TCAGG,an alternative telomeric sequence in insects

The TTAGG repeat, the only determined telomerase-dependent sequence in the Insecta, is generally reputed to be the canonical telomeric motif within the class. By studying the distribution of telomeric DNAs in 30 coleopteran beetles using Southern hybridization, BAL 31 DNA end-degradation assay and fluorescence in situ hybridization, we showed that arrays built of a TCAGG repeat substitute for (TTAGG)n sequences in all tested species within the superfamily Tenebrionoidea. We also provided the experimental evidence that (TCAGG)n repeats represent the terminal sequences on all chromosomes of the model species Tribolium castaneum. (TCAGG)n repeats are therefore promoted as the first sequence-motif alternative to TTAGG-type chromosome ends in insects. Detection of species negative for both TTAGG and TCAGG reveals that, although widespread, these motifs are not ubiquitous telomeric sequences within the order Coleoptera. In addition, Timarcha balearica proved to be a species that harbors (TTAGG)n repeats, but not at telomeric positions, thus further increasing the complexity of telomeric DNAs. Our experiments discarded CTAGG, CTGGG, TTGGG, and TTAGGG variants as potential replacements in TTAGG/TCAGG-negative species, indicating that chromosome termini of these beetles comprise other form(s) of telomeric sequences and telomere maintenance mechanisms.     

Phylogenetic distribution of TTAGG telomeric repeats in insects.

We examined the presence of TTAGG telomeric repeats in 22 species from 20 insect orders with no or inconclusive information on the telomere composition by single-primer polymerase chain reaction with (TTAGG)6 primers, Southern hybridization of genomic DNAs, and fluorescence in situ hybridization of chromosomes with (TTAGG)n probes. The (TTAGG)n sequence was present in 15 species and absent in 7 species. In a compilation of new and published data, we combined the distribution of (TTAGG)n telomere motif with the insect phylogenetic tree. The pattern of phylogenetic distribution of the TTAGG repeats clearly supported a hypothesis that the sequence was an ancestral motif of insect telomeres but was lost repeatedly during insect evolution. The motif was conserved in the "primitive" apterous insect orders, the Archaeognatha and Zygentoma, in the "lower" Neoptera (Plecoptera, Phasmida, Orthoptera, Blattaria, Mantodea, and Isoptera) with the exception of Dermaptera, and in Paraneoptera (Psocoptera, Thysanoptera, Auchenorrhyncha, and Sternorrhyncha) with the exception of Heteroptera. Surprisingly, the (TTAGG)n motif was not found in the "primitive" pterygotes, the Palaeoptera (Ephemeroptera and Odonata). The Endopterygota were heterogeneous for the occurrence of TTAGG repeats. The motif was conserved in Hymenoptera, Lepidoptera, and Trichoptera but was lost in one clade formed by Diptera, Siphonaptera, and Mecoptera. It was also lost in Raphidioptera, whereas it was present in Megaloptera. In contrast with previous authors, we did not find the motif in Neuroptera. Finally, both TTAGG-positive and TTAGG-negative species were reported in Coleoptera. The repeated losses of TTAGG in different branches of the insect phylogenetic tree and, in particular, in the most successful lineage of insect evolution, the Endopterygota, suggest a backup mechanism in the genome of insects that enabled them frequent evolutionary changes in telomere composition.     

Presence of the canonical TTAGG insect telomeric repeat in the Tenthredinidae (Symphyta) suggests its ancestral nature in the order Hymenoptera

Telomeric repeats in two members of the sawfly family Tenthredinidae (Hymenoptera), namely, Tenthredo omissa (Förster, 1844) and Taxonus agrorum (Fallén, 1808) (both have n?=?10), were studied using fluorescence in situ hybridization (FISH). Chromosomes of both species were demonstrated to contain the canonical TTAGG insect telomeric repeat, which constitutes the first report of the (TTAGG)_n telomeric motif for the Tenthredinidae as well as for the clade Eusymphyta and the suborder Symphyta in general. Taken together with the presence of this repeat in many other Holometabola as well as in the hymenopteran families Formicidae and Apidae from the suborder Apocrita, these results collectively suggest the ancestral nature of the (TTAGG)_n telomeric motif in the Hymenoptera as well as its subsequent loss within the clade Unicalcarida and independent reappearance in ants and bees. If this is true, the loss of the TTAGG repeat can be considered as a synapomorphy of the corresponding clade.     

Complex and Tandem Repeat Structure of Subtelomeric Regions in the Taiwan Cricket, <Emphasis Type="BoldItalic">Teleogryllus taiwanemma</Emphasis>

Telomeres of most insects are composed of simple (TTAGG) _n repeats that are synthesized by telomerase. However, in some dipteran insects such as Drosophila melanogaster, (TTAGG) _n repeats or telomerase activity has not been detected. Although telomere structure is well documented in Diptera and Lepidoptera, very limited information is available on lower insect groups. To understand general aspects of telomere function and evolution in insects, we endeavored to characterize structures of the telomeric and subtelomeric regions in a lower insect, the Taiwan cricket, Teleogryllus taiwanemma. FISH analysis of this insect's chromosomes demonstrated (TTAGG) _n repeat elements in all distal ends. Just proximal to the telomeric repeats, the highly conserved 9-kb long terminal unit (LTU) sequences are tandemly repeated. These were observed in four of six chromosomes, three autosomal ends, and one X-chromosomal end. LTU sequences represent about 0.2% of the T. taiwanemma genome. Each LTU contains a core (TTAGG)₈-like sequence (TRLS) and five types of conserved sequences—ST (short telomere associated), J (joint), X, SR (satellite sequence rich), and Y—which vary in length from about 150 bp to 2.7 kb. The LTU sequence is defined as ST–J–TRLS–SR–X–Y–X–Y–X. Most LTU regions may be derived from the ancestral common sequence, which is observed in ST regions six times and at many other LTU sites. We could not find the LTU-like sequence in three other crickets including the closest species, T. emma, suggesting that the LTU in T. taiwanemma has been rapidly amplified in subtelomeric regions through recent evolutional events. It is also suggested that the highly conserved structure of the LTU is maintained by recombination and may contribute to telomere elongation, as seen in dipteran insects. Received: 6 August 2001/Accepted: 10 October 2001     

Distribution of 18S rDNA sites and absence of the canonical TTAGG insect telomeric repeat in parasitoid Hymenoptera

Karyotypes of six species belonging to three main clades of parasitoid Hymenoptera, the superfamilies Ichneumonoidea (Ichneumonidae: Ichneumon amphibolus), Cynipoidea (Cynipidae: Diplolepis rosae) and Chalcidoidea (Eurytomidae: Eurytoma robusta, Eu. serratulae and Eu. compressa, and Torymidae: Torymus bedeguaris) were studied using FISH with 18S rDNA and telomeric (TTAGG)_n probes. Haploid karyotypes of D. rosae, Eu. robusta and Eu. serratulae carried the only 18S rDNA hybridization signal, whereas those of I. amphibolus and Eu. compressa carried three and two rDNA clusters respectively. In addition, three rDNA sites were visualized in the aneuploid female of T. bedeguaris. The number of rDNA clusters in parasitoid Hymenoptera generally correlates to the chromosome number. Apart from the overwhelming majority of the studied species of aculeate Hymenoptera, no hybridization signals were obtained from FISH with the telomeric (TTAGG)_n probe in the examined parasitoid species. These data suggest absence of the canonical (TTAGG)_n insect telomeric motif in the Ichneumonoidea, Cynipoidea and Chalcidoidea, and perhaps in parasitoid Hymenoptera in general.     

Chromosome Analysis and Molecular Characterization of Highly Repeated DNAs in the Aphid Acyrthosiphon Pisum (Aphididae, Hemiptera)

Despite the interest in aphid biology, information on chromatin organization of their holocentric chromosomes is still limited to few species. In order to fill this gap, we have performed an extensive survey on pea aphid mitotic chromosomes using both classical and molecular cytogenetic techniques. Our results after silver, CMA₃ and DAPI-staining, C-banding and fluorescent in situ hybridization (FISH) using 28S rDNA and 5S rDNA as probes evidenced a tendency of repetitive DNAs to be concentrated on the X chromosomes. FISH experiments with the telomeric probe (TTAGG) _n revealed bright hybridization signals on each telomere of all Acyrthosiphon pisum chromosomes. No interstitial signals were seen. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera,Belostoma)

Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome–autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elegans (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elongatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG)_n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere–telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.     

Characterization of TTAGG telomeric repeats,their interstitial occurrence and constitutively active telomerase in the mealybug <Emphasis Type="Italic">Planococcus lilacinus</Emphasis> (Homoptera; Coccoidea)

We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.     

Interstitial telomere‐like repeats in the monocot family Araceae

Combining molecular cytogenetics and phylogenetic modelling of chromosome number change can shed light on the types of evolutionary changes that may explain the haploid numbers observed today. Applied to the monocot family Araceae, with chromosome numbers of 2n = 8 to 2n = 160, this type of approach has suggested that descending dysploidy has played a larger role than polyploidy in the evolution of the current chromosome numbers. To test this, we carried out molecular cytogenetic analyses in 14 species from 11 genera, using probes for telomere repeats, 5S rDNA and 45S rDNA and a plastid phylogenetic tree covering the 118 genera of the family, many with multiple species. We obtained new chromosome counts for six species, modelled chromosome number evolution using all available counts for the family and carried out fluorescence in situ hybridization with three probes (5S rDNA, 45S rDNA and Arabidopsis‐like telomeres) on 14 species with 2n = 14 to 2n = 60. The ancestral state reconstruction provides support for a large role of descending dysploidy in Araceae, and interstitial telomere repeats (ITRs) were detected in Anthurium leuconerum, A. wendlingeri and Spathyphyllum tenerum, all with 2n = 30. The number of ITR signals in Anthurium (up to 12) is the highest so far reported in angiosperms, and the large repeats located in the pericentromeric regions of A. wendlingeri are of a type previously reported only from the gymnosperms Cycas and Pinus. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 15–26.     

Characterisation of an unusual telomere motif (TTTTTTAGGG)n in the plant Cestrum elegans (Solanaceae), a species with a large genome

The characterization of unusual telomere sequence sheds light on patterns of telomere evolution, maintenance and function. Plant species from the closely related genera Cestrum, Vestia and Sessea (family Solanaceae) lack known plant telomeric sequences. Here we characterize the telomere of Cestrum elegans, work that was a challenge because of its large genome size and few chromosomes (1C 9.76 pg; n = 8). We developed an approach that combines BAL31 digestion, which digests DNA from the ends and chromosome breaks, with next‐generation sequencing (NGS), to generate data analysed in RepeatExplorer, designed for de novo repeats identification and quantification. We identify an unique repeat motif (TTTTTTAGGG)_n in C. elegans, occurring in ca. 30 400 copies per haploid genome, averaging ca. 1900 copies per telomere, and synthesized by telomerase. We demonstrate that the motif is synthesized by telomerase. The occurrence of an unusual eukaryote (TTTTTTAGGG)_n telomeric motif in C. elegans represents a switch in motif from the ‘typical’ angiosperm telomere (TTTAGGG)_n. That switch may have happened with the divergence of Cestrum, Sessea and Vestia. The shift in motif when it arose would have had profound effects on telomere activity. Thus our finding provides a unique handle to study how telomerase and telomeres responded to genetic change, studies that will shed more light on telomere function.     

Closely relatedAllium species (Alliaceae) share a very similar satellite sequence

A satellite sequence repeat ofAllium cepa was tested by fluorescent in situ hybridization (FISH) for cross-hybridization to chromosomes of 27 species (in 37 accessions) belonging to 14 sections of four subgenera ofAllium. All investigated species of sect.Cepa, with the two subsects.Cepa andPhyllodolon, revealed clear satellite-specific hybridization signals mainly at their chromosome termini. The tested species belonging to other sections/subgenera revealed no hybridization signals. An exception wasA. roylei, assigned to sect.Oreiprason. Its chromosomes also showed strong terminal hybridization signals. This and other features suggest a close relationship ofA. roylei to the species of sect.Cepa in spite of deviating morphological characters. The divergence between the satellite repeats to species to which theA. cepa repeat cross-hybridized was determined and revealed high degrees of similarity. Therefore, we conclude that this satellite sequence had evolved already in progenitor forms of sect.Cepa and remained unusually well conserved during speciation. This might indicate selection pressure exerted on a secondarily acquired telomere function of the satellite sequence.Dedicated to Dr habil.Peter Hanelt on the occasion of his 65th birthday.     

Characterization of the telomere regions of scleractinian coral, <Emphasis Type="Italic">Acropora surculosa</Emphasis>

Terminal ends of vertebrate chromosomes are protected by tandem repeats of the sequence (TTAGGG). First thought to be vertebrate specific, (TTAGGG) _n has recently been identified in several aquatic invertebrates including sea urchin (Strongylocentrotus purpuratus), bay scallop (Argopecten irradians), and wedgeshell clam (Donax trunculus). We analyzed genomic DNA from scleractinian corals, Acropora surculosa, Favia pallida, Leptoria phrygia, and Goniastrea retiformis to determine the telomere sequence. Southern blot analysis suggests the presence of the vertebrate telomere repeats in all four species. Treatment of A. surculosa sperm DNA with Bal31 exonuclease revealed progressive shortening of the DNA fragments positive for the (TTAGGG)₂₂ sequence, supporting location of the repeats at the chromosome ends. The presence of the vertebrate telomere repeats in corals is evidence that the (TTAGGG) _n sequence is highly conserved among a divergent group of vertebrate and invertebrate species. Corals are members of the Lower Metazoans, the group of organisms that span the gap between the fungi and higher metazoans. Corals are the most basal organism reported to have the (TTAGGG) _n sequence to date, which suggests that the vertebrate telomere sequence may be much older than previously thought and that corals may share a number of genes with their higher relatives.     

Dietary composition and echolocation call design of three sympatric insectivorous bat species from China

Studying the diet of echolocating, insectivorous bats can provide important insights into their foraging behaviors and ecological constraints they are facing. By examining an extensive data set covering a period of 2 years, the present study identifies the dietary composition of three sympatric insectivorous bat species in rural areas of Beijing municipality. Each species clearly has different preferences for particular food items. Greater horseshoe bats, Rhinolophus ferrumequinum, preferred to catch nocturnal, actively flying insects, mostly moths (Lepidoptera), and to a lesser percentage flies (Diptera), beetles (Coleoptera), and flying ants and termites (Hymenoptera). Other nocturnal insects which do not exhibit any perceptible wing movements, such as true bugs (Homoptera), or strictly diurnal insects that hardly ever fly in the dark, such as grasshoppers (Orthoptera) and dragon- and damselflies (Odonata), were never found in droppings of horseshoe bats. Large mouse-eared bats, Myotis chinensis, preferentially glean relatively large terrestrial prey of the order Coleoptera (mostly carabid beetles) and Orthoptera, whereas greater tube-nosed bats, Murina leucogaster, consume predominantly smaller, diurnal Coleoptera (mostly soldier beetles, Cantharidae, and ladybugs, Coccinellidae). Our findings also indicate previously not described, significant spectro-temporal differences in the echolocation signals of M. chinensis and M. leucogaster. The results suggest that in our study area the dramatic differences in the dietary composition of these three bat species are mainly based upon differences in their foraging behaviors, including differences in their echolocation signal structure. The dietary data provide important background information for conservational efforts, such as habitat protection.     

Conservation of (TTAGG)(n) telomeric sequences among ants (Hymenoptera,Formicidae)

To determine the telomere sequence in Tapinoma nigerrimum, we carried out in situ hybridization using TTAGGG and TTAGG repeat polymerase chain reaction (PCR)-generated probes. No hybridization signals were found when TTAGGG was used as a probe. However, strong signals were observed at the end of the chromosomes with the TTAGG probe. Southern blot analysis carried out on genomic DNA using TTAGG as a probe showed a strong hybridization signal even under highly stringent conditions. Similar results were obtained in Southern blot analysis carried out on genomic DNA of 19 species of ants belonging to three different subfamilies. In accordance with all the results shown in this article, the TTAGG repeat seems to be the major component of the telomere sequence in the majority of ant species.     

Attraction of a kleptoparasitic sphaerocerid fly (Norrbomia frigipennis) to dung beetles (Phanaeus spp. andCanthon sp.)

Norrbomia frigipennis (Diptera: Sphaeroceridae) is phoretic on dung-feeding scarab beetles (Coleoptera: Scarabaeidae). In this study we investigate the attractiveness of three beetle species,Phanaeus ignius, P. vindex, andCanthon pilularis, to the fly. Stationary, moving-dead, and live beetles were used. More flies were attracted toPhanaeus. However, this attractiveness may be due to the larger average size ofPhanaeus. A preference for larger individuals was found withinPhanaeus, though not withinC. pilularis. Flies mounted beetles on the thorax and the elytra at similar rates.Phanaeus males that possesed horns did not attract more flies than did hornless ones, and there was no effect of host sex on attractiveness. In hornedPhanaeus, about 11–16% of the flies mounting those beetles landed on the horn.     

Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences

A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. in situ hybridization experiments showed dispersed organization of the sequences over all chromosomes of H. vulgare and the wild barley species H. bulbosum, H. marinum and H. murinum. Southern blot hybridization revealed different levels of polymorphism among barley species and the RFLP data were used to generate a phylogenetic tree for the genus Hordeum. Our data are in a good agreement with the classification system which suggests the division of the genus into four major groups, containing the genomes I, X, Y, and H. However, our investigation also supports previous molecular studies of barley species where the unique position of H. bulbosum has been pointed out. In our experiments, H. bulbosum generally had hybridization patterns different from those of H. vulgare, although both carry the I genome. Based on our results we present a hypothesis concerning the possible origin and phylogeny of the polyploid barley species H. secalinum, H. depressum and the H. brachyantherum complex.     

Genome organization of repetitive elements in the rodent,Peromyscus leucopus

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)_n was present in 87% of the clones, (CT)_n was present in 59% of the clones, and (AT)_n and (GC)_n each was represented in our sample by a single clone (0.05%). (TCC)_n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)_n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.