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Karyotypes of Entedon cionobius Thomson, 1878 and Entedon cioni Thomson, 1878 (Hymenoptera: Eulophidae) were studied using DNA-binding ligands with different base specificity (propidium iodide, chromomycin A3, methyl green and DAPI; all these ligands, except for the last one, were used for the first time in parasitic wasps), C-banding, fluorescence in situ hybridization (FISH) with a 45S rDNA probe and 5-methylcytosine immunodetection. Female karyotypes of both species contain five pairs of relatively large metacentric chromosomes and a pair of smaller acrocentric chromosomes (2n = 12). As in many other Hymenoptera, males of both Entedon Dalman, 1820 species have haploid chromosome sets (n = 6). Fluorochrome staining revealed chromosome-specific banding patterns that were similar between the different fluorochromes, except for the CMA3- and PI-positive and DAPI-negative band in the pericentromeric regions of the long arms of both acrocentric chromosomes. The obtained banding patterns were virtually identical in both species and allowed for the identification of each individual chromosome. C-banding revealed a pattern similar to DAPI staining, although centromeric and telomeric regions were stained more intensively using the former technique. FISH detected a single rDNA site in the same position on the acrocentric chromosomes as the bright CMA3-positive band. Immunodetection of 5-methylcytosine that was performed for the first time in the order Hymenoptera revealed 5-methylcytosine-rich sites in the telomeric, centromeric and certain interstitial regions of most of the chromosomes.
Hymenoptera, Eulophidae, Entedon, chromosomes, karyotypes, base-specific fluorochromes, fluorescence in situ hybridization (FISH) , DNA methylation
Parasitic wasps are a very diverse, taxonomically complicated and economically important group of insects (
Currently, it is becoming obvious that DNA methylation plays a crucial role in the regulation of gene activity (
The present study used molecular cytogenetic techniques to examine two closely related species of parasitic Hymenoptera that belong tothe genus Entedon Dalman, 1820 of the family Eulophidae. This genus comprises about 170 described species (http://www.nhm.ac.uk/research-curation/research/projects/chalcidoids/database/index.dsml, on February 8, 2012); however, critical revision of the group is needed. Both studied species, namely, Entedon cionobius Thomson, 1878 and Entedon cioni Thomson, 1878, though being slightly habitually different, belong to the same species group, Entedon cioni, and attack larvae of the genus Cionus Clairville, 1798 (Coleoptera: Curculionidae) in Europe (
Both Entedon cionobius and Entedon cioni are gregarious endoparasitoids of beetle larvae of the genus Cionus. Host larvae potentially containing broods of Entedon species were collected on different plants of the genus Scrophularia Linnaeus, 1753 (Lamiales: Scrophulariaceae) in Kiev, Ukraine, in May 2010. Weevil larvae were fed with leaves of the host plant in the laboratory until the emergence of the mature parasitoid larvae. For every brood, a few larvae were retained for identification purposes. Larvae that were reared to adults were subsequently identified by A.V. Gumovsky. Voucher specimens were deposited in the collection of the Institute of Zoology of the National Academy of Sciences of Ukraine.
Preparation of chromosomesChromosomal preparations were obtained from cerebral ganglia of prepupae generally following the protocol developed by
Chromosome spreads were stained with combinations of different fluorochromes, including CMA3/DAPI and methyl green (MG)/CMA3 (
CMA3/DAPI staining. The slide was flooded with chromomycin staining solution (0.5 mg/ml in McIlvaine’s buffer (pH 7.0) containing 5 mM MgCl2), covered with a coverslip, and incubated at room temperature in the dark overnight. The coverslip was then removed, and the slide was briefly rinsed with distilled water and air-dried. The slide was then flooded with DAPI solution (1 μg/ml in McIlvaine’s buffer), covered with a coverslip, and stained in the dark at room temperature for 15 min. The coverslip was then removed, and the slide was briefly rinsed with distilled water before being air-dried. The preparation was then mounted in a 1:l mixture of glycerol/McIlvaine’s buffer containing 2.5 mM MgCl2 and sealed with rubber cement. The slide was aged prior to examination by storing in the dark at 30–37°C for a minimum of one day.
MG/CMA3 staining. The slide was stained with CMA3 solution (see above) for approximately one hour and then briefly rinsed with distilled water and air-dried. The preparation was then counterstained for l0–20 min with MG solution in McIlvaine’s buffer (3.5 μg/ml), briefly rinsed with distilled water, and then air-dried. The slide was then mounted in glycerol and sealed with rubber cement. The preparation was stored for one day at 37°C prior to analysis by epifluorescence microscopy.
PI/DAPI staining. The slide was stained with a PI and DAPI mixture (1 mg/ml and 0.5 mg/ml, respectively, in McIlvaine’s buffer) for 20 min with 10 min of pre- and post-incubation in McIlvaine’s buffer. The slide was then briefly rinsed with distilled water, air-dried and mounted in VECTASHIELD anti-fading medium (Vector Laboratories).
MG/DAPI staining. The slide was stained with MG solution in McIlvaine’s buffer (0.35 mg/ml) for 15–30 min and rinsed with distilled water and air-dried. The preparation was then stained with DAPI solution (see above) for 5 min in the dark, rinsed with distilled water, and then air-dried. The slide was then mounted in a mixture of glycerol and McIlvaine’s buffer (1:1).
Fluorescence in situ hybridization (FISH)Plasmid pTa 71 containing the full DNA sequence of the 45S rRNA gene of wheat was used as the probe for visualizing ribosomal genes (
C-banding was carried out as described by
5-methylcytosine was detected according to the protocol described by
Metaphase plates were studied and photographed using an Olympus BX-61 epifluorescence microscope fitted with Cool Snap Ropper Scientific black-and-white CCD camera. The obtained images were processed using the VideoTesT-Kario 1.5 software (Ista-VideoTesT, Russia).
ResultsThe overall chromosomal morphology of both Entedon cionobius and Entedon cioni was very similar. Diploid female karyotypes of these parasitic wasps were comprised of five pairs of relatively large metacentric chromosomes (approximately 10 μm in length) and a pair of smaller acrocentric chromosomes (approximately 5 μm) (2n = 12). Males had haploid chromosome sets (n = 6). The DAPI banding patterns in Entedon cionobius and Entedon cioni were virtually identical. From those patterns, we were able to identify all of the chromosomes within the karyotypes. The DAPI banding patterns demonstrated intensive staining of the pericentromeric regions together with a few weaker interstitial bands on all of the metacentric chromosomes. Additionally, the short arms of the acrocentric chromosomes were also intensively stained (Fig. 1).
Female karyotypes of Entedon cionobius and Entedon cioni. Inverted DAPI staining. Bar = 10 μm.
A similar banding pattern was observed when using PI and CMA3 staining. However, there were narrow but distinct pericentromeric DAPI-negative but PI- and CMA3-positive bands on the long arms of the acrocentric chromosomes (Figs 2 and 3). CMA3/MG staining also demonstrated analogous GC-rich bands in this region (Fig. 4). MG/DAPI staining revealed the same banding patterns as DAPI staining alone (data not shown).
2 PI/DAPI-stained male metaphase plate of Entedon cioni. a PI staining b DAPI staining c superposition of a and b 3 CMA3/DAPI-stained female metaphase plate of Entedon cionobius. a CMA3 staining b DAPI staining c superposition of a and b 4 MG/CMA3-stained female metaphase plate of Entedon cionobius 5 FISH with 45S rDNA probe on a female metaphase plate of Entedon cionobius. Green labels indicate hybridization signals. The chromosomes were counterstained with DAPI. Arrows on Figures 2-5 indicate DAPI-negative, PI- and CMA3-positive NORs on acrocentric chromosome 6. Bar = 10 μm.
FISH using a 45S rDNA probe (Fig. 5) demonstrated distinct pericentromeric signals on the long arms of the acrocentric chromosomes. Thus, the 45S rDNA is located in the same position as the bright CMA3-positive bands that were visualized after CMA3/DAPI and CMA3/MG staining.
The C-banding pattern (Fig. 6) was analogous to the fluorochrome banding pattern, although it differed in the intensity of staining at the centromeric and telomeric regions. Weakly stained smaller interstitial C-bands were also revealed.
5-methylcytosine immunodetection with fluorochrome-labeled antibodies revealed distinct positive signals in the telomeric regions of most chromosomes. Additionally, a few weaker centromeric and interstitial signals could also be seen. However, the nucleolus organizer region did not demonstrate visible positive signals (Fig. 7).
After DNA denaturation during FISH and 5-methylcytosine detection, DAPI counterstaining demonstrated a banding pattern that was rather similar to both the DAPI- and C-banding patterns (Figs 5, 7). Therefore, it was possible to identify most chromosomes after performing FISH or 5-methylcytosine immunodetection.
6 Female karyotype of Entedon cioni. C-banding pattern and Giemsa staining 7 Female karyotype of Entedon cioni. Indirect immunodetection of 5-methylcytosine. a 5-methylcytosine distribution along chromosomes b DAPI counterstaining c superposition of a and b. Bar = 10 μm.
The diploid karyotype of the previously studied Entedon species (
Structural heterogeneity of chromosomes is characteristic of many animals, including insects (
The rDNA sites are highly conserved in all eukaryotic organisms (
Currently, 5-methylcytosine localization has been attempted only for mammalian and plant chromosomes (
In many organisms where the distribution of 5-methylcytosine on chromosomes has been studied, high levels of DNA methylation in heterochromatic segments were detected, usually in telomeric and centromeric regions. The occurrence of intense DNA methylation in the analogous heterochromatic regions of Entedon chromosomes is therefore not surprising. However, highly functionally active chromosomal regions, such as NORs, are not intensively methylated in many organisms (
The present study supports previous hypotheses and provides new insights into the chromosomal structure of parasitic Hymenoptera. Specifically, several different DNA-binding ligands, such as propidium iodide, chromomycin A3 and methyl green, were used for the first time for cytogenetic study of these insects. However, the similarity in banding patterns obtained through these ligands confirms that, unlike in mammals and some other groups, the bands represent differences in packing density along chromosomes instead of differences in base composition. The karyotype structure of the Entedon species, and the family Eulophidae in general, appeared to be relatively conserved. However, we were able to demonstrate an unusual position for the nucleolus organizer in both examined species. For the first time in the order Hymenoptera, our data also visualize the presence of 5-methylcytosine in Entedongenomes in detectable amounts and its non-random distribution along the chromosomes. This allows the use of 5-methylcytosine immunodetection for the investigation of sex determination, cell differentiation and epigenetic regulation of Hymenoptera genomes.
The present study was partly supported by the research grants no. 10-04-01521 and 11-08-00716 from the Russian Foundation for Basic Research (RFBR), no. F35/002 from the State Foundation for Fundamental Research (SFFR) (Ukraine), and from the Alexander von Humboldt Foundation (Germany).