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Insufficient oxygen supply represents a relevant issue in several fields of human physiology and medicine. It has been suggested that the implantation of photosynthetic cells can provide oxygen to tissues in the absence of a vascular supply. This approach has been demonstrated to be successful in several in vitro and in vivo models; however, no data is available about their safety in human patients. Here, an early phase-1 clinical trial (ClinicalTrials.gov identifier: NCT03960164, https://clinicaltrials.gov/ct2/show/NCT03960164) is presented to evaluate the safety and feasibility of implanting photosynthetic scaffolds for dermal regeneration in eight patients with full-thickness skin wounds. Overall, this trial shows that the presence of the photosynthetic microalgae Chlamydomonas reinhardtii in the implanted scaffolds did not trigger any deleterious local or systemic immune responses in a 90 days follow-up, allowing full tissue regeneration in humans. The results presented here represent the first attempt to treat patients with photosynthetic cells, supporting the translation of photosynthetic therapies into clinics. Clinical Trial Registration: www.clinicaltrials.gov/ct2/show/NCT03960164, identifier: NCT03960164., Competing Interests: JTE is co-founder of SymbiOx Inc., a startup company that owns IP for the technology described here. During the conduct of the trial, MB, RC-O and FC were full-time employees of SymbiOx Inc., while AE-Z, XM and CW were part-time employees. All SymbiOx team members were financed with a R&D grant obtained from the Chilean Ministry of Economics (CORFO 18PIDE98887). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Obaíd, Camacho, Brenet, Corrales-Orovio, Carvajal, Martorell, Werner, Simón, Varas, Calderón, Guzmán, Bono, San Martín, Eblen-Zajjur and Egaña.)

For many years, parasitic B chromosomes have been considered genetically inert elements. Here we show the presence of ten protein-coding genes in the B chromosome of the grasshopper Eyprepocnemis plorans. Four of these genes (CIP2A, GTPB6, KIF20A, and MTG1) were complete in the B chromosome whereas the six remaining (CKAP2, CAP-G, HYI, MYCB2, SLIT and TOP2A) were truncated. Five of these genes (CIP2A, CKAP2, CAP-G, KIF20A, and MYCB2) were significantly up-regulated in B-carrying individuals, as expected if they were actively transcribed from the B chromosome. This conclusion is supported by three truncated genes (CKAP2, CAP-G and MYCB2) which showed up-regulation only in the regions being present in the B chromosome. Our results indicate that B chromosomes are not so silenced as was hitherto believed. Interestingly, the five active genes in the B chromosome code for functions related with cell division, which is the main arena where B chromosome destiny is played. This suggests that B chromosome evolutionary success can lie on its gene content.

Schielzeth et al. (2014) concluded that attractive grasshopper singers have significantly smaller genomes thus suggesting a possible role for sexual selection on genome size. Whereas this conclusion could still be conceivably valid, it is not supported by the data presented due to some technical flaws. In addition, the interpretation of the results, speculating on the possible presence of B chromosomes, is not justified., (© 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.)

B chromosomes constitute a heterogeneous mixture of genomic parasites that are sometimes derived intraspecifically from the standard genome of the host species, but result from interspecific hybridization in other cases. The mode of origin determines the DNA content, with the B chromosomes showing high similarity with the A genome in the first case, but presenting higher similarity with a different species in the second. The characid fish Moenkhausia sanctaefilomenae harbours highly invasive B chromosomes, which are present in all populations analyzed to date in the Parana and Tietê rivers. To investigate the origin of these B chromosomes, we analyzed two natural populations: one carrying B chromosomes and the other lacking them, using a combination of molecular cytogenetic techniques, nucleotide sequence analysis and high-throughput sequencing (Illumina HiSeq2000). Our results showed that i) B chromosomes have not yet reached the Paranapanema River basin; ii) B chromosomes are mitotically unstable; iii) there are two types of B chromosomes, the most frequent of which is lightly C-banded (similar to euchromatin in A chromosomes) (B1), while the other is darkly C-banded (heterochromatin-like) (B2); iv) the two B types contain the same tandem repeat DNA sequences (18S ribosomal DNA, H3 histone genes, MS3 and MS7 satellite DNA), with a higher content of 18S rDNA in the heterochromatic variant; v) all of these repetitive DNAs are present together only in the paracentromeric region of autosome pair no. 6, suggesting that the B chromosomes are derived from this A chromosome; vi) the two B chromosome variants show MS3 sequences that are highly divergent from each other and from the 0B genome, although the B2-derived sequences exhibit higher similarity with the 0B genome (this suggests an independent origin of the two B variants, with the less frequent, B2 type presumably being younger); and vii) the dN/dS ratio for the H3.2 histone gene is almost 4-6 times higher for B chromosomes than for A chromosome sequences, suggesting that purifying selection is relaxed for the DNA sequences located on the B chromosomes, presumably because they are mostly inactive.

Parasitic supernumerary (B) chromosomes show high capability to spread across populations. But the existence of abrupt discontinuities in their distribution demands an explanation. The grasshopper Eyprepocnemis plorans plorans harbour supernumerary chromosomes in all natural populations hitherto analyzed from the Circum-Mediterranean region, with the single exception of the headwaters of the Iberian Segura River and several of its tributaries. To ascertain the causes of this distribution pattern, we analyze here the genetic structure of five natural populations collected in this zone (two +B and three -B), by means of ISSR markers. We found significant population structure, with two kinds of populations coinciding with +B and -B ones, separated by strong barriers to gene flow. This gives strong support to the hypothesis that the non-B populations precede B origin, and that B-carrying individuals from coastal zones have been able to colonize upstream areas, until geographical barriers (usually narrow canyons and arid areas surrounding them) impeded their advance.

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

Much is known about the abundance of transposable elements (TEs) in eukaryotic genomes, but much is still unknown on their behaviour within cells. We employ here a combination of cytological, molecular and genomic approaches providing information on the intragenomic distribution and behaviour of non-long terminal repeat (LTR) retrotransposon-like elements (RTE). We microdissected every chromosome in a single first meiotic metaphase cell of the grasshopper Eyprepocnemis plorans and polymerase chain reaction (PCR) amplified a fragment of the RTE reverse transcriptase gene with specific primers. PCR products were cloned and 139 clones were sequenced. Analysis of molecular variance (AMOVA) showed significant intragenomic structure for these elements, with 4.6 % of molecular variance being found between chromosomes. A maximum likelihood tree built with the RTE sequences revealed the frequent presence of two or more elements showing very high similarity and being located on the same chromosome, thus suggesting intrachromosome movement. The 454 pyrosequencing of genomic DNA gave strong support to the microdissection results and provided evidence for the existence of 5' truncated elements. Our results thus indicate a tendency of RTE elements to reinsert into the same chromosome from where they were transcribed, which could be achieved if retrotranscription and insertion takes place immediately after transcription.

Simple sequence repeats (SSRs), also known as microsatellites, are one of the prominent DNA sequences shaping the repeated fraction of eukaryotic genomes. In spite of their profuse use as molecular markers for a variety of genetic and evolutionary studies, their genomic location, distribution, and function are not yet well understood. Here we report the first thorough joint analysis of microsatellite motifs at both genomic and chromosomal levels in animal species, by a combination of 454 sequencing and fluorescent in situ hybridization (FISH) techniques performed on two grasshopper species. The in silico analysis of the 454 reads suggested that microsatellite expansion is not driving size increase of these genomes, as SSR abundance was higher in the species showing the smallest genome. However, the two species showed the same uneven and nonrandom location of SSRs, with clear predominance of dinucleotide motifs and association with several types of repetitive elements, mostly histone gene spacers, ribosomal DNA intergenic spacers (IGS), and transposable elements (TEs). The FISH analysis showed a dispersed chromosome distribution of microsatellite motifs in euchromatic regions, in coincidence with chromosome location patterns previously observed for many mobile elements in these species. However, some SSR motifs were clustered, especially those located in the histone gene cluster.

The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements.

Cactophilic Drosophila species provide a valuable model to study gene-environment interactions and ecological adaptation. Drosophila buzzatii and Drosophila mojavensis are two cactophilic species that belong to the repleta group, but have very different geographical distributions and primary host plants. To investigate the genomic basis of ecological adaptation, we sequenced the genome and developmental transcriptome of D. buzzatii and compared its gene content with that of D. mojavensis and two other noncactophilic Drosophila species in the same subgenus. The newly sequenced D. buzzatii genome (161.5 Mb) comprises 826 scaffolds (>3 kb) and contains 13,657 annotated protein-coding genes. Using RNA sequencing data of five life-stages we found expression of 15,026 genes, 80% protein-coding genes, and 20% noncoding RNA genes. In total, we detected 1,294 genes putatively under positive selection. Interestingly, among genes under positive selection in the D. mojavensis lineage, there is an excess of genes involved in metabolism of heterocyclic compounds that are abundant in Stenocereus cacti and toxic to nonresident Drosophila species. We found 117 orphan genes in the shared D. buzzatii-D. mojavensis lineage. In addition, gene duplication analysis identified lineage-specific expanded families with functional annotations associated with proteolysis, zinc ion binding, chitin binding, sensory perception, ethanol tolerance, immunity, physiology, and reproduction. In summary, we identified genetic signatures of adaptation in the shared D. buzzatii-D. mojavensis lineage, and in the two separate D. buzzatii and D. mojavensis lineages. Many of the novel lineage-specific genomic features are promising candidates for explaining the adaptation of these species to their distinct ecological niches., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Chromosome painting with DNA probes obtained from supernumerary (B) and sex chromosomes in three species of fish genus Characidium (C. gomesi, C. pterostictum and C. oiticicai) showed a close resemblance in repetitive DNA content between B and sex chromosomes in C. gomesi and C. pterostictum. This suggests an intraspecific origin for B chromosomes in these two species, probably deriving from sex chromosomes. In C. oiticicai, however, a DNA probe obtained from its B chromosome hybridized with the B but not with the A chromosomes, suggesting that the B chromosome in this species could have arisen interspecifically, although this hypothesis needs further investigation. A molecular phylogenetic analysis performed on nine Characidium species, with two mtDNA genes, showed that the presence of heteromorphic sex chromosomes in these species is a derived condition, and that their origin could have been unique, a conclusion also supported by interspecific chromosome painting with a CgW probe derived from the W chromosome in C. gomesi. Summing up, our results indicate that whereas heteromorphic sex chromosomes in the genus Characidium appear to have had a common and unique origin, B chromosomes may have had independent origins in different species. Our results also show that molecular phylogenetic analysis is an excellent complement for cytogenetic studies by unveiling the direction of evolutionary chromosome changes.

Heterochromatin protein 1 (HP1) is a highly conserved family of eukaryotic proteins required for heterochromatic gene silencing and euchromatic gene transcription regulation. In addition, HP1 is involved in chromatin organization and protection of chromosome integrity during cell division. Here, we present a cytological and molecular analysis of the effects of HP1 knockdown in Eyprepocnemis plorans, a grasshopper species polymorphic for supernumerary heterochromatic chromosomes. Our results revealed contrasting effects of HP1 knockdown on gene activity. While the Bub1 gene decreased in expression level in HP1 knockdown animals, NOR activity, rRNA and, contrarily to previous reports in Drosophila, Hsp70 gene expression remained unchanged. Furthermore, HP1 knockdown resulted in abnormal chromatin condensation, chromosomal bridges, higher frequency of macrospermatids, loss of muscle mass and hemolymph amount as well as a low number of dividing cells and survival reduction. All these phenotypes are very likely due to the chromatin condensation disruption observed for almost all kinds of chromatin.

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.

R2 non-LTR retrotransposons exclusively insert into the 28S rRNA genes of their host, and are expressed by co-transcription with the rDNA unit. The grasshopper Eyprepocnemis plorans contains transcribed rDNA clusters on most of its A chromosomes, as well as non-transcribed rDNA clusters on the parasitic B chromosomes found in many populations. Here the structure of the E. plorans R2 element, its abundance relative to the number of rDNA units and its retrotransposition activity were determined. Animals screened from five populations contained on average over 12,000 rDNA units on their A chromosomes, but surprisingly only about 100 R2 elements. Monitoring the patterns of R2 insertions in individuals from these populations revealed only low levels of retrotransposition. The low rates of R2 insertion observed in E. plorans differ from the high levels of R2 insertion previously observed in insect species that have many fewer rDNA units. It is proposed that high levels of R2 are strongly selected against in E. plorans, because the rDNA transcription machinery in this species is unable to differentiate between R2-inserted and uninserted units. The B chromosomes of E. plorans contain an additional 7,000 to 15,000 rDNA units, but in contrast to the A chromosomes, from 150 to over 1,500 R2 elements. The higher concentration of R2 in the inactive B chromosomes rDNA clusters suggests these chromosomes can act as a sink for R2 insertions thus further reducing the level of insertions on the A chromosomes. These studies suggest an interesting evolutionary relationship between the parasitic B chromosomes and R2 elements.

The B24 chromosome variant emerged several decades ago in a Spanish population of the grasshopper Eyprepocnemis plorans and is currently reaching adjacent populations. Here we report, for the first time, how a parasitic B chromosome (a strictly vertically transmitted parasite) expands its geographical range aided by high gene flow in the host species. For six years we analyzed B frequency in several populations to the east and west of the original population and found extensive spatial variation, but only a slight temporal trend. The highest B24 frequency was found in its original population (Torrox) and it decreased closer to both the eastern and the western populations. The analysis of Inter Simple Sequence Repeat (ISSR) markers showed the existence of a low but significant degree of population subdivision, as well as significant isolation by distance (IBD). Pairwise Nem estimates suggested the existence of high gene flow between the four populations located in the Torrox area, with higher values towards the east. No significant barriers to gene flow were found among these four populations, and we conclude that high gene flow is facilitating B24 diffusion both eastward and westward, with minor role for B24 drive due to the arrival of drive suppressor genes which are also frequent in the donor population.

B chromosomes are considered to be genetically inert elements. However, some of them are able to show nucleolus organizer region (NOR) activity, as detected by both cytological and molecular means. The grasshopper Eyprepocnemis plorans shows a B chromosome polymorphism characterized by the existence of many B variants. One of them, B24, shows NOR activity in about half of B-carrying males in the Torrox population. Molecular data have suggested the recent origin for B chromosomes in this species, and on this basis it would be expected that NOR activity was widespread among the different B variants. Here we test this hypothesis in four different B chromosome variants (B1, B2, B5, and B24) from 11 natural populations of the grasshopper E. plorans covering the south and east of the Iberian Peninsula plus the Balearic Islands. We used two different approaches: (1) the cytological observation of nucleoli attached to the distal region of the B chromosome (where the rDNA is located), and (2) the molecular detection of the rDNA transcripts carrying an adenine insertion characteristic of B chromosome ITS2 sequences. The results showed NOR expression not only for B24 but also for the B1 and B2 variants. However, the level of B-NOR expression in these latter variants, measured by the proportion of cells showing nucleoli attached to the B chromosomes, was much lower than that previously reported for B24. This suggests the possibility that structural or genetic background conditions are enhancing the expressivity of the rDNA in the B24 variant.

The Ku70 protein is involved in numerous cell functions, the nonhomologous end joining (NHEJ) DNA repair pathway being the best known. Here, we report a novel function for this protein in the grasshopper Eyprepocnemis plorans. We observed the presence of large Ku70 foci on the centromeres of meiotic and mitotic chromosomes during the cell cycle stages showing the highest centromeric activity (i.e., metaphase and anaphase). The fact that colchicine treatment prevented centromeric location of Ku70, suggests a microtubule-dependent centromeric function for Ku70. Likewise, the absence of Ku70 at metaphase-anaphase centromeres from three males whose Ku70 gene had been knocked down using interference RNA, and the dramatic increase in the frequency of polyploid spermatids observed in these males, suggest that the centromeric presence of Ku70 is required for normal cytokinesis in this species. The centromeric function of Ku70 was not observed in 14 other grasshopper and locust species, or in the mouse, thus suggesting that it is an autapomorphy in E. plorans.

The grasshopper Eyprepocnemis plorans subsp. plorans harbors a very widespread polymorphism for supernumerary (B) chromosomes which appear to have arisen recently. These chromosomes behave as genomic parasites because they are harmful for the individuals carrying them and show meiotic drive in the initial stages of population invasion. The rapid increase in B chromosome frequency at intrapopulation level is thus granted by meiotic drive, but its spread among populations most likely depends on interpopulation gene flow. We analyze here the population genetic structure in 10 natural populations from two regions (in the south and east) of the Iberian Peninsula. The southern populations were coastal whereas the eastern ones were inland populations located at 260-655 m altitude. The analysis of 97 ISSR markers revealed significant genetic differentiation among populations (average G(ST) = 0.129), and the Structure software and AMOVA indicated a significant genetic differentiation between southern and eastern populations. There was also significant isolation by distance (IBD) between populations. Remarkably, these results were roughly similar to those found when only the markers showing low or no dropout were included, suggesting that allelic dropout had negligible effects on population genetic analysis. We conclude that high gene flow helped this parasitic B chromosome to spread through most of the geographical range of the subspecies E. plorans plorans.

We analyze here the presence and abundance of three types of transposable elements (TEs), i.e. Gypsy, RTE and Mariner, in the genome of the grasshopper Eyprepocnemis plorans. PCR experiments allowed amplification, cloning and sequencing of these elements (EploGypI, EploRTE5, EploMar20) from the E. plorans genome. Fluorescent in situ hybridization (FISH) showed that all three elements are restricted to euchromatic regions, thus being absent from the pericentromeric region of all A chromosomes, which contain a satellite DNA (satDNA) and ribosomal DNA (rDNA), and being very scarce in B chromosomes mostly made up of these two types of repetitive DNA. FISH suggested that EploGypI is the most abundant and EploMar20 is the least abundant, with EploRTE5 showing intermediate abundance. An estimation of copy number, by means of quantitative PCR, showed that EploGypI is, by far, the most abundant element, followed by EploRTE5 and EploMar20, in consistency with FISH results. RNA isolation and PCR experiments on complementary DNA (cDNA) showed the presence of transcripts for the three TE elements. The implications of the preferential location of these TE elements into euchromatin, the significance of TE abundance in the giant genome of this species, and a possible relationship between TEs and B chromosome mutability, are discussed.

Ninety individuals of the characid fish Astyanax fasciatus (Cuvier, 1819) were collected at Água da Madalena stream (Botucatu, São Paulo, Brazil) and analyzed for diploid chromosome number 2n and karyotype composition as well as for the chromosomal location of the 5S and 18S ribosomal DNA (rDNA). Whereas no chromosome differences were associated with sex, three different karyomorphs with diploid chromosome numbers 2n=46, 2n=48 and 2n=50 were found. No intermediate 2n numbers were discovered. The 2n=50 karyomorph showed some differences in 18S rDNA location compared to the two other karyomorphs. Finally, all specimens with the 2n=46 karyomorph showed the presence of a partly heterochromatic macro supernumerary chromosome, which was absent in all individuals with the two other karyomorphs. All these results suggest that indviduals of the three different karyomorphs are not likely to hybridize in the examined populations. Our findings strongly suggest the presence of three separate species (sensu biological species concept) easily diagnosed on the basis of differences in the diploid chromosome numbers and other chromosomal markers.

B-chromosomes are frequently argued to be genetically inert elements, but activity for some particular genes has been reported, especially for ribosomal RNA (rRNA) genes whose expression can easily be detected at the cytological level by the visualization of their phenotypic expression, i.e., the nucleolus. The B(24) chromosome in the grasshopper Eyprepocnemis plorans frequently shows a nucleolus attached to it during meiotic prophase I. Here we show the presence of rRNA transcripts that unequivocally came from the B(24) chromosome. To detect these transcripts, we designed primers specifically anchoring at the ITS-2 region, so that the reverse primer was complementary to the B chromosome DNA sequence including a differential adenine insertion being absent in the ITS2 of A chromosomes. PCR analysis carried out on genomic DNA showed amplification in B-carrying males but not in B-lacking ones. PCR analyses performed on complementary DNA showed amplification in about half of B-carrying males. Joint cytological and molecular analysis performed on 34 B-carrying males showed a close correspondence between the presence of B-specific transcripts and of nucleoli attached to the B chromosome. In addition, the molecular analysis revealed activity of the B chromosome rDNA in 10 out of the 13 B-carrying females analysed. Our results suggest that the nucleoli attached to B chromosomes are actively formed by expression of the rDNA carried by them, and not by recruitment of nucleolar materials formed in A chromosome nucleolar organizing regions. Therefore, B-chromosome rDNA in E. plorans is functional since it is actively transcribed to form the nucleolus attached to the B chromosome. This demonstrates that some heterochromatic B chromosomes can harbour functional genes.

Chromosome painting (CP) with a probe of B chromosome obtained by microdissection and fluorescence in situ hybridization (FISH) with probes of As51 satellite DNA, C( o )t-1 DNA, and 18S and 5S rDNA confirmed sharing of some repetitive DNA but not rDNA between A and B chromosomes in the fish Astyanax scabripinnis. Meiotic analysis revealed a pachytene B chromosome bivalent nearly half the size of its mitotic configuration, suggesting a self-pairing of B chromosome arms. Such an isochromosome nature of somatic B chromosome was further evidenced by CP and FISH. All the findings obtained suggest (i) intraspecific origin of B chromosome, and (ii) evolutionary enrichment of repetitive DNA classes, especially those contained in the C( o )t-1 and the As51 probes, in B chromosome. However, the precise origin of B chromosome in the present species remains to be elucidated by further molecular cytogenetic analysis because of painting of some A chromosome regions with the B chromosome-derived probe.

We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers.

B chromosomes are dispensable chromosomes found in >2000 eukaryotic species, usually behaving as genomic parasites. Most B chromosomes seem to be made up of the same kind of DNA sequences present in the A chromosomes. This sequence similarity makes it difficult to obtain specific molecular probes that may permit B-presence diagnosis without cytogenetic analysis. We have developed a sequence-characterized amplified region (SCAR) marker for B chromosomes in the grasshopper Eyprepocnemis plorans, which specifically amplifies a 1510-bp DNA fragment exclusively in B-carrying individuals. Fluorescent in situ hybridization and fiber FISH analyses showed that this marker is a tandemly repeated DNA sequence closely intermingled with 45S rDNA. PCR reactions showed the presence of SCAR-like sequences in the A chromosomes, but in two separate fragments, supporting the intraspecific origin of B chromosomes in this species. SCAR marker DNA sequence showed to be identical in B chromosome variants from several localities from Spain and Morocco, and it was very similar to those found in B chromosome variants from Greece and Armenia. This strongly suggests that this sequence was already present in the ancestral B chromosome of this species. In addition, the scarce sequence variation observed among several B variants from very distant populations suggests either a functional constraint or, more likely, a recent and unique origin for B chromosomes in this species., (© 2011 by the Genetics Society of America)

We analyze the prevalence of B chromosomes in 1,601 species of orthopteran insects where chromosome number and shape are known. B chromosomes have been reported in 191 of these species. Bs are not uniformly distributed among orthopteran superfamilies, with evident hotspots in the Pyrgomorphoidea (32.3% of species carrying Bs), Grylloidea (14.9%), Acridoidea (14.6%) and Tetrigoidea (14.3%). As expected under the theory of centromeric drive, we found a correlation between B chromosome presence and A chromosome shape-Bs are more frequent in karyotypes with more acrocentric A chromosomes. We also found that Bs are less common in species with high chromosome numbers and appear to be most common at the modal chromosome number (2n = 24). Study effort, measured for each genus, was not associated with B prevalence, A chromosome shape or A chromosome number. Our results thus provide support for centromeric drive as an important and prevalent force in the karyotypic evolution of Orthoptera, just as it appears to be in mammals. We suggest that centromeric drive may provide a mechanistic explanation for White's principle of karyotypic orthoselection.

In addition to the standard set of chromosomes (A), about 15% of eukaryote genomes carry B chromosomes. In most cases, B chromosomes behave as genomic parasites being detrimental for the individuals carrying them and prospering in natural populations because of transmission advantages (drive). B chromosomes are mostly made up of repetitive DNA sequences, especially ribosomal DNA (rDNA), satellite DNA and mobile elements. In only two cases have B chromosomes been shown to carry protein-coding genes. Although some B chromosomes seem to have derived from interspecific hybridisation, the most likely source of B chromosomes is the host genome itself, but the specific A chromosome being the B ancestor has not been identified in any B-containing species. Here, we provide strong evidence for B chromosome ancestry in the migratory locust, based on the location of genes for the H3 and H4 histones in the B chromosome and a single A chromosome pair (i.e. the eighth in order of decreasing size). The high DNA sequence similarity of A and B chromosome H3-H4 genes supports B-origin from chromosome 8. The higher variation shown by B sequences, compared to A sequences, suggests that B chromosome sequences are most likely inactive and thus less subjected to purifying selection. Estimates of time of divergence for histone genes from A and B chromosomes suggest that B chromosomes are quite old (>750,000 years), showing the B-chromosome ability to persist in natural populations for long periods of time.

The Geographic Mosaic Theory of Coevolution predicts the occurrence of mosaics of interaction-mediated local adaptations and maladaptations. Empirical support to this prediction has come mostly from specialist interactions. In contrast, local adaptation is considered highly unlikely in generalist interactions. In this study, we experimentally test local adaptation in a generalist plant-pollinator geographic mosaic, by means of a transplant experiment in which plants coming from two evolutionary hotspots and two coldspots were offered to pollinators at the same four localities. Plants produced in the hotspots attracted more pollinators in all populations, whereas coldspot plants attracted fewer pollinators in all populations. Differences in adaptation were not related to genetic similarity between populations, suggesting that it was mainly due to spatial variation in previous selective regimes. Our experiment provides the first strong support for a spatially structured pattern of adaptation and maladaptation generated by a generalist free-living mutualism.

The B24 chromosome in the Torrox population of the grasshopper Eyprepocnemis plorans is recurrently attached to a nucleolus in diplotene cells, indicating the activity of its distally located ribosomal DNA (rDNA). The frequency of males expressing the B chromosome nucleolus organizer region (B-NOR) almost doubled in 4 years. The likelihood of expressing the B-NOR increased with the B number and, in males expressing it, about 20% of their cells showed a nucleolus attached to the B. When active, the B-NOR contributed more than 25% of total cell nucleolar area (NA). Within males expressing the B-NOR, total cell NA did not differ between cells showing the active or inactive B-NOR, suggesting that total cell NA is tightly regulated in this species. However, this parameter tended to increase in this population from 1999 to 2004, in parallel to the neutralization process which is taking place in this population. Finally, an analysis of A chromosome NOR interdependence for activity revealed a positive correlation among autosomes but a negative correlation between autosomes and the X chromosome, the manifestation of which depends on B-NOR activity. These results are discussed in the context of the nucleolus as a sensor of the stress caused by parasitic B chromosomes.

Acquisition of knowledge of the nature and DNA content of B chromosomes has been triggered by a collection of molecular techniques, one of which, microdissection, has provided interesting results in a number of B chromosome systems. Here we provide the first data on the molecular composition of B chromosomes in Locusta migratoria, after microdissection of the B and X chromosomes, DNA amplification by one (B) or two (X) different methods, and chromosome painting. The results showed that B chromosomes share at least two types of repetitive DNA sequences with the A chromosomes, suggesting that Bs in this species most likely arose intraspecifically. One of these repetitive DNAs is located on the heterochromatic distal half of the B chromosome and in the pericentromeric regions of about half of the A chromosomes, including the X. The other type of repetitive DNA is located interspersedly over the non-centromeric euchromatic regions of all A chromosomes and in an interstitial part of the proximal euchromatic half of the B chromosome. Chromosome painting, however, did not provide results sufficiently reliable to determine, in this species, which A chromosome gave rise to the B; this might be done by detailed analysis of the microdissected DNA sequences.

We analyse chromosome location of H3 and H4 histone gene clusters by fluorescence in-situ hybridization (FISH) in 35 species of Acrididae grasshoppers belonging to seven subfamilies. As in other organisms, H3 and H4 co-localized in the same chromosome region in the 11 species where double FISH was performed with the H3 and H4 DNA probes. Chromosome location of H3-H4 histone gene clusters showed high regularity in the species analysed, with all of them carrying a single H3-H4 cluster in an autosome which, in most cases, was located interstitially in the proximal chromosome third. In 17 out of the 21 species with 2n masculine = 23 acrocentric chromosomes, the H3-H4-carrying autosome was about eighth in order of decreasing size. Two of the four exceptions changed H3-H4 localization to proximal (Pezotettix giornae) or distal (Tropidopola graeca) in the eighth-sized autosome, but the remainder (the two Eyprepocnemis species) showed the H3-H4 cluster distally located in the second-sized autosome. All 14 species with 2n masculine = 17 chromosomes (including three long metacentric autosome pairs, five acrocentric autosome pairs and an acrocentric X chromosome) carried an interstitial H3-H4 cluster in the short arm of the smallest of the three long metacentric pairs. These results suggest that chromosome location of H3-H4 histone gene clusters seem to be highly conservative in Acrididae grasshoppers. The change in H3-H4 location from the acrocentric medium-sized autosome in the 2n masculine = 23 karyotype to the long metacentric autosome in the 2n masculine = 17 karyotype is most parsimoniously explained by common ancestry, i.e. by the involvement of the H3-H4-carrying acrocentric in the centric fusion that gave rise to the smallest of the three long metacentric autosomes of 2n masculine = 17 species.

An adaptive role of corolla shape has been often asserted without an empirical demonstration of how natural selection acts on this trait. In generalist plants, in which flowers are visited by diverse pollinator fauna that commonly vary spatially, detecting pollinator-mediated selection on corolla shape is even more difficult. In this study, we explore the mechanisms promoting selection on corolla shape in the generalist crucifer Erysimum mediohispanicum Polatschek (Brassicaceae). We found that the main pollinators of E. mediohispanicum (large bees, small bees and bee flies) discriminate between different corolla shapes when offered artificial flowers without reward. Importantly, different pollinators prefer different shapes: bees prefer flowers with narrow petals, whereas bee flies prefer flowers with rounded overlapping petals. We also found that flowers with narrow petals (those preferred by bees) produce both more pollen and nectar than those with rounded petals. Finally, different plant populations were visited by different faunas. As a result, we found spatial variation in the selection acting on corolla shape. Selection favoured flowers with narrow petals in the populations where large or small bees are the most abundant pollinator groups. Our study suggests that pollinators, by preferring flowers with high reward, exert strong selection on the E. mediohispanicum corolla shape. The geographical variation in the pollinator-mediated selection on E. mediohispanicum corolla shape suggests that phenotypic evolution and diversification can occur in this complex floral trait even without specialization.

Background and Aims: Floral rewards may be associated with certain morphological floral traits and thus act as underlying factors promoting selection on these traits. This study investigates whether some traits that are under pollinator-mediated selection (flower number, stalk height, corolla diameter, corolla tube length and corolla tube width) in the Mediterranean herb E. mediohispanicum (Brassicaceae) are associated with rewards (pollen and nectar)., Methods: During 2005 the phenotypic traits and the visitation rate of the main pollinator functional groups were quantified in 720 plants belonging to eight populations in south-east Spain, and during 2006 the same phenotypic traits and the reward production were quantified in 400 additional plants from the same populations., Key Results: A significant correlation was found between nectar production rate and corolla tube length, and between pollen production and corolla diameter. Visitation rates of large bees and butterflies were significantly higher in plants exhibiting larger flowers with longer corolla tubes., Conclusions: The association between reward production and floral traits may be a factor underlying the pattern of visitation rate displayed by some pollinators.

The acrocentric macro B chromosomes of Rhammatocerus brasiliensis (Acrididae, Gomphocerinae) and Xyleus discoideus angulatus (Romaleidae, Romaleinae) are highly similar to the X chromosome in each species in terms of morphology, size, and pycnosis. However, the results of FISH experiments using 45S and 5S rDNA probes suggest that in both species the B chromosomes are most likely of autosomal origin. In R. brasiliensis, the B chromosome presented 5S rDNA but not 45S rDNA, in resemblance to the L(2), L(3), M(5) and S(11) autosomes, but the X chromosome lacks both rDNA families. In X. d. angulatus, 45S rDNAs is absent from the B chromosome, whereas the X chromosome contains one of the two 45S rDNA clusters in the genome. The occurrence of B chromosomes in all nine R. brasiliensis populations analyzed indicates that they are widely distributed in Northeastern Brazil, and the small amount of interpopulation variation found for B chromosome prevalence suggests the existence of high gene flow, presumably due to the abundance of this grasshopper species on several types of vegetation and its relatively high flight capability.

We report here, for the first time, the chromosome complement, number and location of the nucleolar organizer regions (NORs) revealed by silver staining (AgNO(3)) and fluorescent in situ hybridization (FISH) in five Neotropical gomphocerine species: Rhammatocerus brasiliensis, R. brunneri, R. palustris, R. pictus and Amblytropidia sp. The objective of this study was to summarize available data and propose a model of chromosome evolution in Neotropical gomphocerines. All five species studied showed chromosome numbers consisting of 2n = 23,X0 in males and 2n = 24,XX in females. Amblytropidia sp. was the only species showing a bivalent (M(8)) with megameric behavior during meiosis. The rDNA sites were restricted to autosomal pairs, i.e. the pericentromeric region of the S(9) chromosome, the consensus NOR location in all five species. R. brasiliensis was the only species showing additional NORs on M(4) and M(6) pairs which, likewise the S(9) NOR, were active in all cells analyzed. Comparison of these results with those reported previously in Palearctic gomphocerine species suggests higher resemblance of Neotropical species with the Old World species also possessing 23/24 chromosomes. Evolutionary mechanisms responsible for the observed interspecific variation in NOR location in this group are discussed.

We investigate regularities and restrictions in chromosome location of ribosomal RNA genes, analysed by fluorescent in situ hybridization (FISH), and their phenotypic expression assessed by nucleolus formation at first meiotic prophase cells, analysed by silver impregnation, in 49 grasshopper species. High variation was found for rDNA location between species within most genera analysed. The mean haploid number of rDNA loci detected by FISH was 2.47, but some species had up to 10 loci. Chromosome distribution of rDNA loci differed between the Gomphocerinae and Oedipodinae subfamilies, most loci being proximal to the centromere in the former and distal to it in the latter. Chromosomes 2, 3 and X frequently carried rDNA in Gomphocerinae species with 2n male symbol=17 chromosomes, whereas chromosomes 6 and 9 were the most frequent rDNA locations in the Oedipodinae. About 13% of the 126 rDNA loci detected by FISH were silent, although this figure might be even higher. The comparison of FISH and silver-impregnation results also suggested the existence of cryptic NORs, i.e. those forming small nucleoli with no apparent presence of rDNA revealed by FISH. This was especially clear after the same cells in two species were sequentially treated with both silver impregnation and FISH. The abundance of silent and cryptic loci might thus suggest that rDNA spreads through grasshopper genomes by the Dubcovsky and Dvorak mechanism-that is, the transposition of a few rRNA genes to new chromosome locations, their amplification giving rise to new NORs, and the elimination of the old NORs. The cryptic NORs might correspond to nascent NORs, i.e. a few rRNA gene copies moved to new locations, whereas the inactive rDNA loci might correspond to those being in the process of elimination.

The number of nucleoli and nucleolar area were measured in meiotic cells from males of the grasshopper Eyprepocnemis plorans collected in three natural populations. Number of nucleoli per cell showed no significant correlation among cells in different meiotic stages, but there was strong positive correlation for nucleolar area between leptotene and interkinesis cells in individuals from distant populations (Salobreña in Spain, and Smir in Morocco). No correlation was, however, observed for both parameters between the meiotic stages analysed in individuals from the population of Torrox (Spain). The number of nucleoli at leptotene was about double the number at interkinesis, as expected from the double ploidy level at leptotene and the corresponding double number of rDNA clusters. Leptotene nucleolar area, however, was about fourfold that in interkinesis, presumably due to higher requirements for ribosome biogenesis in meiosis I than meiosis II. In Torrox, diplotene cells showed a lower number of nucleoli but larger nucleolar area than in leptotene cells, suggesting an increase in nucleolus size during prophase I. Significant differences were found among populations for nucleolar area but not for number of nucleoli, the smallest nucleolar area being observed in Torrox, which is the population harbouring the most parasitic B chromosome variant. No clear effects on nucleolar area or number of nucleoli were associated with the B-chromosome number. However, B-chromosome effects on the nucleolar area were apparent in the Torrox population when data were analysed with respect to a B-chromosome odd-even pattern in leptotene and interkinesis cells. However, in diplotene cells no odd-even pattern was observed for both nucleolar parameters, suggesting that the increase in nucleolar size from leptotene to diplotene dilutes the leptotene odd-even pattern. The rDNA distally located in the B chromosome was associated with a nucleolus in 6.5% out of the 247 diplotene cells analysed. The implications of these findings are discussed in the context of B chromosomes as stress-causing genome parasites and the nucleolus as a sensor of stress.

Fluorescence immunostaining for the phosphorylated H2AX histone (gammaH2AX) in the grasshopper Eyprepocnemis plorans has shown abundance of gammaH2AX in the nuclei of round and elongating spermatids, suggesting that DNA double-strand breaks (DSBs) occur regularly during spermiogenesis. Immunofluorescence patterns for Ku70, a DNA-repair protein participating in the non-homologous end-joining (NHEJ) pathway, showed that this protein is present in round and elongating spermatids, implying that the NHEJ DNA-repair pathway operates during chromatin compaction in spermiogenesis. In addition, during the final stages of spermiogenesis, the Ku70 protein concentrates on the region forming the sperm tail. Since Ku70 was also abundant in spermatid tails, it is reasonable to assume that Ku70 might play a novel function in sperm-tail formation. The analysis of Ku70 immunofluorescence patterns in 13 other grasshopper species also showed the presence of this protein in the nucleus and tail of elongating spermatids, indicating that this is a general characteristic in grasshoppers.

Paleontological and phylogenetic studies have shown that floral zygomorphy (bilateral symmetry) has evolved independently in several plant groups from actinomorphic (radially symmetric) ancestors as a consequence of strong selection exerted by specialized pollinators. Most studies focused on unraveling the developmental genetics of flower symmetry, but little is known about the adaptive significance of intraspecific flower shape variation under natural conditions. We provide the first evidence for natural selection favoring zygomorphy in a wild population of Erysimum mediohispanicum (Brassicaceae), a plant showing extensive continuous variation in flower shape, ranging from actinomorphic to zygomorphic flowers. By using geometric morphometric tools to describe flower shape, we demonstrate that plants bearing zygomorphic flowers received more pollinator visits and had the highest fitness, measured not only by the number of seeds produced per plant but also by the number of seeds surviving to the juvenile stage. This study provides strong evidence for the existence of significant fitness differences associated with floral shape variation in E. mediohispanicum, thus illuminating a pathway for the evolution of zygomorphy in natural populations.

We have analysed B chromosome frequency for three consecutive years, B transmission rate at population and individual levels, clutch size, egg fertility and embryo-adult viability in a natural population of the grasshopper Eyprepocnemis plorans containing two different B chromosome variants, i.e. B(2) and B(24), the second being derived from the first and having replaced it in nearby populations. From 2002 to 2003 the relative frequency of both variants changed, although the differences did not reach significance. A mother-offspring analysis showed no significant effect of any of the two B variants on clutch size, egg fertility or embryo-adult viability, but B(24) was more efficiently transmitted than B(2) through males from the 2002 season, which explains the observed frequency change. Controlled crosses, at individual level, showed significant drive through some females for B(24) but not for B(2), suggesting that this difference in transmission rate might also be important for the substitution process. The analysis of relative fitness for B(2) and B(24) carriers for all fitness components, as a whole, showed a significantly better performance of B(24)-carrying individuals, suggesting that the cumulative effect of these slight differences might contribute to the replacement of B(2) by B(24).

B chromosome variation in nine Moroccan populations of the grasshopper Eyprepocnemis plorans was analysed for 3 consecutive years. In addition to B1, which was the predominant B chromosome in all nine populations, we found 15 other B variants, albeit at very low frequency. Eight variants were found in adults caught in the wild, four appeared in adults reared in the laboratory and seven were found in embryo progeny of controlled crosses between a 0B male and a B-carrying female. Some variants were found in more than one kind of material. At least the seven B variants that appeared in embryo progeny of females carrying a different B type arose de novo through mutation of the maternal B chromosome. The mutation rate of B chromosomes was 0.73%, on average, which explains the high variety of morphs and banding patterns found. The most frequent de novo mutations observed in these chromosomes were centromere misdivision with or without chromatid nondisjunction, which generates iso-B-chromosomes or telocentric Bs, respectively, as well as translocations with A and B chromosomes and deletions. But the whole variation observed, including that found in adult individuals, suggests that other mutations such as duplications, inversions and centric fusions do usually affect B chromosomes. Finally, B chromosome mutation rate was remarkably similar in both Moroccan and Spanish populations, which suggests that it might be dependent on B chromosome intrinsic factors., (March 2004)

Analysis of chromosome localization of three molecular markers, 18S-5.8S-28S rDNA, 5S rDNA and a 180 bp satDNA, showed that B chromosomes in the grasshopper Eyprepocnemis plorans originated independently in Eastern (Caucasus) and Western (Spain and Morocco) populations. Eastern B chromosomes are most likely derived from the smallest autosome, which is the only A chromosome carrying the three markers, in coincidence with Caucasian B chromosomes. Western B chromosomes, however, lack 5S rDNA and are most likely derived from the X chromosome, which is the only A chromosome carrying the two remaining markers, always in the same order with respect to the centromere, as the B chromosome.

The chromosomal localization of ribosomal DNA, and a 180 bp satellite DNA isolated from Spanish Eyprepocnemis plorans specimens, has been analysed in five Eyprepocnemidinae species collected in Russia and Central Asia. Caucasian E. plorans individuals carried each of the two DNAs, but the rDNA was limited to only two chromosomes (S(9) and S(11)) in sharp contrast to Spanish specimens that show 4-8 rDNA clusters and to Moroccan specimens which carry rDNA in almost all chromosomes. The four remaining species, however, lacked the 180 bp tandem repeat, and showed rDNA clusters in one (S(9) in Thisoicetrinus pterostichus), two (S(9) and S(10) in Eyprepocnemis unicolor; M(8) and S(11) in Heteracris adspersa), or three (S(9), S(10), and S(11) in Shirakiacris shirakii) chromosome pairs. The implications of these findings for the evolution of these two chromosome markers in this group of species are discussed.

One of the conceivable evolutionary pathways of a parasitic B chromosome is its integration into the host genome through translocation to an A chromosome. To investigate this possibility, we analyze here the nature, meiotic behavior and genetic effects of a spontaneous interchange between a medium-sized autosome and a B chromosome, found in one male caught in a Moroccan population of the grasshopper Eyprepocnemis plorans and the offspring of controlled crosses with six different females. Most metaphase I cells analyzed (115 out of 118) showed a trivalent with chiasmata in both the interstitial and pairing segments, which predicted about half of genetically unbalanced spermatozoa. The analysis of 234 embryos sired by this male on six females showed the lethality of some meiotic products paralleled to a decrease in egg fertility (0.541 +/- 0.051, compared to the 0.879 +/- 0.017 shown by females mated to standard males). These results suggest that the cost of the B-A interchange on host fitness, in terms of gametic inviability, highly diminishes the possibility of frequency increase for the interchange to reach a polymorphic status, which is the first and indispensable step to reach fixation and thus integration of the B chromosome DNA into the host genome.

The double FISH analysis of two repetitive DNAs (a satellite DNA and ribosomal DNA) in 12 natural populations of the grasshopper Eyprepocnemis plorans collected at the south (Granada and Málaga provinces) and south-east (Albacete and Murcia provinces) of the Iberian Peninsula has shown their wide-spread presence throughout the whole genome as well as extensive variation among populations. Both DNAs are found in most A chromosomes. Regularly, both DNAs occurred in the S11 and X chromosomes, rDNA in the S10 and satDNA in the L2 and M3. No correlation was found between the number of satDNA and rDNA clusters in the A genomes of the 12 populations analysed, and both figures were independent of the presence of B chromosomes. The genomic distribution of both DNAs showed no association with the geographical localization of the populations analysed. Finally, we provide evidence that the supernumerary chromosome segment proximally located on the S11 chromosome is, in most cases, the result of satDNA amplification but, in some cases, it might also derive from amplification of both satDNA and rDNA.

The grasshopper Eyprepocnemis plorans harbours an extremely widespread polymorphism for supernumerary (B) chromosomes, which is found in almost all circum-Mediterranean and Caucasian populations hitherto analysed. B chromosomes in this species have been shown to evolve through several stages of parasitic and near-neutral nature, presumably because of an arms race between the standard (A) and B chromosomes. This intragenomic conflict can either be solved with the extinction of the neutralised B chromosome or, more interestingly, with the replacement of the neutralised B by a mutant version being parasitic again and thus prolonging B chromosome life. This species thus provides a complete view of the long-term life-cycle of parasitic B chromosomes.

Parasites and hosts are involved in a continuous coevolutionary process leading to genetic changes in both counterparts. To understand this process, it is necessary to track host responses, one of which could be an increase in sex and recombination, such as is proposed by the Red Queen hypothesis. In this theoretical framework, the inducible recombination hypothesis states that B-chromosomes (genome parasites that prosper in natural populations of many living beings) elicit an increase in host chiasma frequency that is favoured by natural selection because it increases the proportion of recombinant progeny, some of which could be resistant to both B-chromosome effects and B-accumulation in the germline. We have found a clear parallelism between host recombination and the evolutionary status of the B-chromosome polymorphism, which provides explicit evidence for inducible recombination and strong support for the Red Queen hypothesis.

A previous study showed that in the haplodiploid solitary wasp Trypoxylon albitarse, most individuals carry one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, indicating a possible regularization of B-chromosome meiotic behaviour and its integration into the A genome. In a new sampling, we have analysed 15 populations (including 9 out of the 10 previously analysed) to test the evolution of this integration process. The new results provide a direct report of the invasion process in the Porto Firme population, where B frequency has dramatically increased in only four generations. In the populations from the Viçosa region, however, B frequency has remained stable, although the principal B type, the metacentric one, has increased in frequency at the expense of the acrocentric one in several populations. The implications of these new results on the hypothesis of the integration of these B chromosomes, as regular members of the A genome, are discussed.