is not controlled by one or a few cis-acting centers or loci.     

A comparative study of male meiosis in Drosophila melanogaster and D. virilis

Male meiosis in D. melanogaster cytologically follows the usual pattern, whereas in D. melanogaster and in D. virilis oocytes the chromosomes clump into a karyosphere at early meiotic prophase and remain so up to metaphase I.Male meiosis in D. virilis spermatocytes has an intermediate character: a part of the chromatin clumps together in a karyosphere at early prophase, whereas the other part of the chromatin remains diffuse all through prophase. At the end of prophase, the diffuse chromatin becomes integrated into the karyosphere before metaphase I. During the meiotic divisions the chromosomes have the same clumped aspect as those in Drosophila oocytes and thus differ strikingly from the dividing chromosomes in D. melanogaster spermatocytes.In D. virilis spermatocytes the nucleolus exhibits changes during the meiotic prophase that may be related to synthetical activities. The DNA specific staining with the fluorochrome DAPI reveals the existence of extrachromosomal DNA in the later prophase. Other striking differences in meiotic events between the two Drosophila species concern the centrioles and spermiogenesis.