Corresponding author: Mónica Gabriela Chirino ( email@example.com )
Academic editor: Seppo Nokkala
© 2017 Lucila Belén Salanitro, Anabella Cecilia Massaccesi, Santiago Urbisaglia, María José Bressa, Mónica Gabriela Chirino.
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: Salanitro LB, Massaccesi AC, Urbisaglia S, Bressa MJ, Chirino MG (2017) A karyotype comparison between two species of bordered plant bugs (Hemiptera, Heteroptera, Largidae) by conventional chromosome staining, C-banding and rDNA-FISH. Comparative Cytogenetics 11(2): 239-248. https://doi.org/10.3897/CompCytogen.v11i2.11683
A cytogenetic characterization, including heterochromatin content, and the analysis of the location of rDNA genes, was performed in Largus fasciatus Blanchard, 1843 and L. rufipennis Laporte, 1832. Mitotic and meiotic analyses revealed the same diploid chromosome number 2n = 12 + X0/XX (male/female). Heterochromatin content, very scarce in both species, revealed C-blocks at both ends of autosomes and X chromosome. The most remarkable cytological feature observed between both species was the different chromosome position of the NORs. This analysis allowed us to use the NORs as a cytological marker because two clusters of rDNA genes are located at one end of one pair of autosomes in L. fasciatus, whereas a single rDNA cluster is located at one terminal region of the X chromosome in L. rufipennis. Taking into account our results and previous data obtained in other heteropteran species, the conventional staining, chromosome bandings, and rDNA-FISH provide important chromosome markers for cytotaxonomy, karyotype evolution, and chromosome structure and organization studies.
Largus, Heteroptera, C-banding, rDNA-FISH, holokinetic chromosomes, karyotype comparison
All species of Hemiptera studied so far present holokinetic chromosomes (i.e. without a primary constriction). Kinetic activity is restricted to the chromosome ends and the chromosomes can be regarded as telokinetic during male meiosis, but holokinetic activity is recognized in mitosis. Meiotic behaviour is slightly different depending on whether we are dealing with autosomal bivalents, sex chromosomes, m chromosomes or autosomal univalents. As a rule, autosomal bivalents are chiasmatic and segregate reductionally, whereas sex and m chromosomes are achiasmatic and divide equationally at first male meiotic division. Besides, sex chromosomes do not present a defined position at metaphases I and II. Several reports on C-positive heterochromatin in true bugs showed that C-bands are terminally located (
At present, the seven species cytogenetically studied of Largidae possess a low diploid chromosome number, ranging between 11 and 17 autosomes, a X0/XX sex chromosome system (male/female), except for one species, and a pair of m chromosomes, excluding the genus Largus Hahn, 1831 (
The genus Largus comprises 61 taxonomically described species and most of them are distributed in America, where its geographic distribution ranges from the north of the United States to the south of Argentina. Although they are more diverse and abundant in tropical and subtropical areas, in Argentina there are only seven species recorded (
The main aim of this study was to describe the karyotype of L. fasciatus Blanchard, 1843 and examine the structure of its holokinetic chromosomes by means of C- banding and fluorescent in situ hybridization (FISH) with 18S rDNA probes. Using these data we performed a detailed comparison of the content and distribution of constitutive heterochromatin and the location of rDNA gene clusters between L. fasciatus and L. rufipennis collected from several fields in Argentina.
Adults and nymphs of L. fasciatus (12 males and 2 females) and L. rufipennis (6 males) were collected from 1995 to 2009 in several fields from Buenos Aires and Entre Ríos in Argentina (Table
Species, locality, geographical coordinates, and number of adults’ collected and examined of Largus for chromosomal analyses discriminated by gender.
|Species||City and Province from Argentina||Coordinates (DMS)||N° of individuals|
|Largus fasciatus||Tornquist, Buenos Aires||38°05'45"S, 62°13'25"W||11 males, 2 females|
|Largus rufipennis||Isla Martín García, Buenos Aires||34°11'03"S, 58°14'58"W||1 male|
|Sierra de los Padres, Buenos Aires||37°56'50"S, 57°46'40"W||3 males|
|Santa Catalina, Buenos Aires||34°46'11"S, 58°27'19"W||1 male|
|Ceibas, Entre Ríos||33°30'02"S, 58°48'16"W||1 male|
The captured specimens were swollen in freshly prepared fixative (methanol: glacial acetic acid, 3:1). In the laboratory their gonads were dissected out in 70% ethanol. Cells of gonads were dissociated in a drop of 45% acetic acid, prepared by the squash technique, and stored at -20º C until use. Chromosome preparations were removed from freezer, dehydrated in an ethanol series, and air-dried. For mitotic and meiotic analyses, the chromosome preparations were stained with 5% Giemsa solution following conventional procedures. Heterochromatin content and distribution were analysed by means of C-bands according to
Spread chromosome preparations were made in a drop of 60% acetic acid with the help of tungsten needles and the spreading on the slide was performed using a heating plate at 45° C as described in
Preparations were observed under high power magnification using a Leica DMLB epifluorescence microscope equipped with a Leica DFC350 FX CCD camera and Leica IM50 software, Version 4.0 (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). Black-and-white images of chromosomes were recorded separately for each fluorescent dye with the CCD camera. Images were pseudo-coloured (light blue for DAPI and red for Cy3) and processed with Adobe Photoshop CS6 Version 6.1 (1999–2012) software.
Based on the observation of metaphase I autosomal bivalents (AA) and the identification of the sex univalent we described the male karyotype of L. rufipennis as 2n = 6AA + X0 (see
The C-banding pattern in L. rufipennis and L. fasciatus showed discrete C-positive bands terminally located in all autosomes and the X chromosomes, which were observed in all stages of mitosis and meiosis (Fig.
FISH experiments with the 18S rDNA probe revealed differences in the location of the probe signals between both species analysed (Fig.
Karyotypes of L. rufipennis (a–b) and L. fasciatus (c–e) stained with 5% Giemsa. a diakinesis b metaphase I c oogonial metaphase (2n = 12 + XX) d diakinesis e metaphase I. X = sex chromosome. Scale bar: 10 μm.
C-banding in chromosomes of L. rufipennis (a–c) and L. fasciatus (d–f) stained with DAPI. a spermatogonial metaphase b pachytene c metaphase I d oogonial promethaphase e spermatogonial metaphase f diakinesis. X = sex chromosome. Scale bar: 10 μm.
Largus rufipennis and L. fasciatus, the two species herein analysed, showed similar karyotypes composed of six pairs of autosomes, a simple sex chromosome system (X0/XX), and the same location and distribution of constitutive heterochromatin. The main cytogenetic difference between both species was detected in the location of the rDNA clusters. Two signals were located at a subterminal position of an autosomal bivalent of L. fasciatus but only one signal was observed at one end of the X chromosome of L. rufipennis. Taking into account the data on the chromosomal location of rDNA clusters in other heteropteran species along with our results, the NORs chromosome location varies among several congeneric species, i.e. Belostoma Leach, 1815 (Nepomorpha), Triatoma Laporte, 1832, Panstrongylus Berg, 1879, Rhodnius Stål, 1859 (Cimicomorpha), and Dysdercus Guérin-Méneville 1831 (Pentatomomorpha) (
From the cytogenetic point of view, Largidae is an interesting heteropteran family because of its low diploid chromosome number and the large chromosome size observed in most of the species (
The use of different cytogenetic techniques will be very useful in further integrative studies because a group-level taxonomy followed by a reliable association among different data sets is fundamental to allow a more precise evaluation of the processes involved in the karyotype evolution and the interrelationships among different species.
Taking into account the data on the number and location of rDNA clusters in L. rufipennis and L. fasciatus, we can observe two different patterns of rDNA distribution. As a result, the rDNA clusters revealed by rDNA-FISH are very useful tools for the study of the karyotype structure and chromosome evolution in groups with holokinetic chromosomes due to it can contribute to understand the karyotype evolution and taxonomic relationships among several taxa.
This work was funded by grants PIP 2015 Nº 11220150100347 of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and PICT 0604 of Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) from Argentina.