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Distinct lineages of the grasshopper Chorthippus parallelus (Orthoptera: Acrididae) form well-known hybrid zones (HZs) both in the Pyrenees and the Alps mountain ranges in South Europe. These HZs represent unique experimental systems to identify "key genes" that maintain genetic boundaries between emerging species. The Iberian endemism C. p. erythropus (Cpe) and the subspecies C. p. parallelus (Cpp), widely distributed throughout the rest of Europe, overlap and form the Pyrenean HZ. Both subspecies differ morphologically, as well as in behavioral, mitochondrial, nuclear, and chromosomal traits, and in the strains of the maternally transmitted bacterial endosymbiont Wolbachia infecting them. This results in either unidirectional and bidirectional cytoplasmic incompatibility between both grasshopper subspecies, pointing out that Wolbachia clearly affects gene expression in the infected individuals. Here we explore how Wolbachia may modify the expression of some major genes involved in relevant pathways in Cpp in the Pyrenean HZ. We have analyzed, through molecular biomarkers, the physiological responses in C. parallelus individuals infected by Wolbachia, with particular attention to the energy metabolism, the immune system response, and the reproduction. qPCR was used to evaluate the expression of selected genes in the gonads of infected and uninfected adults of both sexes, since this tissue constitutes the main target of Wolbachia infection. Transcriptional analyses also showed differential sex-dependent responses in most of the analyzed biomarkers in infected and noninfected individuals. We identified for the first time new sensitive biomarkers that might be involved in the reproductive barrier induced by Wolbachia in the hybrid zone., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

Recent advances in next generation sequencing (NGS) have greatly increased our understanding of non-coding tandem repeat (TR) DNA. Here we show how TR DNA can be useful for the study of hybrid zones (HZ), as it serves as a marker to identify introgression in areas where two biological entities come in contact. We used Illumina libraries to analyse two subspecies of the grasshopper Chorthippus parallelus , which currently form a HZ in the Pyrenees. We retrieved a total of 152 TR sequences, and used fluorescent in situ hybridization (FISH) to map 77 families in purebred individuals from both subspecies. Our analysis revealed 50 TR families that could serve as markers for analysis of this HZ, using FISH. Differential TR bands were unevenly distributed between chromosomes and subspecies. Some of these TR families yielded FISH bands in only one of the subspecies, suggesting the amplification of these TR families after the geographic separation of the subspecies in the Pleistocene. Our cytological analysis of two TR markers along a transect of the Pyrenean hybrid zone showed asymmetrical introgression of one subspecies into the other, consistent with previous findings using other markers. These results demonstrate the reliability of TR-band markers for hybrid zone studies.

The process of species formation is characterized by the accumulation of multiple reproductive barriers. The evolution of hybrid male sterility, or Haldane's rule, typically characterizes later stages of species formation, when reproductive isolation is strongest. Yet, understanding how quickly reproductive barriers evolve and their consequences for maintaining genetic boundaries between emerging species remains a challenging task because it requires studying taxa that hybridize in nature. Here, we address these questions using the meadow grasshopper Pseudochorthippus parallelus, where populations that show multiple reproductive barriers, including hybrid male sterility, hybridize in two natural hybrid zones. Using mitochondrial data, we infer that such populations diverged some 100,000 years ago, at the beginning of the last glacial cycle in Europe. Nuclear data show that contractions at multiple glacial refugia, and post-glacial expansions have facilitated genetic differentiation between lineages that today interact in hybrid zones. We find extensive introgression throughout the sampled species range, irrespective of the current strength of reproductive isolation. Populations exhibiting hybrid male sterility in two hybrid zones show repeatable patterns of genomic differentiation, consistent with shared genomic constraints affecting ancestral divergence or with the role of those regions in reproductive isolation. Together, our results suggest that reproductive barriers that characterize late stages of species formation can evolve relatively quickly, particularly when associated with strong demographic changes. Moreover, we show that such barriers persist in the face of extensive gene flow, allowing future studies to identify associated genomic regions., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Photovoltaic optoelectronic tweezers are a useful platform with many applications in optical manipulation and nanotechnology. They are based on electrical forces associated with the bulk photovoltaic effect presented by certain ferroelectric crystals, such as Fe doped lithium niobate. This manipulation technique has experienced huge developments in recent years, although its use in biology and biomedicine is still scarce. Recently, a novel strategy has been reported that extends the platform capabilities to the manipulation of polar droplets, such as water and aqueous bio-droplets, promising great potential for biological applications. In this work, we are taking this challenge, addressing the manipulation of cells and macromolecules contained inside the droplets by optoelectronic ferroelectric platforms. On the one hand, experiments of photoelectric induced migration of DNA and sperm droplets have been successfully developed and the corresponding droplet dynamics have been analyzed in depth. From this analysis, parameters of the biomaterial such as its concentration and its electrical charge have been evaluated, showing the sensing capabilities of the platform. In fact, the charge of sperm cells has been demonstrated to be negative, and the relative sperm concentration of the samples determined. On the other hand, experiments on the light-induced merging of two droplets have been carried out. Specifically, sperm droplets are mixed with droplets containing acridine orange, a convenient dye for visualization purposes. The spermatozoa become clearly visible in the final droplet through fluorescence imaging. The results point out the multiple possibilities of application of the optoelectronic ferroelectric platform in biology and biomedicine including the development of "lab on a chip" devices. Hence, these capabilities introduce these platforms as an efficient tool in biotechnology., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

The theory of evolution is one of the greatest scientific achievements in the intellectual history of humankind, yet it is still contentious within certain social groups. Despite being as robust and evidence-based as any other notable scientific theory, some people show a strong reluctance to accept it. In this study, we used the Measure of Acceptance of the Theory of Evolution (MATE) and Knowledge of Evolution Exam (KEE) questionnaires with university students from four academic degree programs (Chemistry, English, History, and Biology) of ten universities from Spain to measure, respectively, acceptance and knowledge of evolutionary theory among third-year undergraduate students (nMATE = 978; nKEE = 981). Results show that acceptance of evolution is relatively high (87.2%), whereas knowledge of the theory is moderate (5.4 out of 10) although there are differences across degrees (Biology>Chemistry>History>English), and even among various universities (ranging from 4.71 to 5.81). Statistical analysis reveals that knowledge of evolutionary theory among Biology students is partially explained by the relative weight of evolutionary themes within the curriculum, suggesting that an increase in the number of hours dedicated to this topic could have a direct influence on students' knowledge of it. We also found that religion may have a significant-although relatively small-negative influence on evolutionary theory acceptance. The moderate knowledge of evolution in our undergraduate students, together with the potential problem of acceptance in certain groups, suggests the need for a revision of the evolutionary concepts in the teaching curricula of our students since primary school., Competing Interests: The authors have declared that no competing interest exist.

The generation and manipulation of small aqueous droplets is an important issue for nano- and biotechnology, particularly, when using microfluidic devices. The production of very small droplets has been frequently carried out by applying intense local electric fields to the fluid, which requires power supplies and metallic electrodes. This procedure complicates the device and reduces its versatility. In this work, we present a novel and flexible, to the best of our knowledge, electrodeless optoelectronic method for the production of tiny droplets of biologically friendly aqueous fluids. Our method takes advantage of the photoinduced electric fields generated by the bulk photovoltaic effect in iron-doped lithium niobate crystals. Two substrate configurations, presenting the polar ferroelectric axis either parallel or perpendicular to the active surface, have been successfully tested. In both crystal geometries, small droplets on the femtoliter scale have been obtained, although with a different spatial distributions correlated with the symmetry of the photovoltaic fields. The overall results demonstrate the effectiveness of the optoelectronic method to produce femtoliter droplets, both with pure water and with aqueous solutions containing biological material.

Wolbachia is an intracellular endosymbiont that can produce a range of effects on host fitness, but the temporal dynamics of Wolbachia strains have rarely been experimentally evaluated. We compare interannual strain frequencies along a geographical region for understanding the forces that shape Wolbachia strain frequency in natural populations of its host, Chorthippus parallelus (Orthoptera, Acrididae). General linear models show that strain frequency changes significantly across geographical and temporal scales. Computer simulation allows to reject the compatibility of the observed patterns with either genetic drift or sampling errors. We use consecutive years to estimate total Wolbachia strain fitness. Our estimation of Wolbachia fitness is significant in most cases, within locality and between consecutive years, following a negatively frequency-dependent trend. Wolbachia spp. B and F strains show a temporal pattern of variation that is compatible with a negative frequency-dependent natural selection mechanism. Our results suggest that such a mechanism should be at least considered in future experimental and theoretical research strategies that attempt to understand Wolbachia biodiversity.

Wolbachia is a well-known endosymbiotic, strictly cytoplasmic bacterium. It establishes complex cytonuclear relations that are not necessarily deleterious to its host, but that often result in reproductive alterations favoring bacterial transmission. Among these alterations, a common one is the cytoplasmic incompatibility (CI) that reduces the number of descendants in certain crosses between infected and non-infected individuals. This CI induced by Wolbachia appears in the hybrid zone that the grasshoppers Chorthippus parallelus parallelus (Cpp) and C. p. erythropus (Cpe) form in the Pyrenees: a reputed model in evolutionary biology. However, this cytonuclear incompatibility is the result of sophisticated processes of the co-divergence of the genomes of the bacterial strains and the host after generations of selection and coevolution. Here we show how these genome conflicts have resulted in a finely tuned adjustment of the bacterial strain to each pure orthopteroid taxon, and the striking appearance of another, newly identified recombinant Wolbachia strain that only occurs in hybrid grasshoppers. We propose the existence of two superimposed hybrid zones: one organized by the grasshoppers, which overlaps with a second, bacterial hybrid zone. The two hybrid zones counterbalance one another and have evolved together since the origin of the grasshopper's hybrid zone.

Wolbachia are endosymbiotic bacteria of arthropods and nematodes that can manipulate the reproduction of various host organisms to facilitate their own maternal transmission. Moreover, Wolbachia's presence in host germ cells may contribute to the many cases of lateral gene transfer from Wolbachia to host genomes that have been described. A previous study in Chorthippus parallelus, a well-known orthopteroid forming a hybrid zone in the Pyrenees, identified Wolbachia sequences from two major supergroups in the genomes of infected and uninfected Chorthippus parallelus parallelus (Cpp) and Chorthippus parallelus erythropus (Cpe) subspecies. In this study, we map the Wolbachia genomic inserts to specific regions on the chromosomes of Cpp and Cpe by fluorescent in situ hybridization (FISH) using tyramides to increase the accuracy and detection of these insertions. Additionally, we consider some of the possible roles that these bacterial inserts play in the organization and function of the grasshopper genome, as well as how they can serve as markers for phylogenetic relationships of these organisms.

Hybrid zones and the consequences of hybridization have contributed greatly to our understanding of evolutionary processes. Hybrid zones also provide valuable insight into the dynamics of symbiosis since each subspecies or species brings its unique microbial symbionts, including germline bacteria such as Wolbachia, to the hybrid zone. Here, we investigate a natural hybrid zone of two subspecies of the meadow grasshopper Chorthippus parallelus in the Pyrenees Mountains. We set out to test whether co-infections of B and F Wolbachia in hybrid grasshoppers enabled horizontal transfer of phage WO, similar to the numerous examples of phage WO transfer between A and B Wolbachia co-infections. While we found no evidence for transfer between the divergent co-infections, we discovered horizontal transfer of at least three phage WO haplotypes to the grasshopper genome. Subsequent genome sequencing of uninfected grasshoppers uncovered the first evidence for two discrete Wolbachia supergroups (B and F) contributing at least 448 kb and 144 kb of DNA, respectively, into the host nuclear genome. Fluorescent in situ hybridization verified the presence of Wolbachia DNA in C. parallelus chromosomes and revealed that some inserts are subspecies-specific while others are present in both subspecies. We discuss our findings in light of symbiont dynamics in an animal hybrid zone.

Wolbachia is an obligate endosymbiont whose spread depends mainly on its capacity to alter host reproduction by, for instance, cytoplasmic incompatibility. Several mathematical models have been developed to explain the dynamics of bacterial spread, because of its applied interest. However, some aspects of the host's and bacterium's biology have not been considered in modelling: for instance, changes in Wolbachia proportions during the host's life cycle have been observed in several species, including Drosophila sp., Nasonia sp. and Aedes sp. (Diptera), but also in the grasshopper Chorthippus parallelus (Orthoptera), the species studied in this article. These changes influence the proportion of incompatible crosses and, consequently, infection prevalence in subsequent generations. In this paper, we are interested in ascertaining whether these changes in the infection proportions during the host's life cycle can influence the dynamics of the spread of these bacteria. We have examined its consequences using a mathematical model to predict the evolution of Wolbachia infection frequencies. The simulations were validated by experimental field data from C. parallelus. The main outcome is that those changes above mentioned might affect long-term infection spread, with possible consequences for the current distribution of Wolbachia and the way it affects its host's reproduction.

We evaluate for the first time the effect of Wolbachia infection, involving two different supergroups, on the structure and dynamics of the hybrid zone between two subspecies of Chorthippus parallelus (Orthoptera) in the Pyrenees. Wolbachia infection showed no effects on female fecundity or a slight increment in females infected by F supergroup, although in the last case it has to be well established. Cytoplasmic incompatibility (CI) is confirmed in crosses carried out in the field between individuals from a natural hybrid population. This CI, registered as the relative reduction in embryo production (sh ), was of sh = 0.355 and sh = 0.286 in unidirectional crosses involving B and F supergroups, respectively. CI also occurred in bidirectional crosses (sh = 0.147) but with a weaker intensity. The transmission rates of the two Wolbachia strains (B and F) were estimated by the optimization of a theoretical model to reach the infection frequencies observed in certain population. To fit this scenario, both supergroups should present transmission rates close to 1. Further, we have simulated the infection dynamics, and hence, the capacity of Wolbachia to structure the population of the host insects and to affect to reproduction and genetic introgression in the hybrid zone. This represents a first example of the influence of Wolbachia in an insect natural hybrid zone., (© 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.)

This chapter describes the various methods derived from the protocol of standard fluorescent in situ hybridization (FISH) that are used in human, animal, plant, and microbial studies. These powerful techniques allow us to detect and physically map on interphase nuclei, chromatin fibers, or metaphase chromosomes probes derived from single-copy genes to repetitive DNA sequences. Other variants of the technique enable the co-localization of genes and the overall comparison of the genome among individuals of the same species or of different taxa. A further variant detects and localizes bacteria on tissues and cells. Overall, this offers a remarkable multiplicity of possible applications ranging from strict physical mapping, to clinical and evolutionary studies, making it a powerful and informative complement to other molecular, functional, or genomic approaches.

We have recently detected the endosymbiont Wolbachia in multiple individuals and populations of the grasshopper Chorthippus parallelus (Orthoptera: acrididae). This bacterium induces reproductive anomalies, including cytoplasmic incompatibility. Such incompatibilities may help explain the maintenance of two distinct subspecies of this grasshopper, C. parallelus parallelus and C. parallelus erythropus, which are involved in a Pyrenean hybrid zone that has been extensively studied for the past 20 years, becoming a model system for the study of genetic divergence and speciation. To evaluate whether Wolbachia is the sole bacterial infection that might induce reproductive anomalies, the gonadal bacterial community of individuals from 13 distinct populations of C. parallelus was determined by denaturing gradient gel electrophoresis analysis of bacterial 16S rRNA gene fragments and sequencing. The study revealed low bacterial diversity in the gonads: a persistent bacterial trio consistent with Spiroplasma sp. and the two previously described supergroups of Wolbachia (B and F) dominated the gonad microbiota. A further evaluation of the composition of the gonad bacterial communities was carried out by whole cell hybridization. Our results confirm previous studies of the cytological distribution of Wolbachia in C. parallelus gonads and show a homogeneous infection by Spiroplasma. Spiroplasma and Wolbachia cooccurred in some individuals, but there was no significant association of Spiroplasma with a grasshopper's sex or with Wolbachia infection, although subtle trends might be detected with a larger sample size. This information, together with previous experimental crosses of this grasshopper, suggests that Spiroplasma is unlikely to contribute to sex-specific reproductive anomalies; instead, they implicate Wolbachia as the agent of the observed anomalies in C. parallelus.

The cytoplasmic incompatibility induced by the bacterial endosymbiont Wolbachia is attributed to chromatin modification in the sperm of infected individuals and is only 'rescued' by infected females after fertilization. Chorthippus parallelus is a grasshopper with 2 subspecies that form a hybrid zone in the Pyrenees in which this Wolbachia-generated cytoplasmic incompatibility has recently been described. The analysis of certain cytogenetic traits (sex chromosome-linked heterochromatic bands, nucleolar organizing region expression, spermatid size and morphology, and number of chiasmata formed) in pure and hybrid Chorthippus parallelus that are infected and not infected by this bacterium indicates that the infection affects some of these traits and, in the case of the spermatids, reveals a synergism between the infection and the hybrid condition. These results are interpreted as being secondary effects of the chromatin modification induced by Wolbachia which thereby support this model of modification/rescue. The possible effects of these cytogenetic variations on affected individuals are also considered., (Copyright © 2012 S. Karger AG, Basel.)

Two subspecies of the meadow grasshopper Chorthippus parallelus meet in a secondary hybrid zone in the Pyrenees. We have recently detected Wolbachia infection in this extensively studied species. C. p. parallelus (Cpp) and C. p. erythropus (Cpe) harbour bacteria from the B and F supergroups, but they differ noticeably in the incidence and type of infection present in their populations. We can distinguish different regional infection patterns that are associated with the distribution of pure and hybrid C. parallelus individuals. The northern pattern of low-level infection characterizes Cpp populations, whereas the southern pattern of high-level infection affects Cpe locations. These patterns converge in the hybrid zone and generate a third infection pattern featuring an extremely high degree of co-infection with the two Wolbachia types in hybrids. The distribution of Wolbachia among the studied populations encourages us to consider the possibility that this bacterium has a significant influence on the origin, maintenance and dynamics of this hybrid zone, given the reproductive alterations that are often associated with Wolbachia.

Several species of the South American genus Akodon present fully fertile XY females besides XX ones. To analyze the possibility of a Sry mutation as the cause of sex reversal in A. azarae and A. boliviensis, we determined the sequence of the Sry gene in 2 males and 3 XY females from each of these species. The Sry gene sequence was also studied in A. dolores, a species that does not have XY females. In inter-specific comparisons, the percentage identities with respect to the region analyzed varied between 96.8% and 97.9%. An ORF of 543 nucleotides was identified, and the predicted Sry proteins comprised 180 amino acids, with an HMG domain of 83 amino acids. Our results indicate that female sex reversal in A. azarae and A. boliviensis cannot be explained by sequence differences in the Sry region analyzed here, which includes the complete ORF and, together with previous results concerning the inheritance of the XY condition, show that Sry mutation is not the basis of sex reversal., (Copyright 2010 S. Karger AG, Basel.)

A study of the variation in pattern and frequency of constitutive heterochromatin and nucleolar organizing regions of the X chromosomes of male Chorthippus parallelus grasshoppers in 25 populations within the Iberian peninsula requires us to revise our interpretation of the biogeography and evolutionary history of this species. Hybridization between the subspecies Cp erythropus and Cp parallelus, previously only known from populations in the Pyrenean cols, is shown to extend at least 400 km further into north-west Spain. A novel X-chromosome variant is described that appears to be close to fixation in 18 populations, mainly from the centre and south of Spain. Our findings indicate a possible independent origin for each of three distinct, nonderivative X variants present in Spain: the northern Cp erythropus and Cp parallelus variants, and a central-southern Cp erythropus variant. The first two are distinguished by interstitial and distal C bands, respectively, whereas the central-southern form has neither. This central-southern form is probably the current representative of the ancestral Iberian X variant. The pattern of variation supports the hypotheses of multiple refugia for Iberian populations and that more hybrid zones exist between these chromosomal variants.

In mouse epidermal carcinogenesis, the latest stage of malignant progression involves the transition from squamous cell carcinoma to a highly aggressive type of tumor with spindle morphology. In this work, we have isolated a minor epithelial cell subpopulation (CarC-R) contained in the highly malignant spindle carcinoma cell line CarC. CarC-R exhibited a drastic reduction in tumorigenicity when compared with CarC, but CarC-R-induced tumors were mainly sarcomatoid, although they subsequently reverted to the epithelial phenotype when tumor explants were recultured in vitro. Several single-cell clones with either stable epithelial or fibroblastic phenotypes were isolated from an explanted CarC-R tumor (CarC-RT). All these cell lines contained the same specific point mutation in H-Ras codon 61, but while CarC spindle cells had lost the normal H-Ras allele, it was retained in CarC-R- and CarC-RT-derived cell lines. Furthermore, CarC cells have inactivated p16INK4a and p19INK4a/ARF transcription, while CarC-R and CarC-RT clones expressed p19 mRNA and protein but not p16. Altogether, these results suggest that CarC-R represents a precursor stage to CarC in malignant progression. Spectral karyotyping analysis revealed that CarC-R was highly aneuploid and contained many chromosomal abnormalities. In contrast, CarC had a diploid or tetraploid modal chromosome number and contained a specific T(14;15) translocation in all of the analysed metaphases. The T(14;15) translocation was present in only a minority (1.9%) of CarC-R cells, but it was widely spread in CarC-RT and its derived cell clones, regardless of their epithelial or fibroblastic phenotype, indicating that T(14;15) segregates with malignancy.

Numerous non-rigid and bow-shaped cationic fluorochromes exist which bind to chromatin DNA, but only some of them are able to selectively label pericentromeric heterochromatin (C-bands) in metaphase chromosomes. Likewise, some DNA ligands allow the recognition of adenine-thymine sequences establishing hydrogen bonds with acceptor atoms in the minor groove. In the present study, we have employed cationic fluorochromes based on a variety of chemical groups to analyze comparatively their potential to demonstrate C-bands, as well as the relationship between this feature and structural parameters of fluorochromes such as curvature radius and hydrogen-bonding ability. Only fluorochromes that bind DNA by hydrogen bonds demonstrated the characteristic C-banding pattern in mouse metaphase chromosomes (together with a weak G/Q-banding), whereas the other (non-hydrogen-bonding) cationic fluorochromes produced uniform emission without any visible banding.

The distribution of the vertebrate telomeric sequence T2AG3 in three species of the rodent genus Akodon was examined by FISH with a peptide nucleic acid probe. In addition to the expected telomeric hybridization, non-telomeric signals were observed in the three species. In A. dolores, centromeric signals were visible in two of the four biarmed autosome pairs featuring Robertsonian polymorphism, indicating the retention of at least part of the telomeric sequences during the fusion process, and an interstitial signal of lower intensity was observed in the short arm of another. In A. boliviensis, a strong signal was observed near the centromeric end of the first chromosome pair. The first pair of A. azarae (homologous to the first pair of A. boliviensis) showed a similar but markedly amplified signal, and a subcentromeric signal in the X chromosome corresponding to a heterochromatic region; additionally, interstitial signals of lower intensity were present in one to four chromosomes in the majority of cells examined., (Copyright 2004 S. Karger AG, Basel)

The grasshopper species Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) form a hybrid zone in northern Spain. These species probably diverged while isolated in southern refugia during one of the recent ice ages, and are clearly distinguished by morphology and male calling song. However, in contrast to other Chorthippus taxa that form hybrid zones in Europe, these two species cannot be reliably distinguished on the basis of characteristics of the karyotype such as heterochromatin banding patterns and composition, as revealed by C-banding and fluorochrome staining. Silver staining also reveals the presence of two autosomal nucleolar organiser regions (NORs) in both species. However, differentiation between C. brunneus and C. jacobsi was revealed on the X chromosome using fluorescent in situ hybridisation (FISH). C. brunneus individuals showed additional rDNA sequences on the X chromosome that were not observed in any C. jacobsi individuals. These sequences are not transcribed, indicating either mutational silencing of an ancestral NOR on the X chromosome, or the transposition of non-functional sequences from the autosomes. The implications of these results for the evolution of NOR number in Chorthippus are discussed.

N-Bromosuccinimide (NBS) is a known protein reagent able to modify amino acids and proteins, resulting in oxidation of tryptophan, tyrosine and histidine residues, as well as sulfhydryl, alcohol and phenol groups. These properties make NBS a suitable reagent to selectively block certain amino acid residues in biochemistry, and also permit the histochemical detection of proteins by oxidative deamination followed by the Schiff reaction. In this paper we show that, under ultraviolet excitation, NBS selectively reveals the cytoplasmic granules of mammalian eosinophils and chicken heterophils, rendering considerable white--blue fluorescence, in a remarkable fluorogenic reaction which rapidly increases at the beginning of the observation. This emission slightly decays afterwards and then remains almost stable still yielding a high level of emission after 10min of continuous excitation. Possible mechanisms underlying these results are discussed and we propose NBS as a very suitable fluorogenic reagent for the microscopical detection and analysis of proteins.

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric heterochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.

We describe the fluorescence properties and cytochemical applications of the aromatic diamidine M&B 938. Treatment of cell smears (chicken blood, Ehrlich ascites tumor, rat bone marrow, mouse mast cells, and Trypanosoma cruzi epimastigotes) with aqueous solutions of M&B 938 (0.5-1 microgram/ml at pH 6-7; UV excitation) induced bright bluish-white fluorescence in DNA-containing structures (interphase and mitotic chromatin, AT-rich kinetoplast DNA of T. cruzi), which was abolished by previous DNA extraction. DNA was the unique fluorescent polyanion after staining with M&B 938 at neutral or alkaline pH, other polyanions such as RNA and heparin showing no emission. M&B 938-stained mouse metaphase chromosomes revealed high fluorescence of the AT-rich centromeric heterochromatin, and strong emission of heterochromatin in human chromosomes 1, 9, 15, 16, and Y was found after distamycin A counterstaining. On agarose gel electrophoresis, M&B 938-stained DNA markers appeared as fluorescent bands. The 1.635-KBP fragment from DNA ladder revealed a higher emission value than that expected from linear regression analysis. Spectroscopic studies showed bathochromic and hyperchromic shifts in the absorption spectrum of M&B 938 complexed with DNA, as well as strong enhancement of fluorescence at 420 nm. In the presence of poly(dA)-poly(dT), the emission of M&B 938 was 4.25-fold higher than with DNA; no fluorescence was observed with poly(dG)-poly(dC). Experimental results and considerations of the chemical structure suggest that the minor groove of AT regions of DNA could be the specific binding site for M&B 938, which shows interesting properties and useful applications as a new DNA fluorochrome.

The Ribosomal DNAs of Helianthus annuus and H. argophyllus were analysed. Total DNA from single individuals of six cultivated lines, one wild ecotype ofH. annuus, and three ecotypes of H. argophyllus, were digested with various restriction enzymes. Hybridisation of Southern blots with sunflower ribosomal probes containing most of the interspacer regions (R3) or the 25 s coding region (R2) reveals different patterns from those expected: while no difference between H. annuus and H. argophyllus had been observed in previous rDNA RFLP analysis, our study clearly distinguished the two species on the basis of two different patterns when using R3 and BamHI, BstYI, or EcoRI/BamHI. Furthermore, the sum of the fragment weights of the BamHI restriction patterns was much greater than that of the rDNA entire unit-weight space. The co-existence of different rDNA units within single individuals is proposed as a model to explain these results. Four rDNA units were distinguished, which differed in their state of methylation and by the presence of mutations at two BamRI restriction sites. H. annuus individuals displayed two types of rDNA units while H. argophyllus individuals displayed four types.

C-banded slides stained with DAPI or chromomycin A3 show different banding patterns between human and L929 mouse cell line metaphase chromosomes, which are also different from those obtained with standard Giemsa C-banding or fluorochrome staining. Human metaphase chromosomes pretreated for C-banding and stained with DAPI show simultaneous C- and DA-DAPI banding patterns, whilst the mouse metaphase chromosomes show both C-banding and G/Q banding like patterns. However, the chromomycin A3 staining of pre-C-banded metaphase chromosomes reveals conspicuous R-banding in man that is absent in mouse. Chromatin species-specific structural factors would explain these results, which prevent simple comparisons of R-, G-, and C-bands among different organisms. The markers induced by this technique may be of practical use for chromosome identification in human-mouse somatic cell hybridization cultures.

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pretreatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.

Mitotic chromosomes from individuals of a Pyrenean hybrid zone between two subspecies of the grasshopper Chorthippus parallelus have been pretreated as for C-banding and subsequently stained with 4,6-diamidino-2-phenylindole (DAPI) and (or) Chromomycin A3 (CMA). The results, which are different from those obtained with Giemsa C-banding or standard DAPI - CMA treatments show (i) hidden heterochromatic heterogeneity that may be correlated with the existence of distinct families of repetitive DNAs, (ii) information about the possible independent origin of the three detected types of heterochromatin, and (iii) a further marker difference between the sex chromosomes of these two subspecies. This last result leads us to discuss the possible differential rates of evolution of sex chromosomes and autosomes in these subspecies and provides us with a new tool for the study of the structure and dynamics of this hybrid zone.

The employment of certain DNA-specific fluorescent stains on unbanded and C-banded chromosomes of two species of grasshoppers shows remarkable differences among C-heterochromatic regions supposed to be similar in their base pair composition, according to their response to the standard fluorescence techniques. The possible interspersion of the opposite DNA base pairs in these regions as well as the role played by proteins in chromosome banding are discussed.

The fluorescent properties of the antibiotic distamycin A were investigated in a range of materials including Trypanosoma cruzi epimastigotes, chicken erythrocytes, calf thymus DNA and synthetic polynucleotides using both microscopic and spectroscopic techniques. A bright blue-white fluorescence was observed from kinetoplast DNA and chromatin after treatment with distamycin A under ultraviolet (365 nm) excitation. Considerable enhancement of distamycin A fluorescence (emission peak at 455 nm under 320-340 nm excitation) was found in the presence of DNA and poly(dA-dT).poly(dA-dT). We discuss a possible explanation for this unexpected fluorescent emission, as well as its implications for microscopic and fluorimetric studies.

Fixed mitotic chromosomes of A. tornosi have been analysed by means of C-banding, DA-DAPI and Chromomicin A3 fluorescence, as well as by digestion in situ with Alu I, Hae III, Hinf I and Hind III restriction endonucleases. From the results obtained at least nine types of chromatin can be distinguished in A. tornosi. Some C-band positive areas (constitutive heterochromatin) which show a characteristic fluorescence pattern are digested by specific endonucleases, whilst others are undigested. C-band negative areas (euchromatin) are digested by some restriction endonucleases but not by others. Regions digested are supposed to contain highly repetitive DNAs. It is noteworthy, however, that the heterochromatin associated with NORs is not attacked by any of the enzymes we used, while regions believed to contain AT-rich DNA (DA-DAPI positive) are digested by Hae III that cleaves the GG decreases CC base sequence target.

The fluorescence analysis carried out in Triturus alpestris alpestris and Triturus alpestris cyreni subspecies has revealed differences related to the content and distribution of AT-rich, GC-rich and non-fluorescent heterochromatic bands. These results provide new evidence on the chromosome differentiation undergone in their phylogenetic splitting.