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  1. 1. Micron 36 (2005) 359–367 Ultrastructural characterization of the hemocytes of Culex quinquefasciatus (DIPTERA: Culicidae) F.A. Braynera,b,*, H.R.C. Araujoa,b, M.G.S. Cavalcantia,b, L.C. Alvesa,b, C.A. Peixotoa,b,1 ´a ˜ Departamento de Biologia Celular e Ultraestrutura, Centro de Pesquisas Aggeu Magalhaes (FIOCRUZ), Av. Moraes Rego s/n, Recife 50670-420, Brazil b ´ Laboratorio de Imunopatologia Keizo Asami (LIKA) da Universidade Federal de Pernambuco, Recife, Brazil Received 20 May 2004; revised 30 November 2004; accepted 30 November 2004Abstract Six hemocytes cell types from Culex quinquefasciatus were identified by light and transmission electron microscopy: They areprohemocytes (9.3%), spherulocytes (1.6%), adipohemocytes (0.8%), oenocytoids (4.6%), plasmatocytes (43.4%) and granulocytes (40.3%).The prohemocytes were the smallest hemocytes encountered in the hemolymph, displaying a large and centrally located nucleus, almostfilling the whole cell. The spherulocytes, which were small hemocytes, presented small and numerous spherules with a lamellar pattern andan electron-dense core. Rare adipohemocytes were observed in the C. quinquefasciatus hemolymph, presenting large nucleus with an evidentnucleolus, cytoplasm containing rough endoplasmic reticulum (RER), mitochondriae and lipid inclusions. C. quinquefasciatus oenocytoidsshowed homogeneous cytoplasm with several granules, completely or partially filled with amorphous material. These cells showed abundantsmooth endoplasmic reticulum (SER) and dense mitochondriae. By light microscopy analysis we identified two morphological types ofplasmatocytes, granular and agranular. However, ultrastructural investigation revealed that the granular cells contained lipid inclusionbetween RER membranes, instead of membrane-bounded granules. The granulocytes presented a fusiform or circular profile and displayed aunique and very complex process of granules formation, including organization of polysomes inside vesicles that protrude from the Golgisystem, synthesis of a proteinaceous material, condensation of the granule matrix and recycling of endoplasmic membranes. Intenseendocytic pathways were also observed in the granulocytes.q 2005 Elsevier Ltd. All rights reserved.Keywords: Culex quinquefasciatus (Insecta); Hemocytes; Light microscopy; Electron microscopy; Morphology; Mosquito1. Introduction immune responses, like phagocytosis, nodulation and encapsulation (Pech and Strand, 2000). However, accord- In insects, the immune system includes both humoral ing to Lavine and Strand (2002) this subdivision of theand cellular components. Humoral defenses involve the insect immune system into cellular and humoralproduction of antimicrobial peptides (Lowenberger, responses is somewhat arbitrary since humoral factors2001), reactive free radical intermediates of oxygen or affect hemocyte function and on the other hand,nitrogen (Vass and Nappi, 2001), and the complex hemocytes are an important source of many humoralenzymatic cascades that regulate coagulation or melani- molecules.zation of hemolymph (Muta and Iwanaga, 1996). The hemocytes have the ability to defend insects againstIn contrast, cellular defense refers to hemocyte-mediated pathogens, parasites and other foreign bodies, which entered in the hemocoel. These defense reactions are mediated by phagocytosis, encapsulation, wound repair and coagulation * Corresponding author. Address: Laboratorio de Imunopatologia Keizo ´ (Lavine and Strand, 2002; Falleiros et al., 2003). TheAsami (LIKA) da Universidade Federal de Pernambuco, Recife, Brazil.Tel.: C55 81 3301 2540; fax: C55 81 3453 2449. population of circulating hemocytes is an important tool to E-mail addresses: (F.A. Brayner), understand the host–parasite interactions, since (C.A. Peixoto). ment in the total and differential number of hemocyte may 1 Tel.: C55 81 3301 2557; fax: C55 81 3453 2449. contribute to the protection against a specific parasite0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. (Nappi and Christensen, 1986; Christensen et al., 1989;doi:10.1016/j.micron.2004.11.007 Da Silva et al., 2000).
  2. 2. 360 F.A. Brayner et al. / Micron 36 (2005) 359–367 Studies by means transmission electron microscopy pH 7.2 and post-fixed with osmium tetroxide in cacodylate(TEM) have produced hemocyte classification by identifi- buffer for 1 h. After dehydration in graded acetone series,cation of seven morphological types: prohemocytes, the cells were embedded in EMBED 812/Araldite (Electronplasmatocytes, granulocytes, spherulocytes, adipohemo- Microscopy Sciences, Fort Washington, PA).cytes, oenocytoids and coagulocytes (Beeman et al., 1983; ˇHypsa and Grubhoffer, 1997; Giulianini et al., 2003; 3. ResultsFalleiros et al., 2003). However, several authors havefound between three and four hemocyte types in different ´ Six morphological types of the circulating cells can begenera of mosquitoes (Hernandez et al., 1999; Da Silva recognized in the hemolymph of adult C. al., 2000; Hillyer and Christensen, 2002; Hillyer et al., They are prohemocytes, spherulocytes, adipohemocytes,2003). Culex quinquefasciatus is a culicid mosquito that oenocytoids, plasmatocytes and granulocytes.transmits several pathogens, including Wuchereria ban-crofti and arboviruses, such as St Louis encephalitis (SLE)in Western States and Oropouch virus in the north of Brazil 3.1. Prohemocytes(Da Silva et al., 2000). Conversely, there are very fewpapers about the morphological characterization of hemo- Prohemocytes are the smallest cells encountered in thecytes of C. quinquefasciatus and this is due in large part to hemolymph, displaying a spherical profile with w5–8 mmthe small size of these insects, which is difficult to perform in diameter, which represents 9.3% of the total hemocytemanipulative experiments. The aim of the present study was population. The large and centrally located nucleusto characterize by the first time distinct morphological types almost fills the whole cell, so that the cytoplasmof hemocytes of C. quinquefasciatus by transmission occupies only a narrow area around the nucleuselectron microscopy. (Figs. 1A and 2A and B). The chromatin is scattered, and in some cells the presence of nucleoli is evident. Only a few organelles can be seen, with a conspicuous2. Materials and methods development of rough endoplasmic reticulum (RER) and mitochondriae (Fig. 2A and B).2.1. Insects 3.2. Spherulocytes Laboratory bred C. quinquefasciatus (Recife strain) wereused throughout this study. Adults were maintained in cages Spherulocytes shows an oval cell profile with average(30!30!30 cm3) at room temperature (27G3 8C) with diameter of 8–10 mm, displaying a round nucleus and a85G10% relative humidity and fed with 10% (w/v) sucrose condensed chromatin with a large nucleolus (Fig. 2C). Severalsolution. small spherules (1–1.5 mm in diameter) (Figs. 1B and 2C and D) containing a lamellar pattern with an electron-dense2.2. Hemocytes characterization core region (Fig. 2C and D). The cytoplasm also contained other organelles such as RER and mitochondriae (Fig. 2C). For light microscopy (LM), the adult insects were washed The spherulocytes represented 1.6% of the total PBS and placed on ice (1–2 min) for immobilization. Thehemolymph of 10 insects (4-day-old) was obtained by 3.3. Adipohemocytesperfusing the thorax with anticoagulant II solution (Meadet al., 1986) and bled directly on to a glass slide and allowed Adipohemocytes are rare small and elongated cellsdry in natural air conditions for 20–30 min. Cells were then measuring 8–15 mm in length. In the adipohemocytesfixed in methanol for 10 min. After natural air-drying of the observed in this study, a round nucleus could befixative, hemocytes were stained with Giemsa (diluted 1:9 in observed (Fig. 1C). Inside the cytoplasm several largebuffered distilled water) for 10–15 min and slides were rapidly lipid vesicles and mitochondriae (Figs. 1C and 2E and F)washed with buffered distilled water (Da Silva et al., 2000; were observed. They were the less frequent hemocytes,Silva et al., 2002). After air drying the slides were dehydrated with 0.8%.and mounted in Entellan. For transmission electron microscopy (TEM), hemo- 3.4. Oenocytoidslymph of at least 300 insects (4-day-old) was collected froma punctured thorax perfused directly with fixation solution Oenocytoids presents a round shape, approximatellyand the obtained hemolymph was pooled and centrifuged at 6–13 mm in diameter, with small and eccentric nucleus500g for 5 min. The pellet was resuspended and fixed in 4% (Figs. 1D and 3A) or oval with chromatin showing lumps ofglutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, over- condensation. The ultrastructure revealed a cytoplasm ˇnight (Hypsa and Grubhoffer, 1997). The samples were rich in vacuoles, some filled with a hetrogeneous electron-washed in 5% sucrose solution in 0.2 M cacodylate buffer, dense material and others completely empties. It presents
  3. 3. F.A. Brayner et al. / Micron 36 (2005) 359–367 361Fig. 1. Light microscopy of hemocytes from C quinquefasciatus. (A) A prohemocyte with a large nucleus (thin arrow) containing a prominentnucleolus (arrowhead). (B) A spherulocyte showing an oval profile and numerous spherules within the cytoplasm (thin arrows). (C) An adipohemocyteshowing an irregular cell profile and lipid vesicles in the cytoplasm (thin arrows). (D) A oenocytoid with a round eccentric nucleus (thin arrow). (E) Agranular plasmatocyte exhibiting a central nucleus with nucleolus (thin arrow). In the cytoplasm, vacuoles (short arrows) and granules (arrowheads) areshown. Observed also a phylopodium process (open arrow). (F) An agranular plasmatocyte spreading a lobular pseudopodia (arrowhead). Note also theeccentric nucleus (thin arrow). (G) A granulocyte displaying a large mass of heterochomatin (long arrow) inside the nucleus. Observe also severalgranules in the cytoplasm (arrowheads) and a short phylopodium (large arrow). (H) A granulocyte with multiple pseudopodia (short arrow). Observealso the large nucleus and several dense compact granules (arrowheads). BarsZ5 mm.
  4. 4. 362 F.A. Brayner et al. / Micron 36 (2005) 359–367Fig. 2. (A–F) Electron micrographs of hemocytes from C. quinquefasciatus. (A and B) Prohemocytes displaying spherical profiles with a large central nucleus(N) containing nucleolus (Nu) and a thin cytoplasm with few organelles. Note lumps of heterochromatin (arrows). Mitochondriae (m) and RER (arrowheads)are also indicated. (C) A spherulocyte with numerous spherules showing a round nucleus with condensed chromatin (N) and nucleolus (Nu), RER complex(thin arrow) and mitochondrion (m). (D) Magnification of the spherules showing a lamellar pattern with an electron-dense core region (arrows). (E–F)Adipohemocytes with oval profile showing large nucleus (N) containing evident nucleolus (Nu). Observe cytoplasm with several lipid vesicles inside (stars)and mitochondriae (m). BarsZ0.5 mm.a homogeneous cytoplasm with abundant SER and dense philopodia and pseudopodia (Fig. 1E and F). The majoritymitochondriae. Sparce RER is also present (Fig. 3A). They of the plasmatocytes were mononucleated but someare presented 4.6% of the total circulating hemocytes. binucleated cells were occasionally observed. In electron micrographs agranular plasmatocytes presented a lobated3.5. Plasmatocytes nucleus with evident nucleoli in a pericentral position (Fig. 3B). The chromatin was finely distributed but some The plasmatocytes were the most cellular types fre- heterochromatin clumps were present. Within the cytoplasmquently observed, representing 43.4%. Two types of several elongated and round mitochondria were observed.plasmatocytes were observed, granular and agranular The reticular cytoplasm showed well-developed RER,plasmatocytes. These cells are very polymorphic, varying Golgi system and some vacuoles. Also patches of smoothfrom spindle-shaped to round cells, w6–22 mm in diameter. endoplasmic reticulum SER were present at the cell polesThe plasma membrane exhibit irregular processes, (Fig. 3B). The granular plasmatocytes showed elongated or
  5. 5. F.A. Brayner et al. / Micron 36 (2005) 359–367 363Fig. 3. (A) An oenocytoide showing an eccentric lobated nucleus (N) with clumps of hetrochromatin (short arrows). Several cytoplasmic vacuoles arepresent, some filled with a heterogeneous electron-dense material (large arrowheads) and others completely empty (thin arrows). Note also electron-dense mitochondriae (arrows), abundant SER (small arrowheads) and few RER (open short arrow). (B) An agranular plasmatocyte showing a lobatednucleus (N) with nucleolus (Nu) in a pericentral position. Some heterochromatin lumps are present (arrows). Within the cytoplasm several elongatedand round mitochondria are present (thin arrows). Note the well-developed RER (arrowheads) and isles of SER were presented (stars). (C) A granularplasmatocyte showing elongated nucleus (N) with a scattered mass of heterochromatin. Abundant RER (arrowheads), lipid inclusions located amongendoplasmic membranes (thin arrows), and electron-dense inclusions (open arrow) are indicated. Part of adjacent granular plasmatocyte contains lipidinclusions (thin arrow). BarsZ0.5 mm.circular nucleus displaying a scattered mass of hetero- 3.6. Granulocyteschomatin, and in some cells a large nucleolus could beobserved (not shown). The cytoplasm was rich in RER and Granulocytes present a circular to fusiform profileGolgi system. The numerous granules seen by LM were w8–13 mm in diameter, which represents 40.3% of theobserved by EM with no limiting membrane, similar to total hemocyte population. The plasma membrane iscellular inclusions, showing a homogeneous matrix and irregular displaying pseudopodia and philopodia in itslocated among endoplasmic membranes. Some of the surface (Fig. 1G and H). In electron micrographs, theseinclusions show an electron-dense core, possibly indicating hemocytes show important morphological characteristics.a dual constitution (Fig. 3C). The lobated nucleus has a large mass of heterochomatin and
  6. 6. 364 F.A. Brayner et al. / Micron 36 (2005) 359–367Fig. 4. A granulocyte showing a lobated nucleus (N) with a large mass of heterochomatin (large short arrows) and an active nucleolus (Nu). Endocytic processwith the presence of numerous coated vesicles (short arrows) and coated pits (open short arrow) and mitochondria (m). The cytoplasm contains sparce RER(thin arrows), abundant free ribosomes (small arrows) with formation of numerous polysomes (arrowheads). A well-developed Golgi complex displays vesicleproduction (G). The following steps of the granule synthesis are indicated. (1) Large vesicles containing engulfed cytoplasm with polysomes (arrowheads), freeribosomes (small arrow) and occasional mitochondria (m). (2) Vesicles containing large empty vacuoles (V) and ribosomes, organized as small polysomes(arrowheads). (3) Initial synthesis of a dense proteinaceous matrix (M) by polysomes (arrowheads). (4) Fusion of multiple polysomes (arrowheads) andformation of the granule matrix (M). (5) Condensation of the granule with production small vesicles in order to eliminate ribosomes and recycle endoplasmicmembranes (r). (6) A mature granule inside the cytoplasm. (7) A granule being exocytosed by the cell. BarZ0.5 active nucleolus and a clear endocytic process with the final stage, the granule expells the ribosomes and producespresence of numerous coated vesicles and coated pits is small vesicles in order to condense the proteinaceous matrixpresent. Round or elongated mitochondria are also detected. and recycle intracellular membranes. (6) The matureThe cytoplasm contains dilated RER, formation of poly- granule is then free inside the cytoplasm. (7) Finally, thesomes and abundant ribosomes. Also, a well-developed granule is exocysed by the cell.Golgi complex displays large vesicle production (Fig. 4). Synthesis of a second type of granule is also shown.All the steps of the granule synthesis machinary can be A vesicle containing several sites of membranes ofobserved, as follows: (1) large vesicles containing engulfed endoplasmic membranes producing a very electron-densecytoplasm with free ribosomes and occasional mitochon- matrix was observed (Fig. 5). A large electron-densedria; (2) some vesicles contain large empty vacuoles granule is shown, probably due to the fusion of the productspossibly, due to the membrane fusion with other empty of several synthesis sites.vacuoles. Also, ribosomes are organized as numerous large It is important to note that near to this synthesis granulepolysomes, some of which show the formation of other machinery several elongated or round mitochondria aresmall polysomes. (3) Initial synthesis of a proteinaceous present. In addition, in many other granulocytes we foundmatrix inside the polysomes; (4) fusion of multiple mitochondria inside the vesicle of synthesis of the granulepolysomes and formation of the granule matrix. (5) At the (Figs. 4 and 5).
  7. 7. F.A. Brayner et al. / Micron 36 (2005) 359–367 365Fig. 5. A granulocyte showing a lobated nucleus (N) with a large mass of heterochromatin (short arrows) and an active nucleolus (Nu). Several elongatedmitochondria (m) are also present. The cytoplasm contain sparce RER (thin arrows), abundant free ribosomes (small arrows) and formation of numerouspolysomes (arrowheads). The following steps of the granule machinary synthesis are indicated. (1) Not shown. (2) Vesicle containing empty vacuoles (V)and free ribosomes organized as numerous polysomes, some of which are small polysomes (arrowheads). Note the elongated mitochondrion attached to thevesicle membrane (open short arrow). (3) Initial synthesis of a dense proteinaceous matrix by polysomes (M). Note also the multiple polysomes(arrowheads). (4) Fusion of multiple polysomes and formation of the granule matrix (M). Multiple polysomes (arrowheads) are also present. (5)Condensation of granule and recycle of endoplasmic membranes (r). (6) Not shown. (7) A granule exocyted by the cell. Note also a second type of granulesynthesis: a vesicle containing several sites of synthesis of very an electrondense matrix (small short arrows) and the formation of a large granule inside(white asterisk). BarZ0.5 mm.4. Discussion recognized as the spherulocyte. Recently, Hillyer et al. (2003) described only four morphological cell types in C. quinquefasciatus adults possess six different types of Armigeres subalbatus, a natural vector of Japanese ence-hemocytes, which vary considerably in their morphology and phalitis and filarial nematodes: granulocytes, oenocytoids,size, namely: prohemocytes, spherulocytes, adipohemo- adipohemocytes and thrombocytoids.cytes, oenocytoids, plasmatocytes and granulocytes. Silva In the present study, we observed that prohemocyteset al. (2002) studying Anastrepha obliqua instar larva, which displayed unmistakable characteristics such as small sizeis a member of the Diptera Order, described a similar range of and large nuclear–cytoplasmic ratio, with a morphologymorphological types of hemocytes by LM analysis. Analo- identical to that described in previous hemocyte studiesgous results were also obtained by Kaaya and Ratcliffe ˇ (Hypsa and Grubhoffer, 1997; Falleiros et al., 2003;(1982) who performed an important morphological com- Giulianini et al., 2003).parison of hemocytes from several medical important Some studies have described spherulocytes as hemocytes-dipterans, including the C. quinquefasciatus. However, in containing large membrane-bound spherules that deform thethese LM studies, they did not identify the hemocyte cellular surface. In C. quinquefasciatus, these cells presented
  8. 8. 366 F.A. Brayner et al. / Micron 36 (2005) 359–367similar organelles, showing moderated electron density with of a proteinaceous material, and condensation of thean electron dense core, as described for other species granule matrix with recycling of endoplasmic membranes.(Beeman et al., 1983; Falleiros et al., 2003). However, the Synthesis of a second type of granule was also observed,size of spherules of these hemocytes was smaller than those occurring inside vesicles almost free of ribosomes, whichdescribed previously and did not protrude from the cellular contained an electron-dense matrix probably composed ofsurface. Giulianini et al. (2003) observed a similar spherule substances other than proteins. On the other hand,size in Cetonischema aeruginosa larvae (Coleoptera, granular plasmatocytes showed no such machinery forScarabaeidae). synthesis of granules, instead they display several small Rarely, adipohemocytes were observed in the present lipid inclusions among ER membranes. So, we not agreework, mostly because of the instability of these cells. ˇ with Hypsa and Grubhoffer (1997) since plasmatocytesBesides large lipid vesicles, other cellular organelles could (granular and granular) and granulocytes presented abe readily identified, i.e., scarce RER and mitochondria. completely different morphology, and thus may belong toConversely, Hillyer et al. (2003) described that these cells a distinct class of hemocytes, however, functional studieswere the second most common cells obtained from naive are necessary to confirm this.mosquitoes. In our study, adipohemocytes presented similarsize to granulocytes and oenocytoids, whereas Hillyer et al.(2003) found that these cells were w30 mm in diameter,being several times the size of granulocytes and oenocy-toids. Some authors do not regard the adipohemocyte as a Acknowledgementsdistinct hemocyte type, since they considered their mor-phology to be very similar to that of granulocytes, and also The authors are grateful to Rafael Padilha, Raimundoindicated that these cells are most similar to the fat body ´ Pimentel and Sergio Santos for the precious technicalcells (Kaaya and Ratcliffe, 1982). However, we did not support. This work has been supported by Fundacao ¸˜observe any similarity between these two cellular types and Oswaldo Cruz (FIOCRUZ).therefore further studies are necessary to clarify thiscontroversial matter. Oenocytoids have been described as cells typically Referencescontaining a greater number of dense granules located at ˇthe cell periphery (Hyps a and Grubhoffer, 1997; Beeman, S.C., Wilson, M.E., Bulla Jr., L.A., Consigli, R.A., 1983.Giulianini et al., 2003). In EM analyses, we detected Structural characterization of the hemocytes of Plodia interpunctella.that in oenocytoids from C. quinquefasciatus these Journal of Morphology 175, 1–16.granules were completely or partially filled with Christensen, B.M., Huff, B.M., Miranpuri, G.S., Harris, K.L.,amorphous material. Christensen, L.A., 1989. Hemocyte population changes during the immune response of Aedes aegypti to inoculated microfilariae of By LM analysis we identified two morphological types Dirofilaria immitis. Journal of Parasitology 75, 119–123.of plasmatocytes, granular and agranular. However, ´ Da Silva, J.B., Albuquerque, C.M.R., Araujo, E.C., Peixoto, C.A., Hurd, H.,ultrastructural investigation revealed that the granular 2000. Immune defense mechanisms of Culex quinquefasciatuscells do in fact contain lipid inclusions between RER, (Diptera: Culicidae) against Candida albicans infection. Journal ofrather than membrane-bounded granules. In Triatoma Invertebrate Pathology 76, 257–262. ´ Falleiros, A.M.F., Bombonato, M.T.S., Gregorio, E.A., 2003. Ultrastruc-infestans agranular and granular plasmatocytes were tural and quantitative studies of hemocytes in the sugarcane borer, ˇdescribed by LM and EM analysis (Hypsa and Grubhoffer, Diatraea saccharalis (Lepidoptera: Pyralidae). Brazilian Archives of1997). These authors demonstrated that granular plasma- Biology and Technology 46, 287–294.tocytes contained granules composed by more or less Giulianini, P.G., Bertolo, F., Battistella, S., Amirante, G.A., 2003.regularly arranged fibrils, through very dense to appar- Ultrastructure of the hemocytes of Cetonischema aeruginosa larvae (Coleoptera, Scarabeidae): involvement of both granulocytes andently amorphous matrix. According to the Hypsa and ˇ oenocytoids in vivo phagocytosis. Tissue and Cell 35, 243–251.Grubhoffer (1997) terminology, different types of prohe- ´ ´ Hernandez, S., Lanz, H., Rodrıguez, M.H., Torres, J.A., Martınez- ´mocytes generate two cellular lineages: granular or Palomo, A., Tsutsumi, V., 1999. Morphological and cytochemicalagranular plasmocytes. In their opinion, the term granulo- characterization of female Anopheles albimanus (Diptera: Culicidae)cyte is unacceptable, considering that granulocytes are hemocytes. Journal of Medical Entomology 36, 426–434. Hillyer, J.F., Christensen, B.M., 2002. Chracterization of hemocytes fromcommonly regarded as cells closely related to plasmato- the yellow fever mosquito, A. aegypti. Histochemistry and cell Biologycytes or even differentiated from them. However, in our 313, 117– it was shown that cells termed granulocytes were Hillyer, J.F., Schmidt, S.L., Christensen, B.M., 2003. Hemocyte-mediatedquite different from plasmatocytes in several aspects. The phagocytosis and melanization in the mosquito Armigeres subalbatusresults of the present work indicate that the process of following immune challenge by bacteria. Cell and Tissue Research 313, 117–127.granule formation in granulocytes is unique and very ˇ Hypsa, V., Grubhoffer, L., 1997. Two hemocyte populations in Triatomacomplex, including organization of polysomes insides infestans: ultrastructural and lectin-binding characterization. Foliavesicles, which protrude from the Golgi system, synthesis Parasitologica 44, 62–70.
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