A step to the gigantic genome of the desert locust: chromosome sizes and repeated DNAs.

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.

Non-random expression of ribosomal DNA units in a grasshopper showing high intragenomic variation for the ITS2 region.

We analyse intragenomic variation of the ITS2 internal transcribed spacer of ribosomal DNA (rDNA) in the grasshopper Eyprepocnemis plorans, by means of tagged PCR 454 amplicon sequencing performed on both genomic DNA (gDNA) and RNA-derived complementary DNA (cDNA), using part of the ITS2 flanking coding regions (5.8S and 28S rDNA) as an internal control for sequencing errors. Six different ITS2 haplotypes (i.e. variants for at least one nucleotide in the complete ITS2 sequence) were found in a single population, one of them (Hap4) being specific to a supernumerary (B) chromosome. The analysis of both gDNA and cDNA from the same individuals provided an estimate of the expression efficiency of the different haplotypes. We found random expression (i.e. about similar recovery in gDNA and cDNA) for three haplotypes (Hap1, Hap2 and Hap5), but significant underexpression for three others (Hap3, Hap4 and Hap6). Hap4 was the most extremely underexpressed and, remarkably, it showed the lowest sequence conservation for the flanking 5.8-28S coding regions in the gDNA reads but the highest conservation (100%) in the cDNA ones, suggesting the preferential expression of mutation-free rDNA units carrying this ITS2 haplotype. These results indicate that the ITS2 region of rDNA is far from complete homogenization in this species, and that the different rDNA units are not expressed at random, with some of them being severely downregulated.

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.

Differences in ribosomal DNA distribution on A and B chromosomes between eastern and western populations of the grasshopper Eyprepocnemis plorans plorans.

Distribution of ribosomal DNA (rDNA) on standard (A) and supernumerary (B) chromosomes of the grasshopper Eyprepocnemis plorans was analysed in specimens collected in Turkey and Armenia, belonging to the E. p. plorans subspecies, and in South Africa, belonging to the E. p. meridionalis subspecies. The latter individuals showed rDNA loci in chromosomes 9 and 11 only, whereas those from Armenia carried it in chromosomes 9 and 11 or else in chromosomes 9-11, depending on the population. The specimens from Turkey carried it in chromosomes 1, 9-11 and X. A comparison of this pattern with those previously observed in populations from Spain, Morocco, and Greece (belonging to E. p. plorans) suggests the existence of two evolutionary patterns in rDNA chromosome location in A chromosomes of this subspecies: eastern populations showing rDNA restricted to the small (9-11) chromosomes (as in E. p. meridionalis and other closely related taxa within the Eyprepocneminae subfamily) and western populations carrying rDNA in most A chromosomes (Spain) or all of them (Morocco). The intermediate pattern discerned in geographically intermediate populations (in Greece and Turkey), with rDNA also being located on the X chromosome, suggests a possible east-west cline. Additional support for east-west differentiation in the rDNA location pattern comes from the analysis of B chromosomes. In eastern populations, including Daghestan, Armenia, Turkey, and Greece, B chromosomes are composed mostly of rDNA, whereas in western populations (Spain and Morocco) they contain roughly similar amounts of rDNA and a 180-bp tandem repeat (satDNA), the latter being scarce in eastern Bs.

The odd-even effect in mitotically unstable B chromosomes in grasshoppers.

The odd-even effect, by which B chromosomes are more detrimental in odd numbers, has been reported in plants and animals. In grasshoppers, there are only a few reports of this effect and all were referred to as traits related to the formation of aberrant meiotic products (AMPs). Here we review the existing information about B chromosome effects on AMPs, chiasma frequency and the number of active nucleolus organizer regions (NORs) per cell. Polysomy for A chromosomes and B chromosomes are two kinds of chromosome polymorphism frequently found in grasshoppers. In some aspects, e.g. meiotic behaviour and mitotic instability leading to individual mosaicism (in the case of mitotically unstable Bs), polysomic As show similar characteristics to B chromosomes. In fact, polysomy is regarded as one of the main mechanisms for B chromosome origin. Here we review some features of meiotic behaviour in known cases of polysomy and mitotically unstable Bs in grasshoppers, in looking for possible causes for the odd-even effect. In all these traits, the odd-even effect was apparent, although its appearance was not universal in any case, with variation among species or populations within the same species. The equational division and lagging of the extra chromosomes, when univalents, could favour the appearance of abnormal meiotic products, and the formation of bivalents, when there are two or more extra chromosomes, inhibits this process. Therefore, the odd-even effect might be a consequence of the concomitant operation of both aspects of extra chromosome meiotic behaviour. The possibility that the odd-even effect might result from an increase in cell stress generated by odd numbers is suggested.

Rapid suppression of drive for a parasitic B chromosome.

The persistence of parasitic B chromosomes in natural populations depends on both B ability to drive and host response to counteracting it. In the grasshopper Eyprepocnemis plorans, the B24 chromosome is the most widespread B chromosome variant in the Torrox area (Málaga, Spain). Its evolutionary success, replacing its ancestral neutralized B variant, B2, was based on meiotic drive in females, as we showed in a sample caught in 1992. In females collected six years later, mean B24 transmission ratio (k(B)) was 0.523, implying a very rapid decrease from the 0.696 observed in 1992. This shows that B24 neutralization is running very fast and suggests that it might most likely be based on a single gene of major effect.

Host recombination is dependent on the degree of parasitism.

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.

Causes of B chromosome variant substitution in the grasshopper Eyprepocnemis plorans.

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).

Changes in NOR activity pattern in the presence of supernumerary heterochromatin in the grasshopper Eyprepocnemis plorans.

Nucleolus organizer region (NOR) activity was analysed in four types of males of the grasshopper Eyprepocnemis plorans, possessing two kinds of supernumerary heterochromatin: a B chromosome and a supernumerary chromosome segment proximally located on the smallest autosome (S11). In males lacking extra heterochromatin, the four active NORs located on the S9, S10, S11, and X chromosomes showed independent activity patterns, but several kinds of dependence appeared in the presence of supernumerary heterochromatin. Furthermore, temporal changes in NOR activity were observed during the first 2 weeks of adult life in standard males but not in males carrying supernumerary heterochromatin. It is suggested that all these effects are related to the DNA content of both types of extra heterochromatin.

Male and female segregation distortion for heterochromatic supernumerary segments on the S8 chromosome of the grasshopper Chorthippus jacobsi.

The mode of inheritance of supernumerary segments located on three different chromosome pairs was investigated in controlled crosses with specimens of the grasshopper Chorthippus jacobsi. While extra segments located on chromosomes M5 and M6 showed Mendelian inheritance, that on S8 did not. Thus, the two supernumerary heterochromatic chromosome segments located distally on the S8 chromosome accumulated through non-Mendelian transmission through both sexes. The observed transmission patterns may be explained by gametic selection for spermatozoa carrying segmented S8 chromosomes, in addition to meiotic drive for segmented S8 chromosomes in heterozygous females. The significance of these findings for the maintenance of these polymorphisms in natural populations is discussed.

Unusually high amount of inactive ribosomal DNA in the grasshopper Stauroderus scalaris.

Fluorescence in-situ hybridization (FISH) was employed to determine the chromosomal location of the ribosomal DNA cistrons in spermatocytes of two populations of the grasshopper Stauroderus scalaris. The results showed that paracentromeric C-bands, which in this species constitute about 50% of the total chromatin, contain substantial amounts of rDNA in all chromosomes. However, silver impregnation showed the presence of a single active nucleolus organizing region (NOR) in chromosome 3 of primary spermatocytes, indicating an extremely high amount of silent rDNA across the whole genome of this species in the two geographically distant populations analysed. The significance of such an unusual phenomenon is discussed.

Achiasmate segregation of X and B univalents in males of the grasshopper Eyprepocnemis plorans is independent of previous association.

B chromosomes proved to be more frequent in males than females of the grasshopper Eyprepocnemis plorans collected from the population in Jete (Granada, Spain) in 1992. The meiotic behaviour of the X and B univalents was analysed in a high number of 1B males collected from this population in 1991 and 1992, and in males from another population (Salobreña, Granada, Spain) for comparison. These two chromosomes showed a significant tendency to migrate to opposite poles in the Jete population, during the 2 years analysed, but separated randomly in the Salobreña population. Thus, sex differences in the B frequency in Jete seemed to be due to the non-random X-B segregation during male meiosis. The analysis of association patterns between the two univalents over several stages of the first meiotic division indicated a heterochromatic affinity rather than association by chiasmata because most X-B associations had resolved by metaphase I. The X and B chromosomes share two different DNA sequences, so that some associations during prophase I undoubtedly involve homologous DNA sequences. The frequency with which X and B migrated to opposite poles at anaphase I in Jete, however, did not show any significant dependence on previous association at zygotene, diplotene or metaphase I.

The B chromosomes of the grasshopper Eyprepocnemis plorans and the intragenomic conflict.

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.

Undertransmission of a supernumerary chromosome segment through heterozygous females possessing B chromosomes in the grasshopper Eyprepocnemis plorans.

The transmission ratio (ks) for a supernumerary chromosome segment was studied in a total of 54 heterozygous females collected from two Spanish natural populations of the grasshopper Eyprepocnemis plorans. Our analysis clearly demonstrated that ks is negatively dependent on the number of B chromosomes in the female. The possible mechanisms by which B chromosomes may cause undertransmission of the supernumerary segment, and the implications of this phenomenon for the maintenance of this extra chromosome segment, are discussed.

Transmission analysis of mitotically unstable B chromosomes in Locusta migratoria.

Seventeen controlled crosses in which the mitotically unstable B chromosome of Locusta migratoria was carried by one parent only have provided evidence that B chromosomes are significantly eliminated during sexual transmission in males, at a mean rate that almost counteracts the premeiotic accumulation derived from mitotic instability during germ line development. On the other hand, B chromosomes are significantly accumulated in females, presumably by their preferential migration to the secondary oocyte during the first meiotic division. These results substantially change the current knowledge about this B chromosome system, because the main B accumulation occurs in females and not in males, as was hitherto thought. Furthermore, this case shows that the maintenance of a single B system in natural populations may be the result of many different forces and mechanisms acting for and against B chromosomes.

Cloning and sequence analysis of an extremely homogeneous tandemly repeated DNA in the grasshopper Eyprepocnemis plorans.

Digestion of total nuclear DNA of the grasshopper Eyprepocnemis plorans with seven different restriction endonucleases (REs), and subsequent agarose gel electrophoresis, has shown the presence of highly repetitive DNA yielding the typical ladder-like banding pattern. The most clear pattern was produced by DraI, the monomer being some 180 bp. This repeat unit was subsequently cloned and sequenced. Bidirectional sequencing of five randomly chosen clones showed exactly the same nucleotides in all 180 positions. The possible explanations for such an extreme homogeneity of this tandem repeat are discussed in the light of current hypotheses on repetitive DNA function and molecular drive mechanisms.

POLYMORPHISM REGENERATION FOR A NEUTRALIZED SELFISH B CHROMOSOME.

Long-run evolution of B chromosomes is mainly made up by an evolutionary arms race between these selfish genetic elements and the standard genome. The suppression of B drive is one of the clearest expressions of genome defense against B chromosomes. After drive neutralization, the B is condemned to extinction unless a new variant showing drive can emerge and replace it. This paper reports the first empirical evidence for the substitution of a neutralized B variant by a new selfish B variant. Such a polymorphism regeneration has recently taken place in a natural population of the grasshopper Eyprepocnemis plorans.

Population variation in the A chromosome distribution of satellite DNA and ribosomal DNA in the grasshopper Eyprepocnemis plorans.

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.

Population differences in the expression of nucleolus organizer regions in the grasshopper Eyprepocnemis plorans.

Fluorescence in situ hybridization revealed the presence of ribosomal RNA genes in paracentromeric regions of all A chromosomes and in the distal half of B chromosomes in embryonic cells from Moroccan specimens of the grasshopper Eyprepocnemis plorans. The expression of these genes was monitored by the presence of nucleoli attached to each chromosome bivalent in diplotene cells from males collected from two different Moroccan populations and was compared to previous data of Spanish populations. Whereas only the nucleolus organizer regions (NORs) on S9-S11 and X chromosomes were active in the Spanish specimens, Moroccan individuals showed NOR activity in all chromosomes. The rRNA genes on the B chromosome were inactive in both populations. The S9 and S10 NORs were less active in Moroccan specimens than in Spanish specimen, which might be partly explained by the negative inter-dependence for expression of the S10 NOR with respect to those on L2 and X chromosomes. On the other hand, the X NOR was more active in Moroccan specimens than in Spanish specimens, and this might be partly due to the positive effect that the presence of B chromosomes has on the expression of this NOR. The implications of these observations on current models of NOR activity regulation are discussed.