Ellen M. Rasch

Post-zygotic modifications and intra- and inter-individual nucleolar organizing region variations in fish: report of a case involvingLeporinus friderici

Silver nitrate staining, a rapid and efficient method, has proven to be excellent for nucleolar organizing region (NOR) studies in fish. Some fish appear to have only two NOR-bearing chromosomes in their karyo-type, whereas others probably have several. In the present study we analyzed the NORs ofLeporinus friderici, a species that, on the basis of previous studies, has been considered as representative of species with NORs carried by a single chromosome pair. The analyses were performed by a combination of three methods,i.e. silver nitrate staining, staining with the GC-specific fluorochrome chromomycin A3, andin situ hybridization with digoxigenin-labeled probes. The results showed that, although more frequent and conspicuous in a single chromosome pair, the NORs of this species are present in multiple chromosomes. Intra- and inter-individual variations observed by the three methods strongly suggest the occurrence of post-zygotic modifications involving NORs. NOR identification in fish, almost exclusively performed by the silver nitrate method, is currently being re-evaluated by methods such as chromomycin A3 staining andin situ hybridization, which may provide important information leading to a better understanding of chromosome evolution in these animals.

Reproductive behavior and the maintenance of all-female Poecilia

SynopsisThere are four members involved in the breeding complexes of poeciliid fishes found in the freshwaters of northeastern Mexico: males and females of a bisexual species, and diploid and triploid unisexuals. Both unisexuals reproduce by gynogenesis, i.e., an asexual type of reproduction where the sperm triggers egg development but the male genome is excluded to produce clonal offspring. The three types of females are closely related, which suggests that they are potential competitors since all three require the service of the same males. The potential for competition is compounded by a highly skewed sex ratio in favor of females. On the average the unisexuals comprise about 30% of the Poecilia females. This high frequency coupled with a close genetic relatedness to their bisexual hosts, raises the question of how the unisexuals are maintained in nature.Other investigators who work with bisexual/unisexual complexes in the related genus, Poeciliopsis, have postulated that male dominance hierarchies are responsible for restricting the access of subordinate males to their conspecific females. Consequently, these subordinate males mate with unisexual females. The current report tests whether or not this hypothesis applies to bisexual/unisexual complexes of Poecilia.We have found that linear dominance hierarchies appear to function in the defense of home ranges and do not restrict access of males to females. Dominant males exhibit less mating activity than subordinate males towards females. Previous reports showed that males are reproductively competent throughout the year, whereas females show striking asynchrony in their reproductive readiness. Such asynchrony limits the proportion of receptive females at any one time. Consequently, there are more males ready to mate than there are females receptive to their mating attempts. This may lead to mating frenzies. We postulate that these indiscriminate matings maintain the fertility of both unisexuals. When the relative reproductive outputs of adult females are compared, both unisexuals appear as fit as their bisexual congeners.

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish, Poecilia formosa and its related, triploid unisexuals

SummaryAmounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of 3H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.

Reduction of competition between bisexual and unisexual females ofPoecilia in northeastern Mexico

SynopsisBreeding compexes of poeciliid fishes with a bisexual and two unisexual species were studied for mechanisms permitting Sympatric coexistence. The unisexuals are gynogenetic and thereby sexually dependent on the males of the bisexual species for sperm to initiate development, but inheritance is entirely maternal.Bisexual females are more abundant in headwater localities; unisexuals increase in downstream localities. Males were 10 – 18% of the total poeciliid population, regardless of the relative proportions of bisexual to unisexual females. Downstream localities were typified by greater habitat diversity, including a variety of backwater pools. The unisexuals showed a marked preference for such pools. Both field and laboratory studies showed that all three types of females as well as males preferred shaded areas with a gravel substrate.Although the four types of fish were found together, nearest neighbour data indicated that each type of female preferred its own kind. Males courted throughout the year and were indiscriminate in their choice of mates. Despite the skewed sex ratio, males were not in short supply because only a few females were sexually receptive at a given time. No significant differences existed between bisexuals and unisexuals in their relative reproductive outputs, but they were asynchronous.

Apomictic Reproduction in the Amazon Molly, Poecilia formosa, and Its Triploid Hybrids

The Amazon molly, Poecilia formosa, initially described in 1859 by Girard, is the first vertebrate in which unisexuality was recognized (Hubbs and Hubbs, 1932). Poecilia formosa is intermediate in form between two sexual species, Poecilia latipinna (LeSueur) and Poecilia mexicana Steindachner and is thought to have arisen in nature as a hybrid of these species (Hubbs and Hubbs, 1932; Hubbs, 1955, 1964; Abramoff et al., 1968; Prehn and Rasch, 1969; Balsano et al., 1972). Poecilia formosa is not a true thelytokous parthenogen as defined by White (1973); instead, it reproduces by gynogenesis, a mechanism that is sperm dependent. Males of related sexual species provide sperm, which serve only to activate the egg. Functional syngamy does not occur. Thus, developing embryos are derived from diploid ova that contain only maternal chromosomes and each daughter is a genetic copy of its mother (Meyer, 1938; Hubbs and Hubbs, 1946; Kallman, 1962a,b,c; Hubbs, 1964; Rasch et al., 1965; Darnell et al.,1967; Schultz, 1969; Uzzell, 1970; White, 1973). The obligatory dependence on sperm that is a hallmark of gynogenetic reproduction compels P. Formosa to behave in nature as a sexual parasite on closely related, sympatric bisexual species (Hubbs, 1964). Although a variety of poeciliid males can “father” P. formosa in the laboratory (Hubbs and Hubbs, 1946), the survival and success in nature of these all-female forms require their coincident distribution with at least one of the bisexual host species.

Determination of DNA content in the nurse and follicle cells from wild type and mutant Drosophila melanogaster by DNA-Feulgen cytophotometry

SummaryDNA replication patterns in the nurse and follicle cells of wild type and a female sterile mutant, fs(1)1304, of Drosophila melanogaster have been studied by DNA-Feulgen cytophotometry, using a cell dispersal technique that allowed the measurement of DNA amounts in individual nuclei from egg chambers of known developmental stages. DNA-Feulgen values associated with various ovarian nuclei from egg chambers at different stages of development were used to assess a base line DNA content for ovarian tissues and to estimate the extent of DNA replication in the nurse cells and follicle cells of growing and mature egg chambers. Our data show that both the nurse and follicle cells undergo multiple cycles of endonuclear DNA replication and that there may be selective amplification as well as underreplication by portions of the genome in these highly polyploid, ovarian cells. Alternative models are proposed to account for the DNA replication patterns observed. Comparisons of DNA-Feulgen levels in wild type ovarian nuclei with those found for the fs(1)1304 mutant and its heterozygote in the balanced stock fs/FM3, show that equivalent DNA levels are present in follicle cell nuclei from all three types of females. Nurse cell nuclei in the homozygous fs stock, however, fail to achieve the same high DNA levels observed in both fs/FM3 and wild type nurse cell nuclei. Although the nuclei of follicle cells in ovaries from fs/fs females appear morphologically like those surrounding egg chambers in wild type ovaries, nurse cell nuclei from mutant females show a more compacted organization of their chromatin than found for nurse cell nuclei from wild type ovaries at similar developmental stages. Our findings suggest that a major effect of the fs(1)1304 mutation may be on the coiling behavior of chromatin and the conformation of DNA-protein moieties in both nurse cell and follicle cell nuclei. These changes in chromatin structure apparently are manifest by perturbations in DNA replication patterns and normal gene function in these biosynthetically active cells.

Cytophotometric evidence of variation in genome size of desmognathine salamanders

SummaryThe amount of DNA per haploid genome, the C-value, is often directly correlated with nuclear and cell volume, but inversely correlated with cell replication rate. Also, rates of cellular growth sometimes appear to be correlated with organismal developmental rates and life history patterns. Among vertebrates, salamanders exhibit the greatest variation in genome size. In the present study we have examined interspecific and intraspecific variation in blood cell DNA levels in the genus Desmognathus, which shows greater variation in life history traits than any other salamander genus. Specimens of Desmognathus quadramaculatus, D. Monticola, D. ochrophaeus and D. wrighti were collected from nature at two localities in the southern Appalachian Mountains. Estimates of genome size in pg of DNA were obtained from blood smears by DNA-Feulgen cytophotometry, using erythrocyte nuclei of Xenopus laevis as an internal reference standard of 6.35 pg DNA per cell. C-values of Desmognathus are the smallest in the order Caudata. Although significant variation in DNA levels was found among the four species, the differences were small, and do not support previously proposed relationships between C-value and life-history variation.

Cytophotometric studies on cells from the ovaries of otu mutants of Drosophila melanogaster

SummaryAmounts of chromosomal DNA were estimated for Feulgen-stained, ovarian cells from flies carrying certain mutant alleles of the otu (ovarian tumor) gene. Epithelial sheath cells and lumen cells were found to contain the diploid (2C) amount of DNA and therefore served as internal, cytophotometric standards. Mitotically active follicle cells over young tumors-from homozygous otu1 females contained either the 2C or 4C amounts of DNA; whereas, the tumor cell population contained 2C, 4C and 8C nuclei and many intermediate values. Egg chambers also occur in homozygous otu7 females. Follicle cells above these oocytes undergo a maximum of four cycles of endomitotic DNA replication. The accompanying nurse cells (PNC) contain polytene chromosomes. These undergo a maximum of 12 endonuclear replication cycles. The PNCs show the expected levels of DNA for the first 6 cycles and the fraction failing to replicate during subsequent cycles may be as small as 10%. Lower than expected levels of DNA were detected in PNCs from an otu1/otu3 ovary, reflecting roughly 20% underreplication. The latter PNCs may have been interrupted before DNA synthesis was concluded. No simple model of genomic underreplication accounts for the several different patterns of DNA behavior observed for various otu mutants.

Sperm availability in naturally occurring bisexual-unisexual breeding complexes involvingPoecilia mexicana and the gynogenetic teleost,Poecilia formosa

SynopsisTesticular maturation indices (TMI) were determined for wild-caught males ofPoecilia mexicana by quantitative analysis of the frequency of ongoing stages of spermatogenesis and spermatid differentiation in sections of testes stained with the Feulgen reaction for DNA. In nature, males maintain essentially constant levels of sperm production throughout the year and show no significant variations in mean TMI values associated with season, microhabitat, standard body length, or rank in male dominance hierarchies. Winter males or males isolated from females in the laboratory show accumulation of mature spermatophores. These findings suggest that fluctuations in the microstructure of wild populations ofPoecilia from northeastern Mexico may be due primarily to differences in the temporal scheduling of female reproductive cycles and not to limitations imposed by sperm availability or male reproductive competence. The data are discussed in terms of competitive interactions within unisexual-bisexual breeding complexes involving the Amazon mollyP. formosa, its related triploid hybrids, and the bisexual species,P. mexicana.

Nucleoprotein cytochemistry during oogenesis in a unisexual fish,Poecilia formosa

SummaryCytochemical methods and electron microscopy were used to study changes in the chemical composition of nuclear, nucleolar and perinuclear bodies during the early stages of oocyte development inPoecilia formosa, an apomictic species of fish that produces only female offspring. Prominent accumulations of ribonucleoprotein (RNP) occur in nucleoli and appear on either side of the nuclear envelope during diplotene. In certain planes of section, RNP material seems to be in transit across this interface.En bloc acid extractions or RNAse treatment abolished basophilia and markedly reduced the electron density of both nucleoli and cytoplasmic nucleolar-like bodies. DNA-specific fluorescent probes such as mithramycin failed to reveal nucleolar cores in poeciliid oocytes, although the same procedures showed unequivocal localization of GC-rich DNA cores within multiple nucleoli of diplotene oocytes fromXenopus laevis or the rainbow trout,Salmo gairdneri. Also, cytological hybridization studies, utilizing [3H]rRNA as a probe for nucleolar oocytes. Feulgen-stained pachytene oocytes ofP. formosa have twice the number of chromosome strands seen in similar stages of oocytes from two, related bisexual species,P. mexicana andP. latipinna. Although the bivalent nature of these chromosomes could not be resolved with the light microscope, configurations resembling, but not identical to, synaptonemal complexes were identified by electron microscopy.