Research Article |
Corresponding author: Natalia V. Golub ( nvgolub@mail.ru ) Academic editor: Christina Nokkala
© 2015 Natalia V. Golub, Victor B. Golub, Valentina G. Kuznetsova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Golub NV, Golub VG, Kuznetsova VG (2015) Variability of 18rDNA loci in four lace bug species (Hemiptera, Tingidae) with the same chromosome number. Comparative Cytogenetics 9(4): 513-522. https://doi.org/10.3897/CompCytogen.v9i4.5376
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Male karyotypes of Elasmotropis testacea (Herrich-Schaeffer, 1835), Tingis cardui (Linnaeus, 1758), T. crispata (Herrich-Schaeffer, 1838), and Agramma femorale Thomson, 1871 (Heteroptera, Cimicomorpha, Tingidae) were analyzed using conventional chromosome staining and FISH with 18S rDNA and (TTAGG)n telomeric probes. The FISH technique was applied for the first time in the Tingidae. In spite of the fact that all species showed the same chromosome number (2n = 12 + XY), they have significant differences in the number and position of rDNA loci. FISH with the classical insect (TTAGG)n probe produced no signals on chromosomes suggesting telomeres in lace bugs to be of some other molecular composition. Tingidae share absence of the (TTAGG)n telomeric sequence with all so far studied taxa of the advanced true bug infraorders Cimicomorpha and Pentatomomorpha.
Karyotype, sex chromosomes, FISH, rDNA, (TTAGG)n, Hemiptera , Heteroptera , Cimicomorpha , Tingidae
Tingidae (lace bugs) are a large widespread family of herbivorous bugs including 2200 species belonging to 280 genera. The family is subdivided into two, Tinginae and Cantacaderinae, or into three (Vianaidinae in addition) recent subfamilies; the subfamily Tinginae is the largest and the most diverse subfamily of lace bugs. Tingidae are placed in the Cimicomorpha, but their relationships within this large infraorder are not entirely clear (
Many studies have proven that chromosome alterations are significant for species evolution and then, cytogenetics can be a useful tool for evolutionary, taxonomic, phylogenetic and speciation studies (
Cytogenetic data on members of the Tingidae are scarce and only involve species of the Tinginae. Currently, chromosome information of 29 species, belonging to 18 genera, i.e., approximately 1% and 6% respectively is known (
All previous investigations of lace bugs have been carried out using conventional chromosome staining techniques. Identification of individual chromosomes in karyotypes is a difficult task in the case of true bugs because of morphologically uniform holokinetic chromosomes. However, with the use of C-banding technique,
In the past decades, fluorescence in situ hybridization (FISH) has increased the resolution of the true bugs’ cytogenetics. Thanks to this technique, the analysis of the karyotypes has become more informative and comprehensive. In true bugs, ribosomal genes are commonly used as markers for the physical mapping of their chromosomes (reviewed in
Here, the first FISH-based study for the characterization of tingid karyotypes is presented. We describe the karyotypes of Elasmotropis testacea (Herrich-Schaeffer, 1835), Tingis cardui (Linnaeus, 1758), T. crispata (Herrich-Schaeffer, 1838), and Agramma femorale Thomson, 1871 after FISH with an 18S rDNA probe. Note that for two last species, the standard karyotype is reported for the first time.
Additionally, we used FISH with a (TTAGG)n probe to analyze whether the classical “insect” telomeric motif (TTAGG)n is present in the lace bug species. Previous studies on species of two cimicomorphan families (Miridae and Cimicidae) showed the absence of this telomeric repeat (
The material studied is presented in Table
Species | Number of males/chromosome plates studied | Locality and date of collection | Host plant |
---|---|---|---|
Elasmotropis testacea | 2/37 | Russia, Republic of Bashkortostan, South-Ural state natural reserve, env. of village Terekly, 12 km ENE of settl. Arhangelskoe, 54°26'N, 56°57'E, alt. 269 m, 5.08.2014 | Echinops sp. (Asteraceae) |
Tingis cardui | 2/19 | Russia, Republic of Bashkortostan, South-Ural state natural reserve, env. of settl. Inzer, 54°13'N, 57°34'E, alt. 349 m, 4.08.2014 | Carduus sp. (Asteraceae) |
T. crispata | 3/143 | Russia, Tolyatti, 53°31'N, 49°25'E, alt. 95 m, 13.08.2014 | Artemisia vulgaris Linnaeus, 1753 (Asteraceae) |
Agramma femorale | 2/23 | Russia, Republic of Bashkortostan, South-Ural state natural reserve, env. of village Revet’, 54°11'N, 57°37'E, alt. 285 m, 10.08.2014 | Juncus sp. (Juncaceae) |
Lace bug species were collected in 2014 by V. Golub in Republic of Bashkortostan, Russia. Only male adult specimens were analyzed. In field, the specimens were fixed immediately after capturing in 3:1 fixative (96% ethanol: glacial acetic acid) and stored at 4 °C. In laboratory, testes were dissected in a drop of 45% acetic acid and squashed. The cover slips were removed using dry ice. Prior to staining, the preparations were examined by phase contrast microscopy. Chromosome staining techniques applied were a Feulgen-Giemsa method as described in
Tingis crispata, 2n = 14 (12A + XY)
Published data: absent
During the diffuse stage, the autosomes were de-condensed whilst the X and Y chromosomes appeared to be fused and heteropycnotic (Fig.
1–6 Male meiosis in Tingis crispata (conventional staining): 1 diffuse stage 2 early diakinesis, two-chiasmate bivalent is indicated by arrow 3 early MI 4 mature MI 5 early AI 6 MII. Sex chromosomes are indicated by arrowheads 7–10 Meiotic chromosomes in Tingidae species after FISH with an 18S rDNA probe: 7 diakinesis in T. crispata 8 first prometaphase in T. cardui 9 MI in Elasmotropis testacea 10 first prometaphase in A. femorale. Sex chromosomes are indicated by arrowheads; autosomally located signals are indicated by arrows.
The 18S rDNA FISH resulted in appearance of a comparatively small interstitial signal in the larger sex chromosome (presumably, the X) and a larger subterminal signal in the smaller sex chromosome (presumably the Y) (Fig.
Tingis cardui, 2n = 14(12A + XY)
Published data: 2n = 14(12A + XY) (
At first prometaphase subjected to 18S rDNA FISH, eight elements were present, including six autosomal bivalents and X and Y chromosomes which lied separately from each other. The bivalents constituted a series decreasing in size, and sex chromosomes were of different size. The subterminally located 18S rDNA sites were revealed on both homologues of a medium-sized autosomal bivalent (Fig.
Elasmotropis testacea, 2n = 14(12A + XY)
Published data: 2n = 14(12A + XY) (
At first metaphase subjected to 18S rDNA FISH, eight elements were present, including six autosomal bivalents which formed a ring with a pseudobivalent of the X and Y chromosomes located in its center. The bivalents constituted a series decreasing in size, and sex chromosomes were of similar size. The subterminally located 18S rDNA sites were revealed in a medium-sized bivalent (Fig.
Agramma femorale, 2n = 14(12A + XY)
Published data: absent
At first prometaphase subjected to 18S rDNA FISH, eight elements were present, including six autosomal bivalents and X and Y chromosomes which lied separately from each other. The bivalents constituted a series decreasing in size, sex chromosomes could not be told apart because of their similar size. The 18S rDNA signals were dispersed all over one of the two sex chromosomes (Fig.
FISH with a (TTAGG)n telomeric probe
In none of the species studied, the (TTAGG)n telomeric probe produced fluorescent signals.
Like other true bugs, Tingidae have holokinetic chromosomes (
For insects with holokinetic chromosomes the low number of chiasmata is characteristic and is considered as a result of a specific structure of holokinetic bivalents (
In “standard” meiosis, during the first division all the chromosomes reduce in number (reductional division), whereas during the second division the chromatids separate (equational division), and this pattern is named “pre-reduction” (
In groups with holokinetic chromosomes, the main problem is to identify individual chromosomes and chromosomal regions in karyotypes. Different cytogenetic techniques, e.g. C-banding, DNA-specific fluorochrome staining, AgNO3 staining, make possible only a few markers to be revealed in true bugs’ karyotypes (
In order to reveal additional chromosomal markers and gain deeper insights into the evolution of the Tingidae, we have applied FISH with 18S rDNA and telomeric (TTAGG)n probes to the four species from the present study. This is the first time that the lace bugs have been the subject of a molecular cytogenetic study. Physical location of genes remains very poorly studied in true bugs. Out of approximately 40.000 described species (
A very similar variation holds for the four tingid species possessing the same karyotype, 2n = 12 + XY, including two closely related species of the genus Tingis Fabricius, 1803. Our findings suggest that chromosomal divergence can occur among seemingly conserved karyotypes and may play a role in reproductive isolation and speciation of the family Tingidae. Males of T. crispata were found to have rDNA sites on both sex chromosomes, interstitial on the larger and subterminal on the smaller. Since in the XY true bugs species the larger of the two sex chromosomes is conventionally taken as the X (e.g.
Changes in the number and location of rDNA loci are a well-known phenomenon in eukaryotic organisms, including true bugs (e.g.
The majority of insect species is known to share the telomeres composed of the pentanucleotide TTAGG repeat which is considered as an ancestral telomeric motif in this large group of Arthropoda (
The study was performed in the frames of the state research projects Nos 01201451099 and 01201351193, and financially supported by the Russian Foundation for Basic Research (grants no. 14-04-01051-a, 15-04-02326-a). We thank Dr. B. Anokhin (Zoological Institute RAS, St. Petersburg) for technical assistance with FISH.