Heredity74 (1995)369—375 Received26 May 1994

Chromosomal markers in three species of the Mytilus (Moflusca: )

A. MARTINEZ-LAGE, A. GONZALEZ-TIZON & J. MENDEZ Departamento de Bio/ogia Ce/u/ar y Molecular, Areade Genética,Universidad de La Coruña, 15071 La Coruña, Spain

Theanalysis of C-banding, NOR and fluorochrome staining was carried out in three species of European , Mytilus edulis, M. galloprovincialis and M. trossulus. The results obtained allow us to detect changes in the constitutive heterochromatin within the genus Mytilus. The existence of chromosomal markers permit us to identify and distinguish, at the cytogenetical level, these three types of mussel.

Keywords:C-bands,chromosome markers, CMA3 bands, heterochromatin, Mytilus, NORs.

along various coastal regions of the British Isles. Subse- Introduction quently, in France, Britain and Ireland, M. galloprovin- Thegenus Mytilus is the subject of an important cialis has been found to be intermixed with M. edulis, controversy about the systematic status of the different producing hybrid forms (Skibinski & Beardmore, forms within it. In this genus, taxonomic studies were 1979). M. trossulus is distributed in the Baltic Sea. In initially developed in order to establish a systematic the Danish straits, M. trossulus is intermixed with M. relationship between M edulis and M. gaioprovincialis edulis (Váinölä & Hvilsom, 1991). and, later, among other forms of . At first, the On the other hand, studies using Mytilus cyto- studies were focused on morphological criteria and genetics have shown that the diploid number is 28 morphometric parameters (internal and external shell chromosomes (Ahmed & Sparks, 1970; Ieyama & characteristics, the anterior adductor muscle features, Inaba, 1974; Thiriot-Quiévreux & Ayraud, 1982; etc.). However, as pointed out by Seed (1968), such Moynihan & Mahon, 1983; Dixon & Flavell, 1986; characters are enormously plastic, being influenced by Pasantes et a!., 1990), and were confined to the factors such as the age of the mussels, the density of the descriptions of the nucleolar organizer regions and the population, the tidal level and habitat type, and even location of heterochromatin in M. edulis (Dixon et a!., then they have not allowed identification of the differ- 1986; Dixon & McFadzen, 1987). In M. galboprovin- ent types of mussels. Later, samples of mussels from cialis a 2 x SSC banding pattern has been induced by different geographical regions in the northern and Méndez et a!. (1990) and NORs were described by southern hemispheres wereelectrophoretically MartInez-Expósito et al. (1994) in four populations of analysed. Different loci were studied, but none of them La Coruña (Galicia, NW Spain) and cytogenetic was truly diagnostic; namely none allowed assignment characterization by means of C-banding, fluoro- of a particular sample to a particular species. However, chromes and restriction endonucleases has been the results obtained after studying several loci per indi- carried out by MartInez-Lage eta!. (1994). vidual and a great number of individuals per popula- In the present paper we describe the differences tion have allowed us to characterize different among M. edubis, M. galboprovinciabis and M. trossulus populations and to discriminate between different on the basis of C-, NOR and fluorescence banding in mussel types within the genus Mytilus (Koehn, 1991; order to detect possible chromosome markers which Gosling, 1992). will allow us to identify, at the individual level, the According to these data, it is considered that there different mussel types. are three species of mussel within the genus Mytilus distributed along European coasts. M. edulis is distri- Materialsand methods buted along the Atlantic coast of Europe; M. ga/b- pro vincialis appears to be distributed along the Takinginto account the distribution of the genus Mediterranean coast, the French Atlantic coast and Mytilus along the European coasts, we have collected samples from places where hybrid forms have not been *Correspondence detected. So, individuals of M. edubis were collected

369 1995The Genetical Society of Great Britain. M gaioprovincialis;and(v) M.gaioprovinciauisshows some 8appearsinM.edulis andM.trossulusbutnotin and M.trossulus;(iv)thetelomeric C-bandofchromo- C-band ofchromosome6 failstoappearinMedulis absent inM.galloprovincialis; (iii)theintercalary shows telomericC-bands ontheparm,whichare us; (ii)chromosome3fromMedulisandM.trossulus are replacedbyaninterstitialoneinM.galloprovincia- in chromosome1ofM.edulisandMtrossulus,which presence ofonecentromericandtwotelomericbands 9 (Fig.2;Table1).Thesedifferencesare:(i)the main differencesinvolvedchromosomes1,3,6,8and of theC-bandsinthesespecies.Weobservedthat tion ofC-bands;thesearelocatedonchromosomes1, 3, 5,6,7,8,12and13.Table1showsthelocalization and 13.MedulisM.trossulusshowthesameloca- C-bands appearonchromosomes1,3,5,6,7,9,12 previously reportedbyMartInez-Lageeta!.(1994), C-bands distribution.InM.gaioprovincialis,as In thesethreespecies,somedifferencesexistinthe C-banding pairs ofsubmeta-subtelocentricchromosomes(Fig.1). M. trossuluscomprisesixpairsofmetacentricandeight The karyotypesofMedulis,M.galloprovincialisand Results Giemsa karyotype For fluorescence,theNikonfiltersemployedwereUV- graphed withaNikonmicrophotAFXmicroscope. methods developedbySchweizer(1976,1980). mycin A3(CMA3),dystainicin(DA)and4'-6 using quinacrine(Casspersoneta!.,1968);chromo- Black (1980).Fluorescenceanalysiswascarriedout nitrate stainingwasperformedaccordingtoHowell& for constitutiveheterochromatinlocalization;meta- from 1A (AD/DAPI),By-lA(CMA3)andB-2A(AO). diamidine-phenyl indole(DAPI)accordingtothe Sorensen's buffer(0.06M,pH6.5)for5mm.Silver phases werestainedwithacridineorange(AO)in M. trossulus. galloprovincialis, andfivefemalessevenmalesof males ofM.edulis,10femalesand Martinez-Lage eta!.(1994),fromsixfemalesandfive zation andculturewascarriedoutasdescribedby (Mecklenburger Bay,Germany).Theprocessoffertili- Spain) andindividualsofMtrossulusfromDahme galloprovincialis fromRIadeBetanzos(LaCoruña, 370 A.MARTINEZ-LAGEETAL. Metaphase chromosomeswereobservedandphoto- The C-bandingmethod(Sumner,1972)wasused Zoutelande (Zeeland,Holland),individualsofM. M gaioprovincialis, andfromtwotofive inMtrossu- NORs, whichvariedfrom two tofourinM.edulisand analysed. Wehavedetected avariablenumberofAg- Forty NOR in theotherspecies. a telomericC-bandonchromosome9,whichisabsent galloprovincialjs andM.trossulus. Fig. 1GiemsametaphaseplatesfromMytilusedulis,M. silver staining 1995 TheGenetical SocietyofGreatBritain,Heredily, 74,4. larval metaphasesofeach musselspecieswere I I . .eJo a,, a 'a 1 I- vr •0

C.a CHROMOSOMAL MARKERS IN GENUS MYTIWS 371 0 -t P P C I- — galloprovincialis from RIa de Betanzos (La Coruña, O•Q I I zation and culture was carried out as described by )l -'4

Martinez-Lage et a!. (1994), from six females and five fl_JDC. I I •

galloprovincialis, and five females and seven males of -. The C-banding method (Sumner, 1972) was used I for constitutive heterochromatin localization; meta- I Sorensen'sphases were buffer stained (0.06 with M, acridinepH 6.5) fororange 5 mm. (AO) Silver in - I' U) I I I using quinacrine (Cassperson et a!., 1968); chromo- a A ' Metaphase chromosomes were observed and photo- I I It- 'C IIIDCT a U I I I I 3 . * 3 * The karyotypes of M edulis, M. galloprovincialis and "C pairsM. trossulus of submeta-subtelocentric comprise six pairs of chromosomes metacentric and (Fig. eight 1). _ I- I.

Fig. 2 C-banding and comparative idiograms of the three European mussel species. e, Mytilus edulis; g, M. — previously reported by MartInez-Lage et a!. (1994), gaioprovincialis; t, M. trossulus; chro. a no., chroniosonie number; ,absence tionand 13.of C-bands;M edulis theseand M. are trossulus located showon chromosomes the same loca- 1, of banding. lus. The silver-stained karyotypes of the three species DA/DAPI produced a negative response on the Fig. 1 Giemsa metaphase plates from Mytilus edulis, M. show chromosomal pairs 6 and 7 (submeta-sub- chromosomes, which was accompanied by a decrease main differences involved chromosomes 1, 3, 6, 8 and telocentrics) with telomeric NORs (Fig. 3a,c,e). In M of the staining in the telomeric regions of chromosomes 9 (Fig. 2; Table 1). These differences are: (i) the trossulus, a third additional NOR appears located on 6 and 7 from the three species (Fig. 3g,h). Similarly, 0 presence of one centromeric and two telomeric bands the p arm of metacentric chromosome 8 (Fig. 3e, Table staining (Fig. 3i) revealed a negative response in each in chromosome 1 of M. edulis and M trossulus, which of the species. are replaced by an interstitiala telomeric one C-band in M. on chromosome galloprovincia- 9, which is absent 2). Fluorochromestaining Discussion absent in M. galloprovincialis; (iii) the intercalary TelomericCMA3 bands were found on chromosomes Thegenus Mytilus is a complex of closely related C-band of chromosome 6NOR failslarval metaphasesto appear of each in mussel M species edulis were 6 and 7 in each one of the three mussel species (Fig. species which share the same basic karyotype in terms and M. trossulus; (iv) the telomericanalysed. We have C-band detected a variable of chromo- number of Ag- 3b,f,g; Table 2). In M gaioprovincialis, a third telo- of external chromosome morphology. When we some 8 appears in M. edulisNORs, and which M. varied trossulus from two to four but in M. not edulis inand meric CMA3bandappears located on the p arm of examined metaphases from M. edulis, M. galloprovin- M gaioprovincialis; and (v)M gaioprovincialis,M. gaioprovinciauis and from two to five inshows M trossu- chromosome 3 (Fig. 3d). cialis and M. trossulus, we observed an uniformity of

1995 The Genetical Society of Great Britain, Heredily, 74, 4. 1995 The Genetical Society of Great Britain, Heredity, 74,4. 372 A.MARTINEZ-LAGE ETAL.

Table I Distribution of C-bands in mussel species chromosomal rearrangements such as fusions or fissions which would alter the morphology of the C. no. M. edulis M. gallopro M. trossulus chromosome complement. Such types of changes have been described by Mayr et al. (1985) in mammals and * by John et a!. (1985) in grasshoppers. However, in lp,t *** — lp,i — mussels, we have not observed differences in the Ic — ** * chromosome length or morphology between the lq,t * species studied; our results suggest that this mechanism 3p,t ** * is not involved in the differentiation of the karyotypes. 3q,t ** ** The second mechanism postulates the transformation 5q,i ** — ** of heterochromatin to euchromatin (or the reverse), 6q,i *** ** 6q,t which would involve internal transformation changes. *** * This process seems to be uncommon, but it has been 7q,t ** * 8p,t — described in rats by Yosida & Sagai (1975), in orthop- *** — 9q,t — terans by Camacho et a!. (1981) and in crocodiles by *** **'K ** 12q,i ** ** ** King et a!. (1986). Initially, our results suggest that the 13q,t differences in the C-banding patterns between M. edu!is—M trossulus and M. galloprovincialis could be dullbands; —no , strongbands; 'u', intermediate bands; *, produced by a heterochromatin—euchromatin trans- banding; p, band on p arm; q, band on q arm; t, telomeric band; c, centromeric band; i, interstitial band; C. no., formation process. As we have pointed out, there are chromosome number. changes in the heterochromatin but chromosomal rearrangements do not exist. On the other hand, the C-bands of the Mytilus chromosome number, relative chromosome size and species analysed in this paper do not show centromeric centromere position. location (except for chromosome 1 from M. edulis and According to our previous report on M. ga/lopro- M. trossulus). To explain the location and distribution vincialis (MartInez-Lage et al., 1994), C-banding of the heterochromatic regions, Macgregor & Sessions analysis confirms that these three species possess small (1986) propose that the centromeres are C-band initia- amounts of constitutive heterochromatjn which is tion sites from which a heterochromatin transference located, mainly, in the telomeres and interstially, and takes place towards the telomeres and, therefore, very rarely located on centromeres. After C-banding, karyotypes with more telomeric heterochromatin have chromosomes from M. edulis and M. trossulus exhibit an older phylogenetic status. In this sense, the presence 11 blocks or regions of constitutive beterochromatin, of more telomeric bands in M. edulis than in M. gallo- and M. galloprovincialis only shows nine hetero- provincialis corroborates the phylogenetic data which chromatin regions (Fig. 2; Table 1). The more remark- indicate that M edulis has an older evolutionary status. able differences in C-bands among these species are The analysis of NOR regions revealed the functional related to chromosome 1 from M. galloprovincialis, location on the telomeres of chromosomes 6 and 7 in which only shows an interstitial C-band, whereas M. each one of the mussel species. In M. trossulus a third edulis and M. trossulus show two telomeric and one additional single NOR was located on the telomere of centromeric C-band (Fig. 2; Table 1). Furthermore, in metacentric chromosome 8. A clear coincidence M. galloprovincialis, the loss of the heterochromatic between C-banding and NORs is detected in chromo- C-bands on the p arms of chromosomes 3 and 8 and somes 6 and 7 from the three species and in chromo- the gain of the interstitial C-band on chromosome 6 some 8 from M. trossulus. Furthermore, CMA3 and the telomeric one on chromosome 9 reflect some staining reveals a positive response on the NOR changes in the heterochromatin of these species in the regions of chromosomes 6 and 7 in the three species. A course of divergence from M edulis. third positive CMA3 band appears on the short arm of It is assumed that the differences in the amount and chromosome 3 in M galloprovincialis, but it does not location of constitutive heterochromatin is a mechan- appear to be an active NOR. The additional NOR ism of karyotype differentiation. In this sense, some detected in M. trossulus appears to be CMA3 negative. models have been proposed to explain these changes Some possible mechanisms have been postulated to or variations in heterochromatin. King (1980) and, explain the appearance of new NORs in species. One later, other authors predicted that the differences in of the most common suggests the activation of latent C-bands could be attributed to the addition or loss of NORs, which take over the primary nucleolar function heterochromatin; this process may be accompanied by under certain conditions or in certain genomes (King,

1995 The Genetical Society of Great Britain, Heredity, 74,4. CHROMOSOMAL MARKERS IN GENUS MYT/LUS 373

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i• I1_ D.C —j — 4-.' Fig. 3 NORs and CMA3 bands in 4 C Mytilus edulis (a,b), M. galloprovincialis (c,d) and M. trossulus (e,f). In (3e) the wide arrow identifies the additional single NOR. (g,h) absence of staining on the telomeric regions of chromo- somes 6 and 7 from M. edulis after DA/ DAPI staining. (i) metaphase from Ps'!, galloprovincialis after quinacrine staim ing.

1980; Verma & Raina, 1981; Fernández-Piqueras et trossulus is CMA3 negative). In this sense, we hope that a!., 1983; Cabrero et a!., 1987). However, we do not in situ hybridization analysis will clarify this situation. think that the additional NOR of M. trossulus consti- The existence of an additional NOR in M trossulus tutes a latent NOR, because this region is shown to allows us to distinguish between this species and M. have a different heterochromatin from the NORs of M. edulis. As both species show the same location and edulis and M gaioprovincialis (chromosomes 6 and 7 distribution of C- and CMA3 bands, let us suppose are CMA3 positive, whereas chromosome 8 from M. that they have the same evolutionary status. The NOR

1995 The Genetical Society of Great Britain, Heredity, 74,4. 374 A.MARTIN EZ-LAGE ETAL.

Table 2Distribution of NORs and CMA3 bands in mussel have taken place in the evolution of this genus result species from qualitative modifications in the constitutive heterochromatin. We hope that studies of satellite DNA M. edulis M gallopro M. trossulus and in situ hybridization will help us to elucidate the C. no.NORCMA3NOR CMA3NORCMA3 role of the constitutive heterochromatin and NORs in

* .. the course of karyotypic divergence of the Mytilus 3p,t — — — * * * * * species studied in this paper. 6q,t * * * * * * 7q,t * * 8p,t — — — — Acknowledgements

'i', presenceof band; —,absenceof band; C. no., chromosome Weare very grateful to Dr H. Hummel, who permitted number; p, band on p arm; q, band on q arm; t, telomeric us to use his laboratory at Delta Instituut voor Hydro- band. biologisch Onderzoek of Yerseke (Holland), and to Miss B. Carril for her technical assistance. This work has been supported by a D.G.I.C.Y.T. 90/0323 (Spain).

of M. trossulus has a different heterochromatin status from the other NORs; this is therefore evidence of its References uniqueness in this species. The additional CMA3 band ARMED,M. AND SPARKS, A. K. 1970. Chromosome number, detected in M. galloprovincialis and the additional structure and autosomal polymorphism in the marine NOR in M trossulus can be considered as chromo- mussels Mytilus edulis and Mytilus californianus. Biol. somal markers which allow us to identify and Bull., 138, 1—13. distinguish the three types of mussels. CABRERO, J., ALCHE, J. D. AND CAMACHO, 1. P. M. 1987. Effects of B On the other hand, staining with AD/DAPI reveals chromosomes on the activity of nucleolar organizer an absence of banding on the chromosomes of the regions in the grasshopper Eyprepocnemis plorans: activa- genus Mytilus. The negative staining observed in the tion of a latent nucleolar organizer region on a B chromo- telomeric areas of chromosomes 6 and 7 (Fig. 3g,h) some fused to an autosome. Genorne, 29, 116—121. had already been observed by Dixon & Flavell (1986) CAMACHO, J. P. M., CABRERO, J. AND VISERAS, n. 1981. C-hetero- chromatin variation in the genus Eumigus (Orthoptera: in M. edulis when they treated the chromosomes with Pamphagoidea). Genetica, 56, 185—188. Giemsa staining and/or borate buffer treatment. The CASPERSSON, T, FABER, S., FOLEY, G. E., KUDYNOWSKI, J., MODEST, E. existence of these areas, which are coincident with the J., SIMONSSON, F., WAGH, U. AND ZECH, L. 1968. Chemical locations of active NORs, led us to suppose that a differentiation along metaphase chromosomes. Exp. Cell correlation between chromatin decondensation and Res., 49, 2 19—222. transcriptional activity must exist in these organisms. DIXON, D. R. AND FLAvELL, N. 1986. A comparative study of the Furthermore, the fact that these telomeric regions were chromosomes of Mytilus edulis and Mytilus galloprovin- shown to be C positive/CMA3 positive/NOR positive! cialis. .1. Mar. Biol. Ass. UK., 66, 2 19—228. AD/DAPI negative, confirms the absence of AT-rich DIXON, DR. AND MCFADZEN, I. R. B. 1987. Heterochromatjn in the base pairs, in opposition to the data reported by Dixon interphase nuclei of the common mussel Mytilus edulLs L. & McFadzen (1987) from M edulis that C positive! J. Exp. Mar. Biol. Ecol., 112, 1—9. NOR positive regions were AT-rich. The negative DIXON, D. R., McFADZEN. I. R. B. AND SISLEY, K. 1986. Hetero- chromatic marker regions (nucleolar organisers) in the response after Qbandingseems to reflect the non- chromosomes of the common mussel, Mytilus edulis existence of AT-rich regions along the chromosome (: Pelecypoda). .1. Exp. Mar. Biol. Ecol., 97, structure of these organisms. However, taking into 205—2 12. account the results obtained after in situ digestion with FERNANDEZ-PIQUERAS, J., RODRIGUEZ CAMPOS, A., SENTI S CASTAO, restriction endonucleases (unpublished data), we could C. AND ROJO GARCIA, E. 1983. Chromosomal location of the suppose that AT sequences are interspersed in the active NORs in the Steropleurus martorelli complex. genomes of these species and, consequently, there Genetica, 61,9—12. could exist a certain degree of genome compartmental- GOSLING, F.M. (ED.) 1992. The Mussel Mytilus. Elsevier Press, ization. Amsterdam. In conclusion, the distribution of C-bands and the HOWELL, W M. AND BLACK, D. A. 1980. Controlled silver-staining existence of chromosomal markers allow us to of nucleolus organizer regions with a protective colloidal developer, a 1-step method. Experientia, 36, 1014—1015. distinguish these mussel species, and open up new IEYAMA, H. AND INABA, A. 1974. Chromosome numbers of ten possibilities to clarify the systematic status of the genus species in four families of Pteriomorphia (Bivalvia). Venus, Mytilus. We consider that the karyotypic changes which 33, 129—137.

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JOHN, B., KING, M., SCHWEIZER, D. AND MENDELAK, M. 1985. MOYNIHAN, u. pANDMAFION, G. A. 1. 1983. Quantitative karyo- Equilocality of heterochromatin distribution and hetero- type analysis in the mussel Mytilus edulis L. Aquaculture, chromatin heterogeneity in acridid grasshoppers. 33, 30 1—309. Chromosoma, 91,185-200. PASANTES, 3., MART(NEZ-EXPOSITO, M. 3., MARTINEZ-LAGE, A. AND KING, M. 1980. C-banding studies on Australian hylid frogs: MENDFz, j.1990.Chromosomes of Galician mussels. .J. secondary constriction structure and the concept of Mo!!. Stud., 56, 123—126. euchromatin transformation. Chromosoma, 80, 191—217. SCHWEIZER, D. 1976. Reverse fluorescent chromosome KING, M., HONEYCUTI', R. AND CONTRERAS, N. 1986. Chromosomal banding with chromomycin and DAPI. Chromosoma, 58, repatterning in crocodiles: C, G and N-banding and the in 307—324. situ hybridization of 18S and 26S rRNA cistrons. SCHWEIZER, D. 1980. Simultaneous fluorescent staining of R Genetica, 70, 191—201. bands and specific heterochromatic regions (DA-DAPI KOEHN, R. K. 1991. The genetics and of species in bands) in human chromosomes. Cytogenet. Cell Genet., the genus Mytilus. Aquaculture, 94,125—145. 27, 190—193. MACGREGOR, H. C. AND SESSIONS, S. K. 1986. The biological SEED, R. 1968. Factors influencing shell shape in the mussel significance of variation in satellite DNA and hetero- Mytilus edulis. J. Mar. Biol. Ass. UK., 48, 56 1—584. chromatin in newts of the genus Triturus: an evolutionary SKIBINSKI, D. 0. F AND BEARDMORE, J. A. 1979. A genetic study of perspective. Phil. Trans. R. Soc. B, 312, 243—259. intergradation between Mytilus edulis and Mytilus galbo- MARTINEZ-EXPO5ITO,M. J., PASANTES, J. J. AND MENDEZ, j.1994. pro vincialis. Experientia, 35, 1442—1444. NOR activity in larval and adult mussel Mytilus ga/b- SUMNER, A. T. 1972. A simple technique for demonstrating provincialisL. J. Exp. Mar. Biol. Ecol., 94,155—165. centromeric heterochromatin. Exp. Cell Res., 75, MARTINEZ-LAGE, A., GONZALEZ-TIZON, A. AND MENDEZ, j.1994. 304—306. Characterization of different chromatin types in Mytilus THIRIOT-QUTEYREUX, C. AND AYRAUD, N. 1982. Les caryotypes de galboprovincialis L. after C-banding, fluorochrome and quelques espèces de bivalves et de gastéropodes marins. restriction endonuclease treatments.Heredity, 72, Mar. Biol., 70, 165—172. 242—249. vAINOLA, R. AND HvILSOM, M. M. 1991. Genetic divergence and a MAYR, B., SCHWEIZER, D., MENDELAK, M., KRUTZLER, J., SCHLEGER, hybrid zone between Baltic and North Sea Mytilus w., KALAT, M. AND AUER, H. 1985. Levels of conservation and populations (; Mollusca). Biol. J. Linn. Soc., 43, variation of heterochromatin and nucleolus organizers in 127—148. the Bovidae. Can. J. Genet. Cytol., 27, 665—682. VERMA, R. C. AND RAINA, S. N. 1981. Cytogenetics of Crotolaria. MENDEZ, 3., PASANTES, 3. J. AND MARTINEZ-EXPOSITO, M. j.1990. V. Supernumerary nucleoli in C. agatiflora (Leguminosae). Banding pattern of mussel (Mytilus galloprovincialis) Genetica, 56, 75—80. chromosomes induced by 2 x SSC/Gienisa-stain treat- YOSIDA, T. H. AND SAGAI. T. 1975. Variation of C-bands in ment. Mar. Biol., 106,375—377. several subspecies of Rattus rattus. Chromosoma, 50, 283—300.

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