Research Article |
Corresponding author: Natalia V. Golub ( nvgolub@mail.ru ) Academic editor: Christina Nokkala
© 2016 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 VB, Kuznetsova VG (2016) Further evidence for the variability of the 18S rDNA loci in the family Tingidae (Hemiptera, Heteroptera). Comparative Cytogenetics 10(4): 517-527. https://doi.org/10.3897/CompCytogen.v10i4.9631
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As of now, within the lace bug family Tingidae (Cimicomorpha), only 1.5% of the species described have been cytogenetically studied. In this paper, male karyotypes of Stephanitis caucasica, S. pyri, Physatocheila confinis, Lasiacantha capucina, Dictyla rotundata and D. echii were studied using FISH mapping with an 18S rDNA marker. The results show variability: the major rDNA sites are predominantly located on a pair of autosomes but occasionally on the X and Y chromosomes. All currently available data on the distribution of the major rDNA in the Tingidae karyotypes are summarized and shortly discussed. Our main concern is to clarify whether the chromosomal position of rDNA loci can contribute to resolving the phylogenetic relationships among the Tingidae taxa.
Karyotype, FISH, major rDNA cluster, lace bugs, Cimicomorpha , Hemiptera
The true bug family Tingidae is a relatively large and widespread group of phytophagous (sap-sucking) insects, some of which are important agricultural and forestry pests. The insects of this family are commonly known as the lace bugs due to a reticulation of the pronotum and fore wings. The family Tingidae is included in the true bug infraorder Cimicomorpha (Hemiptera, Heteroptera) and considered as the closest relative to the family Miridae, lace bugs being either placed within the superfamily Miroidea (
The relationships within the Tingidae are not entirely clear (
Like other Heteroptera, lace bugs possess holokinetic chromosomes characterized by a non-localised centromere (
Until recently, only conventional chromosome staining techniques were used for the Tingidae. The first attempt to use a differential staining protocol was made by
A molecular hybridization technique such as fluorescence in situ hybridization (FISH) is a very useful method for studying molecular structure of chromosomes and differentiating separate chromosomes in different species. The chromosomal location of the rRNA genes is currently the most widely exploited marker in comparative cytogenetics of the Heteroptera (for a review see
In the context of the above studies, we examined here the location of the 18S rDNA loci through FISH in six further species from the genera Stephanitis Stål, 1873, Physatocheila Fieber, 1861, Dictyla Stål, 1874 and Lasiacantha Stål, 1873. The standard karyotypes of four species, Stephanitis caucasica, S. pyri, Physatocheila confinis and Dictyla rotundata were studied for the first time.
The lace bug species used here were collected in 2015 in the Teberda Nature Reserve, North Caucasus and in Voronezh Province, Russia (Table
Species | Number of males examined | Host plant, date and locality of collection |
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Dictyla echii (Schrank, 1782) | 6 | Echium sp., 22-26.07.2015, Teberda Nature Reserve, North Caucasus, Russia. |
D. rotundata (Herrich-Schaeffer, 1835) | 9 | Echium sp., 27.07.2015, Teberda Nature Reserve, North Caucasus, Russia. |
Lasiacantha capucina (Germar, 1837) | 3 | Thymus sp., 02.08.2015, Teberda Nature Reserve, North Caucasus, Russia. |
Physatocheila confinis (Horváth, 1906) | 3 | Crataegus sp., 2.08.2015, Teberda Nature Reserve, North Caucasus, Russia. |
Stephanitis caucasica Kiritshenko, 1939 | 12 | Rhododendron caucasicum Pallas, 1786, 30.07.2015, Teberda Nature Reserve, North Caucasus, Russia. |
S. pyri (Fabricius, 1775) | 8 | Malus sp., Pyrus sp., 15.08.2015, Voronezh Prov., Russia. |
Only males were used in chromosome analysis. The specimens were fixed in the field in 3:1 Carnoy solution (96% ethanol: glacial acetic acid) and stored at 4°C. In the laboratory, testes were dissected out in a drop of 45% acetic acid and squashed on the slide. The cover slips were removed using dry ice. The preparations were stained using a Feulgen-Giemsa method by
Chromosome slides were analyzed under a Leica DM 6000 B microscope. Images were taken with a Leica DFC 345 FX camera using Leica Application Suite 3.7 software with an Image Overlay module.
Stephanitis caucasica, 2n = 14 (12 + XY)
Published data: absent
At spermatocyte metaphase I (MI), six bivalents of autosomes and X and Y univalent chromosomes are present suggesting diploid karyotype of 2n = 14 (12 + XY). All bivalents are of similar size. The sex chromosomes show different sizes, the larger being presumably the X, and are situated alongside each other (Fig.
Meiotic chromosomes of the Tingidae species with 2n = 12 + XY studied using conventional staining technique and 18S rDNA FISH. 1–3 Stephanitis caucasica 1, 2 conventional staining: MI (1), AI (2) 3FISH: MI4, 5 Stephanitis pyri 4 conventional staining: MI5FISH: MI6, 7 Physatocheila confinis 6 conventional staining: MI/AI transition 7FISH: early MI8, 9 Dictyla rotundata 8 conventional staining: MI9FISH: MI10 Dictyla echiiFISH: early MI11 Lasiacantha capucinaFISH: prophase I. rDNA FISH signals are indicated by arrows. X and Y chromosomes are indicated by arrowheads. Bar = 10µm.
18S rDNA FISH resulted in bright signals on an autosomal bivalent at MI. The signals are most likely located subterminally on each homolog. Sex chromosomes are placed very close to each other (Fig.
Stephanitis pyri, 2n = 14 (12 + XY)
Published data: absent
At spermatocyte MI, six bivalents of autosomes and X and Y univalent chromosomes are present suggesting diploid karyotype of 2n = 14 (12 + XY). All bivalents are of similar size. The sex chromosomes show slightly different sizes, the larger being presumably the X, and are situated alongside each other (Fig.
18S rDNA FISH resulted in bright signals on an autosomal bivalent at MI. The signals are located interstitially on each homolog. The sex chromosomes are mutually co-orientated on the spindle (Fig.
Phisatocheila confinis, 2n = 14 (12 + XY)
Published data: absent
At spermatocyte MI/AI transition, six bivalents of autosomes and X and Y univalent chromosomes are present suggesting diploid karyotype of 2n = 14 (12 + XY). All bivalents are of similar size. The sex chromosomes show distinctly different sizes, the larger being presumably the X. The sex chromosomes segregate ahead of the autosomes (Fig.
18S rDNA FISH resulted in bright signals on an autosomal bivalent at MI. The signals are located interstitially on each homolog (Fig.
Dictyla rotundata, 2n = 14 (12 + XY)
Published data: absent
At spermatocyte MI, six bivalents of autosomes and X and Y univalent chromosomes are present suggesting diploid karyotype of 2n = 14 (12 + XY). All bivalents are of similar size. The sex chromosomes show a similar size and are situated alongside each other (Fig.
18S rDNA FISH resulted in bright signals on an autosomal bivalent at MI. The signals are located interstitially on each homolog (Fig.
Dictyla echii, 2n = 14 (12 + XY)
Published data: 2n = 14 (12 + XY) in
At early spermatocyte MI, there are six bivalents of autosomes and X and Y univalent chromosomes. All bivalents are of similar size. The sex chromosomes show a similar size and are placed not far from each other. Bright 18S rDNA FISH signals are located at one end of each sex chromosome (Fig.
Lasiacantha capucina, 2n = 14 (12 + XY)
Published data: 2n = 14 (12 + XY) in
At spermatocyte prophase I, there are six bivalents of autosomes which have diffuse structure at this stage. The X and Y chromosomes are positively heteropycnotic and placed very close to each other. Bright 18S rDNA FISH signals are located interstitially on each homolog of a bivalent (Fig.
Comparative karyotype analysis of six lace bug species was achieved using standard chromosome staining along with the 18S rDNA FISH marker. All species were found to have 2n = 14 (12 + XY). The karyotypes of Stephanitis caucasica, S. pyri, Physatocheila confinis and Dictyla rotundata were studied for the first time. The karyotypes of Dictyla echii and Lasiacantha capucina were previously studied by
The results of this study confirmed the assumption of the high degree of karyotype conservation for the Tingidae (
Despite the relative conservatism of the karyotype structure in general, some lace bug species clearly differ in size of sex chromosomes. For example, X and Y chromosomes appear noticeably heteromorphic in size in Physatocheila confinis, while they are evenly-sized in Dictyla rotundata and D. echii (Figs
Some other true bug families also demonstrate interspecies difference in size of sex chromosomes (
In the Heteroptera, the major rRNA gene FISH has yielded a significant body of literature (
Species | Karyotype | 18S rDNA-bearing chromosomes | The chromosomal location of 18S rDNA clusters | References |
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Agramma femorale Thomson, 1871 | 12 + XY | X | Subterminal |
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Dictyla echii (Schrank, 1782) | 12 + XY | XY | Subterminal both on X and Y | Present paper |
D. rotundata (Herrich-Schaeffer, 1835) | 12 + XY | AA | Interstitial | Present paper |
Elasmotropis testacea testacea (Herrich-Schaeffer, 1830) | 12 + XY | AA | Subterminal |
|
Lasiacantha capucina (Germar, 1837) | 12 + XY | AA | Interstitial | Present paper |
Physatocheila confinis (Horvath, 1906) | 12 + XY | AA | Interstitial | Present paper |
Stephanitis caucasica Kiritshenko, 1939 | 12 + XY | AA | Subterminal | Present paper |
S. pyri (Fabricius, 1775) | 12 + XY | AA | Interstitial | Present paper |
Tingis crispata (Herrich-Schaeffer, 1838) | 12 + XY | X,Y* | Interstitial on X, subterminal on Y |
|
Tingis cardui (Linnaeus, 1758) | 12 + XY | AA** | Interstitial |
|
Despite the same chromosome number, the 18S rDNA clusters were found to vary in number (one or two in diploid karyotype) and location (sex chromosomes or autosomes) in lace bug species. The rDNA signals were observed either on the X chromosome as in Agramma femorale, or on both sex chromosomes as in Tingis crispata and Dictyla echii, or on a pair of autosomes as in the remaining species. The congeneric species can demonstrate both similarity and dissimilarity in the rDNA location pattern. For example, both studied Stephanitis species (S. caucasica and S. pyri) were found to have rDNA clusters on autosomes. A different situation arises with genera Tingis Fabricius, 1803 and Dictyla, where the congeneric species have rDNA either on autosomes or on sex chromosomes. Different mechanisms have been appointed to play a role in the rDNA evolutionary dynamics, particularly the transposition of the rRNA genes to new chromosome location in closely related species without changes in chromosome number (e.g.,
Besides, the interspecific differences were found in the position of 18S rDNA clusters within chromosomes – subterminal or interstitial, and such differences are occurring likewise in congeneric species (Table
The results presented here show that the major rDNA loci in the lace bug karyotypes may be considered as essential cytological markers to compare karyotypes of phylogenetically related species and to disclose chromosomal differentiation in species with similar karyotypes. This is likewise true for the species of the subfamily Triatominae (Reduviidae) which share the karyotype of 2n = 12 + XY and show extremely high dynamics of rDNA clusters, with the variation observed both between and within the species (
Based on the currently available data, the autosomal major rRNA gene location appears prevalent in the Tingidae being found in 6 genera out of the 7 genera tested. The occurrence of major rDNA sites in autosomes of the Tingidae is similar to the pattern that is most frequent in the order Heteroptera (e.g.,
In summary, the interspecific similarities and differences in the distribution of the major rDNA clusters make them promising markers for the further study of chromosome evolution in lace bugs. However, because of insufficient taxon sampling, the currently available data are inadequate to clarify the phylogenetic relationships within the Tingidae.
The study was performed due to the state research project No 01201351193 and was 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.