Mercedes Ruiz-Estévez

B-Chromosome Ribosomal DNA Is Functional in the Grasshopper Eyprepocnemis plorans

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 B24 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 B24 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.

DNA Amount of X and B Chromosomes in the Grasshoppers Eyprepocnemis plorans and Locusta migratoria

We analyzed the DNA amount in X and B chromosomes of 2 XX/X0 grasshopper species (Eyprepocnemis plorans and Locusta migratoria), by means of Feulgen image analysis densitometry (FIAD), using previous estimates in L. migratoria as standard (5.89 pg). We first analyzed spermatids of 0B males and found a bimodal distribution of integrated optical densities (IODs), suggesting that one peak corresponded to +X and the other to –X spermatids. The difference between the 2 peaks corresponded to the X chromosome DNA amount, which was 1.28 pg in E. plorans and 0.80 pg in L. migratoria. In addition, the +X peak in E. plorans gave an estimate of the C-value in this species (10.39 pg). We next analyzed diplotene cells from 1B males in E. plorans and +B males in L. migratoria (a species where Bs are mitotically unstable and no integer B number can be defined for an individual) and measured B chromosome IOD relative to X chromosome IOD, within the same cell, taking advantage of the similar degree of condensation for both positively heteropycnotic chromosomes at this meiotic stage. From this proportion, we estimated the DNA amount for 3 different B chromosome variants found in individuals from 3 E. plorans Spanish populations (0.54 pg for B1 from Saladares, 0.51 pg for B2 from Salobreña and 0.64 for B24 from Torrox). Likewise, we estimated the DNA amount of the B chromosome in L. migratoria to be 0.15 pg. To automate measurements, we wrote a GPL3 licensed Python program (pyFIA). We discuss the utility of the present approach for estimating X and B chromosome DNA amount in a variety of situations, and the meaning of the DNA amount estimates for X and B chromosomes in these 2 species.

Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans

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.

B1 was the ancestor B chromosome variant in the western Mediterranean area in the grasshopper Eyprepocnemis plorans.

We analyzed the distribution of 2 repetitive DNAs, i.e. ribosomal DNA (rDNA) and a satellite DNA (satDNA), on the B chromosomes found in 17 natural populations of the grasshopper Eyprepocnemis ploransplorans sampled around the western Mediterranean region, including the Iberian Peninsula, Balearic Islands, Sicily, and Tunisia. Based on the amount of these repetitive DNAs, 4 types of B variants were found: B1, showing an equal or higher amount of rDNA than satDNA, and 3 other variants, B2, B24 and B5, bearing a higher amount of satDNA than rDNA. The variants B1 and B2 varied in size among populations: B1 was about half the size of the X chromosome in Balearic Islands, but two-thirds of the X in Iberian populations at Alicante, Murcia and Albacete provinces. Likewise, B2 was about one-third the size of the X chromosome in populations from the Granada province but half the size of the X in the populations collected at Málaga province. The widespread geographical distribution of the B1 variant makes it the best candidate for being the ancestor B chromosome in the whole western Mediterranean region.

B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans

The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2A+B) and the other being specific to B chromosomes (ITS2B). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2B amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITSA+B amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed.

Preferential Occupancy of R2 Retroelements on the B Chromosomes of the Grasshopper Eyprepocnemis plorans

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.

Sesquiterpenoids Isolated from Two Species of the Asteriscus Alliance

Investigation of the aerial parts of two Spanish members of the Asteriscus alliance, Asteriscus graveolens subsp. stenophyllus and Asteriscus schultzii, afforded four new sesquiterpene lactones containing a humulene skeleton (1-4) and one new sesquiterpene lactone of the asteriscanolide type (5). Their chemical structures were determined on the basis of the HRMS and from 1D and 2D NMR spectroscopic studies. Both species showed different profiles of sesquiterpenoid constituents. A. schultzii did not show humulene or asteriscane sesquiterpenes, suggesting a resemblance to the genus Pallenis, another member of the Asteriscus alliance. A literature review on chemical isolates from the Asteriscus alliance supported the placement of A. schultzii in the genus Pallenis. The isolated components (1-5) were assessed for cytotoxicity against the HL-60 and MOLT-3 leukemia cell lines, with compound 1 showing activity in both biological assays (IC50 value range 4.1-5.4 μM).

Transient Microgeographic Clines during B Chromosome Invasion

The near-neutral model of B chromosome evolution predicts that the invasion of a new population should last some tens of generations, but the details on how it proceeds in real populations are mostly unknown. Trying to fill this gap, we analyze here a natural population of the grasshopper Eyprepocnemis plorans at three time points during the last 35 years. Our results show that B chromosome frequency increased significantly during this period and that a cline observed in 1992 had disappeared in 2012 once B chromosome frequency reached an upper limit at all sites sampled. This indicates that, during B chromosome invasion, transient clines for B chromosome frequency are formed at the invasion front on a microgeographic scale. Computer simulation experiments showed that the pattern of change observed for genotypic frequencies is consistent with the existence of B chromosome drive through females and selection against individuals with a high number of B chromosomes.

B chromosomes in the grasshopper Eyprepocnemis plorans are present in all body parts analyzed and show extensive variation for rDNA copy number

B chromosomes in the grasshopper Eyprepocnemis plorans are considered to be mitotically stable, because all meiotic (primary spermatocytes and oocytes) or mitotic (embryos, ovarioles, and gastric caecum) cells analyzed within the same individual show the same B chromosome number. Nothing is known, however, about body parts with somatic tissues with no mitotic activity in adult individuals, constituting the immense majority of their body. Therefore, we investigated whether B chromosomes are present in 8 non-mitotically active somatic body parts from both sexes in addition to ovarioles and testes by PCR analysis of 2 B-specific molecular markers. We also elucidated the number of B chromosomes that an individual carried through quantifying the B-located rDNA copy number by qPCR. Our results indicated the amplification of both B-specific markers in all analyzed body parts. However, we found high variation between males for the estimated number of rDNA units in the B chromosomes. These results demonstrate the presence of B chromosomes in all body parts from the same individual and suggest a high variation in the rDNA content of the B chromosomes carried by different individuals from the same population, presumably due to unequal crossovers during meiosis.

Identification and Characterization of TALE Homeobox Genes in the Endangered Fern Vandenboschia speciosa

We report and discuss the results of a quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression patterns of seven three amino acid loop extension (TALE) homeobox genes (four KNOTTED-like homeobox (KNOX) and three BEL1-like homeobox (BELL) genes) identified after next generation sequencing (NGS) and assembly of the sporophyte and gametophyte transcriptomes of the endangered fern species Vandenboschia speciosa. Among the four KNOX genes, two belonged to the KNOX1 class and the other two belonged to the KNOX2 class. Analysis of the deduced amino acid sequences supported the typical domain structure of both types of TALE proteins, and the homology to TALE proteins of mosses, lycophytes, and seed plant species. The expression analyses demonstrate that these homeodomain proteins appear to have a key role in the establishment and development of the gametophyte and sporophyte phases of V. speciosa lifecycle, as well as in the control of the transition between both phases. Vandenboschia speciosa VsKNAT3 (a KNOX2 class protein) as well as VsBELL4 and VsBELL10 proteins have higher expression levels during the sporophyte program. On the contrary, one V. speciosa KNOX1 protein (VsKNAT6) and one KNOX2 protein (VsKNAT4) seem important during the development of the gametophyte phase. TALE homeobox genes might be among the key regulators in the gametophyte-to-sporophyte developmental transition in regular populations that show alternation of generations, since some of the genes analyzed here (VsKNAT3, VsKNAT6, VsBELL4, and VsBELL6) are upregulated in a non-alternating population in which only independent gametophytes are found (they grow by vegetative reproduction outside of the range of sporophyte distribution). Thus, these four genes might trigger the vegetative propagation of the gametophyte and the repression of the sexual development in populations composed of independent gametophytes. This study represents a comprehensive identification and characterization of TALE homeobox genes in V. speciosa, and gives novel insights about the role of these genes in fern development.