Karyotype features of trematode Himasthla elongata
© Solovyeva et al. 2016
Received: 11 February 2016
Accepted: 12 April 2016
Published: 29 April 2016
Trematodes have a complex life cycle with animal host changes and alternation of parthenogenetic and hermaphrodite generations. The parthenogenetic generation of the worm (rediae) from the first intermediate host Littorina littorea was used for chromosome spreads production. Karyotype description of parasitic flatworm Himasthla elongata Mehlis, 1831 (Digenea: Himasthlidae) based on fluorochrome banding and 18S rDNA mapping.
Chromosome spreads were obtained from cercariae embryos and redial tissue suspensions with high pressure squash method.74.4 % of the analysed spreads contained 12 chromosome pairs (2n = 24). Chromosome classification was performed according to the morphometry and nomenclature published. H. elongata spread chromosomes had a rather bead-like structure. Ideograms of DAPI-banded chromosomes contained 130 individual bands. According to flow cytometry data, the H. elongata genome contains 1.25 pg of DNA, so one band contains, on average, 9.4 Mb of DNA. Image bank captures of individual high-resolution DAPI-banded chromosomes were provided. Differential DAPI- and CMA3-staining revealed the chromatin areas that differed in AT- or GC-content. Both dyes stained chromosomes all along but with varying intensities in different areas. FISH revealed that vast majority (95.0 %) of interphase nuclei contained one signal for 18S rDNA. This corresponded to the number of nucleoli per cell detected by observations in vivo. The rDNA signal was observed on one or two homologs of chromosome 10 in 72.2 % of analysed chromosome spreads, therefore chromosome 10 possessed the main rDNA cluster and minor ones on chromosomes 3 and 6, that corresponds with AgNOR results.
Himasthla elongata chromosomes variations presented as image bank. Differential chromosome staining with fluorochromes and FISH used for 18S rDNA mapping let us to conclude: (1) Himasthla elongata karyotype is 2n = 24; (2) chromosome number deviates from the previously studied echinostomatids (2n = 14–22); (3). Chromosome 10 possesses the main rDNA cluster with the minor ones existing on chromosomes 3 and 6.
The digenetic trematodes, or flukes, are ones of the most common and abundant of parasitic worms. They act as parasites on all classes of vertebrates, especially marine fishes, and nearly every organ of the vertebrate body can be parasitised by some kind of trematode, adult or juvenile . Many trematode species are the causative agents for massive zoonosises. The list of flukes infectious to humans is quite large, and because of their importance, numerous investigations have been initiated, especially regarding parasite-host interactions . Human parasites as model objects require appropriate laboratory conditions. The subclass Digenea comprises about 18000 species and it is possible to find a safe alternative for parasite research. Genus Himasthla is an example of a safe research option. There are 25 presently described species of Himasthla Mehlis, 1831 (Digenea: Himasthlidae ). Just two of them were found in fishes and one in humans; all three cases seemed to be accidental infections . However, most are studied quite insufficiently, excepting Himasthla elongata (Mehlis, 1831), which became a new model for ecological, immunologic and molecular investigations [4–7].
H. elongata is common in the coastal ecosystems of northern European seas. Like other trematodes, it has a complex lifecycle dependent on of host and parthenogenetic (redia, cercaria) and hermaphrodite generations. The first intermediate hosts of this parasite are intertidal snails of the Littorina (Gastropoda, Prosobranchia) genus and the second intermediate hosts are the intertidal bivalves, Mytilus edulis and Cerastoderma edule; gulls are its final hosts .
Trematode metacercariae parasitising bivalve molluscs may influence the vital functions of the hosts, lowering their resistance to unfavourable environmental factors and, in the case of intensive infection, even causing their death and resulting in mass mollusc mortality .
Cytogenetic studies of parasites are useful not only for understanding systematics, but also the basic mechanisms underlying parasitic agents. Among invertebrates, chromosome mapping has been carried out for a few model organisms due to methodological difficulties.
H. elongata becomes a model for zoological and molecular studies, but it has not been studied at the cytogenetic level. The lack of knowledge about karyotype makes problems for genomic investigation. Moreover, propelled by ever-increasing throughput and decreasing costs, next generation sequencing (NGS) has produced a growing number of genomes and transcriptomes sequenced (existing in databases) and it looks like the H. elongata genome will be sequenced soon. Newly sequenced genome should be assembled and attached to a chromosome’s physical map, thus it is necessary to acquire the data on its karyotype. The purpose of the current study is the description of H. elongata chromosomes. DNA sequence mapping is most convenient for carrying out counterstaining chromosome bands with fluorochromes. DAPI excitation emission varies in proportion to the AT-content of DNA and chromatin condensation level . We elaborated protocols for chromosome preparation and fluorescence in situ hybridisation (FISH), enabling conservation of the typical pattern of DAPI-banding for all components of the karyotype. To establish the position of GC-rich bands on individual chromosome sets, a GC-specific fluorochrome, chromomycin A3 (CMA3), was used for staining in addition to DAPI counterstaining. The present study is focused on karyotype description of the parasitic flatworm, H. elongata, based on fluorochrome banding and 18S rDNA mapping.
Sampling site and collection of parasites
A collection of periwinkles infected with H. elongata was obtained from, along with cell suspension preparation carried out at the White Sea Biological Station of the Zoological Institute of the Russian Academy of Sciences situated in the Chupa Inlet, the Kandalaksha Bay of the White Sea (66°20′N; 33°38′E) during July and early August in 2012 and 2013. Digeneans were identified on the basis of their mollusc hosts and their morphological  and molecular (18S rDNA) features. At least 8 snails with only H. elongata infection i.e. 8 populations of H. elongata parthenogenetic larvae obtained from their hosts were used in cytogenetic experiments.
Live materials were observed under a Leika DM2500 microscope. Dark field illumination at low magnification (objective 10×) was realised by use of the Ph3 phase diaphragm in combination with closed differential interference contrast (DIC) prisms. DIC was utilized for observations under the 100× DIC objective.
Metaphase chromosome spread preparation
H. elongata parthenitae - rediae were obtained in amount of several hundreds individuals from each L. littorea snails and washed from the host tissue by three exchanges of seawater filtered through a 0.22 mm Millipore filter. The worms were incubated in Leiboviz L-15 (Sigma) medium with 0,01 mg/ml gentamycin (PanECO, Russia) and 0.1 % colchicine (PanECO, Russia) for 4 h at room temperature and treated with hypotonic solution (5 mM KCl) for 40 min, then fixed with Carnoy’s solution (methanol:glacial acetic acid mixture; 3:1). Fixed rediae were repeatedly passed through the syringe with a 22 G needle. The suspension was placed in 15 ml tubes and kept still for 3–5 min to sediment large fragments. The top phase was collected and centrifuged three times at 2.5 krpm for 10 min with the three changes of fixative and stored at −20 °C until slide preparation. Chromosome spreads were prepared according to classic cytogenetical protocols used for trematodes [10–15] with convenient air-drying method, along with more recent techniques, such as high-pressure squash preparation [16, 17]. The convenient air-drying method was performed as follows: 4 or 5 drops of cell suspension were carefully placed onto slides which had been previously chilled in ice water for maintaining a thin film of water at the time when the drops fall on the slide from a height of about 20 cm. Slides were air-dried and then stored at −20 °C until staining. A modified protocol from Deng et al.  was performed the next way: the washed slides were placed an a stainless steel bar inside a moist chamber. 30 μl of cell suspension were dropped on each slide Then the moist chamber was at 50 °C in thermostat until fixative drying.
High-pressure squash preparation
Whole H. elongata rediae were fixed after colchicine and hypotonic solution treatment and used for spreading chromosomes. On average 50 rediae from different snails were used for slides preparations. The suspension of dissociated into small pieces worms’ tissues was dropped on slides containing Carnoy’s solution on the surface. The spread cells were coated with a 50.0 % propionic acid drop and then covered by 24 × 24 mm cover slips immediately after fixative evaporation. A mechanical vise was used to evenly apply pressure to further flatten chromosomes on the preparation. Approximately 150 kg/cm2 of pressure through the precision vise was gradually applied during 90–120 second intervals. At that point, the slides were placed into liquid nitrogen and the cover slips were removed. Afterwards, the slides were dehydrated in a series of ethanol (70.0, 80.0, and 100.0 %), air-dried and kept in −20 °C until staining.
The slides were stained in a 3.0 % solution of Giemsa dye (Merck, USA) in phosphate buffer solution (pH 6.8) for 12 min and flushed with flowing water.
Fluorescent in situ hybridization with 18S rDNA probe
As the H. elongata genome is not yet sequenced and 18S rDNA is quite conservative, a small subunit ribosomal probe was generated by polimerase chain reaction (PCR) using the 18Sa forward primer (AACCTGGTTGATCCTGCCA) and the 18Sb reverse primer (GATCCTTCTGCAGGTTCACCTAC) . The PCR product was sequenced in order to confirm its attribution to 18S rDNA and submitted to GenBank (KU886143). The analysis of a 18S rDNA probe sequence was performed with the BLAST tool . Isolation of genomic DNA was performed according to Winnenpeninx . The probes were labelled with biotin-14-dUTP under appropriate conditions. Slide pretreatment was performed according to Khodyuchenko et al.  with modifications: chromosome preparations were digested with 100 μg/ml RNase A in 2 × SSC for 1 h at 37 °C and washed twice in 2 × SSC for 5 min each, then prefixed with 2.0 % PFA for 15 min, and then washed with 1 × PBS three times for 5 min at a time and incubated in 0.1 % Triton X-100 for 10 min and washed again with 1 × PBS. Hybridisation at 37 °C for 18 h was followed by the washes, which included 0.2 × SSC (3 × 5 min, 60 °C) and 2 × SSC (3 × 5 min, 42 °C). Probe signals were detected with streptavidin – Alexa Fluor 594 conjugate (Life technologies, USA) in blocking solution. The slides were counterstained with Slow Fade Gold Antifade with DAPI (Molecular Probes, USA).
Double Chromomycin A3 - DAPI staining
The slides were stained with Chromomycin A3 (CMA3) based on Schweizer  with several modifications. The stock solution of chromomycin A3 (Sigma-Aldrich) (1 mg/ml) was prepared by dissolution in deionised water without stirring for several days at 4 °C in the dark. Older solutions tend to stain better. Working solution of CMA3 (0.5 mg/ml) was prepared by dissolving (1:1) the stock solution in Mcllvaine’s buffer (pH = 7.0) with 5 mM MgCl2. Slides were rinsed in McIlvaine’s buffer and placed in CMA3 working solution under a coverslip and stained in the dark at RT for 1 to 2 hours. To remove the coverslip, the slides were briefly washed in McIlvaine’s buffer and air-dried. After that a few drops of DAPI solution are placed on slides and covered with a coverslips. Slides are stained in the dark for 20–30 min at RT, rinsed in McIlvaine’s buffer and air-dried. DAPI stock solution (1 mg/ml) is prepared on deionized water and can be stored frozen in dark for a year. DAPI working solution (0.8mkg/ml) is prepared on McIlvaine’s buffer (pH = 7.0) usually fresh before use. Then the slides were mounted in ProLongR Gold antifade (Invitrogen) and sealed with nail polish. Stained slides were aged for 3 to 5 days in the dark at 4 °C to stabilise CMA3 fluorescence before examination.
Chromosome spreads were examined with a Leica Fluorescence Microscope DMI 6000B (Leica Wetzlar GmbH, Germany) at the Development of Molecular and Cellular Technologies Resource Centre at Saint-Petersburg State University. Images were taken with a 100×/1.4 oil immersion objective using appropriate filter cubes fluorescent dyes, like CMA3(430–480 nm), Alexa 594 and DAPI(360–390 nm), and recorded using a monochrome-cooled CCD camera. Karyological data of H. elongata (relative length and centromeric index) were calculated in 64 best spreads out of 100 evaluated spreads with Image Tool 3.0 software . The centromere position on the chromosomes was classified according to the nomenclature of Levan et al. . Negative images of DAPI-stained chromosomes were enhanced in Adobe Photoshop version 4 as described before .
Frequencies of chromosomes with dissociated sister chromatids in H. elongata chromosome spreads (N = 100)
Number of chromosomes with dissociated SCs in each spread
% of spreads with chromosomes with dissociated SCs
Relative length (means ± SD) and centromeric index of H. elongata chromosomes
Relative length, %
Centromeric index, %
14.6 ± 1.7
37.2 ± 2.8
12.6 ± 2.1
14.9 ± 2.3
10.8 ± 2.0
42.2 ± 3.8
9.4 ± 1.4
20.2 ± 3.1
8.4 ± 1.5
21.4 ± 2.9
8.3 ± 1.6
15.8 ± 2.6
8.2 ± 1.4
14.8 ± 2.8
7.0 ± 1.1
24.2 ± 3.4
6.5 ± 1.0
24.5 ± 2.9
5.6 ± 1.0
27.1 ± 3.6
5.2 ± 0.9
20.2 ± 2.8
4.1 ± 1.9
20.7 ± 2.3
Distribution of different types of homologous sister chromatid segregation in H. elongata chromosomes (100 spreads analysed)
Homologous chromosomes combinations
Dissociated and conjugated SCs
The chromosome pairs are distinguished by different types of sister chromatid segregation patterns: e.g. pairs 4, 5, and 8–11 are represented mostly in form with dissociated SCs, while some pairs are represented mostly by forms with conjugated SCs – pairs 3, 7, and 12. It appears that chromosomes prefer to segregate in the approximate order: 10 > 9 > 8, and so on.
There was variability in staining between homologs and sister chromatids. A clear example is seen on chromosome 4 (Fig. 5d). This kind of chromosome variability could be associated with the recombination that takes place during the sexual process between adult worms inside definitive host birds.
The goal of the work was the description of the H. elongata chromosome spreads, good enough for further cytogenetic and molecular researches.
In case of using conventional methods, metaphase cell accumulation observed only after prolonged 4–6 h of colchicine treatment of redia in culture medium and only clumpy metaphases unsuitable for future cytogenetic investigations obtained. It was impossible to determine more than one half of the metaphase chromosome set, while separate chromosomes were too condensed for bands identification. Up to 0.5 % per preparations of prometaphase cells suitable for band detection were obtained by suspending rediae after 4 h and 6 h of colchicine treatment. Thus, it was necessary to find the appropriate method for obtaining a larger amount of a better prometaphase or metaphase spreads. So in vivo observations helped to detect multiple dividing cells in several rediae tissues and cercarial embryos. High-pressure squash preparation used to improve chromosome spreads resolution. This delicate approach allows to get unbroken even fragile structures of polytene chromosomes. The results reproduced in multiple experiments at H. elongata spreads without significant chromosome damages.
There are morphologic polymorphisms in the number of spreads revealed varying levels of sister chromatid segregation (Tables 1 and 3) and discordance among homologous chromosomes. Partial sister chromatid segregation in H. elongata cells is always accompanied by a pronounced asymmetry in the level of chromatin condensation and (or) the band pattern. The phenomenon of homologue discordance is well established for human chromosomes. It was shown that the discordance minimum values (6.7 – 14.2 %) were observed at metaphase after GTG chromosome banding (G-banding with trypsin-Giemsa), whereas prometaphase chromosomes varied in size and had greater homolog discordance – up to 29.8 % after GTG and 64.2 % RBG (R-banding using bromodeoxyuridine and Giemsa) banding . Homologue discordance has been widely discussed in the literature in terms of embryogenesis  and tissue specific stem cell differentiation , as well as asymmetry in cell division and tissue differentiation during embryogenesis [35–37]. Thus, the image bank for H. elongata karyotype component was made in order to detect all probable variabilities in chromosome morphology.
Representative rows for H. elongata individual chromosomes (Fig. 4) served for identification of individual chromosomes of prometaphase and metaphase sets. Individual chromosome variation was estimated by the following criteria: cohesion and incomplete sister chromatid segregation, banding pattern, centromeric heterochromatin and the possibility of distinguishing terminal bands by DAPI-staining. A more detailed description of homologs discordance and sister chromatids’ segregation variability requires additional study. Right now, we point out that one spread can contain homologs with resolutions corresponding to the extreme right and left positions in the chromosome identification rows (Fig. 5). The chromosome shapes are the superposition of two events: (1) the degree of condensation, which reflects mitosis advance; (2) the extent of sister chromatids’ segregation.
Centromeric heterochromatin blocks’ size variations, different from other morphological changes, may potentially reflect population variability. The source of such polymorphism could be found with the determination of species major tandem repeat, the main constituent of heterochromatic regions. It is possible that the first steps of H. elongata genome sequencing will bring up a major tandem repeat of this species. Such a repeat will serve as a reliable probe to assess heterochromatin block variability. Right now, it may only be supposed that possible heterozygosity of large heterochromatin blocks visible in many chromosome sets could be associated with cross fertilisation in adult flukes. The intercellular variation observed in heterochromatin does not exclude the possibility of polymorphism in parthenogenetic clonal populations.
Noticeable banding pattern along H. elongata chromosome arms visible after differential DAPI- and CMA3-staining. It is widely accepted that clear G- and R- blocks, typical for higher vertebrate chromosomes, are heterochromatic and euchromatic regions, respectively . In the current work such banding is obtained for the flatworm karyotype for the first time. Echinostoma caproni is the nearest H. elongata relative, whose genome size is known – 0.85 pg . Unfortunately, E. caproni karyotype is performed by Giemsa staining and C bands (centromeric) were described  and no detailed ideograms were made.
From 40.0 to 95.0 % of ribosomal DNA is in the inactive state in animal cells . FISH identification of genes in heterochromatin requires increased chromosome deproteinisation to improve hybridization conditions. Chromosome mapping of inactive rDNA clusters is a common problem of animals cytogenetics with no universal solution so far. Even short-term treatment with pepsin and proteinase K led to the complete disappearance of the chromomere patterns on H. elongata chromosomes. We used the 18S rDNA FISH protocol without any protease treatment. All signals detected with rDNA FISH were also revealed by Ag-staining. Thus, there are at least 3 nucleoli organizing chromosomes in H. elongata karyotype.
Chromosome number described for Echinostomatidae Looss, 1899 family representatives before recent phylogenetic update 
Chromosome number (2n)
Barsiene, Kiseliene, 1990 
Staneviciute et al. 2015 
Mutafova, Kanev, 1984 
Mutafova et al. 1987 
Barsiene et al. 1990 
Mutafova, Kanev, 1983 
Richard, Voltz, 1987 
Terasaki et al. 1982 
Terasaki et al. 1982 
Barsiene, Kiseliene, 1990 
Mutafova et al. 1986 
Mutafova, et al. 1991 
Barsiene et al. 1990 
Mutafova, 1994 
Mutafova, 1994 
Aleksandrova, Podgornova, 1978 
Aleksandrova, Podgornova, 1978 
Mutafova et al. 2001 
In the current work karyotype of the first representative of Himastla genus determined. It is shown that H. elongata karyotype consists of two pairs of large chromosomes and 10 pairs of smaller-sized chromosomes. Differential DAPI- and CMA3-staining revealed the AT- and GC-rich chromosome bands. The identification of nucleoli organizing chromosomes was performed with 18S rDNA FISH and Ag-staining. The main rDNA cluster was observed on chromosome 10 with minor examples on chromosomes 3 and 6.
This work was supported by the Russian Foundation for Basic Research (grants № 05-04-49156-а, 11-04-01700, 15-04-01857), the Russian Science Foundation (grant no.15-15-20026), and the granting program for “Molecular and Cell Biology” of the Presidium of Russian Academy of Sciences (№ 01.2.00 955639). Editing and publishing costs have been paid for by a grant from the Russian Science Foundation (grant №15-15-20026). We deeply appreciate our colleagues, especially Prof. Kirill Galaktionov and Dr. Ivan Levakin, of the Zoological Institute RAS White Sea Biological Station “Kartesh” for provided support and constant attention. We also thank Dr. Nikolai Aksenov of the Flow cytometry and sorting group at Institute of Cytology RAS for kind advises and help with genome size evaluation. The work was partially carried out at the Development of Molecular and Cellular Technologies Resource Center at St. Petersburg University (project № 1.50.1042.2014).
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