morphological and molecular taxonomical study of euglena ... · morphological and molecular study...

JKAU: Sci., Vol. 26 No. 2, pp: 3-18 (2014 A.D./ 1435 A.H.) DOI:10.4197/Sci.26-2.1

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Morphological and Molecular Taxonomical Study of Euglena viridis Ehren and Euglena gracilis Klebs Growing

in Aleppo, Syria

Montaha Sheikh Al-Ashra, Mohamed Abiad, Ahmad Allahem

Dept. of Zoology, Faculty of Sciences, University of Aleppo, Syria E-mail: [email protected]

Abstract: In spite of the big progress in taxonomy by means of the classical methods, they could not help dealing with many issues especially when studying similar species. This led scientists to use the genotype. Molecular biological data came to emphasize the given taxonomical data which is reasonable since that all the characteristics in an organism reflect the molecular reservoir the organism has “the DNA”. Matter produced by organisms is remarkable. Evolutionary optimized properties such as in Euglena (E.) Euglena descends to the protozoa, It mostly occurs in still water of puddles, ponds, and lakes especially in water with high levels of organic nutrients, Species of genus Euglena are very important for the food chain, and are used as indicators of water quality and in many academic studies. In this study two species were isolated (E. viridis and E. gracilis ) and cultivated by using The differential modified media (HUT, Cramer and Myers). E. viridis was cultivated on Hutner's modified medium (HUT), while E. gracilis was Cultivated on Cramer and Myers modified medium. Both species were classified morphologically and Molecularly using species-specific primers. A local taxonomic key was developed. Key words: E. viridis, E. gracilis, HUT, Cramer and Myers.

4 Montaha Sheikh Al-Ashra et al

Introduction

Euglena is classified as: Kingdom Protista, Sub Kingdom Protozoa, Phylum Sarcomastigopora, Subphylum Mastigophora, Order Euglenida, Family Euglenidae[1]. The name Euglena comes from a Greek word meaning “eyeball organism’’ [2]. The Euglenoid genus is mostly common worldwide. Motile cells are ovoid-cylindrical to narrowly fusiform, frequently with a narrow, elongated, posterior caudus[3]. All green cells have an orange to red stigma unassociated with chloroplasts. There is a single emergent flagellum from a ventral canal opening, and a very short second flagellum retained in the reservoir, with its tip opposite to the paraflagellar swelling of the emergent flagellum[4].

Cytokinesis has been the only way proven of asexual reproduction for Euglena spp[5]. Cell division was described in motile and palmelloid cells, and in certain cyst types [6]. Chloroplast number ranges from one to many, with or without pyrenoids[7]. Euglena mostly occurs in still water of puddles, ponds, and lakes, especially in water with high levels of organic nutrients. It may also occur in and on sediments of river banks[8]. Euglena inhabits shaded or sunny areas, in hard or soft water, of low pH =0.9 to high pH =over 8.0[9].

Euglena gracilis showed some promise after the purification of the waste water with growing conditions pH= 4, and temperature 30–35°C[10]. Species studied (mostly Euglena) have both a vitamin B12 (cyanocobalamin) and a vitamin B1 (thiamine) requirement in nature[11]. Euglena gracilis strain Z is the first euglenoid to have its chloroplast DNA genome sequenced[12].

Euglena gracilis strain Z is used in medical science as a bioassay for vitamin B12 in blood plasma, and it may one day become useful in aquaculture, making it a nutritious food item for the mass production of cultured marine fish larvae [13]. An aquaculture activity system including fish and microalgae, which will be an essential component of Bioregenerative Life Support Systems (BLSS) in space to sustainable development for aqueous environments. Cultivation system of Protozoa is the highest standard in aquaculture activity system, they form the base of the pyramid of power and the beginning of the food chain[14].

Morphological and Molecular Study of Euglena spp. E. viridis and E. gracilis 5

New culturing techniques of micro- organisms caused remarkable progress in the field of Protozoa as at Euglenoids following physiological, genetic and biochemistry process.

Euglena Cultivation needs patience and experience, it is inexpensive and can be maintained long time by renew of media, getting a pure isolates of them is not easy, but repetition of Cultivate and work in sterile conditions can provide pure isolates of Euglena for Molecular study. It was difficult to distinguish the species of Euglena, but by a local taxonomic key which was made depending on an international taxonomic keys we distincted between E. viridis and E. gracilis.

In this study two species were isolated and cultivated ( E. viridis and E. gracilis Klebs (strainZ) by cultivation techniques, using the differential modified media (HUT, Cramer and Myers). E. viridis was cultivated on Hutner's modified medium (HUT)[15], E. gracilis was cultivated on Cramer and Myers modified medium[16] and they have been classified Morphologically and Molecularly using species-specific primers.

Materials and Methods

Organism and culture conditions

Samples of waste water were collected in bottles from El Hamadania area in Aleppo. pH and temperature were measured. Euglena gracilis cells were grown on modified Cramer-Meyer medium as shown in Table1, under the following growth conditions: 24 ±1 ºC; 12:12 h light: dark regime with cool-white fluorescent light at 70 μmol photons m-2s-1 irradiance. Euglena viridis cells were grown on modified HUT medium as shown in Table 2, under the following growth conditions: 20–22ºC; 10:14h light: dark regime with cool white fluorescent tubes which provided approximately 30 μmol photons m -2s-1 irradiance. The two mediums were modified to the optimal medium for growth to increase the numbers of Euglena cells.


Cell number determination

Cell movement was stopped with formalin 3%, Cellular density was evaluated using a Neubauer chamber through a microscope.

Cells in the exponential phase of growth were used in all experiments.

Culture conditions

Euglena cells were maintained autotrophically. Cells were harvested at 4°C, suspended in cold STE buffer (0.37 M Sucrose, 10 mM Tris – HCl, 50 mM EDTA [ethylene diamine tetraacetic acid], pH 7.6), and centrifuged at 4000 rpm for 10 min. The pellets were suspended in 4 volumes of cold STE and used immediately for chloroplast isolation.

Isolation of chloroplasts

Euglena chloroplasts were isolated by modification of the procedure of Manning et al. (1971) [17]. Autotrophically grown cells were destroyed by SLB (Solution Lyses Buffer). The resulting suspension was centrifuged at 4000 rpm for 10 min, the pellet was resuspended in 20 vol (all volumes are based on the original packed-cell volume) of STE and allowed to stand for 10 min. The upper 3/4 of the suspension was decanted through 10 layers of cheesecloth to remove unbroken cells and cell debris, and the remaining solution was discarded. The decanted suspension was centrifuged at 4000 rpm for 10 min and the pellet was suspended in 2 volumes of STE. Four volumes of 2.2 M sucrose were added and the suspension was centrifuged at 14500 rpm for 30 min in the eppendorf rotor.

The green pad that layered on top of the sucrose solution was removed and suspended in STE and centrifuged at 4000 rpm for 10 min; the pellet was suspended in STE. Another sucrose flotation was performed, and the floating pad was again suspended in STE and centrifuged at 4000 rpm for 10 min. This pellet was washed two more times. These chloroplasts were suspended in 0.3 M KCl-0.05 M EDTA (pH=7.6) and centrifuged at 1000 rpm for 5 min. This washing was repeated twice, and the pellet was finally suspended in an equal volume of 0.15 M NaCl- 0.10 M EDTA -0.05 M Tris HCl (pH=9.0) and stored at -20°C.


Isolation of DNA by kt Promega

Tissue Culture Cells

1) The cell or pellet of chloroplast was centrifuged at 14500 rpm for 10 seconds.

2) This pellet was washed with PBS, vortexed and then 600μl of Nuclei Lysis Solution was added and mixed by pipetting.

Lysis and Protein Precipitation

3) 3μl of RNase Solution was added to the cell or pellet of chloroplast and mixed. It was incubated for 15–30 minutes at 37°C and cooled to room temperature.

4) 200μl of Protein Precipitation Solution was added. Then it was vortexed and chilled on ice for 5 minutes.

5) It was centrifuged at 14500 rpm for four minutes.

DNA Precipitation and Rehydration

6) Supernatant was transfered to a fresh tube containing 600μl isopropanol at room temperature.

7) It was mixed gently by inversion.

8) It was centrifuged at 14500 rpm for one minute.

9) Supernatant was removed and added 600μl 70% ethanol at room temperature and mixed.

10) It was centrifuged again as in step six.

11) The ethanol was aspirated and the pellet was air-dried for 15 minutes.

12) DNA rehydrated in 100μl of DNA rehydration solution overnight at 4°C.

DNA Amplification by Polymerase Chain Reaction (PCR)

All primers were synthesized by Sigma Company. E. viridis specific primer for ycf9 gene (accession no. AY047487) are as follows: V1: 5′-CAACCAACGTCTTTTATCAG and V2: 5′-CTAATCAACTTAGGCGTA. E. gracilis specific primer for rpoA gene from chloroplast complete genome (accession no. X70810) are as


follows: P1: 5′-CTCTTCTAAACCTAAAAAGTTGTGAAC; P2: 5′-AAG [18].

Table 1. Cramer-Meyer modified medium Material Per Liter modified

(NH4)2HPO4 1g NH4H2PO4 KH2PO4 1g

MgSO4.7H2O 0.2gCaCl2 0.02 g

Sodium Citrate(Na3C6H507.2H2O) 0.8 gFe2(SO4)3.nH20 3 mg Fen(SO4)3.nH20

MnCl2.4H2O 1.8 mgCoSO4.7H2O 1.5 mg CoSO4 ZnSO4.7H2O 0.4 mg

Na2MoO4.2H2O 0.2 mg Na2MoO4 CuSO4.5H2O 0.02 mg CuSO4

H3BO3 2.48 mgThiamine HCl(VitaminB1) 0.5 mg 0.8 mg

Cyanocobalamin ( Vitamin B12) 0.02 mg 0.7 mg Glucose 10 gPH=3.4

Table 2. HUT modified medium

Exemplary modified Material Amount-quantityKH

2PO

42 mg

MgSO4· 7H

2O 2.5 mg

Sodium acetate 40 mgPotassium citrate 4 mg

Polypeptone 60 mg peptone Yeast extract 40 mgVitamin B

120.05 µg 50 µl (1g/L)

Thiamine HCl 0.04 mgDistilled water 100 mL

pH 6.4

PCR amplification reactions was carried out in 25μl volume reactions mix, according to the following protocol: 12.5 μl of 2X Taq Master Mix, 1 μl of MgCl2(50mM), 2 μl of P1, P2 (25 mM), 3.5 μl of water and 4µl of DNA. PCR tubes were carried out on PCR apparatus (eppendorf). The amplification program consisted of: For E.viridis cycles were as follows: 95°C/2min (1x); 95°C/30 sec, 58°C/30 sec, and 72°C/1min (30x); and 72°C/5min (1x). For E. gracilis cycles were as follows: 95°C/2min (1x); 95°C/30 sec, 55°C/30 sec, and 72°C/1min (30x); and 72°C/5min (1x).


Electrophoresed through a 1% agarose gel, visualized by ethidium bromide staining; amplified DNA products were run on a 1% TBE agarose gel. The gel was stained with 4 μl ethidium bromide after the mixture was cooled. After the gel was allowed to be solidified, 7 μl of amplified DNA fragments, 3 μl of Bromphenolxylencyanolblau (Ficoll) and 5μl of molecular 500 or 1000-bp loading marker ladder (provided from ROTH) were independently added in wells made by special comb and loaded in the gel, then the gel was run under 100 volts for 30 min. The gel was viewed under UV illumination and photographed using gel documentation system.

Results

Cells of E. viridis was appeared on HUT modified medium and arrived to exponential phase of growth after two weeks. Cellular density was evaluated using a neubauer chamber by microscope and it was (45 x 103 cells/ml) Figure 1.

The morphological taxonomicy was supported by using analysis molecular and species - specific primers for E. viridis [18] and after PCR reaction products were separated in agarose gels and visualized by ethidium bromide staining for E. viridis and bands appeared in (amplified sample was repeated to confirm the result) Figure 2.

1. E. viridis, p1=V1 ,p2=V2 ( specific primers V1,V2) 375 pb

2. E. viridis, p1=V1 ,p2=V2 ( specific primers V1,V2) 375 pb

Cells of E. gracilis appeared on Cramer and Myers medium and arrived to exponential phase of growth after three weeks, Cellular density was evaluated using a Neubauer chamber by microscope, it was (2 x 106 cells/ml) Figure 3.

A morphological classification was supported by molecular analysis using species specific primers for E. gracilis [18] and after PCR reaction, products were separated in agarose gels and visualized by ethidium

bromide staining. E. gracilis bands appeared (amplified the sample

was repeated to confirm the result) Figure 4.

1- E. gracilis , p1= P1, p2= P2 ( specific primers P1,P2)350 pb


2- E. gracilis, p1= P1, p2= P2 ( specific primers P1,P2) 350 pb

Fig. 1: Culture of E. viridis on HUT modified medium 4X (image of search)

Fig. 2. PCR reaction products were separated in agarose gels and visualized

by ethidium bromide staining for E. viridis, 1, 2 =375 pb


Fig. 3. A-B Cultures of E. gracilis on Cramer and Myers modified medium

4X,40X(image of search)

Fig. 4 . PCR reaction products were separated in agarose gels and visualized

by ethidium bromide staining for E. gracilis 1, 2 = 350 pbA


Discussion

The qualities of individuals of Euglena viridis coincided with the results showing by Dillard[19] and Kim et al.[20] but differed in dimensions. It was (30-89) µm long, (11-22) µm broad[19] and it was (55-68) µm long, (11-19 ) µm broad [20].

The qualities of individuals of Euglena gracilis coincided with those of Dillard[19]; Kim et al.[20] and Kudo.[21] but differed in dimensions. It was (31-68) µm long, (6-18) µm broad[19]; (46-84) µm long, (11-19 ) µm broad[20], and (35-55) µm long, (6-25) µm broad[21]. This is due to the different conditions in places of samples collection; intensity of light, difference of night and day length, pH, and of temperature.

Cells of E. viridis appeared on HUT modified medium [1], while

Cells of E. gracilis appeared on Cramer and Myers modified medium [17].

Species Synopsis

A local taxonomic key was made depending on international taxonomic keys [19,20], based on some characteristics of organelles as chloroplasts shape, dimensions of cell, length of the Flagellum, and paramylon grains.

A local taxonomic key for E. viridis and E. gracilis: 1- Cell with > 1chloroplast …………………….......................………..……......2

2a- Chloroplasts long and flattened, ribbon-like…………....….…3a

2b- Chloroplasts with paramylon-sheathed pyrenoids….……..….3b

3a- Chloroplasts arranged in stellate groups ……………….…......4a

3b- Chloroplasts discoid to oval, the margins smooth……………4b

4a- Cell spindle-shaped………………………………….….….….5a

4b- Cell nearly cylindrical without haematochrome….……..……5b

5a- Cell 13-18 W., 30-75 L,; paramylon bodis ovoid………..E. viridis

5b- Cell 18-25 W., 40-80 L …………………………...…… E. gracilis


Euglena viridis Ehrenberg ,1830 (Figures 5,6)

Synonyms: E. viridis var. lacustris Francee 1897, E. viridis var. stagnalis France 1897, E. viridis var. mucosa Lemmennann 1910, E. viridis var. purourea Playfair 1921, E. stellata Mainx 1926, E. viridis var. lefevrei Chadefaud 1937, E. viridis var. cuntabrica Pringsheim 1956, E . viridis var. maxima Philipose l982.

Cells are 30-75µm long, 13-18 µm broad, spindle-shaped to cylindrical, anterior end rounded, posterior end tapering gradually to a point. Flagellum exceeding cell length, with moderate euglenoid Movement; Nucleus spherical, shifted posteriorly. Chloroplasts 7-12, elongate bands converging to area anterior to the nucleus, giving rise to a satellite formation, axial with centrally located pyrenoid, pyrenoid in central portion of the chloroplast, covered by other cellular organelles, numerous, small, oval or rod- like paramylon bodis and Stigma present.

Fig. 5: A: E. viridis 40X (image of search), B: Sketch of an Euglena viridis cell [20].

Flagellum

ChloroplastA

20µm

Nucleus

B

20µm

A

10µm

B

10µm


Fig. 6: A: E. viridis 40X (image of search), B: Sketch of an Euglena viridis cell [21].

Euglena gracilis Klebs,1883 (Figure 7)

Synonyms: E. agilis Carter 1865, E. gracilis var. zumsteinii Lwoff 1932, E. hiemalis Matvienko1938, E. gracilis var, urophora Chadefaud et Provasoli 1939, E. gracilis var. bacillaris Pringsheim et Hovasse 1950, E. gracilis var. luxunans Pringsheim 1955, E. gracilis var. saccharophila Pringsheim 1955.

Cells are 40-80µm long, 18-25 µm broad, metabolic, cylindrical or spindle-shaped, slightly attenuated and rounded anteriorly, narrowing posteriorly and terminated by short, non-hyaline obtuse peak. Chloroplasts are brilliant, rather few (8-14 in the cell ), parietal, big, discoid with slightly lobed margins, saucer-shaped in ventral view; each with single pyrenoid doubly sheathed with paramylon caps. Paramylon grains small, oval, elliptical or rod-like, few to fairly numerous, scattered throughout the cell. Nucleus is spherical or elliptical in shape and located in center of the cell or slightly posterior to the center. Flagellum was about one half of the body length, stigma distinct and nearly cup-shaped, relatively small. With marked euglenoid movement, swimming and rotating. Locomotion was clearly metabolic.

Fig. 7. A-D E. gracilis Cells 40X (image of search), E Sketch of an Euglena gracilis cell

[5].


Conclusions

1. Morphological characters of local Euglena spp. were similar to Euglena species around the world, except the dimensions; because of changing environment conditions, lightning, pH, temperate, etc.

2. Shape, number and position of the chloroplasts are some of the most important classical taxonomic features.

3. HUT is a special and differential medium for E. viridis. 4. Cramer-Meyer is a special and differential medium for E. gracilis. 5. Environmental conditions, light, temperature and pH affect growth and

abundance of Euglena. 6. Components of medium play major role in growth of specific species of

Euglena. 7. Molecular data agreed with the given classical taxonomic data.

Acknowledgements

I would like to thank the University Administration and Zoology Department Faculty of Sciences in Aleppo university for their financial support, their continuous help and support during the preparation of this work.

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Protozoologists, U.S.A. 600 (1985). [2] Brocklesby, J., Views of the microscopic world. Pratt. Woodford, Co., New York, 146 (1851). [3] Pringsheim, E. G., Contributions towards a monograph of the genus Euglena. Nova Acta

Leopoldina 18:1–168 (1956). [4] Johnson, L. P. and Jahn, T. L., Cause of the green–red color change in Euglena rubra.

Physiological Zoology 15:89–94 (1942). [5] Leedale, G. F., Euglenoid flagellates Prentice Hall, Englewood Cliffs, NJ, 242 (1967). [6] Johnson, L. P., Euglenae of Iowa. Transactions of the American Microscopical Society

63:97–135 (1944). [7] Zakrys´, B. and Walne, P. L., Floristic, taxonomic and phytogeographic studies of green

Euglenophyta from the southeastern United States, with emphasis on new and rare species. Algological Studies 72:71–114 (1994).

[8] Say, P. J. and Whitton, B. A. Changes in flora down a stream showing a zinc gradient. Hydrobiologia 76:255–262 (1980).

2

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[10] Waygood, F. R., Hussain, A., Godavari, H. R., Tai, Y. C. and Badour, S. S. Purification and reclamation of farm and urban wastes by Euglena gracilis: photosynthetic capacity, effect of pH hydrogen-ion concentration, temperature acetate and whey. Ecological and Biological Series A Environmental Pollution.23:179–215 (1980).

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[13] Hayashi, M., Toda, K. and Kitaoka, S., Enriching Euglena with unsaturated fatty acids. Bioscience, Biotechnology and Biochemistry, 57:352–353 (1993).

[14] Kitaya, Y., Azuma, H. and kiyota, M ., Effects of temperature, CO2/O2 concentrations and light intensity on cellular multiplication of microalgae, Euglena gracilis. Advances in Space Research 35, 1584–1588, Japan (2005).

[15] Hutner, S. H., Zahalsky, A. C., Aaronson, S., Baker, H. and Frank, O., Culture media for Euglena gracilis. In Methods in Cell Physiology. Vol. 2, Ed. by Prescott, D. M., Academic Press, New York, p. 217-228 (1966).

[16] Cramer, M. and Myers, J., Growth and hotosynthetic characteristics of Euglena gracilis. Arch. Microbiol. 17, 384–402 (1952).

[17] Manning, J. E., Wolstenholme, D. R., Ryan, R. S., I1untert, J. A. and Richards, O. C., Circular Chlor-oplast DNA from Euglena gracilis (electron microscopy/density gradient centrifugation/mitochondrial DNA). Proc. Nat. Acad. Sci. USA, 68, No.6, 1169-1173 (1971).

[18] Sheveleva, E. V., Giordani, N. v. and Hallick, R.B., Identification and comparative analysis of the chloroplast -subunit gene of DNA-dependent RNA polymerase from seven Euglena species. Nucleic Acids Research., 30, No. 5 1247–1254 (2002).

[19] Dillard, G. E., Freshwater algae of the southeastern United States. Part 7. Pigmented Euglenophyceae. Bibliotheca Phycologica, Vol. 106. Cramer, Stuttgart, 134 p. + 20 plates and captions (2000).

[20] Kim, J.T., Boo, S.M. and Zakrys, B., Floristic and taxonomic accounts of the genus Euglena (Euglenophyceae) from Korean fresh waters. Algae, 13: 173-197 (1998).

[21] Kudo, R. R., Protozoology. 5th ed. Charles C.Thomas, Illinois. 1174 (1966). [22] Zakrys´, B. and Walne, P. L., Floristic, taxonomic and phytogeographic studies of green

Euglenophyta from the southeastern United States, with emphasis on new and rare species. Algological Studies, 72:71–114 (1994).


Euglenaلجنس اوجزيئية لنوعي تصنيفية مظھريةدراسة )E. viridis وE. gracilis( النامي في حلب، سوريا

أحمد الالحمو نتھى شيخ العشرة، محمد أبيض، م

قسم علم الحياة الحيوانية، كلية العلوم، جامعة حلب، سورية

معايير على باالعتماد التصنيف علم أنجزه الذي الكبير التقدم رغم :المستخلص

يتعلق عندما وخاصة المعضالت من بالكثير البت يستطع لم فإنه تقليديةال التصنيف

على لالعتماد اللجوء تم فقد السبب ولھذا .المتشابھة األنواع بين بالتمييز األمر

الجزيئية األحياء مجال في المعارف في الحاصل التقدم ومع .النووي الطابع دراسة

أصبح فقد متاحا حيث. التصنيفية المشاكل حل في الجزيئية المعطيات اعتماد حاليا

وإن. الجزيئية األحياء علم حقول في جدا مفيدةو فعالة تقانة PCRالـ تقنية تعتبر

ھذه ومن. بالمالحظة وجديرة ھامة مسألة الحية الكائنات طريق عن اإلنتاجية مسألة

حلقة تشكل التي الھامة األولية كائناتال من يعد الذي Euglenaالـ جنس الكائنات

يمكن ال البيئة في متنوعة بوظائف تقوم أفراده أن كما .الغذائية السالسل في أساسية

المياه لتلوث مؤشرا Euglenaالـ جنس أنواع بعض وتعد ھذا. عنھا االستغناء

جاء لذلك األكاديمية الدراسات في أھمية لھا أن كما .بتنقيتھا فتساھم ةالعضوي بالمواد

وتنمية عزال الكائنات ھذه على الضوء ليلقي البحث ھذا وتنميطا ھذا في تمو. جزيئيا

.Euglena E.) gracilis, E الجنس لنوعي محلي تصنيفي مفتاح وضع البحث

virids (معدلة تفريقية أوساط على وزراعتھا أفرادھا عزل عدب )HUT و

Cramer and Myers(. الجزيئية األحيائية المعطيات استعمال أدى وقد )تقانة

للنوعين المتخصصة نوعيةال البادئات واستخدام الكھربائي والرحالن PCRالـ


الكائن به يتميز ما كل أن حيث الموضوعة التصنيفية المعطيات تأكيد إلى) السابقين

DNA الـ خاص وبشكل عضوية جزيئات من يملكه لما انعكاس ھو صفات من

.الوراثية المادة

morphological and molecular taxonomical study of euglena ... · morphological and molecular study...

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