Research Article |
Corresponding author: Xiao-dong Zheng ( xdzheng@ouc.edu.cn ) Academic editor: Valeria Specchia
© 2018 Jin-hai Wang, Xiao-dong Zheng.
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:
Wang J-h, Zheng X-d (2018) Cytogenetic studies in three octopods, Octopus minor, Amphioctopus fangsiao, and Cistopus chinensis from the coast of China. Comparative Cytogenetics 12(3): 373-386. https://doi.org/10.3897/CompCytogen.v12i3.25462
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To provide markers to identify chromosomes in the genome of octopods, chromosomes of three octopus species were subjected to NOR/C-banding. In addition, we examined their genome size (C value) to submit it to the Animal Genome Size Database. Silver staining revealed that the number of Ag-nucleoli was 2 (Octopus minor (Sasaki, 1920)), 2 (Amphioctopus fangsiao (d’Orbigny, 1839)) and 1 (Cistopus chinensis Zheng et al., 2012), respectively, and the number of Ag-nucleoli visible was the same as that of Ag-NORs on metaphase plates in the same species. In all analyzed metaphases, Ag-NORs were mainly located terminally on the long arms of chromosomes 3 (3rd) of O. minor and on the short arms of chromosomes 4 (4th) of A. fangsiao, whereas only one of the chromosomes 23 (23rd) was found Ag-NORs of C. chinensis. C-bands were localized predominantly in the centromeric regions of chromosomes in the three species, while other conspicuous stable C-bands were observed in terminal regions, including the Ag-NORs. That means these three chromosome pairs (3rd, 4th and 23rd) could be considered species-specific cytogenetic markers. The mean C values of O. minor, A. fangsiao and C. chinensis were 7.81±0.39 pg (0.070 pg per unit length), 8.31±0.18 pg (0.068 pg per unit length) and 5.29±0.10 pg (0.038 pg per unit length), respectively, and results showed that C values of the three species were not proportional to the relative length of the chromosomes. These cytogenetic characteristics will provide more theoretical foundation for further researches on chromosome evolution in octopods.
octopods, karyotype, Ag-NORs, C-bands, genome size, flow cytometry
Genetics and cytology combine to establish cytogenetics, mainly from the perspective of cytology, especially from a chromosome structure and function as well as the relationship between chromosomes and other organelles, to elucidate the mechanism of inheritance and variation. Cytogenetic analysis usually includes chromosome karyotype, band type, flow karyotype analysis and fluorescence in situ hybridization. Previous published reviews on chromosomal studies of molluscs were greatly increased since cytogenetic techniques including silver-staining, C-and G-banding and have begun to be applied to molluscan chromosomes (
Octopods, such as Octopus minor (Sasaki, 1920), Amphioctopus fangsiao (d’Orbigny, 1839) and Cistopus chinensis Zheng et al., 2012 are cephalopod species. The previous chromosome analysis of cephalopods had revealed 2n=60 or 92 in most species studied, including O. minor, A. fangsiao, C. chinensis, two sepiids (Sepia esculenta and S. lycidas Gray, 1849) and three loliginids (Heterololigo bleekeri Natsukari, 1984, Sepioteuthis lessoniana Blainville, 1824 and Photololigo edulis (Hoyle, 1885)) (
As an important part of the study of cytogenetics, more and more genome sizes (C values) have been revealed. Although the genome sizes of 281 mollusks have been submitted to the Animal Genome Size Database (http://www.genomesize.com. Accessed December 25, 2017) while there just have been 6 species of cephalopod C values that can be obtained from the database: O. bimaculatus (
To develop octopus chromosome markers, the present research has first completed NOR/C-banding. Also we examined their C values to submit to the Animal Genome Size Database. This is a basic work for molecular cytogenetic research of octopods. It is expected to lay a solid theoretical foundation for further researches on chromosome evolution in octopods.
The published information of cephalopod genome size. BA: Biochemical Analysis, FCM: Flow Cytometry, BFA: Bulk Fluorometric Assay, CGS: Complete Genome Sequencing, FIA: Feulgen Image Analysis Densitometry.
Species | Origin | Internal standard | Method | C value (pg)/genome size (Gb) | References |
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O. vulgaris | sperm | Not specified | BA/CGS | 5.15 pg/2.5–5 Gb |
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O. minor | Haemocytes | G. domesticus | FCM | 7.82±0.56 pg | This study |
O. bimaculatus | sperm | Strongylocentrotus purpuratus | BFA | 4.30 pg |
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O. bimaculoides | Not specified | Not specified | BFA/CGS | 2.93 pg/3.2 Gb |
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A. fangsiao | Haemocytes | G. domesticus | FCM | 8.23±0.42 pg | This study |
C. chinensis | Haemocytes | G. domesticus | FCM | 5.13±0.38 pg | This study |
H. maculosa | – | – | CGS | 4.5 Gb |
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S. officinalis | – | – | CGS | 4.5 Gb |
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L. plei | sperm | S. purpuratus | BFA | 2.80 pg |
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L. pealeii | – | – | CGS | 2.7 Gb |
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E. scolopes | Haemocytes, Sperm | G. domesticus | FIA/CGS | 3.75 pg/3.7 Gb |
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I. paradoxus | – | – | CGS | 2.1 Gb |
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A. dux | – | – | CGS | 4.5 Gb |
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N. pompilius | – | – | CGS | 2.8–4.2 Gb |
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Collection of samples and chromosome slides preparation based on the previous published paper (
Preparation of cell suspension
Ten individuals (5 males and 5 females) of each species were used for collecting hemolymph. All subjects were handled according to the guidelines put forth by the EU Directive 2010/63/EU for cephalopod welfare (
Then hemolymph was withdrawn from the heart or cephalic aorta of each octopus using a disposable syringe, and the hemolymph was immediately transferred into a 1.5 ml centrifuge tube containing precooled (4 °C) phosphate-buffered saline (PBS) (v/v=1:1). Mixed samples were centrifuged for 12 min at 300g and 4 °C, and then the hemocytes were resuspended twice in PBS according to above. The last suspension containing 300 μl PBS was added to another tube containing 900 μl precooled (–20 °C) anhydrous ethanol (v/v=1:3), fixing at least 3 hours at 4 °C. The fixed cell was washed twice in PBS after it was suspended with PBS up to 1 ml. Before the flow cytometry detection, a moderate propidium iodide solution (PI, 20–30 μg/ml) was added to the suspensions, staining for 2 hours at 4 °C in dark.
Flow cytometry analysis
Previous studies have shown that the genome size (C value) of chickens (Gallus domesticus) was 1.25 pg (
C value was measured using a model PA flow cytometer (Beckman Coulter Cytomics FC 500 MPL), in principle, at least 15,000 cells were measured in each sample. The blue light of 488 nm was first excited, and the fluorescence of PI was detected by the emission wave length of 625±10 nm. The present study used chicken standard sample as calibration instrument, and then used it as the internal standard, by comparing multiple relationships between the standard sample (chicken red blood cells) and the pending sample (octopus hemolymph) peak, calculating the C values.
After silver staining was performed, the number of Ag-nucleoli was between 1–3 in interphase nuclei of three species (Fig.
Ag-nucleoli of interphase. a 1, 2 or 3 normal nucleolus organizer regions (NORs) in interphase of O. minor and A. fangsiao, and the two species mainly contain 2 NORsb 1 or 2 NORs in interphase of C. chinensis, and most of them contain 1 NORs . Scale bar: 5 μm.
The C-band karyotype analysis indicated that there were 31 C-bands (O. minor), 25 C-bands (A. fangsiao) and 25 C-bands (C. chinensis) in three species of octopods, respectively. C-bands were consistently localized in the centromeric regions of most chromosomes in the three species, but which varied in size (Fig.
Karyotypes and NOR-bearing chromosomes from three species of octopod gills. a The metaphase plate of O. minor b Karyogram of O. minor from (a) showed that Ag-NOR loci were located on the long arms of metacentric chromosome 3 c The metaphase plate of A. fangsiao d Karyogram of A. fangsiao from (c) showed that Ag-NOR loci were located on the short arms of metacentric chromosome 4 e The metaphase plate of C. chinensis f Karyogram of C. chinensis from (e) showed that Ag-NOR loci were located on long arms of subtelomeric chromosome 23. Arrows indicate the NOR-bearing chromosomes. Scale bars: 5 μm.
Combined with the results of silver staining and C-banding, although C-bands were localized predominantly in the centromeric regions of chromosomes in the three octopus species, other conspicuous C-bands were observed in terminal regions, including the Ag-NORs. Therefore, we can select chromosome pairs 3, 4 and 23 as effective species-specific markers to distinguish the three octopods.
In data analysis, we selected about 15,000 cells per sample, then fluorescence intensity representing the relative DNA content was recorded. Figure
Flow cytometry profiles of relative fluorescence intensity of propidium iodide (PI) in octopus hemocytes of b O. minor c A. fangsiao and d C. chinensis with G. domesticus a as standard (St). Scatter plots display the quality and number of cell mass of standard samples and test samples, and the peak figures show the relative DNA content of each sample. b, c and d are just a representative graph of the three species of octopus samples. CN, the number of cells; X-MEAN, mean fluorescence intensity.
Species | Sample no. | Fluorescence intensity | C value/pg | C value (X±SE) /pg | ||
G. domesticus | 0 | 10.4 | 1.25 | 1.25 | ||
O. minor | male | 1 | 63.5 | 7.63 | 7.85±0.47 | 7.81±0.39 |
2 | 60.1 | 7.22 | ||||
3 | 70.4 | 8.46 | ||||
4 | 67.2 | 8.08 | ||||
5 | 65.5 | 7.87 | ||||
female | 6 | 61.3 | 7.37 | 7.76±0.32 | ||
7 | 63.5 | 7.63 | ||||
8 | 67.2 | 8.08 | ||||
9 | 66.1 | 7.94 | ||||
A. fangsiao | male | 1 | 72.3 | 8.69 | 8.33±0.25 | 8.31±0.18 |
2 | 68.9 | 8.28 | ||||
3 | 67.5 | 8.11 | ||||
4 | 68.4 | 8.22 | ||||
female | 5 | 70.2 | 8.44 | 8.30±0.10 | ||
6 | 68.8 | 8.27 | ||||
7 | 68.3 | 8.21 | ||||
8 | 69.0 | 8.29 | ||||
C. chinensis | male | 1 | 43.8 | 5.26 | 5.28±0.08 | 5.29±0.10 |
2 | 44.6 | 5.36 | ||||
3 | 43.3 | 5.20 | ||||
female | 4 | 43.5 | 5.23 | 5.29±0.12 | ||
5 | 44.8 | 5.38 | ||||
6 | 45.0 | 5.41 | ||||
7 | 42.9 | 5.16 |
Based on our previous studies, the chromosome total relative lengths of O. minor, A. fangsiao and C. chinensis were 112.33, 122.77 and 139.20. C. chinensis had the largest relative length, followed by A. fangsiao and O. minor, which was not proportional to the C value. Obviously, C. chinensis had the smallest C value but the largest chromosome relative length. The DNA content of the unit length chromosomes of the three octopods was about 0.070 pg (O. minor), 0.068 pg (A. fangsiao) and 0.038 pg (C. chinensis) respectively. Results suggested that there was no significant positive proportional relationship between the C value and the relative chromosome length. Besides, this study analyzed the C values of 14 species of cephalopods, ranging of 2.20 to 8.23 pg (2.10–7.86 Gb), of which I. paradoxus had the smallest genome size and the largest value from A. fangsiao. Overall, the average genome size of 6 species from Octopoda (3.35–8.23 pg) was higher than that of 8 species from Sepiida and Teuthida (2.20–4.71 pg).
We first carried out silver staining (Ag-NOR) in octopus species, and the results showed that C. chinensis had only one nucleolus organizer region (NOR) which was located terminally at the long arms of a pair of homologous chromosomes. O. minor and A. fangsiao had two NORs which located terminally on a pair of homologous chromosomes. As an effective chromosome marker, polymorphisms in NORs can be observed in interspecies or intraspecies comparisons, including the number, location and size of sliver staining (
The number of C-bands in O. minor was larger than that of the other two octopus species, which was consistent with the Ag-NORs. Although A. fangsiao and C. chinensis had the same number of C-bands, while the former contained more interstitial C-bands. Taking these two points into account, it is confirmed that the O. minor is more advanced in evolution from the chromosome level. Almost all chromosomes of three species of octopods can show C-bands in centromere regions, and it means that heterochromatic blocks are evident in the pericentromeric regions of chromosomes, which is consistent with the traditional view that the centromere region is mainly consisted of heterochromatin. The C-banding results showed that the NOR regions of the three species were all deep-stained C-bands, which also coincided with the common assumption that the NOR regions were composed of heterochromatin. The stable C-bands can be used as markers for chromosome identification, while the higher polymorphic C-band is not suitable as a marker for distinguishing chromosomes, but it can be used as a genetic marker for the study of chromosome polymorphism. In present study, chromosomal markers for identifying three species of octopods were developed by Ag-NORs and C-bands, and it was effective means. Long before that, Martinez-Lage et al. (1995) managed to separate the chromosomes of three shellfish by banding techniques, which confirmed the reliability of this method.
In the present study, due to different survival pressure from geographical distribution (
DNA is linear on the chromosome. According to
In conclusion, the present study combining a previously published paper (
We thank associate Prof. Mingzhuang Zhu, who helped us in the flow cytometer analysis. We thank Yaosen Qian, Qiaozhen Ke and Bing Cai for providing the O. minor and C. chinensis individuals from Rongcheng and Ningde, China. We thank Dr. Diego Orol Gómez for correcting the grammar of the article. This study was supported by research grants from National Natural Science Foundation of China (No31672257) and Key Development Plan of Shandong Province (2016GSF115014).