Tandem Repeat DNA Provides Many Cytological Markers for Hybrid Zone Analysis in Two Subspecies of the Grasshopper Chorthippus parallelus.

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.

Extensive introgression at late stages of species formation: Insights from grasshopper hybrid zones.

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.

Optoelectronic manipulation of bio-droplets containing cells or macromolecules by active ferroelectric platforms.

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.

Acceptance and knowledge of evolutionary theory among third-year university students in Spain.

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.

Optoelectronic generation of bio-aqueous femto-droplets based on the bulk photovoltaic effect.

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.

Geographic and Temporal Variation of Distinct Intracellular Endosymbiont Strains of Wolbachia sp. in the Grasshopper Chorthippus parallelus: a Frequency-Dependent Mechanism?

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.

Chorthippus parallelus and Wolbachia: Overlapping Orthopteroid and Bacterial Hybrid Zones.

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.

Chromosomal localization of Wolbachia inserts in the genomes of two subspecies of Chorthippus parallelus forming a Pyrenean 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.

Wolbachia co-infection in a hybrid zone: discovery of horizontal gene transfers from two Wolbachia supergroups into an animal genome.

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.

FISH methods in cytogenetic studies.

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.

No differences in the Sry gene between males and XY females in Akodon (Rodentia, Cricetidae).

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.

Chromosomal instability and phenotypic plasticity during the squamous-spindle carcinoma transition: association of a specific T(14;15) with malignant progression.

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.

DNA-binding fluorochromes: correlation between C-banding of mouse metaphase chromosomes and hydrogen bonding to adenine-thymine base pairs.

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.

Low levels of chromosomal differentiation between the grasshoppers Chorthippus brunneus and Chorthippusjacobsi (Orthoptera; Acrididae) in northern Spain.

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.

Fluorogenic reaction of blood cells induced by N-bromosuccinimide.

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.